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Open Access

Peer-reviewed

Research Article

Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa

  • Oluwatayo E. Abioye ,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

    abioyethayor@gmail.com

    Affiliation Department of Microbiology, Obafemi Awolowo University, Ife, Nigeria

    ⨯
  • Nolonwabo Nontongana,

    Roles Formal analysis, Investigation, Methodology, Resources, Writing – review & editing

    Affiliation SAMRC Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice, South Africa

    ⨯
  • Charles A. Osunla,

    Roles Formal analysis, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Microbiology, Adekunle Ajasin University, Akungba Akoko, Nigeria

    ⨯
  • Anthony I. Okoh

    Roles Conceptualization, Funding acquisition, Investigation, Resources, Validation, Writing – review & editing

    Affiliation SAMRC Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice, South Africa

    ⨯

Abstract

The current study determines the density of Vibrio spp. and isolates V. cholerae and Vibrio mimicus from fish-anatomical-sites, prawn, crab and mussel samples recovered from fish markets, freshwater and brackish water. Virulence and antibiotic resistance profiling of isolates were carried out using standard molecular and microbiology techniques. Vibrio spp. was detected in more than 90% of samples [134/144] and its density was significantly more in fish than in other samples. Vibrio. cholerae and V. mimicus were isolated in at least one sample of each sample type with higher isolation frequency in fish samples. All the V. cholerae isolates belong to non-O1/non-O139 serogroup. One or more V. cholerae isolates exhibited intermediate or resistance against each of the eighteen panels of antibiotics used but 100% of the V. mimicus were susceptible to amikacin, gentamycin and chloramphenicol. Vibrio cholerae exhibited relatively high resistance against polymyxin, ampicillin and amoxicillin/clavulanate while V. mimicus isolates exhibited relatively high resistance against nitrofurantoin, ampicillin and polymixin. The multiple-antibiotic-resistance-index [MARI] for isolates ranges between 0 and 0.67 and 48% of the isolates have MARI that is >0.2 while 55% of the isolates exhibit MultiDrug Resistance Phenotypes. The percentage detection of acc, ant, drf18, sul1, mcr-1, blasvh, blaoxa, blatem, blaoxa48, gyrA, gyrB and parC resistance-associated genes were 2%, 9%, 14%, 7%, 2%, 25%, 7%, 2%, 2%, 32%, 25% and 27% respectively while that for virulence-associated genes in increasing other was ace [2%], tcp [11%], vpi [16%], ompU [34%], toxR [43%], rtxC [70%], rtxA [73%] and hyla [77%]. The study confirmed the potential of environmental non-O1/non-O139 V. cholerae and V. mimicus to cause cholera-like infection and other vibriosis which could be difficult to manage with commonly recommended antibiotics. Thus, regular monitoring of the environment to create necessary awareness for this kind of pathogens is important in the interest of public health.

Citation: Abioye OE, Nontongana N, Osunla CA, Okoh AI (2023) Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa. PLoS ONE 18(8): e0290356. https://doi.org/10.1371/journal.pone.0290356

Editor: Kumar Venkitanarayanan, University of Connecticut, UNITED STATES

Received: April 6, 2023; Accepted: August 4, 2023; Published: August 24, 2023

Copyright: © 2023 Abioye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: This work was supported by Water Research Commission (Grant number: K5 Number: 2432) https://www.wrc.org.za/ and South Africa and Medical Research Council (SAMRC), South Africa https://www.samrc.ac.za/. AIO received the grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Vibrio cholerae strains that cause cholera epidemics and pandemics are found in marine and freshwater environments [1]. Vibrio mimicus is a close relative of V. cholerae and it causes gastroenteritis with symptoms similar to that of cholera. The vehicles of transmission of the two bacteria are water, fish and seafood contaminated with the bacteria [2–5]. The effective dose of V. mimicus for causing human infection is believed to be between 104–106 similar to that of V. cholerae which is between 103–106 [3]. Vibrio mimicus and V. cholerae have been isolated from the aquatic environment and seafood across the globe [3, 6–10]. They cause economic losses in aquaculture and mariculture globally [8, 10–12]. There have been seven cholera pandemics to date. The first six affected all the continents of the world while the ongoing seventh cholera pandemic is restricted to Africa, Asia and the Americas [13, 14]. Recent findings show that some geographical areas of Africa and Asia continents have become the homeland for cholera and the infection is believed to spread from there to other parts of the world as a result of migration to more urban areas and international travel [15].

The aquatic environment is the reservoir of V. cholerae [7, 16, 17]. Therefore, surveillance of the aquatic faunal and flora for V. cholerae and possibly other important human pathogenic Vibrio spp. such as V. mimicus in Asia and Africa countries is relevant for empirically based awareness creation and necessary restrictions which can help in preventing cholera and vibriosis outbreak. The work of [18] recommended the monitoring of the environment for V. cholerae when cholera outbreak is not ongoing and this could be extended to other human vibrio pathogens for cholera and vibriosis preventive purposes.

Seafood provides good protein for humans however, several kinds of seafood contaminated with pathogenic Vibrio spp. are responsible for major foodborne illnesses globally [9, 19, 20]. The practice of aquaculture and mariculture in the Eastern Cape Province contributes to South Africa’s GDP while commercial and recreational angling are peculiar to the rural and urban areas of the Province [21–23]. Pathogenic human Vibrio spp. have been reported from numerous surface water and wastewater treatment plants in Eastern Cape Province [24–29], however, there is a paucity of information about these pathogens in seafood from surface water resources and fish markets in the province. Of the only two studies on seafood from the province identified in the literature, the work of [30] did not detect V. cholerae and V. mimicus in their fish samples while [31] isolated the two pathogens but the report was only on mollusc.

Eastern Cape province is one of the provinces with high cases of reported cholera in South Africa between 2001 and 2003. The case fatality rate of 1.93 for the 2001–2002 outbreak and 1.18 for the year 2003 were higher than the average case fatality rate of the country [32]. Going by this peculiarity of the province, the aquatic animals in the province’s surface water resources and aquaculture markets could be harbouring pathogenic Vibrio spp. To investigate this hypothesis, the current study isolated V. cholerae and V. mimicus from fish, crab, mud brawn and mussel samples recovered from anglers and seafood markets in Eastern Cape Province when cholera outbreak is not ongoing. The antibiotic resistance profile and virulence capability of the isolates were also investigated.

2. Materials and methods

2.1 Samples and sampling sites

Sampling sites included two fish markets (one in East London and the other in Port Alfred), three freshwater and five brackish water resources. The water resources are used for both commercial and recreational angler activities. Samples were purchased from fish markets and anglers based on availability once a month for one year. Licensed anglers were identified at the water resources during a reconnaissance visit. The arrangement was made with them such that samples were recovered in our presence and immediately transferred into a sterile zip-lock bag. We also arranged with the fish market owners such that samples were purchased as soon as they got back from their fishing trip. Samples were purchased whole except in a few instances when fish heads and intestines have been removed by fishermen before returning from fishing trips. After purchase, samples were transported to the laboratory on ice packs in cooler boxes within six hours of collection.

2.2 Sample processing, and Vibrio density determination

Samples preparation was carried out using Bacteriological Analytical Manual as a guide [33]. The vibrio density determination was done using 3 tubes by 5 tenfold serial dilutions Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method and the presumptive vibrio isolation was carried out as described in one of our previous studies [31]. Briefly, each sample comprised pulled 10 to 12 pieces of the sample. At the laboratory, following aseptic rules, fish samples were dissected to remove different anatomical sites (gill, flesh, intestine and fins which were designated as G, FL, IN and FN respectively) and the mussel sample was deshelled. Each of the pulled sample types [gill, flesh, intestine, fins, deshelled mussel, crab and mud prawn] was mashed in separate sterile mortar and pestle. Afterwards, ten grams of the mashed samples were gently pummeled in 90 ml of a sterile Phosphate Buffer Saline Solution [PBS] in the conical flask to have the first tenfold diluted sample homogenate (10−1). Ten millilitres of the first tenfold serially diluted sample homogenate was added to another 90 mL sterile PBS to have the second tenfold diluted sample homogenate (10−2). The process was repeated up to 10−5. One millilitre aliquot from the first tenfold diluted sample homogenate in the conical flask (10−1) was aseptically transferred into each of three separate test tubes containing sterile alkaline peptone water. The procedure was carried out for the reaming four tenfold serially diluted sample homogenate. The set-up (fifteen tubes per sample) was incubated at 37 °C for 24 hrs. and after the incubation period, total genomic DNA was extracted from turbid tubes using the boiling method as described elsewhere [34]. The total genomic DNA from each turbid tube was subjected to PCR (25 μl reaction) to detect the presence of Vibrio spp. in the turbid MPN tubes using a vibrio genus-specific primer used elsewhere [29]. The Vibrio spp. positive tubes were noted and the equivalent density of Vibrio spp. density was extrapolated from the MPN BAM-Excel sheet [35]. The Gene Technology G-Storm GS1 Thermal Cycler PCR Machine, USA was used for PCR and all media used in this study are Oxoid Cambridge, UK products. One Taq 2X Master Mix Standard Buffer (BioLabs, UK) and primers were supplied by Inqaba Biotec, South Africa. The details of the primer are given in S1 Table. Note that tissue e.g. gill from a fish sample [made up of 10–12 pieces of the same fish] purchased at a sampling site formed one gill sample and this applies to other anatomical sites as well.

2.3 Isolation of presumptive Vibrio spp. their PCR confirmation and delineation

Ten grams of the sample prepared in section 2.2 above were enriched with 90 ml of APW and incubated at 37 °C for 24 hrs. After the incubation period, a loop full of the APW directly underneath the surface pellicle formed was streaked on freshly prepared Thiosulfate–citrate–bile salts–sucrose agar (TCBS, Oxoid Cambridge, UK) and incubated for 24 hrs. After the incubation period, two to ten green and yellow distinct colonies that represent the different colony morphology observed on the TCBS plate were selected as presumptive V. cholerae and V. mimcius isolates respectively. The presumptive isolates were purified by streaking them on sterile 1% NaCl nutrient agar plates, checking the morphological uniformity of colonies along the line of streak coupled with Gram-staining. The presumptive isolates were confirmed as Vibrio spp. using the vibrio genus-specific primers and confirmed isolates were afterwards delineated into V. cholerae and V. mimicus using species-specific primers in a 25 μl PCR reaction. Furthermore, V. choleae isolates were screened for the presence of O1 and O139 strains using specific primers that targeted rfb regions responsible for “O” antigen biosynthesis [36, 37]. The details of the primers and amplification conditions are given in S1 Table.

2.4 Antibiotics susceptibility testing and detection of virulence and antibiotics resistance genes

The antibiotic susceptibility testing was performed by Kirby-Bauer Disk Diffusion Susceptibility Test Protocol [38] and interpreted according to the recommendation of the Clinical Standard Institute (CLSI) [39]. The panel of antibiotics used for the study is as recommended by CLSI and those that have been used against Vibrio isolates in previous studies [24, 25, 29, 40–43]. The antibiotics are: kanamycin (K) 30 μg, amikacin (AK) 30 μg, gentamycin (GM) 4 μg, meropenem (MEM) 10 μg, imipenem (IMI) 10 μg, cefotaxime (CTX) 30 μg, cefuroxime (CXM) 30 μg, ofloxacin (OFX) 5 μg, ciprofloxacin (CIP) 5 μg, Nalidixic acid (NA) 30 μg, Azithromycin (ATH) 15 μg, Nitrofurantoin (NI) 300 μg, Amoxicillin/clavulanate (AUG) 30 μg, Ampicillin (AP) 10 μg, chloramphenicol (C) 30 μg, Polymyxin B (PB) 300 units, cotrimoxazole (TS) 25 μg and trimethoprim (TM) 5 μg. Multiple antibiotics resistance index (MARI) was calculated and interpreted for each isolate, sample type and sampling area as recommended. Isolates, sample type and sampling area with ≥ 0.2 MARI were identified as agents/sources of high risk of antibiotic-resistant vibrio infection [44]. The MARI was calculated using Eq 1 below. E. coli ATCC 25922 was used as the quality control organism. (1) where a = number of antibiotics an isolate exhibited resistant against and b = the total number of antibiotics tested against the isolate.

Multiple antibiotics resistance phenotypes (MARP) i.e. isolates that exhibited resistance against more than one antibiotic and multidrug antibiotics resistance phenotypes (MDRP) i.e. isolates that exhibited resistance against at least one antibiotic in three or more classes of antibiotics that were used for this study [45]. Isolates based on their phenotypic resistance profile were tested for the presence of some antibiotic resistance genes [cmlA, cat, flor, aac, ant, aphA, drf18, sul1, sul 2, mcr-1, blasshv, blaoxa, blatem, blavim, blakpc, blandm, blaimp, blaoxa48, gyrA, gyrB, parc, sxt] in a 25 μl PCR reaction.

Also, to understand the pathogenicity of the confirmed isolates, they were tested for the presence of eleven virulence determinants (details in S1 Table) in a 25 μl PCR reaction. The virulence genes selected are those that have been implicated in cholera and vibriosis caused by V. cholerae and V. mimicus. The multi-virulence gene indexes (MVGI) and the multi-resistance gene indexes of the isolates were also determined using a method reported elsewhere [46]. The antibiotics-resistant genes targeted, primers details and thermal conditions used for all PCR reactions are given in S1 Table.

2.5 Statistics

The Vibrio spp. mean density, MARI, MRGI, MVGI and MDRP was subjected to a normality test using Kolmogorov-Smirnov and Shapiro-Wilk test to determine whether to use a parametric or non-parametric test. Afterwards, an appropriate statistical test was employed to compare the Vibrio spp. density across sample types while the relationship between MARI, MVGI, MRGI and MDRP was determined using an appropriate correlation test. Correlation analysis between MARI, MVGI, MRGI and MDRP was carried out to observe the type of relationship that exists between phenotypic resistance, prevalence of virulence determinants, prevalence of resistance genes and prevalence of multidrug-resistant phenotypes. The differences between the magnitude of antibiotics resistance exhibited [using MARI as the corresponding variable], prevalence of virulence determinants (using MVGI as the corresponding variable) and prevalence of antibiotics resistance genes (using MRGI as the corresponding variable) across the Vibrio spp. (V. cholera and V. mimcicus) isolates, sampling sites, sample types, sample sources and sample class were also statistically determined. The significant level was set at <0.05. SPSS version 25 was used for the statistical analysis.

3. Result

3.1 Vibrio spp. density and distribution of V. cholerae and V. mimicus in samples

The mean Vibrio spp. density across sample types is given in Fig 1 with gill having the highest mean density while mud prawn has the lowest. The density of Vibrio spp. for each sample type and site is given in Fig 1 and Table 1. A total of 29 fish samples which resulted in 29 flesh, 24 fins, 26 gills and 18 intestine samples were recovered from the fish markets and surface water. The crab, mud prawn and mussel samples collected from surface water were 24, 9 and 14 respectively. Of the 17 samples recovered from the fish markets, 41% were positive for one or both V. cholerae and V. mimicus. On the other hand, 25% (3/12) of the fish samples recovered from the surface water were positive for the two Vibrio species. At least one of the anatomical sites of all the fish samples was positive for Vibrio spp. however, Vibrio spp. was not isolated in 3 fins, 4 flesh, 2 intestine and one gill samples. Only 27.59% [8/29] of the fish samples (2 flesh, 3 fin, 2 gill and 2 intestine samples) were positive for V. cholerae while 10.34% (3/29) of the fish samples (2 gill and 2 intestine samples) were positive for V. mimicus. None of the mud prawn samples was positive for V. mimicus but 30% (3/10) of the samples were positive for V. cholera. On the other hand, 7.14% (1/14) and 14.29% (2/14) of the mussel samples were positive for V. mimicus and V. cholera respectively. Furthermore, 12.50% (3/24) and 4.17% (1/24) of the crab samples were positive for V. mimicus and V. cholera respectively. Of the 535 presumptive isolates (FL = 145, FN = 114, G = 165 and IN = 111) recovered from the fish samples, 81% (434/535 [FL = 119, FN = 93, G = 124 and IN = 98]) were confirmed as Vibrio spp. and 3% (21/434 [FL = 7, FN = 8, G = 3 and IN = 3) and 1% (4/434 [FL = 0, FN = 0, G = 2 and IN = 2]) were confirmed as V. cholerae and V. mimicus respectively. Given the above, the prevalence of V. cholera among the population of confirmed Vibrio spp. from the flesh, fin, gill and intestine samples were 5.88%, 8.60%, 2.42% and 3.06% respectively while the prevalence of V. mimicus was 0%, 0%, 1.61% and 2.04% respectively. Seventy-four presumptive isolates were recovered from crab samples out of which 68% (51/75) were confirmed as Vibrio spp. while approximately 11% (8/75) and 3% (2/75) isolates of the confirmed Vibrio spp. were V. cholerae and V. mimicus respectively. The presumptive isolates from mud prawn samples were fifty out of which 76% (38/50) were confirmed as Vibrio spp. An 8% (3/38) and 0% (0/38) of the population of the confirmed Vibrio spp. were V. cholerae and V. mimicus respectively. The presumptive isolates recovered from mussel samples were 211 of which 92% (195/211) were confirmed as members of the vibrio genus. Of those confirmed, 1% (2/195) and 2% (4/195) were confirmed as V. cholerae and V. mimicus respectively. A total of 33 and 9 V. cholerae and V. mimicus respectively were recovered from samples and most of the isolates 59% (26/44) were recovered from fish anatomical sites. All the V. cholerae were confirmed to belong to non-O1/non-O139 serogroup.

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Fig 1. Average Vibrio spp. density per sample type.

Key: FL = flesh, FN = fin, G = gill, IN = intestine.

https://doi.org/10.1371/journal.pone.0290356.g001

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Table 1. Percentage prevalence of Vibrio spp., V. cholerae and V. mimicus in sample types and at sampling sites.

https://doi.org/10.1371/journal.pone.0290356.t001

3.2 Phenotypic and genotypic antibiotics resistance profile of V. cholerae and V. mimicus isolates and detection of virulence genes

The percentage prevalence of isolates that exhibit susceptible, intermediate and resistance phenotypic characteristics is given in Table 2 while the prevalence of virulence determinants and antibiotics resistance determinates are given in Table 3. The phenotypic antibiotics susceptible, intermediate and resistance patterns, MARI, MVGI, MRGI and MDR status of the isolates are given in Fig 2.

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Fig 2. Phenotypic antibiotics susceptible, intermediate and resistance patterns of V. cholerae and V. mimicus isolates.

Key: ID = identity, (FL = flesh, FN = fin, G = gill, IN = intestine), MU = mussel, MP = mud prawn samples, ARP = antibiotics resistance profile, MARI = multiple antibiotic resistance index, MDR = multi-drug resistance status, Y = yes, N = No, black = susceptible, pink = intermediate and red = resistance, MVGI = Multi-virulence gene Index, MRGI = Multi-resistance gene index.

https://doi.org/10.1371/journal.pone.0290356.g002

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Table 2. Percentage prevalence of V. cholerae and V. mimicus that exhibited susceptible, intermediate and resistance phenotypic characteristics against panels of antibiotics.

https://doi.org/10.1371/journal.pone.0290356.t002

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Table 3. Percentage prevalence of detected antibiotics resistance genes and virulence determinants among V. cholerae and V. mimicus population.

https://doi.org/10.1371/journal.pone.0290356.t003

Above 50% of all the isolates were susceptible to kanamycin, amikacin, gentamycin, meropenem, imipenem, cefotaxime, cefuroxime, ofloxacin, ciprofloxacin, nalidixic acid, azithromycin, nitrofurantoin and chloramphenicol. Over fifty per cent of isolates exhibited resistance against polymyxin B, amoxicillin/clavulanate and ampicillin while 20% to 36% of the isolates exhibited resistance against nalidixic acid, azithromycin, nitrofurantoin, cotrimoxazole and trimethoprim. Eighteen per cent, 30% and 48% of the isolates exhibited intermediate characteristics against ofloxacin, kanamycin and imipenem respectively. The MARI for the isolates ranges between 0 and 0.67 and 48% of the isolates have MARI that is above 0.2 while 55% of the isolates exhibit MDR phenotypes.

Of the 22 resistant determinants targeted in this study, 12 (55%) were detected in at least one of the isolates. The percentage detection of acc, ant, drf18, sul1, mcr-1, blasvh, blaoxa, blatem, blaoxa48, gyrA, gyrB and parC were 2%, 9%, 14%, 7%, 2%, 25%, 7%, 2%, 2%, 32%, 25% and 27% respectively. Furthermore, 8 [73%] of the eleven virulence determinants targeted in this study were detected in at least one of the isolates. The percentage detection of the virulence-associated genes in increasing other was ace (2%), tcp (11%), vpi (16%), ompU (34%), toxR (43%), rtxC (70%), rtxA (73%) and hyla (77%). Of the eight virulence-associated genes detected, toxR and ace genes were not detected in V. mimicus but vpi, OmpU tcp, hyla, rtxA and rtxc were detected in 11%, 22% 11%, 89%, 67% and 67% of the V. mimicus isolates. The percentages of V. cholreae isolates that carry the eight genes (vpi, toxR, OmpU tcp, ace, hyla, rtxA and rtxc) were 17.14, 54.29, 37.14, 11.43, 2.86, 74.29, 74.29 and 71.43 respectively. Representative gel pictures showing products of the amplification of the region targeted for each virulence determinant are given in S1–S5 Figs while that for the resistance determinants are given in S6–S8 Figs.

3.3 Resistance-associated genes, virulence-associated genes and phenotypic resistance combinations detected in isolates

The highest number of virulence genes detected concurrently in a V. cholerae isolate was 6 and this is found in just one isolate (Table 4). The highest number of the virulence determinants detected simultaneously in a singular V. mimicus isolate per time was four and this was observed in three V. mimicus isolates (Table 4). The combinations of virulence genes, antibiotic resistance genes and phenotypic resistance observed are given in Table 4. A total of fourteen different combinations of the virulence genes targeted were detected. Only one virulence combination type was common to both V. cholerae and V. mimicus isolates while nine combinations were only found among the V. cholera population, four were found only among V. mimicus population.

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Table 4. Resistance-associated genes, virulence-associated genes and phenotypic resistance profiles of isolates.

https://doi.org/10.1371/journal.pone.0290356.t004

Eleven resistance gene profiles were observed, one profile was common to both V. cholerae and V. mimicus, one was only found among V. mimicus population and nine among only the V. cholera isolates. The highest resistance genes detected concurrently among V. cholerae isolate were five and two for V. mimicus. Twenty-one resistance phenotypes were detected of which one was common to the two Vibrio spp. while sixteen were found among V. cholerae isolates only and four among the V. mimicus population only. The highest concurrent phenotypic resistance detected in a V. cholerae isolate was against ten panels of antibiotics while that for V. mimicus was against thirteen. The distribution of resistance genes, phenotypic resistance and virulence genes combinations (profiles) among V. cholerae and V. mimicus populations is given in S3 Table while the dynamics of resistance for sampling sites, sample types, sources of samples and classes of samples using MARI, MARP AND MDRP as indices is given in S4 Table.

3.4 Statistical analysis

The variables (Vibrio spp. mean density, MVGI, MRGI and MARI) failed Kolmogorov-Smirnov and Shapiro-Wilk tests and thus tools for non-parametric data were used for statistical analysis. Welch ANOVA test result revealed a significant difference in Vibrio spp. density across sample types F[6, 46.37] = 7.62 P < 0.001. Game-Howell post Hoc analysis result given in Table 5 indicated that Vibrio species. density in gill, fin and flesh samples were significantly more than that in mud prawn and crab samples. Other comparisons of density across sample types were not significantly different. Spearman rho correlation analysis carried out determined the relationship between MARI, MVGI, MDRP and MRGI because the four variables failed Kolmogorou-Smirnov and Shapiro-Wilk tests and for the same reason, the Wilcoxon test was used to compare the differences between the three variables (MARI, MVGI and MRGI) across Vibrio spp. while Welch ANOVA and Game-Howell post hoc analysis was used to compare the three variables across sampling sites, sample types, sample sources and sample classes. ANOVA was adopted for the interpretation where Welch tests cannot be performed due to a group having a variance of zero. The result of the correlation test (Table 6) revealed significant positive or negative relationships between variables except for MRGI vs MVGI which is negative but not significant. The inverse relationship between levels of antibiotics resistance (determined by MARI, MRGP and MRGI) among isolates and the prevalence of virulence determinants (determined by MVGI) observed in this study have been reported in earlier studies [47, 48] and it has been shown that the inverse relationship is as a result of fitness trade-off [49]. However, a study [50] has reported no correlation between the variables while another one [51] observed a direct relationship. These observations confirmed a complex relationship between the detection of virulence and antibiotic resistance. To unravel this complexity, [52] carried out a review study and found out that the interplay between virulence, antibiotics resistance and the associated biological costs depends on four main factors which are the bacterial species involved, virulence and resistance mechanism at play, the ecological niche involved, and the host in question. Wilcoxon test showed that the MARI and MRGI of V. mimicus compare to that of V. cholerae were not significantly different but their MVGI were significantly different. The statistical comparison [Table 7] showed that MARI and MRGI of V. cholerae and V. mimicus showed varying levels of significant differences across sampling sites, sample types, sample sources and sample class while MVGI is not significantly different across the aforementioned variables.

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Table 5. Excerpt from Game-Howell Pot Hoc analysis showing sample types with significantly different Vibrio spp. density.

https://doi.org/10.1371/journal.pone.0290356.t005

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Table 6. Correlation analysis to determine the relationship between MARI, MVGI, MRGI and MDRP [n = 44].

https://doi.org/10.1371/journal.pone.0290356.t006

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Table 7. The statistically significant differences of MARI, MRGI and MVGI across sampling sites, sample types, source of sample and sample class.

https://doi.org/10.1371/journal.pone.0290356.t007

4. Discussion

4.1 Vibrio spp. density and the prevalence of V. cholerae and V. mimicus in samples

The density of Vibrio spp. differs numerically across anatomical sites of fish and other aquatic animals studied. The mean density of the Vibrio spp. in the fish anatomical sites except intestinal samples was significantly more than that of the crab and mussel samples. The gill sample has the highest average Vibrio spp. density and this is in concordance with an earlier study [53, 54]. The relatively high occurrence of V. cholera and V. mimicus in fish samples when compared to other samples supports earlier studies that reported that fish is one of the main reservoirs of V. cholera and V. mimicus in the aquatic environment. [1, 20, 55–58]. The low prevalence of V. cholerae and V. mimicus amidst the total number of confirmed Vibrio spp. recovered in this study is similar to earlier studies [55, 56]. This could be due to the largeness of the Vibrio genus which is over 100 species [59].

Cholera-causing V. cholerae serotypes (O1 and O139) are not commonly isolated from seafood as observed in this study nevertheless, the non-O1/non-O139 serogroups isolated have been implicated in human and aquatic animal infections outbreaks [60–64]. The relatively high prevalence of V. cholerae and V. mimicus in market samples than in surface water samples suggests a higher risk of contracting non-O1/non-O139 V. cholera and V. mimicus infections at the fish market in Eastern Cape than at the surface water. As relayed by the owners, the fish sold at the fish markets used for this study are from outside Eastern Cape Province. This calls for a more stringent microbial examination of fish products imported into the province.

4.2 The complexity and diversity of virulence determinants mix in non-O1 and non-O139 V. cholerae and V. mimicus isolates and implication

The current study shows the potential complexity and variation that could be observed in the virulence determinant profile of non-O1/non-O139 strains isolated from a similar geographical location. Horizontal gene transfers and serotype conversion has been linked to this observation. All four tcp+ non-O1/non-O139 isolates were rtx+ while two were toxR+. One of the toxR+ isolates was hylAEl Tor positive while the other was positive for vpi.

The ace gene, a core gene of the CTX elements which encodes enterotoxin detected in one of the isolates, is rarely found in non-O1/non-O139 strains but when detected, it is as a result of serogroup conversion of non-pathogenic environmental strains through horizontal gene transfer mechanisms to pathogenic ones [65]. The tcpA gene which is another core CTX element that encode the major structural unit of toxin-coregulated pilus detected in this study is believed to be a specific O1 and O139 serogroup virulence gene. However, the possibility of the acquisition of the tcp A gene by non-O1/non-O139 via horizontal gene transfer has been posited [66] and this explains its detection in the current study and an earlier study.

The VPI gene detected in 17% of the isolates in this study is an essential virulence gene cluster that enhances the colonization of the human intestine as well as the activity of the infection receptor site for most toxigenic Vibrio strains [67]. The toxin coregulated pilus [TCP], an essential colonization factor, is encoded within the VPI and the highland also encodes several virulence regulators and sialic acid utilization genes [68, 69]. All the vpi-positive isolates in our study harboured the two rtx, toxR, hylAEl Tor and one of them tested positive for tcpA gene as earlier reported [70]. On the other hand, tcpA+, toxR+ vpi- strains were detected and this is in line with studies that reported Vibrio cholerae strain that is positive for cholera toxin but negative for vibrio pathogenicity highland [71, 72] and Vibrio alginolyticus, V. cholerae O1, O139 and non-O1/non-O139 strains that are vpi negative but tcpA positive [70, 73]. Genomic sequence and comparative studies have confirmed the inter-specific and intra-specific differences in the content of the pathogenicity highlands. Also, it has been reported that the three Vibrio cholera highlands can excise from the chromosome and circular intermediates and VPI can be transferred from vpi+ strain to vpi- strain [74–77].

As usually reported, OmpU+ V. cholerae isolates are also ToxR+ since ToxR is needed for the regulation of OmPU gene [78–82]. However, only 61.5% of the OmpU+of non-O1/non-O139 isolates were ToxR+ and the two OmpU+ V. mimicus also lack the ToxR gene in this study. OmpU-ToxR- and OmpU-ToxR+ V. cholerae isolates have been reported [78–83] but OmpU+ ToxR- strains are here reported for the first time. We suspected deletion or outright absence of the ToxR gene in the OmpU+ ToxR- strains since ToxR gene could be chromosomal or plasmid-borne and deletion/acquisition of the gene has been reported [84–90]. The OmpU gene can still be expressed in the presence of the ompT promoter and the absence ToxR gene [88]. Although we did not target ToxT gene in this study the aforementioned explained the possibility of OmpU+ and ToxR- genotypes in Vibrio species that will express OmpU in the absence of ToxR gene. None of the eleven virulence-associated genes was detected in 20% of the V. cholerae isolates in this study. The non-O1/non-O139 strain that lacks key structural and regulatory pathogenicity genes have been reported [65].

Vibrio cholerae virulence genes have been reported in other human vibrio pathogens such as V. alginolyticus and V. mimicus [65, 70]. All the virulence-associated genes detected in the V. cholerae isolates in this study were also detected in V. mimicus isolates except for ToxR and ace genes. The study carried out by [8] on the genomic comparison between environmental V. mimicus and clinical isolates concluded that the genes [ctx, ace, zot and tcp] that is related to intestinal infections are common only with clinical V. mimicus isolates. This explains in part our result on the prevalence of the virulence genes targeted in the V. mimicus isolates. Furthermore, the none detection of the ToxR gene even in the OmpU+ V. mimicus could be explained as earlier discussed for the V. cholerae. Nevertheless, none of the V. mimicus could be called avirulent because they all harboured at list one of the virulence determinant targeted in this study. Even if none of the targeted virulent-associated genes is detected in V. mimicus isolates, the targeted vmh gene for the PCR confirmation of the V. mimicus isolates is a virulence determinant [91]. The gene encodes a heat-labile hemolysin, designated V. mimcus hemolysin [5, 92] based on their findings suggested the gene as the most important virulence factor of the pathogen. The toxR, zot, ctx, VPI, ompU, rtx and hylA genes have been reported in both clinical and environmental V. mimicus isolates [4, 65, 93] however, [94] believed that the tcpA+ V. mimicus strain they reported and which we also isolated in the present study is rare. Our literature search confirmed that the strain is scarce nevertheless, it has been reported by [65] before the report of [94]. The analysis of the result of the virulence determinants in this study and our literature search showed that the peculiarity of virulence determinants among vibrio genus members is gradually diminishing. This supports the position of earlier reports and our study that members of the vibrio genus are evolving in terms of virulence. This evolution might lead to the emergence of pathogenic Vibrio spp. that are more virulent than those earlier known.

Our findings in terms of the complex mix of virulence determinants found in non-O1 and non-O139 V. cholerae strains and recent studies on virulence determinants of non-O1 and non-O139 strains suggested that V. cholerae is undergoing an evolution that might lead to the emergence of highly virulent strains. Atypical El Tor strains that share characteristics of prototype classical, the El Tor biotypes that is not known to exist before 2002 and strains that cause more severe cholera outbreak has been reported [95–102]. Also, the emergence of SA-NAG V. cholerae strain that shares genomic [e.g. presence of rtxC, El Tor and the Classical hemolysin gene] and phenotypic attributes of O139 and O1 serogroups have been reported [28]. The detection of the repeat toxin [rtxC] gene which is one of the genomic characteristics that confirms V. cholerae isolates as of the El Tor biotypes in non-O1 and non-O139 in this study is another pointer to the possible evolution of new V. cholerae [98, 103–105]. Summarily, the PCR detection of El Tor hemolysin gene in 74%, TcpA El Tor in 11% and rtxC genes in 71% of the non-O1/non-O139 isolates analyzed in this study supports the possibility of the emergence of a typical pathogenic SA-NAG V. cholera strains that could be more virulence than the known pathogenic strains. This affirms the need for surveillance for non-O1/non-O139 in clinical and non-clinical environments. The need for a surveillance programme for all Vibrio spp. in the clinical and environmental settings has been emphasized by a study carried out in Germany [106]. The call for this kind of program is due to the non-specific spread of virulence determinants among the members of the genus as well as the ongoing global warming challenge which is predicted to cause an increase in the abundance of Vibrio spp. and their infections in the clinical and non-clinical environment.

4.3 Virulence determinants detected in non-O1 and non-O139 V. cholerae and V. mimicus isolates and their role in pathogenicity

Eight of the eleven virulence genes targeted in this study were detected and of the eight detected, toxR, rtx and hlyA ELTor virulence genes predominate as earlier reported [65]. The rtxA is the most detected of the two rtx genes. Similar to earlier studies, some of the isolates in this study possessed both rtxA and rtxC genes, while neither of the two genes was detected in a few of the isolates [107, 108].

4.3.1 Vibrio cholerae non-O1 and non-O139.

The predominance of the three genes [toxR, rtx and hlyAET] in the non-O1 and non-O139 V. cholera and V. mimicus speaks to the public health significance of the current study. It has been reported that ctxA− tcpA− zot− stn/sto− but HlyAET+ Non-O1 and non-O139 V. cholerae can cause diarrhoea and septicemia in humans, and intestinal and extra-intestinal pathophysiological conditions in the animal model [63, 109].

The detection of rtxA and rtxC in the current study is at per with some earlier studies [28, 65, 108, 110] although a higher prevalence of rtxA and rtxC is recorded in the current study. The actin cross-linking repeats in the toxin gene cluster [rtx] lead to actin depolymerization when cross-linked with Hep-2 and this causes villi effacement, hemorrhagic colitis, and bloody diarrhoea as earlier reported [103, 111]. This suggests the need to screen for the SA-NAG Vibrio cholerae in the diarrhoea cases when common etiological agents [e.g. Escherichia coli and Vibrio cholera O1 and O139 serogroups] are not isolated/detected. It is also needful to screen for this non-O1/non-O139 serogroup when poly-bacterial diarrhoea infection is suspected.

ToxR and HylAEl Tor are the other two prevalence virulence-associated genes in this study. The prevalence of toxR [74.3%] and hylA [74.3%] in our study are similar to that of earlier studies [63, 108] from clinical and environmental samples. These two genes are part of the five [hlyA, hlyU, hlx, toxR and attRS1] virulence-associated genes which were found in the Vibrio cholerae non-O1/non-O139 strains that cause cholera epidemics in Calcutta, India. Surprisingly, all the strains isolated from the epidemics lacked the core virulence determinants [ctx elements and tcp genes] of the toxigenic V. cholerae O1 and O139 [66]. The regulatory protein ToxR controls the expression of the virulence determinants in V. cholerae O1, O139 [66] while the product of hylA causes haemolysis of erythrocytes [112]. It has been posited that Vibrio cholerae non-O1/non-O139 could acquire tcpA and ctxA which could favour the emergence of novel toxigenic strains under the biological and physicochemical selective pressures of the aquatic environment [63]. The detection of tcpA 4/35 [11.4%] and ace 1/35 [2.9%] in the current study supports the position of Ottaviani et al. [2018].

The Outer Membrane Protein U [OmpU] gene is another virulence determinant detected in the non-O1/non-O139 strains in this study. This gene is an adherence factor that has been reported in V. cholerae, V. alginolyticus and V. mimicus and it is one of the genes that are responsible for pathogens ability to colonize the intestine [88, 113–115]. The OmpU and OmpT genes are called pore-forming proteins of the outer membrane and they are regulated by ToxR gene [88].

4.3 Antibiotics susceptibility patterns of V. cholera and V. mimicus isolates from market and surface water samples

4.3.1 Vibrio cholerae.

The antibiotics susceptibility pattern observed in this study varies across Vibrio spp., sample types, source of isolates and sampling sites. The V. cholerae isolates were either susceptible or exhibited intermediate susceptibility to amikacin, gentamycin, ofloxacin and cefuroxime. Gentamycin and amikacin interfere with protein synthesis, ofloxacin affect DNA synthesis while cefuroxime interferes with cell wall synthesis. Earlier studies have shown that V. cholerae non-O1/non-O139 are generally susceptible to the four antibiotics [43, 61, 63, 108, 116–120]. To interpret other susceptibility/resistance patterns for the remaining panel of antibiotics, resistance to an antibiotic was referred to as low to medium when the population that exhibited resistance against it is < 50% [121] and high when the population is > = 50% [122]. Going by this interpretation criteria, the V. cholerae exhibited high resistance to polymyxin, amoxicillin/clavulanate and ampicillin that target the cell wall synthesis but low/medium resistance to the other eleven panels of antibiotics. The eleven antibiotics that V. cholerae exhibited low to medium resistance against can be categorized into three. Less than 10% of the V. choloerae isolates exhibited resistance against the first category which is made up of imipenem, meropenem and cefuroxime; while 10–20% exhibited resistance against the second group which is made up of kanamycin, azithromycin, cefotaxime, chloramphenicol and ciprofloxacin. A greater than 20% but less than 35% of the V. cholerae isolates exhibited resistance against the third group [nalidixic, nitrofurantoin, cotrimoxazole and trimethoprim]. The high resistance exhibited against polymyxin B and ampicillin has been reported and this seems to be common among environmental isolates of V. cholerae non-O1/non-O139 strains [108, 123] and O139 toxigenic and non-toxigenic serogroup [124]. The high resistance to polymyxin B and the high prevalence of hylAElTor virulence determinant confirms that most of our V. cholerae isolates belong to El Tor biotype while others might have lost the El Tor marker [polymyxin B resistance] or belong to classical biotype. Polymyxin B resistance is one of the markers for detecting the El Tor biotypes however, it has been reported that the EL Tor biotype can lose this marker [125]. The El Tor strains compare to the classical strains are more adapted and resilient to the environment, cause a higher infection-to-case ratio and have more asymptomatic carriers [95, 126]. Some studies have reported low resistance of the SA-NAG V. cholerae against amoxicillin/clavulanate [117, 127, 128] however, other studies like ours have shown that different V. cholerae serotypes can exhibit high resistance against amoxicillin/clavulanate [129, 130].

The low/medium resistance against eleven antibiotics [meropenem, imipenem, cefotaxime, kanamycin, azithromycin, chloramphenicol, ciprofloxacin, nalidixic acid, nitrofurantoin, trimethoprim and cotrimoxazole] by the O1/non-O139 strains recorded in this study is similar to most of the earlier reports [54, 108, 110, 117, 118, 120, 127, 128, 131, 132] although with some differences. A low/medium resistance observed against nalidixic acid by [120, 132], azithromycin by [131], cefotaxime by [132], imipenem by [108, 132], cotrimoxazole by [108, 118, 120, 131, 132], chloramphenicol by [116, 120, 132], ciprofloxacin by [116, 120, 132] and kanamycin by [116, 120] was as observed in this study. However, in some other reports, no V. cholerae non-O1/non-O139 isolate exhibited resistance against nalidixic acid [110, 117, 128], meropenem [108, 118], cefotaxime [108, 117, 118], imipenem [116–118], nitrofurantoin [108], cotrimoxazole [110, 117], chloramphenicol [108, 110, 117, 118, 131], ciprofloxacin [108, 110, 117, 118], kanamycin [110] and trimethoprim [128]. The percentage of V. cholerae non-O1/non-O139 strains that exhibited resistance/intermediate status against each of the antibiotics employed for this study was generally high compares to that reported in all the literature consulted.

The antibiotics commonly recommended for the management of vibrio infections fall into four classes of antibiotics [tetracyclines, fluoroquinolones, macrolides, and trimethoprim/sulfamethoxazole] [133–135]. Of the four recommended, three were used in this study and the percentage of V. cholerae non-O1/non-O139 strains that exhibited resistance against members of the three classes was mostly more than what has been reported in similar studies.

Although the V. cholerae population in this study exhibited low resistance against kanamycin, meropenem, imipenem, ofloxacin and ciprofloxacin, a high percentage of them exhibited intermediate susceptibility to five antibiotics. This is worrisome because intermediate susceptibility for an antibiotic means a higher dose than recommended is needed for the antibiotic to be effective [136, 137]. The concern stems from the fact that these antibiotics are categorized as critically important antimicrobials for human medicine [138]. The aforementioned suggests that V. cholerae isolates in this study have the potential to cause cholera-like and vibriosis that might be difficult to manage with commonly recommended and relatively inexpensive antibiotics at the usual dosage. Unfortunately, using higher doses and expensive antibiotics raises resistance, side-effects and economic concerns [139–141].

In light of the aforementioned and the low/medium resistance exhibited by the V. cholerae population against 80% of the concerned antibiotics, we opined that most of the V. cholerae isolates in this study have the potential to cause cholera-like and vibriosis that might be difficult to manage with commonly recommended and relatively inexpensive antibiotics.

4.3.2 Vibrio mimicus.

The phenotypic resistance profile of V. mimicus population was similar to that of V. cholerae except for view differences. The differences include: 1. No V. mimicsu isolate exhibited phenotypic resistance against imipenem, chloramphenicol and ciprofloxacin which V. cholerae population exhibited low/medium phenotypic resistance against, 2. the V. mimicus population exhibited low/medium resistance against cefuroxime which none of the V. cholerae exhibited resistance against, 3. V. mimicus population exhibited high resistance against nitrofurantoin to which V. cholerae exhibited low/medium resistance and, 4. V. mimicus isolates exhibited low/medium resistance against polymyxin B, amoxicillin/clavulanate and ampicillin while the V. cholerae isolates exhibited high resistance against the three antibiotics. Reports on the antibiotics resistance profile of V. mimicus isolates are limited in the literature however, few studies we come across during our literature search reported resistance patterns of the organism against all members of the panels of antibiotics used in this study except amikacin [20, 55, 93, 142–144]. Interestingly, all V. mimicus isolates in this study were susceptible to amikacin. The level of resistance observed against each member of the panel of antibiotics in this study was similar in some instances and differs in others to the earlier reports. High resistance against kanamycin and cotrimoxazole was reported by [142] but no resistance to cotrimoxazole and very low resistance to kanamycin was reported three years later [144]. The current study reports low resistance against the two antibiotics while Gxalo et al., [2021] reported high resistance against kanamycin like Chowdhury et al., [1986]. Also, Nilavan et al., [2021] reported 100% susceptibility, Abdalla et al., [2022] reported low/medium resistance while Raissy et al., [2012] reported high resistance of the environmental V. mimicus isolates to cotrimoxazole. The medium resistance [44%] against ampicillin reported in the current study was the same as what Chowdhury et al., [1986] reported but differs from the very high resistance [>80%] reported by Abdalla et al., [2022]; Gxalo et al., [2021]; Nilavan et al., [2021]; Raissy et al., [2012] and Yamanaka and Aziz, [1989]. This same scenario goes for other antibiotics used against V. mimicus in this study and it suggests that local antibiogram result should inform the choice of antibiotics for the management of V. mimicus infections. In the light of this and considering the intermediate susceptibility result, amikacin, gentamycin and ciprofloxacin are the three antibiotics that can be considered good choice for the management of V. mimicus infections in our study area although this is subject to other factors such as cost and recommendations of the regulatory bodies such as World Health Organization, Center for Disease Control and Prevention and local regulatory bodies in-charge of our study area.

4.4 Genomic bases for the phenotypic antibiotic resistance observed in V. cholerae and V. mimicus

A recent systematic review and meta-analysis [145] pointed out the paucity of information on the presence of resistance genes in Vibrio spp. The frequency of detection of the twenty-two antibiotics resistance determinants focused on in this study varies for V. cholerae and V. mimicus. Forty-three per cent of the V. cholerae isolates and twenty-two per cent of the V. mimicus isolates were positive for at least one of the virulence determinants targeted in this study. The sxt element which is a conjugative-transposon-like mobile gene element that encodes multiple antibiotic resistance [146] was not detected in this study and this is in line with an earlier report [123]. Phenotypic-genotypic/genotypic-phenotypic resistance discrepancy reported in earlier studies was observed in the current study [147, 148]. Of the six resistance genes [cmlA, cat, flor, acc, ant and aphA] that confer resistance against protein synthesis inhibitors, cmlA, cat, flor that coffer resistance against chloramphenicol [149, 150] were not detected. The acc and ant that confers resistance against aminoglycosides [amikacin, kanamycin and gentamycin] [151], were detected. However, all isolates that carry the aminoglycosides resistance genes were either phenotypically susceptible or exhibited intermediate susceptibility to the aminoglycosides while one of the isolates surprisingly exhibited resistance against chloramphenicol. The aphA that encodes kanamycin resistance [152] was not detected in any of the isolates yet five isolates exhibited phenotypic resistance against kanamycin. Eighty-nine per cent [16/18] of the isolates that exhibited resistance against antifolate drug carries one or both of the Drf18 and sul1 gene that encodes antifolate resistance.

The current study supports the few reports that recently detected mcr-1 gene in Vibrio specie [153–155]. One V. cholerae isolate was positive for mcr-1 gene which confers resistance to polymyxin E [colistin], polymyxin B and this gene can cause cross-resistance to other antimicrobials [156–159]. This isolate was only positive for one more of the targeted resistance determinants [gyrB] but interestingly, it is a multidrug-resistant isolate with MARI of 0.56. This observation could be due to the presence of mcr-1 gene that causes cross-resistance to other antimicrobial agents in the isolate. Some bacteria which include V. cholerae are typically resistant to polymyxin [160] however detection of the plasmid-mediated mcr-1 gene in them raises public health concerns in terms of the spread of colistin resistance [161, 162] because colistin is one of the antibiotics of last resort [157].

The information on the prevalence of β-lactamases in non-O1/non-O139 Vibrio cholerae is limited in the literature and we could not find information on the prevalence of β-lactamases in V. mimicus. Of the eight β-lactamses [blashv, blaoxa, blatem, blavim, blakpc, blandm, blaimp and blaoxa48] targeted in this study, four [blashv, blaoxa, blatem, blaoxa48] were detected and of the four, blashv was the most detected while blaoxa48 was the least detected. All the four detected β-lactamses were found in at least one V. cholerae while only blashv and blatem were detected among the V. mimicus isolates. The blaoxa-1 gene was detected in three V. cholerae isolates and one of the three aslo carries blaoxa48. The co-existence of bacteria carrying variants of blaoxa genes within the same ecological niche as observed in this study has been reported [163]. We expected the V. cholerae isolate that was positive for both blaoxa-48 and blaoxa-1 resistance genes to exhibit phenotypic resistance against more members of the β-lactam antibiotics used in this study than the two that carry only the blaoxa-1 because the blaoxa-1 gene is easily deactivated by penems [164]. Also, we do not expect blaoxa-48 positive isolate to exhibit a high level phenotypic resistance against the penems since the blaoxa-48 enzyme only hydrolyses penicillins at a high level, broad-spectrum cephalosporins at a moderate level, carbapenems at a low level but not susceptible to β-lactamase inhibitors [165]. However, surprisingly, the two isolates that were negative for the blaoxa-48 like but positive for blaoxa-1 were resistant to all of the β-lactam antibiotics used in this study except imipenem but the blaoxa-48 and blaoxa-1 positive isolate exhibited resistance to ampicillin and amoxicillin/clavulanate but susceptible to imipenem, meropenem, cefotaxime and cefuroxime. The blashv gene detected in the two V. cholerae isolates that were positive for only blaoxa-1 but not detected in the third isolate that was positive for blaoxa-48 and blaoxa-1 explains this observation because the association of oxacillnases gene with extended-spectrum β-lactamases [ESBL] genes such as blashv often increases the level of resistance to carbapenems [166]. The blashv gene is the most detected of all the eight β-lactamases producing genes targeted in this study but isolates harbouring it exhibited varying patterns [six patterns detected as shown in Table 4] of phenotypic resistance to ampicillin, penicillin, amoxicillin/clavulanate, cefuroxime and cefotaxime. Going by the definition of extended β-lactamases, broad spectrum β-lactamases and penicillinases [167–170], we suggest that our isolates carry different variants of blashv gene which could belong to any of the 149 fully characterized variants recently reported [168] because we noticed that primer used to target blashv is not specific for a particular variant when be BLAST it using National Center for Biotechnology Information PRIMER-BLAST tool. Also, the variants could either belong to the two newly detected variants [blashv-230 with accession number NG 148673.1 detected in E. coli and blashv-231 with accession number NG_148674.1 detected in Klebsiella pneumonia] which were part of the hits returned by the PRIMER-BLAST. The three isolates with blatem gene were resistant to at least one of the penicillin antibiotics used in this study. The blatem gene is one of the Class A β-lactamases that exhibit a broad substrate hydrolysis profile against penicillins, cephalosporins, and, for a few of the enzymes, carbapenems [171].

Two phenotypic resistance patterns against β-lactam antibiotics [cefotaximeR-cefuroximeS and amoxicillin/clavulanateR-ampicillinS] observed in this study are strange because we expect a fourth-generation cephalosporin to be more active than third-generation cephalosporin and a penicillin/beta-lactamase inhibitor to be active than penicillin alone. Also, we did not come across these phenotypes in the literature. A research article report and a review [168, 172] have shown that the blasshv-mediated phenotypic and genotypic resistance could be so complex to explain at times due to various factors. Summarily the factors as pointed out in literature are: 1. the balshv family is highly diverse and complex [a large number of allelic variants including extended-spectrum β-lactamases [ESBL], non-ESBL and several not classified variants exist] in nature, 2. β-lactamase produced in small quantity do not contribute significantly to antibiotic resistance and, 3. overproduction of a β-lactamases could mask the effect of other β-lactamases [168, 173–178]. Furthermore, Vibrio parahaemolyticus isolates that are more susceptible to a third-generation cephalosporin [ceftazidime] than fourth-generation cephalosporin [cefepime] have been reported [179]. Also, an unusual case of resistance to amoxicillin/clavulanate but susceptibility to ampicillin in Enterobacter cloacae isolate has been reported [180]. However, the mechanism[s] for this rather weird phenotypic resistance pattern remains unexplained in the literature.

The genomic basis for quinolone resistance has been linked to mutation at quinolone resistance-determining regions [QRDRs] of gyrA, gyrB and ParC genes [181]. This region was targeted in this study using primers that are specific for mutation at the QRDRs of the three genes. Unfortunately, we had challenges sequencing the resulting amplicons which is the usual practice. Nevertheless, our result was interpreted using the only four articles found in the National Library of Medicine of the NCIB database that used the same primers we used for their studies [182–185]. Their works showed that mutation at the QRDRs always results in phenotypic resistance to quinolones hence we suspect the occurrence of mutation at the QRDRs of the gyr and par genes of 88% [15/17] of our isolates that exhibited resistance against the quinolones. Active efflux pump or qnr gene that were not targeted in this study could be responsible for the phenotypic resistance against quinolones exhibited by the other 22% [2/17]. Active efflux pump or qnr genes have been linked to phenotypic resistance even in the absence of mutation in the gyr and par genes [185, 186]. It was observed that some isolates harbour resistance genes without corresponding phenotypic resistance. For example, the four V. cholerae isolates that were acc and ant positive were susceptible to gentamycin. This kind of phenomenon in which bacterial isolates were susceptible to chloramphenicol despite carrying the chloramphenicol resistance gene has been reported [128].

We noticed a higher rate of phenotypic resistance and detection of resistance genes in isolates from fish samples compared to that from crab, mud prawn and mussel samples. This suggests that Vibrio spp. from fish are of more public health significance. We also observed phenotypic resistance in the absence of corresponding resistance genes and the presence of resistance determinants without corresponding phenotypic resistance in isolates. This suggests that our isolates could be engaging other antibiotics resistance mechanisms such as efflux pump [either singly or in synergy with resistance genes] to archive antibiotics resistance. The energy-dependent efflux of multiple antimicrobial agents from bacterial cells of the Vibrio spp. is a widely recognized resistance mechanism [187] and some of the specific efflux pumps found in vibrio are EmrD-3, VceB and VcrM Multidrug Efflux Pumps [188].

5. Conclusion

The virulence genes, resistance genes and phenotypic resistance heterogeneous profiles observed among our isolates [Table 6] suggest that the three variables [sampling sites, sample types and sources of the samples] got contaminated with V. cholerae and V. mimicus from different sources. The group of isolates from these variables have different profiles for resistant genes, virulence genes and phenotypic antibiotics resistance. Interestingly, to corroborate the aforementioned, the virulence genes, resistance genes and phenotypic resistance profiles is unique for each of the Vibrio spp. isolates as shown in S3 Table. Also, the report on the heterogeneity of the virulence profile of enteropathogenic E. coli from different sources supports our opinion [189]. Microbial source tracking coupled with genomic relatedness study on the isolates is expected to unravel this observation in our future study.

Considering the virulence status of our isolates, the relatively high percentages of multidrug-resistant phenotypes [MDRP], [44.44% for V. mimicus and 65.71% for V. cholerae], multiple antibiotics resistance phenotypes [MARP] [44.44% for V. mimicus and 88.57% for V. cholerae], > 0.2 multiple antibiotics indexes [0.22 for V. cholerae and 0.21 for V. mimicus], and the detection of resistance determinants in our isolates, we propose that the environmental sources of our isolates pose a public health risk in terms of the spread of antibiotics resistant strains of V cholerae non-O1/non-O139 and V. mimicus which could cause vibriosis or cholera-like infections that may be difficult to manage with commonly recommended antibiotics. Thus, the findings of this study support the need for prospective surveillance for antibiotic resistance among bacterial isolates from the environment for understanding and minimizing the spread of antibiotic resistance. The CDC recommends regular surveillance for antibiotic resistance among bacterial isolates from any environment as this is essential for understanding and minimizing the spread of resistance [190].

6. Highlights

  1. The study confirms that fish is one of the main reservoirs of V. cholera and V. mimicus in the aquatic environment
  2. Eastern Cape Province environment harbours multi-drug resistant and virulent non-O1/non-O139 V. cholerae and V. mimicus strains.
  3. The observed resistance and virulence profile of isolates in this study calls for the monitoring of environmental non-O1/non-O139 V. cholerae and V. mimicus
  4. The risk of contracting non-O1/non-O139 V. cholerae and V. mimicus infections is higher at the fish markets than at the surface water in Eastern Cape Province, South Africa
  5. An inverse relationship was observed between antibiotic resistance and virulence as reported for Non-O1, Non-O139 Vibrio cholerae Isolates in an earlier work
  6. OmpU+ and ToxR- strains of non-O1/non-O139 V. cholerae and the prevalence of β-lactamases genes in V, mimicus are reported for the first time in this study

Supporting information

S1 Fig. Gel pictures sample showing PCR duplex amplification products of the specific regions of rtx A and rtx C genes Lane 1 = 100bp molecular marker, lane 2 = negative control, lane 3 = positive control and lanes 4–12 = positive isolates.

https://doi.org/10.1371/journal.pone.0290356.s001

(TIF)

S2 Fig. Gel pictures sample showing PCR duplex amplification products of the specific regions of toxR and ompU genes Lane 1 = 100bp molecular marker, lane 2 = negative control, lanes 4 &3 = ToxR positive isolates, lanes 5&6 = ToxR & ompU positive isolates.

https://doi.org/10.1371/journal.pone.0290356.s002

(TIF)

S3 Fig. Gel pictures sample showing PCR sigleplex amplification product of the specific region of vpi gene Lane 1 = 100bp molecular marker, lane 2 = negative control, lanes 3,11&12 = negative isolates and lanes 4–10 = positive isolates.

https://doi.org/10.1371/journal.pone.0290356.s003

(TIF)

S4 Fig. Gel pictures sample showing PCR sigleplex amplification product of the specific region of hylA gene Lane 1 = 100bp molecular marker, lane 2 = negative control, lane 11 = negative isolate and lanes 3–10; 12&13 = positive isolates.

https://doi.org/10.1371/journal.pone.0290356.s004

(TIF)

S5 Fig.

A: Gel pictures sample showing PCR singleplex amplification product of the specific region of tcp gene Lane 1 = 100bp molecular marker, lane 2 = negative control, lane 3 = isolate that is negative for tcp gene, lane 4–6 = TCP positive isolates B: Gel pictures sample showing PCR singleplex amplification product of the specific region of ace gene Lane 1 = 100bp molecular marker, lane 2 = positive isolate.

https://doi.org/10.1371/journal.pone.0290356.s005

(TIF)

S6 Fig. Gel picture showing DNA amplification bands for BlaSHV, BlaOXA and BlaTEM positive DNA templates.

Lane 1 = 100bp gene ladder, lanes 6 and 16 = BlaOXA positive templates, lanes 16 and 21 = BlaSHV positive and BlaTEM positive respectively, and other lanes = DNA templates that are not positive for any BlaSHV, BlaOXA and BlaTEM resistance determinants.

https://doi.org/10.1371/journal.pone.0290356.s006

(TIF)

S7 Fig. Gel pictures showing DNA amplification bands for sul1, ant, acc, gyrB, and ParC positive DNA templates.

A: Lane 1 = 100bp gene ladder, lane = sul 1 positive isolate; B: Lane 1 = 100bp gene ladder, lane 2 = ant positive isolate, lane 3 = acc positive isolate; C: Lane 1 = 100bp gene ladder, lane 3: parC positive isolate, lanes 4&5 = gyrB positive isolates; every other lane is for templates of isolates that are not positive for any of sul1, ant, acc, gyrB, and ParC genes.

https://doi.org/10.1371/journal.pone.0290356.s007

(TIF)

S8 Fig. Gel picture showing DNA amplification bands for Blaoxa48, gyrA, Drf18 and mcr-1 positive DNA templates.

A: Lane 1 = 100bp gene ladder, lanes 4&5 = gyrA positive isolates; lane 6 = mcr-1 positive isolate; B: Lane 1 = 100bp gene ladder, lanes 2&4 = dfr18 positive isolates, lane 6 = BlaOXA48 positive isolate.

https://doi.org/10.1371/journal.pone.0290356.s008

(TIF)

S1 Table. List of primers for the confirmation V. cholerae serotype and detection of virulence determinants.

Key: AMS = amplicon size, Ref = references.

https://doi.org/10.1371/journal.pone.0290356.s009

(DOCX)

S2 Table. Primer list for the detection of antibiotics resistance determinants [PCR conditions are as reported in the references of the primers].

Key: AMS = amplicon size, Ref = references.

https://doi.org/10.1371/journal.pone.0290356.s010

(DOCX)

S3 Table. Distribution of resistance genes, phenotypic resistance and virulence genes combinations among V. cholerae [n = 34] and V. mimicus [n = 10] population.

Key: Vc = Vibrio cholera, Vm = Vibrio mimicus, Black color = Vc only, Blue colour = Vm Only, Red colour = both.

https://doi.org/10.1371/journal.pone.0290356.s011

(DOCX)

S4 Table. Dynamics of resistance for sampling sites, sample types, sources of samples and class of sample types using MARI, MARP AND MDRP as indices.

Key: n = number of isolates, MARI = multiple antibiotics resistance index, MARP = multiple-antibiotics resistance phenotype, MDRP-Multiple-drug resistance phenotype, Brac-water = brackish water, Crustan = Crustaceans.

https://doi.org/10.1371/journal.pone.0290356.s012

(DOCX)

References

  1. 1. Halpern M, Izhaki I. Fish as Hosts of Vibrio cholerae. Front Microbiol [Internet]. 2017 Feb 28 [cited 2022 Oct 19];8. Available from: http://journal.frontiersin.org/article/10.3389/fmicb.2017.00282/full pmid:28293221
  2. 2. Cholera: Background, Pathophysiology, Etiology. 2023 Jun 14 [cited 2023 Jul 12]; https://emedicine.medscape.com/article/962643-overview
    • 3. Hernández-Robles MF, Natividad-Bonifacio I, Álvarez-Contreras AK, Tercero-Alburo JJ, Quiñones-Ramírez EI, Vázquez-Salinas C. Characterization of Potential Virulence Factors of Vibrio mimicus Isolated from Fishery Products and Water. International Journal of Microbiology. 2021 Feb 10;2021:e8397930.
    • 4. Kay MK, Cartwright EJ, MacEachern D, McCullough J, Barzilay E, Mintz E, et al. Vibrio mimicus Infection Associated with Crayfish Consumption, Spokane, Washington, 2010. Journal of Food Protection. 2012 Apr 1;75[4]:762–4. pmid:22488068
    • 5. Mizuno T, Sultan SZ, Kaneko Y, Yoshimura T, Maehara Y, Nakao H, et al. Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease. The FEBS Journal. 2009;276[3]:825–34. pmid:19143841
    • 6. Eyisi OAL, Nwodo UU, Iroegbu CU. Distribution of Vibrio species in Shellfish and Water Samples Collected from the Atlantic Coastline of South-East Nigeria. J Health Popul Nutr. 2013 Sep;31[3]:314–20. pmid:24288944
    • 7. Islam MS, Zaman MH, Islam MS, Ahmed N, Clemens JD. Environmental reservoirs of Vibrio cholerae. Vaccine. 2020 Feb 29;38:A52–62. pmid:31285087
    • 8. Yu Z, Wang E, Geng Y, Wang K, Chen D, Huang X, et al. Complete genome analysis of Vibrio mimicus strain SCCF01, a highly virulent isolate from the freshwater catfish. Virulence. 2020 Dec 31;11[1]:23–31. pmid:31826705
    • 9. Ebob T Jacqueline, Asikong BEE, Mboto., Ukwuoma CIIC. Prevalence of Vibrio species in Sea Foods and Water Sources in Cross River State. ARRB. 2022 Feb 28;63–78.
      • 10. Onohuean H, Agwu E, Nwodo U. A Global Perspective of Vibrio Species and Associated Diseases: Three-Decade Meta-Synthesis of Research Advancement. Environ Health Insights. 2022 May 18;16:11786302221099406. pmid:35601189
      • 11. Kirigia JM, Sambo LG, Yokouide A, Soumbey-Alley E, Muthuri LK, Kirigia DG. Economic burden of cholera in the WHO African region. BMC International Health and Human Rights. 2009 Apr 30;9[1]:8. pmid:19405948
      • 12. Yang Y, Zhang H, Liu Y, Dong J, Xu N, Yang Q, et al. Identification of Vibrio cholerae as a bacterial pathogen of bluegill sunfish. Aquaculture Reports. 2022 Apr 1;23:101092.
      • 13. Azizi M, Azizi F. History of Cholera Outbreaks in Iran during the 19th and 20th Centuries. Middle East J Dig Dis. 2010 Jan;2[1]:51–5.
      • 14. Cholera worldwide overview [Internet]. 2023 [cited 2023 Jul 12]. https://www.ecdc.europa.eu/en/all-topics-z/cholera/surveillance-and-disease-data/cholera-monthly
        • 15. Gupta SS, Ganguly NK. Opportunities and challenges for cholera control in India. Vaccine. 2020 Feb 29;38:A25–7. pmid:31266674
        • 16. Alam MT, Weppelmann TA, Weber CD, Johnson JA, Rashid MH, Birch CS, et al. Monitoring Water Sources for Environmental Reservoirs of Toxigenic Vibrio cholerae O1, Haiti. Emerg Infect Dis. 2014 Mar;20[3]:356–63. pmid:24571741
        • 17. Mavian C, Paise T, Alam M, Browne C, De Rochars V, Nemberini S, et al. PNAS. 2020 [cited 2022 Oct 22]. Toxigenic Vibrio cholerae evolution and establishment of reservoirs in aquatic ecosystems. https://www.pnas.org/doi/10.1073/pnas.1918763117
          • 18. Rebaudet S, Piarroux R. Monitoring Water Sources for Environmental Reservoirs of Toxigenic Vibrio cholerae O1, Haiti. Emerg Infect Dis. 2015 Jan;21[1]:169–70. pmid:25531032
          • 19. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al. Foodborne Illness Acquired in the United States—Major Pathogens. Emerg Infect Dis. 2011 Jan;17[1]:7–15. pmid:21192848
          • 20. Nilavan E, Vaiyapuri M, Sadanandan Sheela G, Nadella RK, Thandapani M, Kumar A, et al. Prevalence of Vibrio mimicus in Fish, Fishery Products, and Environment of South West Coast of Kerala, India. Journal of AOAC INTERNATIONAL. 2021 Jun 1;104[3]:790–4. pmid:33484252
          • 21. Rouhani QA, Britz PJ. CONTRIBUTION OF AQUACULTURE TO RURAL LIVELIHOODS IN SOUTH AFRICA: A BASELINE STUDY. 2004;110.
          • 22. Hinrichsen E. Guide introduction to aquaculture ineasterncape [Internet]. 2008 [cited 2022 Oct 23]. https://www.dffe.gov.za/sites/default/files/legislations/guideintroductiontoaquaculture_easterncape.pdf
            • 23. South Africa factsheet. Investing in South Africa’s Aquaculture Sector: A future growth sector to be unlocked [Internet]. 2020 [cited 2022 Oct 23]. http://www.investsa.gov.za/wp-content/uploads/2021/03/FACT-SHEET_AQUACULTURE_2020.pdf
              • 24. Igbinosa EO, Obi LC, Okoh AI. Occurrence of potentially pathogenic vibrios in final effluents of a wastewater treatment facility in a rural community of the Eastern Cape Province of South Africa. Research in Microbiology. 2009 Oct 1;160[8]:531–7. pmid:19732825
              • 25. Osuolale O, Okoh A. Isolation and antibiotic profile of Vibrio spp. in final effluents of two wastewater treatment plants in the Eastern Cape of South Africa [Internet]. bioRxiv; 2018 [cited 2022 Oct 23]. p. 330456. Available from: https://www.biorxiv.org/content/10.1101/330456v2
              • 26. Abioye OE, Osunla AC, Okoh AI. Molecular Detection and Distribution of Six Medically Important Vibrio spp. in Selected Freshwater and Brackish Water Resources in Eastern Cape Province, South Africa. Frontiers in Microbiology [Internet]. 2021 [cited 2022 Oct 23];12. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2021.617703 pmid:34149632
              • 27. Osunla AC, Abioye OE, Okoh AI. Distribution and Public Health Significance of Vibrio Pathogens Recovered from Selected Treated Effluents in the Eastern Cape Province, South Africa. Water. 2021 Jan;13[7]:932.
              • 28. Igere BE, Okoh AI, Nwodo UU. Atypical and dual biotypes variant of virulent SA-NAG-Vibrio cholerae: an evidence of emerging/evolving patho-significant strain in municipal domestic water sources. Annals of Microbiology. 2022 Jan 10;72[1]:3.
              • 29. Okoh AI, Sibanda T, Nongogo V, Adefisoye M, Olayemi OO, Nontongana N. Prevalence and characterisation of non-cholerae Vibrio spp. in final effluents of wastewater treatment facilities in two districts of the Eastern Cape Province of South Africa: implications for public health. Environ Sci Pollut Res. 2015 Feb 1;22[3]:2008–17.
              • 30. Fri J, Ndip RN, Njom HA, Clarke AM. Occurrence of Virulence Genes Associated with Human Pathogenic Vibrios Isolated from Two Commercial Dusky Kob [Argyrosmus japonicus] Farms and Kareiga Estuary in the Eastern Cape Province, South Africa. International Journal of Environmental Research and Public Health. 2017 Oct;14[10]:1111. pmid:28946684
              • 31. Abioye OE, Okoh AI. Limpet [Scutellastra cochlear] Recovered From Some Estuaries in the Eastern Cape Province, South Africa Act as Reservoirs of Pathogenic Vibrio Species. Frontiers in public health [Internet]. 2018 Aug 31 [cited 2022 Oct 23];6. Available from: https://pubmed.ncbi.nlm.nih.gov/30234084/
              • 32. Mudzanani L, Ratsaka-Mathokoa M, Mahlasela L, Netshidzivhani P, Mugero C. Cholera [Internet]. 2003 [cited 2022 Oct 24]. https://www.hst.org.za/publications/South%20African%20Health%20Reviews/24%20-%20Chapter%2018.pdf
                • 33. Nutrition C for FS and A. Bacteriological Analytical Manual [BAM]. FDA [Internet]. 2022 Oct 12 [cited 2022 Oct 24]; https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam
                  • 34. Maugeri T, Carbone M, Fera MT, Gugliandolo C. Detection and differentiation of Vibrio vulnificus in seawater and plankton of coastal zone of the Mediterranean Sea. Research in microbiology. 2006 Mar 1;157:194–200.
                  • 35. Nutrition C for FS and A. BAM Appendix 2: Most Probable Number from Serial Dilutions. FDA [Internet]. 2020 Oct 9 [cited 2022 Oct 24]; https://www.fda.gov/food/laboratory-methods-food/bam-appendix-2-most-probable-number-serial-dilutions
                    • 36. Albert MJ, Islam D, Nahar S, Qadri F, Falklind S, Weintraub A. Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR. J Clin Microbiol. 1997 Jun;35[6]:1633–5. pmid:9163504
                    • 37. Hoshino K, Yamasaki S, Mukhopadhyay AK, Chakraborty S, Basu A, Bhattacharya SK, et al. Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunology & Medical Microbiology. 1998 Mar 1;20[3]:201–7. pmid:9566491
                    • 38. Hudzicki J. Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf [Internet]. 2009 [cited 2023 Jul 15]. https://asm.org/getattachment/2594ce26-bd44-47f6-8287-0657aa9185ad/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Protocol-pdf.pdf
                      • 39. CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. 2015;
                        • 40. Letchumanan V, Pusparajah P, Tan LTH, Yin WF, Lee LH, Chan KG. Occurrence and Antibiotic Resistance of Vibrio parahaemolyticus from Shellfish in Selangor, Malaysia. Frontiers in Microbiology [Internet]. 2015 [cited 2022 Oct 28];6. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2015.01417 pmid:26697003
                        • 41. CDC. Antibiotics Tested by NARMS | NARMS | CDC [Internet]. 2019 [cited 2022 Oct 28]. https://www.cdc.gov/narms/antibiotics-tested.html
                          • 42. Yin Y, Yin Y, Yang H, Chen Z, Zheng J, Peng B. Vibrio alginolyticus Survives From Ofloxacin Stress by Metabolic Adjustment. Frontiers in Microbiology [Internet]. 2022 [cited 2022 Oct 28];13. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8966707/ pmid:35369464
                          • 43. Nsofor CA, Kemajou ST, Nsofor CM. Incidence and antibiotic susceptibility pattern of Vibrio species isolated from sea foods sold in Port-Harcourt, Nigeria. 2014;4.
                          • 44. Krumperman P. Multiple Antibiotic Resistance Indexing of Escherichia coli to Identify High-Risk Sources of Fecal Contamination of Foods [Internet]. 1983. Available from:
                          • 45. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection. 2012;18[3]:268–81. pmid:21793988
                          • 46. Deng Y, Xu L, Chen H, Liu S, Guo Z, Cheng C, et al. Prevalence, virulence genes, and antimicrobial resistance of Vibrio species isolated from diseased marine fish in South China. Sci Rep. 2020 Aug 31;10[1]:14329. pmid:32868874
                          • 47. Cepas V, Soto SM. Relationship between Virulence and Resistance among Gram-Negative Bacteria. Antibiotics [Basel]. 2020 Oct 20;9[10]:719. pmid:33092201
                          • 48. MEDICC Review. Determinants of Virulence and Antimicrobial Susceptibility in Non-O1, Non-O139 Vibrio cholerae Isolates [Internet]. 2017 [cited 2023 Jul 19]. https://mediccreview.org
                            • 49. Kishii R, Takei M. Relationship between the expression of ompF and quinolone resistance in Escherichia coli. Journal of Infection and Chemotherapy. 2009 Jan 1;15[6]:361–6. pmid:20012725
                            • 50. Gajdács M, Baráth Z, Kárpáti K, Szabó D, Usai D, Zanetti S, et al. No Correlation between Biofilm Formation, Virulence Factors, and Antibiotic Resistance in Pseudomonas aeruginosa: Results from a Laboratory-Based In Vitro Study. Antibiotics [Basel]. 2021 Sep 20;10[9]:1134. pmid:34572716
                            • 51. Yazdanpour Z, Tadjrobehkar O, Shahkhah M. Significant association between genes encoding virulence factors with antibiotic resistance and phylogenetic groups in community acquired uropathogenic Escherichia coli isolates. BMC Microbiology. 2020 Aug 5;20[1]:241. pmid:32758126
                            • 52. Beceiro A, Tomás M, Bou G. Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World? Clin Microbiol Rev. 2013 Apr;26[2]:185–230. pmid:23554414
                            • 53. Jaksic S, Uhitil S, Bazulic D, Karolyi LG. Occurrence of Vibrio spp. in sea fish, shrimps and bivalve molluscs harvested from Adriatic sea. Food Control. 2002;13[2002]:491–3.
                            • 54. Noorlis A, Ghazali F M, Cheah YK, Zainazor TC. Prevalence and quantification of Vibrio species and Vibrio parahaemolyticus in freshwater fish at hypermarket level. International Food Research Journal. 2011;18:689–95.
                            • 55. Abdalla T, Al-Rumaithi H, Osaili TM, Hasan F, Obaid RS, Abushelaibi A, et al. Prevalence, Antibiotic-Resistance, and Growth Profile of Vibrio spp. Isolated From Fish and Shellfish in Subtropical-Arid Area. Frontiers in Microbiology [Internet]. 2022 [cited 2022 Dec 8];13. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2022.861547 pmid:35464960
                            • 56. Elhadi N, Radu S, Chen CH, Nishibuchi M. Prevalence of Potentially Pathogenic Vibrio Species in the Seafood Marketed in Malaysia. Journal of Food Protection. 2004 Jul 1;67[7]:1469–75. pmid:15270503
                            • 57. Möller L, Kreikemeyer B, Gerdts G, Jost G, Labrenz M. Fish as a winter reservoir for Vibrio spp. in the southern Baltic Sea coast. Journal of Marine Systems. 2021 Sep 1;221:103574.
                            • 58. Senderovich Y, Izhaki I, Halpern M. Fish as Reservoirs and Vectors of Vibrio cholerae. PLoS One. 2010 Jan 6;5[1]:e8607. pmid:20066040
                            • 59. Jones JL. Foodborne diseases. 2017 [cited 2022 Dec 8]. Vibrio. http://dx.doi.org/10.1016/B978-0-12-385007-2.00011-5
                              • 60. Chen Y, Ai X, Yang Y. Vibrio cholerae: a pathogen shared by human and aquatic animals. The Lancet Microbe. 2022 Jun 1;3[6]:e402. pmid:35659898
                              • 61. Deshayes S, Daurel C, Cattoir V, Parienti JJ, Quilici ML, de La Blanchardière A. Non-O1, non-O139 Vibrio cholerae bacteraemia: case report and literature review. Springerplus. 2015 Oct 5;4:575. pmid:26543710
                              • 62. Kumar R, Lalitha KV. Prevalence and Molecular Characterization of Vibrio cholerae O1, Non-O1 and Non-O139 in Tropical Seafood in Cochin, India. Foodborne Pathogens and Disease. 2013 Mar;10[3]:278–83. pmid:23489050
                              • 63. Ottaviani D, Medici L, Talevi G, Napoleoni M, Serratore P, Zavatta E, et al. Molecular characterization and drug susceptibility of non-O1/O139 V. cholerae strains of seafood, environmental and clinical origin, Italy. Food Microbiology. 2018 Jun 1;72:82–8.
                              • 64. Ottaviani D, Leoni F, Rocchegiani E, Santarelli S, Masini L, Di Trani V, et al. Prevalence and virulence properties of non-O1 non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy. International Journal of Food Microbiology. 2009 Jun 1;132[1]:47–53. pmid:19386376
                              • 65. Zago V, Zambon M, Civettini M, Zaltum O, Manfrin A. Virulence-associated factors in Vibrio cholerae non-O1/non-O139 and V. mimicus strains isolated in ornamental fish species. Journal of Fish Diseases. 2017;40[12]:1857–68.
                              • 66. Sharma C, Thungapathra M, Ghosh A, Mukhopadhyay AK, Basu A, Mitra R, et al. Molecular Analysis of Non-O1, Non-O139 Vibrio cholerae Associated with an Unusual Upsurge in the Incidence of Cholera-Like Disease in Calcutta, India. J Clin Microbiol. 1998 Mar;36[3]:756–63. pmid:9508308
                              • 67. Labbate M, Orata FD, Petty NK, Jayatilleke ND, King WL, Kirchberger PC, et al. A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules. Sci Rep. 2016 Nov 15;6[1]:36891. pmid:27845364
                              • 68. Kumar A, Das B, Kumar N. Vibrio Pathogenicity Island-1: The Master Determinant of Cholera Pathogenesis. Front Cell Infect Microbiol. 2020 Oct 6;10:561296. pmid:33123494
                              • 69. López-Pérez M, Grant TA, Haro-Moreno JM, Zaragoza-Solas A, Almagro-Moreno S. Cluster-driven evolution and modularity uncover paths of cholera emergence [Internet]. bioRxiv; 2022 [cited 2022 Dec 29]. p. 2022.09.26.509565. Available from: https://www.biorxiv.org/content/10.1101/2022.09.26.509565v1
                              • 70. Sechi LA, Duprè I, Deriu A, Fadda G, Zanetti S. Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy. Journal of Applied Microbiology. 2000;88[3]:475–81. pmid:10747228
                              • 71. Karaolis DKR, Johnson JA, Bailey CC, Boedeker EC, Kaper JB, Reeves PR. A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. Proceedings of the National Academy of Sciences. 1998 Mar 17;95[6]:3134–9. pmid:9501228
                              • 72. Khouadja S, Roque A, Gonzalez M, Furones D. Vibrio pathogenicity island and phage CTX genes in Vibrio alginolyticus isolated from different aquatic environments. Journal of Water and Health. 2022 Oct 1;20[10]:1469–78. pmid:36308492
                              • 73. Faruque SM, Zhu J, Asadulghani, Kamruzzaman M, Mekalanos JJ. Examination of Diverse Toxin-Coregulated Pilus-Positive Vibrio cholerae Strains Fails To Demonstrate Evidence for Vibrio Pathogenicity Island Phage. Infect Immun. 2003 Jun;71[6]:2993–9.
                              • 74. Murphy RA, Boyd EF. Three Pathogenicity Islands of Vibrio cholerae Can Excise from the Chromosome and Form Circular Intermediates. Journal of Bacteriology. 2008 Jan 15;190[2]:636–47. pmid:17993521
                              • 75. O’Shea YA, Boyd EF. Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction. FEMS Microbiology Letters. 2002 Sep 1;214[2]:153–7. pmid:12351223
                              • 76. Takahashi E, Ochi S, Mizuno T, Morita D, Morita M, Ohnishi M, et al. Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India. Frontiers in Microbiology [Internet]. 2021 [cited 2022 Dec 30];12. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2021.726273 pmid:34489915
                              • 77. Vital Brazil JM, Alves RM, Rivera ING, Rodrigues DP, Karaolis DKR, Campos LC. Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999. FEMS Microbiology Letters. 2002 Sep 1;215[1]:15–21. pmid:12393195
                              • 78. Dua P, Karmakar A, Ghosh C. Virulence gene profiles, biofilm formation, and antimicrobial resistance of Vibrio cholerae non-O1/non-O139 bacteria isolated from West Bengal, India. Heliyon. 2018 Dec 1;4[12]:e01040. pmid:30582054
                              • 79. Kimani RW, Muigai AWT, Sang W, Kiiru JN, Kariuki S. Virulence factors in environmental and clinical Vibrio cholerae from endemic areas in Kenya. African Journal of Laboratory Medicine [Internet]. 2014 Jan 1 [cited 2022 Dec 31];3[1]. Available from: https://go.gale.com/ps/i.do?p=AONE&sw=w&issn=22252002&v=2.1&it=r&id=GALE%7CA405925139&sid=googleScholar&linkaccess=abs pmid:29043171
                              • 80. Schirmeister F, Dieckmann R, Bechlars S, Bier N, Faruque SM, Strauch E. Genetic and phenotypic analysis of Vibrio cholerae non-O1, non-O139 isolated from German and Austrian patients. Eur J Clin Microbiol Infect Dis. 2014 May 1;33[5]:767–78. pmid:24213848
                              • 81. Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, et al. Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates. Appl Environ Microbiol. 2001;67[2]:910–21. pmid:11157262
                              • 82. Waturangi D, Fransisca M. Rapid Detection of Virulence Genes in Vibrio cholerae from Edible Ice in Jakarta. MI. 2009 Dec;3[3]:133–8.
                              • 83. Meena B, Anburajan L, Sathish T, Das AK, Vinithkumar NV, Kirubagaran R, et al. Studies on diversity of Vibrio sp. and the prevalence of hapA, tcpI, st, rtxA&C, acfB, hlyA, ctxA, ompU and toxR genes in environmental strains of Vibrio cholerae from Port Blair bays of South Andaman, India. Mar Pollut Bull. 2019 Jul;144:105–16.
                              • 84. Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK, Levine MM. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. Journal of Experimental Medicine. 1988 Oct 1;168[4]:1487–92. pmid:2902187
                              • 85. Kazi MI, Conrado AR, Mey AR, Payne SM, Davies BW. ToxR Antagonizes H-NS Regulation of Horizontally Acquired Genes to Drive Host Colonization. PLoS Pathog. 2016 Apr 12;12[4]:e1005570. pmid:27070545
                              • 86. Morgan SJ, Felek S, Gadwal S, Koropatkin NM, Perry JW, Bryson AB, et al. The two faces of ToxR: activator of ompU, co-regulator of toxT in Vibrio cholerae. Molecular Microbiology. 2011;81[1]:113–28. pmid:21542860
                              • 87. Wang SY, Lauritz J, Jass J, Milton DL. A ToxR Homolog from Vibrio anguillarum Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation. J Bacteriol. 2002 Mar;184[6]:1630–9. pmid:11872714
                              • 88. Wibbenmeyer JA, Provenzano D, Landry CF, Klose KE, Delcour AH. Vibrio cholerae OmpU and OmpT Porins Are Differentially Affected by Bile. Infection and Immunity. 2002 Jan;70[1]:121. pmid:11748172
                              • 89. Zhang Y, Deng Y, Feng J, Hu J, Chen H, Guo Z, et al. ToxR modulates biofilm formation in fish pathogen Vibrio harveyi. Letters in Applied Microbiology. 2022;74[2]:288–99. pmid:34822732
                              • 90. Zhang Y, Hu L, Osei-Adjei G, Zhang Y, Yang W, Yin Z, et al. Autoregulation of ToxR and Its Regulatory Actions on Major Virulence Gene Loci in Vibrio parahaemolyticus. Frontiers in Cellular and Infection Microbiology [Internet]. 2018 [cited 2022 Dec 31];8. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2018.00291 pmid:30234024
                              • 91. Shinoda S, Nakagawa T, Shi L, Bi K, Kanoh Y, Tomochika K ichi, et al. Distribution of Virulence-Associated Genes in Vibrio mimicus Isolates from Clinical and Environmental Origins. Microbiology and Immunology. 2004;48[7]:547–51. pmid:15272201
                              • 92. Miyoshi S, Sasahara K, Akamatsu S, Rahman MM, Katsu T, Tomochika K, et al. Purification and characterization of a hemolysin produced by Vibrio mimicus. Infect Immun. 1997 May;65[5]:1830–5. pmid:9125568
                              • 93. Gxalo O, Digban TO, Igere BE, Olapade OA, Okoh AI, Nwodo UU. Virulence and Antibiotic Resistance Characteristics of Vibrio Isolates From Rustic Environmental Freshwaters. Frontiers in Cellular and Infection Microbiology [Internet]. 2021 [cited 2022 Dec 23];11. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2021.732001 pmid:34490150
                              • 94. Neogi SB, Chowdhury N, Awasthi SP, Asakura M, Okuno K, Mahmud ZH, et al. Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains. Applied and Environmental Microbiology. 2019 Feb 1;85:e01977. pmid:30446560
                              • 95. Na-Ubol M, Srimanote P, Chongsa-nguan M, Indrawattana N, Sookrung N, Tapchaisri P, et al. Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand. Indian J Med Res. 2011 Apr;133[4]:387–94.
                              • 96. Safa A, Nair GB, Kong RYC. Evolution of new variants of Vibrio cholerae O1. Trends Microbiol. 2010 Jan;18[1]:46–54. pmid:19942436
                              • 97. Safa A, Bhuiyan NA, Murphy D, Bates J, Nusrin S, Kong RYC, et al. Multilocus genetic analysis reveals that the Australian strains of Vibrio cholerae O1 are similar to the pre-seventh pandemic strains of the El Tor biotype. J Med Microbiol. 2009 Jan;58[Pt 1]:105–11. pmid:19074660
                              • 98. Safa A, Sultana J, Cam PD, Mwansa JC, Kong RYC. Vibrio cholerae O1 Hybrid El Tor Strains, Asia and Africa. Emerg Infect Dis. 2008 Jun;14[6]:987–8. pmid:18507925
                              • 99. Safa A, Bhuyian NA, Nusrin S, Ansaruzzaman M, Alam M, Hamabata T, et al. Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes. J Med Microbiol. 2006 Nov;55[Pt 11]:1563–9. pmid:17030917
                              • 100. Safa A, Bhuiyan NA, Alam M, Sack DA, Nair GB. Genomic Relatedness of the New Matlab Variants of Vibrio cholerae O1 to the Classical and El Tor Biotypes as Determined by Pulsed-Field Gel Electrophoresis. J Clin Microbiol. 2005 Mar;43[3]:1401–4. pmid:15750117
                              • 101. Zaw MT, Emran NA, Ibrahim MY, Suleiman M, Awang Mohd TA, Yusuff AS, et al. Genetic diversity of toxigenic Vibrio cholerae O1 from Sabah, Malaysia 2015. J Microbiol Immunol Infect. 2019 Aug 1;52[4]:563–70. pmid:29428381
                              • 102. Zaw MT, Emran NA, Naing DKS, Lin Z. Atypical El Tor: new Vibrio cholerae O1 biotype causing epidemic cholera. Borneo Journal of Medical Sciences [BJMS] [Internet]. 2016 Dec 9 [cited 2022 Dec 27];10[1]. Available from: https://jurcon.ums.edu.my/ojums/index.php/bjms/article/view/566
                              • 103. Chow KH, Ng TK, Yuen KY, Yam WC. Detection of RTX Toxin Gene in Vibrio cholerae by PCR. J Clin Microbiol. 2001 Jul;39[7]:2594–7. pmid:11427575
                              • 104. Iredell JR, Manning PA. Biotype-specific tcpA genes in Vibrio cholerae. FEMS Microbiol Lett. 1994 Aug 1;121[1]:47–54. pmid:7915998
                              • 105. Lin W, Fullner KJ, Clayton R, Sexton JA, Rogers MB, Calia KE, et al. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci U S A. 1999 Feb 2;96[3]:1071–6. pmid:9927695
                              • 106. Schwartz K, Hammerl JA, Göllner C, Strauch E. Environmental and Clinical Strains of Vibrio cholerae Non-O1, Non-O139 From Germany Possess Similar Virulence Gene Profiles. Front Microbiol [Internet]. 2019 Apr 12 [cited 2022 Dec 28];10. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.00733/full pmid:31031724
                              • 107. Bhuiyan NA, Nusrin S, Alam M, Morita M, Watanabe H, Ramamurthy T, et al. Changing genotypes of cholera toxin [CT] of Vibrio cholerae O139 in Bangladesh and description of three new CT genotypes. FEMS Immunology & Medical Microbiology. 2009 Nov;57[2]:136–41. pmid:19732141
                              • 108. Li , Wu Y, Sun X, Ma J, Li X, Liu C, et al. Non-O1/non-O139 Vibrio cholerae bacteraemia in mainland China from 2005 to 2019: clinical, epidemiological and genetic characteristics. Epidemiology & Infection. 2020 ed;148:e186. pmid:32635946
                              • 109. Stypulkowska-Misiurewicz H, Pancer K, Roszkowiak A. Two unrelated cases of septicaemia due to Vibrio cholerae non-O1, non-O139 in Poland, July and August 2006. Weekly releases [1997–2007]. 2006 Nov 30;11[48]:3088. pmid:17213560
                              • 110. Ceccarelli D, Chen A, Hasan NA, Rashed SM, Huq A, Colwell RR. Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay, Maryland [Internet]. 2015 [cited 2022 Dec 22]. https://journals.asm.org/doi/epub/10.1128/AEM.03540-14
                                • 111. Goel AK, Jiang SC. Genetic determinants of virulence, antibiogram and altered biotype among the Vibrio cholerae O1 isolates from different cholera outbreaks in India. Infect Genet Evol. 2010 Aug;10[6]:815–9. pmid:19580888
                                • 112. Mizuno T, Debnath A, Miyoshi S ichi, Mizuno T, Debnath A, Miyoshi S ichi. Hemolysin of Vibrio Species [Internet]. Microorganisms. IntechOpen; 2019 [cited 2022 Dec 28]. https://www.intechopen.com/state.item.id
                                  • 113. Liu X, Gao H, Xiao N, Liu Y, Li J, Li L. Outer Membrane Protein U [OmpU] Mediates Adhesion of Vibrio mimicus to Host Cells via Two Novel N-Terminal Motifs. PLOS ONE. 2015 Mar 5;10[3]:e0119026. pmid:25742659
                                  • 114. Nakasone N, Iwanaga M. Characterization of Outer Membrane Protein OmpU of Vibrio cholerae O1. Infect Immun. 1998 Oct;66[10]:4726–8. pmid:9746570
                                  • 115. Sperandio V, Girón JA, Silveira WD, Kaper JB. The OmpU outer membrane protein, a potential adherence factor of Vibrio cholerae. Infect Immun. 1995 Nov;63[11]:4433–8. pmid:7591082
                                  • 116. Noorlis A, Ghazali FM, Zainazor ZTC, Wong WC, Tunung R, Pui CF, et al. Antibiotic resistance and biosafety of Vibrio cholerae and Vibrio parahaemolyticus from freshwater fish at retail level. 2011;
                                  • 117. Baron S, Larvor E, Chevalier S, Jouy E, Kempf I, Granier SA, et al. Antimicrobial Susceptibility among Urban Wastewater and Wild Shellfish Isolates of Non-O1/Non-O139 Vibrio cholerae from La Rance Estuary [Brittany, France]. Frontiers in Microbiology [Internet]. 2017 [cited 2022 Dec 23];8. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2017.01637 pmid:28955305
                                  • 118. Hirsch N, Gangl A, Schwartz K, Mayer-Scholl A, Hammerl JA, Strauch E. Phenotypic and Genotypic Properties of Vibrio cholerae non-O1, non-O139 Isolates Recovered from Domestic Ducks in Germany. microorganisms. 2020;8[1104]. pmid:32717968
                                  • 119. Okoh EB. Antibiotic Susceptibility Patterns and Plasmid Profile of Vibrio cholerae from Water Samples in Elele Community, Rivers State, Nigeria. J Appl Sci Environ Manage. 2012;16[1]:135–40.
                                  • 120. Wu Q, Vaziri AZ, Omidi N, Hassan Kaviar V, Maleki A, Khadivar P, et al. Antimicrobial resistance among clinical Vibrio cholerae non-O1/non-O139 isolates: systematic review and meta-analysis. Pathogens and Global Health. 2022 Aug 19;0[0]:1–10. pmid:35983997
                                  • 121. Tadesse BT, Ashley EA, Ongarello S, Havumaki J, Wijegoonewardena M, González IJ, et al. Antimicrobial resistance in Africa: a systematic review. BMC Infectious Diseases. 2017 Sep 11;17[1]:616. pmid:28893183
                                  • 122. WHO. Report signals increasing resistance to antibiotics in bacterial infections in humans and need for better data [Internet]. [cited 2022 Dec 15]. https://www.who.int/news/item/09-12-2022-report-signals-increasing-resistance-to-antibiotics-in-bacterial-infections-in-humans-and-need-for-better-data
                                    • 123. Miyazato T, Tamaki Y, Sithivong N, Phantouamath B, Insisiengmay S, Higa N, et al. ANTIBIOTIC SUSCEPTIBILITY AND ITS GENETIC ANALYSIS OF VIBRIO CHOLERAE NON-O1, NON-O139 FROM ENVIRONMENTAL SOURCES IN LAO PEOPLE’S DEMOCRATIC REPUBLIC. TropMedHealth. 2004;32[3]:245–8.
                                    • 124. Yu L, Zhou Y, Wang R, Lou J, Zhang L, Li J, et al. Multiple Antibiotic Resistance of Vibrio cholerae Serogroup O139 in China from 1993 to 2009. PLOS ONE. 2012 Jun 11;7[6]:e38633. pmid:22701685
                                    • 125. Samanta P, Saha RN, Chowdhury G, Naha A, Sarkar S, Dutta S, et al. Dissemination of newly emerged polymyxin B sensitive Vibrio cholerae O1 containing Haitian-like genetic traits in different parts of India. Journal of Medical Microbiology. 2018 Sep 1;67[9]:1326–33. pmid:29927375
                                    • 126. Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. The Lancet. 2004 Jan 17;363[9404]:223–33.
                                    • 127. Baron S, Lesne J, Jouy E, Larvor E, Kempf I, Boncy J, et al. Antimicrobial Susceptibility of Autochthonous Aquatic Vibrio cholerae in Haiti. Frontiers in Microbiology [Internet]. 2016 [cited 2023 Jan 6];7. Available from: https://www.academia.edu/57356769/Antimicrobial_Susceptibility_of_Autochthonous_Aquatic_Vibrio_cholerae_in_Haiti pmid:27818656
                                    • 128. Lepuschitz S, Baron S, Larvor E, Granier SA, Pretzer C, Mach RL, et al. Phenotypic and Genotypic Antimicrobial Resistance Traits of Vibrio cholerae Non-O1/Non-O139 Isolated From a Large Austrian Lake Frequently Associated With Cases of Human Infection. Frontiers in Microbiology [Internet]. 2019 [cited 2022 Dec 22];10. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02600 pmid:31781080
                                    • 129. Adekanmbi AO, Olawuni OG, Olaposi AV. Metal contamination and coexistence of metal and antibiotic resistance in Vibrio species recovered from aquaculture ponds with and without history of antibiotic usage in Southwest Nigeria. Bulletin of the National Research Centre. 2021 Jun 30;45[1]:122.
                                    • 130. Igere BE, Okoh AI, Nwodo UU. Antibiotic Susceptibility Testing [AST] Reports: A Basis for Environmental/Epidemiological Surveillance and Infection Control Amongst Environmental Vibrio cholerae. International Journal of Environmental Research and Public Health. 2020 Jan;17[16]:5685. pmid:32781601
                                    • 131. Laviad-Shitrit S, Sharaby Y, Izhaki I, Peretz A, Halpern M. Antimicrobial Susceptibility of Environmental Non-O1/Non-O139 Vibrio cholerae Isolates. Frontiers in Microbiology [Internet]. 2018 [cited 2022 Dec 23];9. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01726 pmid:30116229
                                    • 132. Luo Y, Wang H, Liang J, Qian H, Ye J, Chen L, et al. Population Structure and Multidrug Resistance of Non-O1/Non-O139 Vibrio cholerae in Freshwater Rivers in Zhejiang, China | SpringerLink [Internet]. 2021 [cited 2022 Dec 23]. https://link.springer.com/article/10.1007/s00248-020-01645-z
                                      • 133. Folster JP, Katz L, McCullough A, Parsons MB, Knipe K, Sammons SA, et al. Multidrug-Resistant IncA/C Plasmid in Vibrio cholerae from Haiti. Emerg Infect Dis. 2014 Nov;20[11]:1951–3. pmid:25340576
                                      • 134. Han F, Walker RD, Janes ME, Prinyawiwatkul W, Ge B. Antimicrobial Susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus Isolates from Louisiana Gulf and Retail Raw Oysters. Appl Environ Microbiol. 2007 Nov;73[21]:7096–8. pmid:17827331
                                      • 135. Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. Cholera. Lancet. 2012 Jun 30;379[9835]:2466–76. pmid:22748592
                                      • 136. Reller LB, Weinstein M, Jorgensen JH, Ferraro MJ. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices. Clinical Infectious Diseases. 2009 Dec 1;49[11]:1749–55. pmid:19857164
                                      • 137. Rodloff A, Bauer T, Ewig S, Kujath P, Müller E. Susceptible, Intermediate, and Resistant—The Intensity of Antibiotic Action. Dtsch Arztebl Int. 2008 Sep;105[39]:657–62. pmid:19626213
                                      • 138. World Health Organization. Critically important antimicrobials for human medicine [Internet]. 6th rev. Geneva: World Health Organization; 2019 [cited 2023 Jan 11]. 45 p. Available from: https://apps.who.int/iris/handle/10665/312266
                                        • 139. Chandy SJ, Naik GS, Balaji V, Jeyaseelan V, Thomas K, Lundborg CS. High cost burden and health consequences of antibiotic resistance: the price to pay. J Infect Dev Ctries. 2014 Sep 12;8[9]:1096–102. pmid:25212073
                                        • 140. Naylor NR, Pouwels KB, Hope R, Green N, Henderson KL, Knight GM, et al. The health and cost burden of antibiotic resistant and susceptible Escherichia coli bacteraemia in the English hospital setting: A national retrospective cohort study. PLoS One. 2019 Sep 10;14[9]:e0221944. pmid:31504046
                                        • 141. World Health Organization. Antibiotic resistance [Internet]. 2020 [cited 2023 Jan 11]. https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
                                          • 142. Chowdhury MA, Aziz KM, Rahim Z, Kay BA. Antibiotic resistance patterns of Vibrio mimicus isolated from human and environmental sources in Bangladesh. Antimicrob Agents Chemother. 1986 Oct;30[4]:622–3. pmid:3789697
                                          • 143. Raissy M, Moumeni M, Ansari M, Rahimi E. Occurrence of Vibrio Spp. in Lobster and Crab from the Persian Gulf. Journal of Food Safety. 2012;32[2]:198–203.
                                          • 144. Yamanaka H, Aziz K. Ecology of Vibrio minus in aquatic environment. Applied and Environmental Microbiology. 1989;
                                          • 145. Onohuean H, Agwu E, Nwodo UU. Systematic review and meta-analysis of environmental Vibrio species—antibiotic resistance. Heliyon. 2022 Feb 1;8[2]:e08845. pmid:35265752
                                          • 146. Beaber JW, Hochhut B, Waldor MK. Genomic and Functional Analyses of SXT, an Integrating Antibiotic Resistance Gene Transfer Element Derived from Vibrio cholerae. J Bacteriol. 2002 Aug;184[15]:4259–69. pmid:12107144
                                          • 147. Davis MA, Besser TE, Orfe LH, Baker KNK, Lanier AS, Broschat SL, et al. Genotypic-Phenotypic Discrepancies between Antibiotic Resistance Characteristics of Escherichia coli Isolates from Calves in Management Settings with High and Low Antibiotic Use ▿. Appl Environ Microbiol. 2011 May;77[10]:3293–9.
                                          • 148. Urmi UL, Nahar S, Rana M, Sultana F, Jahan N, Hossain B, et al. Genotypic to Phenotypic Resistance Discrepancies Identified Involving &beta;-Lactamase Genes, blaKPC, blaIMP, blaNDM-1, and blaVIM in Uropathogenic Klebsiella pneumoniae. IDR. 2020 Aug 18;13:2863–75.
                                          • 149. Bae SH, Yoon S, Kim K, Kim YB, Lee YJ. Comparative Analysis of Chloramphenicol-Resistant Enterococcus faecalis Isolated from Dairy Companies in Korea. Vet Sci. 2021 Jul 27;8[8]:143. pmid:34437465
                                          • 150. Bischoff KM, White DG, Hume ME, Poole TL, Nisbet DJ. The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli. FEMS Microbiology Letters. 2005 Feb 1;243[1]:285–91. pmid:15668031
                                          • 151. Begmatov S, Beletsky AV, Gruzdev EV, Mardanov AV, Glukhova LB, Karnachuk OV, et al. Distribution Patterns of Antibiotic Resistance Genes and Their Bacterial Hosts in a Manure Lagoon of a Large-Scale Swine Finishing Facility. Microorganisms. 2022 Nov;10[11]:2301. pmid:36422370
                                          • 152. Gibreel A, Sköld O, Taylor DE. Characterization of plasmid-mediated aphA-3 kanamycin resistance in Campylobacter jejuni. Microb Drug Resist. 2004;10[2]:98–105. pmid:15256024
                                          • 153. Lei T, Zhang J, Jiang F, He M, Zeng H, Chen M, et al. First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus. International Journal of Food Microbiology. 2019 Nov 2;308:108290. pmid:31442712
                                          • 154. Valdez C, Costa C, Simões M, de Carvalho CCCR, Baptista T, Campos MJ. Detection of mcr-1 Gene in Undefined Vibrio Species Isolated from Clams. Microorganisms. 2022 Feb 8;10[2]:394.
                                          • 155. Wang Q, Sun J, Li J, Ding Y, Li XP, Lin J, et al. Expanding landscapes of the diversified mcr-1-bearing plasmid reservoirs. Microbiome. 2017 Jul 6;5[1]:70. pmid:28683827
                                          • 156. Kai J, Wang S. Recent progress on elucidating the molecular mechanism of plasmid-mediated colistin resistance and drug design. Int Microbiol. 2020 Aug 1;23[3]:355–66. pmid:31872322
                                          • 157. Li B, Yin F, Zhao X, Guo Y, Wang W, Wang P, et al. Colistin Resistance Gene mcr-1 Mediates Cell Permeability and Resistance to Hydrophobic Antibiotics. Frontiers in Microbiology [Internet]. 2020 [cited 2023 Jan 14];10. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.03015 pmid:31998280
                                          • 158. Sherman EX, Hufnagel DA, Weiss DS. MCR -1 confers cross-resistance to lysozyme. The Lancet Infectious Diseases. 2016 Nov;16[11]:1226–7. pmid:27788983
                                          • 159. Xu F, Zeng X, Hinenoya A, Lin J. MCR-1 Confers Cross-Resistance to Bacitracin, a Widely Used In-Feed Antibiotic. mSphere. 2018 Sep 5;3[5]:e00411–18. pmid:30185515
                                          • 160. Aghapour Z, Gholizadeh P, Ganbarov K, Bialvaei AZ, Mahmood SS, Tanomand A, et al. Molecular mechanisms related to colistin resistance in Enterobacteriaceae. Infect Drug Resist. 2019 Apr 24;12:965–75. pmid:31190901
                                          • 161. Xu Y, Zhong LL, Srinivas S, Sun J, Huang M, Paterson DL, et al. Spread of MCR-3 Colistin Resistance in China: An Epidemiological, Genomic and Mechanistic Study. EBioMedicine. 2018 Aug;34:139–57. pmid:30061009
                                          • 162. Zając M, Sztromwasser P, Bortolaia V, Leekitcharoenphon P, Cavaco LM, Ziȩtek-Barszcz A, et al. Occurrence and Characterization of mcr-1-Positive Escherichia coli Isolated From Food-Producing Animals in Poland, 2011–2016. Frontiers in Microbiology [Internet]. 2019 [cited 2023 Jan 14];10. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2019.01753
                                          • 163. Grisold AJ, Luxner J, Bedenić B, Diab-Elschahawi M, Berktold M, Wechsler-Fördös A, et al. Diversity of Oxacillinases and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii from Austria. Int J Environ Res Public Health. 2021 Feb 23;18[4]:2171. pmid:33672170
                                          • 164. Che T, Bethel CR, Pusztai-Carey M, Bonomo RA, Carey PR. The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine *. Journal of Biological Chemistry. 2014 Feb 28;289[9]:6152–64.
                                          • 165. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. Journal of Antimicrobial Chemotherapy. 2012 Jul 1;67[7]:1597–606. pmid:22499996
                                          • 166. Mlynarcik P, Chalachanova A, Vagnerovă I, Holy O, Zatloukalova S, Kolar M. PCR Detection of Oxacillinases in Bacteria. Microbial Drug Resistance. 2020 Sep;26[9]:1023–37. pmid:32212994
                                          • 167. Ghafourian S, Sadeghifard N, Soheili S, Sekawi Z. Extended Spectrum Beta-lactamases: Definition, Classification and Epidemiology. Current Issues in Molecular Biology. 2015 Jan;17[1]:11–22. pmid:24821872
                                          • 168. Liakopoulos A, Mevius D, Ceccarelli D. A Review of SHV Extended-Spectrum β-Lactamases: Neglected Yet Ubiquitous. Frontiers in Microbiology [Internet]. 2016 [cited 2022 Nov 3];7. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2016.01374
                                          • 169. Rawat D, Nair D. Extended-spectrum β-lactamases in Gram Negative Bacteria. J Glob Infect Dis. 2010;2[3]:263–74.
                                          • 170. Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M, Herman L, et al. Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health. FEMS Microbiology Reviews. 2010 May 1;34[3]:295–316.
                                          • 171. Brown NG, Shanker S, Prasad BVV, Palzkill T. Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis. J Biol Chem. 2009 Nov 27;284[48]:33703–12.
                                          • 172. Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC-Antimicrobial Resistance. 2021 Sep 1;3[3]:dlab092.
                                          • 173. Arshad M, Nowalk A, Tamma PD. 14—Multidrug-resistant gram-negative infections in transplant and oncology patients. In: Steinbach WJ, Green MD, Michaels MG, Danziger-Isakov LA, Fisher BT, editors. Pediatric Transplant and Oncology Infectious Diseases [Internet]. Philadelphia: Elsevier; 2021 [cited 2023 Jan 19]. p. 97–102.e3. https://www.sciencedirect.com/science/article/pii/B9780323641982000233
                                            • 174. Cantón R, Morosini MI, Martin O, de la Maza S, de la Pedrosa EGG. IRT and CMT β-lactamases and inhibitor resistance. Clinical Microbiology and Infection. 2008 Jan 1;14:53–62.
                                            • 175. Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended-spectrum β-lactamase production in Enterobacteriaceae: review and bench guide. Clinical Microbiology and Infection. 2008 Jan 1;14:90–103.
                                            • 176. Paterson DL. 313—Infections Due to Other Members of the Enterobacteriaceae, Including Management of Multidrug-Resistant Strains. In: Goldman L, Schafer AI, editors. Goldman’s Cecil Medicine [Twenty Fourth Edition] [Internet]. Philadelphia: W.B. Saunders; 2012 [cited 2023 Jan 19]. p. 1874–7. https://www.sciencedirect.com/science/article/pii/B9781437716047003134
                                              • 177. Schumacher H, Scheibel J, Møller JK. Cross-resistance patterns among clinical isolates of Klebsiella pneumoniae with decreased susceptibility to cefuroxime. Journal of Antimicrobial Chemotherapy. 2000 Aug 1;46[2]:215–21. pmid:10933643
                                              • 178. Shaaban M, Elshaer SL, Abd El-Rahman OA. Prevalence of extended-spectrum β-lactamases, AmpC, and carbapenemases in Proteus mirabilis clinical isolates. BMC Microbiology. 2022 Oct 11;22[1]:247.
                                              • 179. Sun J, Li X, Hu Z, Xue X, Zhang M, Wu Q, et al. Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018–2020. PLOS ONE. 2022 Aug 26;17[8]:e0273700. pmid:36018831
                                              • 180. Brumfitt W, Hamilton-Miller JM, Dixson S, Gargan RA, Gooding A. Phenomenon of resistance to Augmentin associated with sensitivity to ampicillin: occurrence and explanation. J Clin Pathol. 1983 Jun;36[6]:670–3. pmid:6343436
                                              • 181. Johnning A, Kristiansson E, Fick J, Weijdegård B, Larsson DGJ. Resistance Mutations in gyrA and parC are Common in Escherichia Communities of both Fluoroquinolone-Polluted and Uncontaminated Aquatic Environments. Frontiers in Microbiology [Internet]. 2015 [cited 2023 Jan 23];6. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2015.01355
                                              • 182. Baranwal S, Dey K, Ramamurthy T, Nair GB, Kundu M. Role of Active Efflux in Association with Target Gene Mutations in Fluoroquinolone Resistance in Clinical Isolates of Vibrio cholerae. Antimicrob Agents Chemother. 2002 Aug;46[8]:2676–8. pmid:12121955
                                              • 183. Marin MA, Thompson CC, Freitas FS, Fonseca EL, Aboderin AO, Zailani SB, et al. Cholera Outbreaks in Nigeria Are Associated with Multidrug Resistant Atypical El Tor and Non-O1/Non-O139 Vibrio cholerae. PLoS Negl Trop Dis. 2013 Feb 14;7[2]:e2049. pmid:23459673
                                              • 184. Okuda J, Hayashi N, Wakahara Y, Gotoh N. Reduced Expression of the vca0421 Gene of Vibrio cholerae O1 Results in Innate Resistance to Ciprofloxacin. Antimicrob Agents Chemother. 2010 Nov;54[11]:4917–9. pmid:20805389
                                              • 185. Srinivasan VB, Virk RK, Kaundal A, Chakraborty R, Datta B, Ramamurthy T, et al. Mechanism of Drug Resistance in Clonally Related Clinical Isolates of Vibrio fluvialis Isolated in Kolkata, India. Antimicrob Agents Chemother. 2006 Jul;50[7]:2428–32. pmid:16801422
                                              • 186. Hooper DC, Jacoby GA. Mechanisms of drug resistance: quinolone resistance. Ann N Y Acad Sci. 2015 Sep;1354[1]:12–31. pmid:26190223
                                              • 187. Stephen J, Lekshmi M, Ammini P, Kumar SH, Varela MF. Membrane Efflux Pumps of Pathogenic Vibrio Species: Role in Antimicrobial Resistance and Virulence. Microorganisms. 2022 Feb 7;10[2]:382. pmid:35208837
                                              • 188. Andersen JL, He GX, Kakarla P, Kc R, Kumar S, Lakra WS, et al. Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens. International Journal of Environmental Research and Public Health. 2015 Feb;12[2]:1487. pmid:25635914
                                              • 189. Xu Y, Bai X, Jin Y, Hu B, Wang H, Sun H, et al. High Prevalence of Virulence Genes in Specific Genotypes of Atypical Enteropathogenic Escherichia coli. Frontiers in Cellular and Infection Microbiology [Internet]. 2017 [cited 2023 Jan 6];7. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2017.00109 pmid:28421169
                                              • 190. CDC. Antibiotic Treatment | Treatment | Cholera | CDC [Internet]. 2022 [cited 2022 Nov 2]. https://www.cdc.gov/cholera/treatment/antibiotic-treatment.html