https://orcid.org/0000-0002-7626-4796
Figures
Abstract
Globally, the significant risk to food safety and public health posed by antimicrobial-resistant foodborne Salmonella pathogens is driven by the utilization of in-feed antibiotics, with variations in usage across poultry production systems. The current study investigated the occurrence of virulence, antimicrobial resistant profiles, and biofilm-forming potentials of Salmonella isolates sourced from different chicken types. A total of 75 cloacal faecal samples were collected using sterile swabs from layer, broiler, and indigenous chickens across 15 poultry farms (five farms per chicken type). The samples were analysed for the presence of Salmonella spp. using species-specific PCR analysis. Out of the 150 presumptive isolates, a large proportion (82; 55%) were confirmed as Salmonella species, comprising the serovars S. typhimurium (49%) and S. enteritidis (30%) while 21% were uncategorised. Based on phenotypic antibiotic susceptibility test, the Salmonella isolates were most often resistant to erythromycin (62%), tetracycline (59%), and trimethoprim (32%). The dominant multiple antibiotic resistance phenotypes were SXT-W-TE (16%), E-W-TE (10%), AML-E-TE (10%), E-SXT-W-TE (13%), and AMP-AML-E-SXT-W-TE (10%). Genotypic assessment of antibiotic resistance genes revealed that isolates harboured the ant (52%), tet (A) (46%), sui1 (13%), sui2 (14%), and tet (B) (9%) determinants. Major virulence genes comprising the invasion gene spiC, the SPI-3 encoded protein (misL) that is associated with the establishment of chronic infections and host specificity as well as the SPI-4 encoded orfL that facilitates adhesion, autotransportation and colonisation were detected in 26%, 16%, and 14% of the isolates respectively. There was no significant difference on the proportion of Salmonella species and the occurrence of virulence and antimicrobial resistance determinants among Salmonella isolates obtained from different chicken types. In addition, neither the chicken type nor incubation temperature influenced the potential of the Salmonella isolates to form biofilms, although a large proportion (62%) exhibited weak to strong biofilm-forming potentials. Moderate to high proportions of antimicrobial resistant pathogenic Salmonella serovars were detected in the study but these did not vary with poultry production systems.
Citation: Dlamini SB, Mlambo V, Mnisi CM, Ateba CN (2024) Virulence, multiple drug resistance, and biofilm-formation in Salmonella species isolated from layer, broiler, and dual-purpose indigenous chickens. PLoS ONE 19(10): e0310010. https://doi.org/10.1371/journal.pone.0310010
Editor: Raúl Alejandro Alegría-Morán, Universidad Santo Tomas, CHILE
Received: November 19, 2023; Accepted: August 22, 2024; Published: October 28, 2024
Copyright: © 2024 Dlamini 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: SB Dlamini (Grant no: 138276) This work was supported by the National Research Foundation (NRF) Grant no: 138276. The URL of the funders website is https://www.nrf.ac.za The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exists.
1. Introduction
The soaring demand for poultry products has seen production methods shift from extensive to intensive techniques to maximise bird productivity [1]. However, intensive production systems are highly stressful environments that compromise bird immune function resulting in high incidences of disease and poor growth performance. To mitigate the negative impacts of stress and infectious diseases, producers have traditionally relied on the use of antibiotics for growth promotion and therapeutic processes [2]. Unfortunately, the extensive use of antimicrobial agents contributes to the emergence of antimicrobial resistance [3], which threatens poultry production, food safety, and public health [4].
Antimicrobial resistance (AMR) has been reported in Salmonella species, thus contributing a significant risk to food safety and public health globally [5]. The most prevalent zoonotic Salmonella serotypes, comprise Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Typhimurium, and these are responsible for salmonellosis in humans and animals [6]. While some Salmonella serotypes cause self-limiting gastroenteritis in humans, resistant cells most often are associated with more complicated infections, which are challenging to treat especially in vulnerable groups such as children [7], the elderly, and immunocompromised individuals [8]. Although it is generally accepted that Salmonella species occur in the gastrointestinal tract of chickens [9], investigations into the influence of poultry production systems and associated husbandry practices on their prevalence are rather limited. Several studies conducted on Salmonella species in poultry from the study area focused on the prevalence of virulent and AMR Salmonella serovars [10–12], with no consideration of the influence of poultry production systems and management practices. The prevalence and distribution of AMR pathogens in chicken vary with management practices and biosecurity standards across different poultry production systems [13]. Indeed, antimicrobial resistance has been reported to occur less in free-range extensive or semi-intensive rearing systems compared to conventional production systems [14]. In addition, broilers tend to harbor more antibiotic-resistant bacteria compared to layers, which is attributed to the reduced use of antibiotics in the latter [15]. This suggests that the occurrence of AMR in indigenous chickens could be even lower than in broilers and layers because antibiotics are rarely used for native birds reared in free-range extensive production systems [16]. However, indirect transmission of antibiotic resistance among livestock animals, including indigenous chickens, especially in resource-limited settings has been reported [17, 18]. For this reason, it is necessary to monitor the distribution of AMR among different chicken types not only to achieve treatment success but also to track the emergence of AMR pathogens and possible spread to the environment and animal food products. Moreover, bacterial pathogens like Salmonella can develop biofilm structures (extracellular polymeric substances) and multicellular properties, enabling them to better survive chemical compounds and antimicrobial agents [19]. Recent studies have reported a high prevalence of multi-drug resistant biofilm-forming Salmonella serovars in poultry farms and processing facilities globally [20–24], suggesting increased risks for recurring contamination of poultry products. The potential of pathogenic bacteria including Salmonella species to form biofilms affects food safety even for products preserved in appropriate refrigerated storage [25]. Therefore, this study investigated the occurrence of virulence and AMR determinants, and biofilm-forming potentials of Salmonella serovars in intensively (layer and broiler) and semi-intensively reared (dual-purpose, indigenous) chickens in the North West province, South Africa. The study hypothesized that the prevalence of AMR and biofilm-forming potentials of Salmonella spp. would be higher in intensively reared compared to extensively reared birds.
2. Materials and methods
2.1 Sampling strategy
Prior to sample collection, 15 (5 broilers, 5 layers and 5 dual-purpose indigenous chickens) poultry farms were identified and selected. The owners of the farms were approached to participate in the study based on willingness. Ethical clearance for the study was obtained from the North-West University AnimCare Research Ethics Committee (approval no. NWU-00503-20-A5). Informed consent was sourced from the farmers through consent forms since no permits were required. Data on antibiotic treatment history and related husbandry practices were collected through a structured questionnaire (S1 File). The questionnaire elicited information on poultry farmers’ demographic, husbandry practices, antibiotic use, and their knowledge of Salmonella spp. The survey instrument was face and content validated prior to administration by experts in the field of study. The reliability test of the instrument was carried out through the test-re-test reliability procedure by administering the questionnaire to two (2) poultry farmers at an interval of one week. The responses from the two administrations were then correlated and a high correlation coefficient of r = 0.80 was obtained. This confirmed the consistency and reliability of the instrument. Furthermore, a multiple contact strategy was used to eliminate sampling error and to ensure accuracy of responses gathered from the farmers. This approach eliminated the risks of receiving socially desirable responses from the farmers.
The survey results showed that broiler chicken flocks (average size: 5000 birds) were all raised intensively in housing units, most of which had foot baths for biosecurity (S2 File). Layer flocks (average size: 1000 birds) were intensively raised in battery cages. Foot baths were used at entrances of only two of the layer farms. Indigenous chicken flocks (average size: 250 birds) were raised semi-intensively with no biosecurity measures in place. Antibiotic use was highest in broilers, followed by layers while no antibiotics were used in dual-purpose indigenous chickens. Broilers and layers were fed formulated commercial diets with antibiotic growth promoters, while dual-purpose indigenous chickens mostly scavenged for their feed.
For microbiological analysis, the minimum sample size of 75 was determined to be adequate for the study using a previously reported formular [26]. To achieve this, 5 samples were collected from each farm.
2.2 Sample collection
A total of 75 faecal samples were collected from 15 poultry farms (5 layer, 5 broiler, and 5 indigenous dual-purpose chicken) in the Ngaka Modiri Molema District, North West province, South Africa. The faecal samples were collected directly from the cloaca of five randomly selected individual birds using sterile swabs containing multipurpose universal transport medium. The swab samples were immediately placed in the tubes containing the multipurpose universal transport medium and transported on ice to the Microbiology laboratory at the North-West University, for bacterial analysis.
2.3 Salmonella isolation
At the laboratory, swabs were immediately rinsed in 10 mL of 1% (w/v) peptone-water. After rinsing, 0.1 mL aliquots from the peptone-water were inoculated into tubes containing 10 mL of Rappaport Vassiliadis (RV) broth medium and incubated at 42°C for 48 hrs [27]. Following enrichment, a loopful of the broth culture was streaked onto Salmonella-Shigella agar (SSA) plates and aerobically incubated at 37°C for 24 hrs. Lactose non-fermenting colonies without black centres (potentially Shigella spp. and non-hydrogen sulphide producing Salmonella spp.) and lactose-fermenting colonies with large black centres (potentially hydrogen sulphide producing Salmonella spp.) were randomly picked and purified on SSA. Two distinct presumptive isolates per sample were picked, thus a total of 150 presumptive Salmonella isolates were used for further identification tests. All pure isolates were preserved in 60% (v/v) glycerol (Merck, Johannesburg, SA) and stored at −80°C for future use.
2.4 Genomic DNA extraction from presumptive isolates
Overnight cultures of presumptive isolates were prepared, and the genomic DNA (gDNA) was extracted using Zymo Research Genomic DNATM–Tissue MiniPrep kit (Biolab, South Africa) obtained from Inqaba Biotec, South Africa, following the manufacturer guidelines. The quality and purity of gDNA extracted from the isolates was assessed using NanoDrop Lite 1,000 spectrophotometer (model: Thermo Fisher Scientific, USA). High quality gDNA samples were stored at −80°C for further analysis by PCR.
2.5 Molecular identification and confirmation of Salmonella isolates
Presumptive Salmonella isolates were subjected to Salmonella-specific PCR through amplification of invA (284 bp), fliC (559 bp), and Prot6e (185 bp) genes using a DNA thermal cycler (C1000 Touch™, BIO-RAD, South Africa) and oligonucleotides supplied by Inqaba Biotec. The primer sequence for invA gene was used to confirm Salmonella genus while prot6e and fliC genes were used to detect S. enteritidis and S. typhimurium, respectively [28]. This set of primers have been previously used to confirm the identity of the genus Salmonella and distinguish between S. typhimurium and S. enteriditis strains from other Salmonella serotypes [29]. The oligonucleotide primer sequences targeted genes, amplicon sizes and the PCR conditions (annealing temperature) are listed in Table 1. The PCR reactions constituted of 12.5 μL of 2X DreamTaq Green Master Mix, 0.5 μM of each primer, 1 μL of template DNA, and 11 μL RNase-nuclease free PCR water. A no-template DNA tube was used as a negative control while Salmonella Enteriditis (ATCC: 13076TM) and Salmonella Typhimurium (ATCC: 14028TM) reference strains obtained from Sigma Aldrich, SA were used as positive control.
2.6 Detection of virulence genes
Polymerase chain reaction assays were performed to amplify spiC (309 bp), misL (400 bp), and orfL (550 bp) virulence gene fragments. The primer sequences, targeted genes, amplicon sizes as well as the annealing temperature are listed in Table 1. All the PCR reactions were prepared in a final volume of 25 μL constituting of 12.5 μL of 2X DreamTaq Green Master Mix, 0.5 μM of each primer, 1 μL of template DNA, and RNase free water. All amplifications were performed using DNA thermal cycler (C1000 Touch™, BIO-RAD, South Africa). PCR amplicons were held at -4°C until electrophoresis was performed.
2.7 Antimicrobial susceptibility test
The Kirby-Bauer disc (Mast Diagnostics, UK) diffusion technique was used to determine the antimicrobial susceptibility profile of all the Salmonella isolates [31]. The choice of selected antibiotics was based on the standard recommendation by CLSI, particularly antibiotics that are commonly used in the treatment of bacterial infections in both humans and animals. The list of used antibiotics comprised of gentamicin (GM10 μg), amoxicillin (A10 μg), erythromycin (E15 μg), chloramphenicol (C30 μg), tetracycline (T10 μg), trimethoprim (TM25 μg), ampicillin (AP30 μg), trimethoprim-sulfamethoxazole (TS25 μg), and kanamycin (K30 μg) [30]. The tested antibiotics belonged to 6 antimicrobial classes, which includes aminoglycosides, tetracyclines, folate pathway antagonists, phenicols, penicillins, β-lactam combination agents. A loopful of confirmed Salmonella isolates was inoculated into sterile nuclease free water to prepare a 0.5 MacFarland’s solution of competent exponential phase growth cells. Aliquots (0.1 mL) of these cells were evenly spread-plated onto Muller Hinton agar plates [32]. Discs impregnated with CLSI recommended concentrations of the antibiotics were evenly placed on the inoculated plates and incubated aerobically at 37°C for 18 hrs. Following incubation, antibiotic growth inhibition zone diameter around the disc was measured in mm and the data was interpreted using CLSI (2023 version) guidelines. The isolates were classified as sensitive (S), intermediate resistance (I), or resistant (R) to each antibiotic following CLSI criteria [32, 34]. Escherichia coli ATCC 25922 was used as a reference strain because it is a recommended strain for antimicrobial susceptibility test and its quality control guidelines permit greater accuracy in interpreting AMR results [33, 34]. Percentage antibiotic resistance was calculated, and multiple antibiotic resistance (MAR) phenotypes were generated for isolates that were resistant to at least one agent in three or more antimicrobial categories [34, 35].
2.8 Detection of antimicrobial resistance genes
Genomic DNA of Salmonella extracted was used to detect antimicrobial resistance genes. All confirmed Salmonella isolates were screened for the presence of the ant (3”)-la (526 bp), tet (A) (210 bp), tet (B) (659 bp), sul1 (350 bp), and sul2 (720 bp) antibiotic resistance determinants [30]. Primer sequences, target genes, amplicon sizes as well as PCR cycling conditions for the different genes are listed in Table 2. Polymerase chain reactions were carried out in total volumes of 25 μL each, comprising 12.5 μL of 2X DreamTaq Green Master Mix, 0.5 μM of each primer, 1 μL of template DNA and RNase free water. Amplifications were performed using DNA thermal cycler (C1000 Touch™, BIO-RAD, South Africa).
2.9 Phenotypic assessment of biofilm-formation
Microtiter plate assays were employed to assess the ability of Salmonella isolates to form biofilm at different temperatures (4°C, 25°C, and 37°C) over a 24-hour period. Triplicates of 10 μL aliquot of each overnight culture at 105 CFU inoculated into 190 μL of brain-heart infusion broth per well were prepared and incubated [36]. Pseudomonas aeruginosa ATCC 27853 was used as a positive control because it is a strong biofilm-former. Biofilm-formation was quantified by crystal violet (CV) staining and isolates were classified into none, weak, moderate, and strong biofilm-formers using automatic Enzyme-Linked Immunosorbent Assay (ELISA) microtiter plate reader (MB-580, Zhengzhou, China) [37].
2.10 Electrophoresis of DNA and PCR products
Genomic DNA and PCR amplicons were all separated by electrophoresis on 1.5% (w/v) agarose gel containing 0.001 μg/mL ethidium bromide using horizontal Pharmacia Biotech equipment (model Hoefer HE 99X; Amersham Pharmacia Biotech, Sweden). A 100 bp DNA molecular weight DNA marker (Thermo Fisher Scientific, South Africa) was used to confirm the sizes of the amplicons. Each electrophoresis run was conducted at 100 V for 10 min and later 80 V for 1 h using 1X TAE buffer (40 mM Tris, 1 mM EDTA and 20 mM glacial acetic acid, pH 8.0). A ChemiDoc Imaging System (Bio-Rad ChemiDocTM MP Imaging System, UK) was used to capture the images using Gene Snap software, version 6.0022. Agarose gel images were analysed to determine gene sizes.
2.11 Statistical analysis
Optical density data were analysed using the General Linear Models procedure of Statistical Analysis System (SAS) 2010. The treatments were analyzed using a 3 × 4 factorial treatment arrangement in a completely randomized design according to the following model:
Where, Yij = optical density; μ = population mean; Bi = bird type; Tj = incubation temperature; (B × T)ij = interactive effect of bird type and incubation temperature; and Eijk = random error associated with observation ijk, assumed to be normally and independently distributed. Least squares means (LSMEANS) were compared using the probability of difference option in the LSMEANS statement of SAS.
Proportional data (arising from discrete counts) were analysed using the multinomial logistic regression procedure of SAS (2010). In the categorical variable ’bird type,’ the reference category selected was broiler due to the reported extensive use of antibiotic growth promoters in this group. Consequently, there was an anticipation of a higher likelihood of detecting antimicrobial resistance in broilers. For all statistical tests, significance was declared at P < 0.05.
3. Results
3.1 Prevalence of Salmonella in different types of chickens
Thus, out of 150 isolates, 82 (55%) were confirmed to be Salmonella (Table 3, Fig 1). More than half (51%) of the isolates were confirmed as S. typhimurium through amplification of the fliC gene (Table 3, Fig 2). Only a small proportion (32%) of the isolates was confirmed as S. enteritidis using the Prot6e gene PCR and the amplicons for representative isolates are shown in Fig 3. A small proportion (17%) of the confirmed Salmonella isolates were not positive for the fliC and Prot6e genes. The proportions of isolates positive for Salmonella species-specific and virulence genes did not vary (p > 0.05) with bird type.
Lane M = 100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lane 1 = invA gene fragment amplified from Salmonella enteriditis positive control strain (ATCC: 13076TM); Lanes 2–18 = Salmonella species specific invA gene fragments amplified from the isolates; Lanes 19 = Negative control.
Lane M = 100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lane 11 = fliC gene fragment amplified from Salmonella typhimurium positive control strain (ATCC:14028TM); Lanes 1–10 and 12–17 = Salmonella species specific fliC gene fragments amplified from the isolates; Lane 18 = Negative control.
Lane M = 100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lane 1 = Prot6e gene fragment amplified from Salmonella enteriditis positive control strain (ATCC: 13076TM); Lanes 2–16, Salmonella species specific Prot6e gene fragments amplified from the isolates; Lane 17 = Negative control.
3.2 Antimicrobial resistance profiles
A total of 82 confirmed Salmonella isolates were subjected to antimicrobial sensitivity test against a panel of 9 different antimicrobial agents (Table 4). Large proportions of the isolates were most often resistant to erythromycin (62%) and tetracycline (59%). On the contrary, smaller proportions of these isolates were resistant to trimethoprim (32%), amoxicillin (26%), ampicillin (22%), trimethoprim- sulfamethoxazole (18%) and kanamycin (15%). In addition, the isolates exhibited high susceptibility to chloramphenicol and gentamicin with 2% and 1% resistance recorded, respectively. Despite that aminoglycoside such as gentamicin appeared active in vitro against Salmonella isolates, it is not utilized clinically, hence it must be reported as resistant according to CLSI (2023 version) standards. The proportion of Salmonella isolates resistant to tested antibiotics were not influenced (p > 0.05) by the type of bird sampled.
The superscript “a” indicate the percentage of Salmonella isolates that were resistant to the aminoglycosides (gentamicin and kanamycin) and these was reported based on the CLSI standards which stipulate that the above antimicrobial agents should not be reported as susceptible since they are not effective clinically.
Multiple antimicrobial resistance (MAR) phenotypes of isolates were generated (Table 5) using abbreviations on the antibiotic discs. All observed phenotypes were given a specific antibiotypes codes (Ac) with a distinct number to differentiate between biotypes and they ranged between Ac1 and Ac20. Phenotypes Ac1 (16%), Ac2 (10%), Ac4 (10%), Ac10 (13%), and Ac19 (10%) were dominant across isolates. Isolate phenotypes Ac14 –Ac18 were resistant to five or more antibiotics. Phenotype Ac20 was resistant to the highest number (7) of antibiotics (ampicillin, chloramphenicol, erythromycin, trimethoprim-sulfamethoxazole, tetracycline, trimethoprim).
3.3 Detection of resistance genes
A total of 82 confirmed Salmonella isolates from different chicken types were subjected to PCR, targeting ant (3”)-la, sul1, sul2, tet (A), and tet (B) antimicrobial resistance genes (Table 6). Large proportions of the isolates possessed the ant (3”)-la (52%) and Tet (A) (46%) resistance genes. The proportions of isolates carrying sul1 and sul2 resistance genes ranged between 13% and 14% (see gene fragments in Figs 6 and 7, respectively). Agarose gel images of the ant (3”)-la, tet (A), sul1, and sul2 gene fragments are shown in Figs 4–7, respectively. On the other hand, only 9% of the isolates possessed tet (B) resistant gene determinants. The proportion of isolates positive for antimicrobial resistance genes were not influenced (p > 0.05) by bird type.
Lane M=100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 1–18, ant (3”) resistance gene fragments amplified from the Salmonella isolates; Lane 19 = Negative control.
Lane M=100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 1–18, tet (A) resistance gene fragments amplified from the isolates; Lane 19 = Negative control.
Lane M=100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 1–10 and 12–17, sui1 resistance gene fragments amplified from confirmed Salmonella isolates; Lane 11 = Negative control.
Lane M = 100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 1–15, sui2 resistance gene fragments amplified from confirmed Salmonella isolates; Lane 16 = Negative control.
3.4 Virulence genes in Salmonella isolates
The 82 confirmed Salmonella isolates were further screened for the presence of three virulence genes (spiC, misL, and orfL) using PCR. A moderate number (26%) of the isolates possessed the spiC virulent gene (Table 3) whose gene fragments are shown in Fig 8. Only 16% of the isolates harboured the misL virulent gene (Table 1) whose amplicons are shown in Fig 9. In addition, 14% of the isolates harboured the orfL virulent gene (see the gene fragments in Fig 10).
Lane M=100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 1–15, Salmonella species spiC virulent gene fragments; Lane 16 = Negative control.
Lane M=100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 2–14 = Salmonella species misL virulent gene fragments from confirmed Salmonella isolates; Lane 15 = Negative control.
Lane M = 100 bp DNA marker (Thermo Fisher Scientific, South Africa); Lanes 2–15 = orfL virulence gene fragments from confirmed Salmonella isolates; Lane 16 = Negative control.
3.5 Phenotypic assessment of biofilm-formation
Only 69 of the 82 confirmed and preserved Salmonella stock cultures were still viable after long-term storage at -80°C. For this reason, 69 of the isolates were subjected to biofilm-formation analysis using microtiter plate assay. The results revealed that neither chicken type nor incubation temperature influenced biofilm-formation among the tested Salmonella isolates. Based on the biofilm-formation patterns observed, isolates were classified as none, weak, moderate, or strong biofilm-forming strains. Regardless of incubation temperature, larger proportions of the isolates (35 to 62%) were categorized as strong biofilm-formers. The proportion of Salmonella isolates that did not form biofilm ranged between 0 and 35%, while those that had moderate biofilm-forming capacity ranged between 0 and 20%. Between 4 and 35% of isolates were classified as weak biofilm-formers. The proportion of isolates that were able to form biofilms were not influenced (p > 0.05) by bird type.
4. Discussion
4.1 Prevalence of Salmonella in different poultry species
A total of 150 isolates were obtained from 15 layer, broiler, and dual-purpose indigenous chicken farms. Salmonella spp. identity was confirmed in 82 (55%) of the isolates by molecular detection of invA gene fragments. Similar results have been reported in previous studies [6, 38]. The occurrence of Salmonella spp. in poultry is influenced by several factors such as geographic location, prevention/control and biosafety measures of flocks, farm-specific husbandry practices, sampling season, and identification methods. In the current study, broilers had the highest Salmonella prevalence rate (78%), followed by layers (46%) and indigenous chickens (40%), as confirmed through PCR amplification of the invA gene. The observed variations in the occurrence of Salmonella spp. across broilers, layers and indigenous chickens may be attributed to the variation in management (biosecurity, hygiene, and sanitation) of the farms [39]. Despite that broilers and layers are raised under strict biosecurity standards compared to indigenous chickens, broilers had the highest occurrence rate of Salmonella spp. corroborating previous findings [40, 41]. Salmonella enteritidis and S. typhimurium are the most problematic zoonotic Salmonella serotypes that are responsible for serious human health infections globally [42]. In South Africa, they are also the most common serotypes reported in food producing animals including food products of animal origin [43, 44]. Confirmed Salmonella isolates were further identified using Salmonella species-specific gene fragments fliC (S. typhimurium) and prot6e (S. enteritidis). This analysis revealed that the prevalence of S. enteritidis and S. typhimurium serotypes was 49 and 30%, respectively. Only 21% of the confirmed Salmonella isolates were negative for the two tested species-specific genes, suggesting that they could be other serotypes. The prevalence of S. typhimurium varied across layers (57%), broilers (41%), and indigenous chickens (55%). On the other hand, S. enteritidis was detected mostly in indigenous chickens (45%), followed by broilers (28%) and layers (22%). The lower prevalence of both S. typhimurium and S. enteritidis in broilers compared to layers and indigenous chickens can be attributed to the strict biosecurity measures employed for broiler production [14]. Salmonella can infect chickens without any clinical signs, leading to compromised productivity [45]. This highlights the importance of periodically screening chickens for the presence of Salmonella species to assess the risks and develop effective biosecurity measures to control their spread. Such interventions will reduce the incidence of Salmonella in poultry resulting in greater productivity and food safety as well as reduced public health concerns.
4.2 Antimicrobial resistance profiles of Salmonella isolates
Misuse or uncontrolled use of antibiotics in poultry production for growth promotion and prophylaxis has significantly contributed to the development of antimicrobial resistance among bacterial pathogens, including Salmonella [46]. Therefore, it is necessary to assess the antimicrobial resistance profile of isolates against commonly used antibiotics to develop more effective treatment and control strategies [47]. The isolates showed high resistance to erythromycin (62%), tetracycline (59%), and trimethoprim (32%). The observed high prevalence of resistance to certain drugs such as tetracyclines can be attributed to their common use in both animals and humans driven by affordability and accessibility, particularly in developing African countries [48, 49]. In comparison, a lower proportion of the isolates (15–26%) was resistant to amoxicillin, ampicillin, trimethoprim-sulfamethoxazole, and kanamycin, drugs that are not frequently used in the study area [30]. Other antibiotics such as ampicillin and amoxycillin are drugs of choice against Salmonellosis, hence the observed resistance may be attributed to their frequent use [50]. The lowest proportions of resistant isolates were observed for chloramphenicol (2%) and gentamicin (1%) antibiotics, reflecting the uncommon use of both drugs in the study area. Although. Aminoglycosides (gentamicin) may appear active in vitro against Salmonella spp. but are not effective clinically, hence it should not be reported as susceptible [32]. Interestingly, a high number of isolates from indigenous chickens showed resistance to several antibiotics and harboured resistance genes, suggesting potential of indirect transmission of antibiotic resistance. This phenomenon is more commonly reported in developing countries, particularly in poultry production settings characterized by limited resources and inadequate biosecurity measures [15, 18]. Additionally, bacterial pathogens disseminate resistance through horizontal gene transfer [51], and that may contribute to the spread of antimicrobial resistance to the environment and ultimately to indigenous chickens, particularly in areas with poor livestock waste management. In addition, the direct interaction or close proximity of the extensively reared indigenous chickens with other animals such as cattle, broilers, layers, and wild birds could have contributed to the observed findings.
Multiple drug resistance (MDR) is another growing global problem because it reduces disease treatment options [52]. Salmonella isolates assessed in the current study exhibited a high rate of MDR to three or more tested antibiotics classes with MAR index ranging from 0.33 to 0.78. Interestingly, the MAR index values were all above the 0.2 threshold, suggesting that the high-risk source of Salmonella pathogens contamination is where antimicrobial agents are frequently used [53]. This confirms that antibiotic use in poultry production farms does contribute to the spread of resistant Salmonella pathogens. Several factors contribute to MDR development, including unregulated access to antibiotics and/or lack of compliance regarding the amount and type of antimicrobial agents used in poultry production and human medicine [54]. Some Salmonella isolates were resistant to more than five antibiotics, a major cause for concern given that diseases caused by such pathogens often have fatal outcomes [55]. Infections caused by MDR pathogens have severely limited treatment options thus putting animal and human lives at risks [38]. The adverse effects of MDR pathogens are a severe concern in developing countries due to inadequate health systems and limited resources to control them [55]. The detection of multiple resistant Salmonella strains in the three chicken types surveyed in this study has serious public health implications through food chain contamination [39].
4.3 Detection of resistance genes
The observed high rates of isolates AMR is not surprising, since most poultry producers in South Africa have unlimited over the counter access to most antimicrobials [56]. Among the 82 Salmonella isolates in the current study, 52, 46, 13, and 14% were positive for the ant (3”)-la, tet (A), sui1, and sui2 resistant gene determinants, respectively. The results obtained from the analysis of resistance genes are consistent with the findings of the phenotype analysis, especially in the case of tetracycline (59%) and trimethoprim-sulfamethoxazole (18%). Notably, there is a partial alignment between genotypic antibiotic resistance (AMR) and phenotypic antibiotic resistance results, suggesting the potential existence of silent antimicrobial resistance genes, particularly tet (A), tet (B) and ant (3”)-la [57]. This phenomenon, also known as cryptic genes, has been observed in earlier studies [58–60]. The presence of silent antimicrobial resistance genes poses a new challenge in the battle against antimicrobial resistance, as it implies a risk not only with phenotypically resistant pathogens but also with antimicrobial-susceptible pathogens harbouring cryptic genes, as reported previously [61]. This dual risk emphasizes the complexity and potential covert nature of antimicrobial resistance. Despite the above, a small number of the isolates (9%) possessed the tet (B) resistance gene. Inexpensive and accessible antimicrobials such as tetracyclines tend to be abused in animal production and human medicine thus contributing to the development and spread of AMR [49].
Additionally, 56% of the isolates also harboured one of the aminoglycosides resistance genes called ant (3”)-la gene. The above findings corroborate previous findings [62]. In the current study, the ant (3”)-la gene was highly prevalent in layers (65%), followed by indigenous chickens (55%) and broilers (36%) among Salmonella isolates that had not shown phenotypic resistance to gentamicin. This suggests the potential presence of silent antimicrobial resistance genes among Salmonella isolates, posing a potential health risk for poultry producers in the study area. Although the ant (3”)-la gene has been reported in several pathogenic bacterial strains, its prevalence in Salmonella isolates in Africa is not well-documented. In addition, genes harbouring resistance to sulfamethoxazole (sul1 and sul2) were also detected in Salmonella isolates, including those resistant to trimethoprim-sulfamethoxazole. The occurrence of resistant Salmonella spp. suggests a need for alternatives to antibiotics [63], as well as treatment options in the event of disease outbreaks. Poultry production in low to medium income countries should be supported with alternative therapies against AMR pathogens, such as bacteriophage therapy.
4.4 Prevalence of virulence genes among Salmonella isolates
Virulence contributes to the invasiveness, pathogenicity, survival, and proliferation of Salmonella spp. [38]. Genetic determinants responsible for virulence help Salmonella to invade and destroy epithelial cells in host intestines and pave way for the colonization of other cell lines [64]. In the current study, all three screened virulence genes belonged to different Salmonella pathogenicity islands, named SP1-2, SP1-3, and SP1-4 encoding for spiC, misL, and orfL genes, respectively. The most prevalent virulent gene was the spiC, found in 26% of the tested isolates, however, the role of spiC gene in the pathogenesis of Salmonella is yet to be unravelled [65]. The other virulent genes detected were the misL (16%) and the orfL (14%). Both genes have been associated with the survival of Salmonella in host macrophages during an infection [66]. The identification of virulent and AMR Salmonella pathogens raises concerns for both public health and the poultry industry. Antimicrobial-resistant pathogens not only lead to challenging-to-treat infections but also exacerbate infections and elevate the risks of mortality [67]. The detection of virulent genes in Salmonella strain at the farm level demonstrates the role played by healthy chickens in spreading pathogenic Salmonella strains to the environment or food chain leading to public health concerns [12, 68].
4.5 Phenotypic assessment of biofilm-formation
Biofilm-formation is a survival strategy used by pathogenic bacteria to evade harsh environments such as antibiotics and disinfectants while enhancing microorganisms’ pathogenicity [69]. Bacterial biofilm-formation increases the burden of resistant pathogens and threatens food safety, especially when hygiene standards are compromised during food production and processing [70]. In the current study, isolates from different chicken types had similar biofilm-forming capacity. Most isolates were strong biofilm-formers, regardless of incubation temperature. These findings underscore the threat to food safety posed by the potential of Salmonella to form biofilms. Given that meat products are stored in cold facilities to mitigate foodborne poisoning incidents in humans, the observed robust biofilm formation by isolates, even at low temperatures (4°C) typical of meat storage, raises significant concerns for food safety and public health. This heightened biofilm-forming nature at low temperatures increases the risk of meat contamination during storage, potentially leading to elevated morbidity and mortality cases, particularly among children and immunocompromised individuals [71]. This outcome necessitates the search for effective control strategies to ensure food safety and public health.
5. Conclusions
In conclusion, bird type or husbandry practices had no significant effects on the prevalence of AMR Salmonella spp. or resistance determinants. The detection of virulent pathogenic and AMR Salmonella spp. in the different chicken types suggest a public health risk and raises a concern for the South African poultry industry. This is because Salmonella is the most prevalent foodborne pathogen globally, frequently associated with the contamination of poultry products and diseases of economic and public health importance in poultry and humans. The occurrence of pathogenic and MDR Salmonella spp. in chickens suggests the need for careful evaluation of antibiotic use in all poultry production systems. Furthermore, it highlights the need to search for alternatives to prophylactic and therapeutic antibiotics such as bacteriophages.
Supporting information
S1 File. The survey questionare used for assessing poultry farms huasbandry practices.
https://doi.org/10.1371/journal.pone.0310010.s001
(DOCX)
S2 File. Poultry farms husbandry practices and their characteristics.
https://doi.org/10.1371/journal.pone.0310010.s002
(DOCX)
S1 Raw images. All uncropped and unadjusted gel images used in the manuscript.
https://doi.org/10.1371/journal.pone.0310010.s003
(PDF)
Acknowledgments
The authors wish to thank Dr K.P. Montso for his support during the characterization analysis of Salmonella isolates.
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