Evidence of potentially unrelated AmpC beta-lactamase producing Enterobacteriaceae from cattle, cattle products and hospital environments commonly harboring the blaACC resistance determinant

Keduetswe Matloko; Justine Fri; Tshepiso Pleasure Ateba; Lesego G. Molale-Tom; Collins Njie Ateba

doi:10.1371/journal.pone.0253647

Abstract

The occurrence and genetic relatedness of AmpC beta-lactamase producing Enterobacteriaceae isolated from clinical environments, groundwater, beef, human and cattle faeces were investigated. One hundred seventy-seven (177) samples were collected and cultured on MacConkey agar. A total of 203 non-repetitive isolates were characterised using genus/species-specific PCRs and the identified isolates were subjected to antibiotic susceptibility testing. The production of AmpC beta-lactamases was evaluated using cefoxitin disc, confirmed by the D96C detection test and their encoding genes detected by PCR. The D64C extended-spectrum beta-lactamases (ESBL) test was also performed to appraise ESBLs/AmpC co-production. The genetic fingerprints of AmpC beta-lactamase producers were determined by ERIC-PCR. A total of 116 isolates were identified as E. coli (n = 65), Shigella spp. (n = 36) and Klebsiella pneumoniae (n = 15). Ciprofloxacin resistance (44.4–55.4%) was the most frequent and resistance against the Cephem antibiotics ranged from 15–43.1% for E. coli, 25–36.1% for Shigella spp., and 20–40% for K. pneumoniae. On the other hand, these bacteria strains were most sensitive to Amikacin (0%), Meropenem (2.8%) and Piperacillin-Tazobactam (6.7%) respectively. Nineteen (16.4%) isolates comprising 16 E. coli and 3 Shigella spp. were confirmed as AmpC beta-lactamase producers. However, only E. coli isolates possessed the corresponding resistance determinants: bla_ACC (73.7%, n = 14), bla_CIT (26%, n = 5), bla_DHA (11%, n = 2) and bla_FOX (16%, n = 3). Thirty-four (27.3%) Enterobacteriaceae strains were confirmed as ESBL producers and a large proportion (79.4%, n = 27) harboured the bla_TEM gene, however, only two were ESBLs/AmpC co-producers. Genetic fingerprinting of the AmpC beta-lactamase-producing E. coli isolates revealed low similarity between isolates. In conclusion, the findings indicate the presence of AmpC beta-lactamase-producing Enterobacteriaceae from cattle, beef products and hospital environments that commonly harbour the associated resistance determinants especially the bla_ACC gene, nonetheless, there is limited possible cross-contamination between these environments.

Citation: Matloko K, Fri J, Ateba TP, Molale-Tom LG, Ateba CN (2021) Evidence of potentially unrelated AmpC beta-lactamase producing Enterobacteriaceae from cattle, cattle products and hospital environments commonly harboring the bla_ACC resistance determinant. PLoS ONE 16(7): e0253647. https://doi.org/10.1371/journal.pone.0253647

Editor: Abdelazeem Mohamed Algammal, Suez Canal University, EGYPT

Received: April 23, 2021; Accepted: June 10, 2021; Published: July 29, 2021

Copyright: © 2021 Matloko 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 paper and its Supporting information files.

Funding: The authors are grateful to the National Research Foundation of South Africa for their financial support provided to MK under the DST-NRF Internship Programme (2018 – 2019). The North-West University is highly appreciated for providing the space for bench work. This project was also supported in part by an incentive funding for rated researchers funding (grant UID number 109187) awarded to CNA by the National Research Fund of South Africa.

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

1. Introduction

The family Enterobacteriaceae consists of bacterial species frequently isolated from clinical specimens and most often incriminated in a variety of infections [1, 2]. The natural hosts of these organisms are ruminants and therefore, may be found in food, water and the environment from where transmission to humans may occur especially when proper hygienic procedures are compromised. Most Enterbacteriaceae were known to be non-pathogenic, however, over the years some members including strains/serotypes of E. coli, Shigella spp. and Klebsiella pneumoniae have become virulent accounting for diarrheal cases (which is a major burden in low and middle-income countries), other intra-abdominal, urinary tract, and bloodstream infections, as well as hospital and healthcare-associated pneumonia [3]. Pathogenic E. coli strains classified based on their virulence properties including enterotoxigenic E. coli, ETEC; enterohemorrhagic E. coli, EHEC; enteroinvasive E. coli, EIEC; enteropathogenic E. coli, EPEC; enteroaggregative E. coli; EAEC and diffusely adherent E. coli (DAEC), neonatal meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), and avian pathogenic E. coli (APEC) cause mild to severe food poisoning in their hosts as well as may lead to extra-intestinal illnesses [4, 5]. A serotype such as the Shiga toxin-producing E. coli 0157:H7 has been associated with life-threatening diseases such as haemolytic uremic syndrome (HUS), haemorrhagic colitis, and thrombotic thrombocytopenic purpura which may be fatal. Four Shigella spp., S. flexneri; S. dysenteriae; S. boydii and S. sonnie have been described and are thought to have evolved from ancestral non-pathogenic E. coli by the acquisition of a large virulence plasmid [6], that encodes many virulence factors on its ipa-mxi-spa region of which the invasion plasmid antigen (antigens (IpaA, IpaB, IpaC and IpaD proteins) is one of the most essential. These proteins are important for bacterial invasion of epithelial cells as well as immune cell escape [7]. Infection caused by Shigella spp., Shigellosis is more common among children and travellers and approximately 60% of cases are accounted for by S. flexneri, although infection with S. dysenteriae causes greater toxicity. S. dysentariae serotype 1 produces the Shiga toxin which can lead to HUS and eventually cause hemolytic anaemia, uremia and thrombocytopenia with up to 20% mortality. Shigella infection can also activate reactive arthritis [8]. K. pneumoniae on the other hand occurs as an opportunistic pathogen especially in persons with weakened immune systems and cause soft tissue (e.g. cellulitis and necrotizing fasciitis), to life-threatening infections such as pneumonia, septicemia, meningitis, and endophthalmitis as well as are implicated in a significant number of cases of hospital-acquired urinary tract infections [9].

There is an increased report of multidrug resistance globally that is considered a public health threat. Several previous studies revealed the emergence of multidrug-resistant bacterial pathogens from different origins including animals, fish, birds, drinking water and other environmental water sources that may be sources of transmission through the food chain to human consumers resulting in severe illness [10–17]. Enterobacteriaceae have exhibited varied antibiotic resistance mechanisms such as the intrinsic production of beta-lactamases that inactivates beta-lactam antibiotics in predominance thus proving to be a public health threat [18]. Of these, the production of Extended Spectrum Beta Lactamases (ESBL) and AmpC beta-lactamases have been the most prominent [19]. AmpC beta-lactamases are group 1 or class C cephalosporinases mostly encoded on chromosomal sequences of many Enterobacteriaceae and mediate resistance to cephalothin, cefazolin, cefoxitin and most penicillins, as well as enhance the production of β-lactamase inhibitor-β-lactam combinations [20]. Chromosomally encoded AmpC genes are constitutive and typically expressed at low levels, however, in most Enterobacteriaceae these genes are inducible and overexpressed in response to β-lactam or other stimuli conferring resistance to broad-spectrum Cephalosporins including Ceftriaxone, Ceftazidime, and Cefotaxime [21, 22]. Enterobacteriaceae belonging to the CESP group or group II Enterobacteriaceae (Citrobacter, Enterobacter, Serratia and Providencia) alongside Hafnia and Morganella included more recently, are typical examples with chromosomally-encoded AmpC beta-lactamases [11, 22]. However, in other Enterobacteriaceae, such as E. coli and Shigella spp., AmpC β-lactamases are chromosomally encoded and constitutively low or poorly expressed but non-inducible. In these bacterial species, expression of AmpC beta-lactamases is due to hyperproduction as a result of chromosomal ampC gene mutation or expression of plasmid-encoded AmpCs [21]. There is an increasing interest in transmissible (plasmid)-mediated cephalosporinases due to their mobility and potential spread. Species harbouring ampC positive plasmids confer resistance to beta-lactams comparable to their chromosomal equivalents and include E. coli, Salmonella spp., Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Enterobacter aerogenes [23, 24]. Currently, resistance amongst these members of Enterobacteriaceae, particularly E. coli to cephalosporins, beta-lactamase inhibitors and cephamycins is concerning and presents a huge challenge to public health.

Although AmpC β-lactamases usually hydrolyse a wide variety of β-lactam antibiotics except for the carbapenems and cefepime, unfortunately, overproduction of AmpC coupled with the outer membrane porin mutations can reduce susceptibility to carbapenems, particularly in plasmid-mediated AmpC producers [22]. This is a cause for concern, as carbapenems are first-line drugs in cases of antibiotic resistance [25], leaving limited available therapeutic options against MDR pathogens. It is therefore imperative to determine the frequency of occurrence of AmpC beta-lactamase-producing bacteria and the possible transmission of their resistance traits between environments.

Various phenotypic tests including the AmpC disk test, modified three-dimension test (M3DT), boric acid detection test, double disc synergy test (CC-DDS) and the D69C AmpC detection set have been reliably used in the detection of AmpC-producing traits in bacterial strains [26–30]. These tests have been supplemented with genotypic tests especially for the detection of plasmid-mediated AmpC genes which pose a significant threat to public health. Genotypic tests have been described for six ampC gene families (MOX, CIT, EBC, FOX, DHA and ACC-1) for which discriminative phenotypic tests are yet to be developed [31]. Despite the reported evidence on the increasing detection of AmpC beta-lactamases among Enterobacteriaceae worldwide [22, 32, 33], little or no data has been generated for South African strains and this highlights the importance of the present study. In the current study area, strains belonging to the family Enterobacteriaceae have previously been isolated from various sources and their antimicrobial susceptibility profiles determined [34–38]. Moreover, previous studies have indicated that besides humans and livestock, food products can also harbour AmpC beta-lactamases-producing Gram-negative bacteria [2, 22, 30, 39–42]. However, besides the study undertaken by Coertze and Bezuidenhout [43], there is no documented data of AmpC beta-lactamases-producing Enterobacteriaceae in the North-West Province of South Africa. This also raises the need to screen community-derived samples for the resistance traits in the area. The study was therefore aimed to isolate, identify and characterise AmpC beta-lactamase-producing Enterobacteriaceae isolated from cattle/beef products, hospital environments, groundwater and humans and to determine their genetic relatedness.

2. Materials and methods

2.1 Sampling

A variety of samples totalling 177 were randomly collected in Kareefontein, Lichtenburg, Mafikeng, Rustenburg and Zeerust in the North-West Province, of South Africa. Ethical approval for the study was obtained from the North-West University Health Science Ethics Committee (NWU-00728-18-A9) and permission obtained from the North West Provincial Health Department, South Africa. A verbal informed consent was obtained from parents/guardians of minors, from which feacal samples were collected, however documented in the laboratory note book. Fifty-four (54) faecal samples were collected directly from the rectum of cattle using sterile arm-length gloves. Twelve (12) beef samples that comprised wors, mincemeat, beef polony and beef fillet steaks were randomly collected from different butcheries and retail stores. Fifty-four (54) water samples were collected from boreholes in villages following the recommendations for groundwater sampling [44]. Seven (7) human stool samples were collected from patients aged between 4 months and 4 years at the Mafikeng Provincial hospital, North West Province of South Africa. In addition, sixty-four (64) swab samples were collected from clinics around the North-West Province of South Africa. Swab samples were obtained from consultation rooms, reception area, staff kitchen and patient toilets at noon after personnel and patients had been in contact with the surfaces. All samples were collected into appropriate containers and transport media where possible and transported on ice to the laboratory for analysis within six hours of collection. Table 1 shows the numbers of the samples collected from various sources.

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Table 1. Types and numbers of the samples collected during the study.

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2.2 Isolation and identification of members of Enterobacteriaceae

2.2.1 Sample processing.

Beef products, human and cattle faeces were processed as previously described [45, 46]. Briefly, 25 g of cattle and human faeces were homogenized in 225 ml of 0.1% (w/v) buffered peptone water (BPW) while 1 g of beef sample was washed in 10 ml of 2.0% (w/v) BPW (Lab M Limited, UK). The cotton wool swabs were washed in 10 ml of 2% (w/v) peptone water and vortexed. For groundwater samples, aliquots of 100 ml of each sample were aseptically filtered through 0.45 μm pore-size filters (Whatman Laboratory Division, Maidstone, England) as recommended [47].

2.2.2 Isolation of members of Enterbacteriaceae.

One hundred microliters of each suspension was spread-plated on MacConkey agar with crystal violet (Biolab, SA) for the isolation of Enterbacteriaceae [48]. Membrane filters were also aseptically inoculated on the agar. Plates were incubated aerobically at 37 °C for 24 hours. One to three colonies per positive plate depending on differing but typical morphological characteristics of Enterobacteriaceae were sub-cultured on fresh agar for purity. A 25% glycerol stock was prepared from a fresh overnight pure culture in tryptic soy broth and preserved at −80°C for future analysis.

2.2.3 Molecular identification of members of Enterbacteriaceae.

Bacterial chromosomal DNA was extracted from presumptive Enterobacteriaceae isolates using the CTAB method [49]. As an internal control, bacterial 16S rRNA gene of all presumptive isolates was amplified by PCR. using the universal oligonucleotide primer sequences 27F 5’-AGA GTT TGA TCM TGG CTC AG-3’ and 1492R 5’-TAC GGY TAC CTT GTT ACG ACT T-3’ Polymerase reaction assays were also used for genus/species-specific identification of Enterobacteriaceae of interest: E. coli, Klebsiella pneumoniae, and Shigella spp. (Table 2 consist of all oligonucleotide primers used in the study). PCR assays were performed in a C1000 Touch Thermal Cycler (Bio-Rad, California, USA) and for each reaction, a total volume of 25 μl containing 12.5 μl DreamTaq Master Mix (1X PCR buffer, 2 mM MgCl₂, 0.6 units of Taq DNA polymerase and 0.2 mM of each dNTPs), 11 μl of nuclease-free water, 0.5 μl oligonucleotide primer set (0.1μM final reaction concentration) and 1 μl of DNA template was prepared. E. coli ATCC 35218 and other in-house strains previously confirmed positive for the other targeted strains were used as controls. Otherwise mentioned, all DNA and amplicons resulting from PCR assays in this study were resolved by electrophoresis on a 1% (w/v) agarose gel stained with 0.001mg/ml ethidium bromide (BioRad, UK). Gel electrophoresis was carried out at 70V and 250A for 90 minutes in 1 X TAE buffer (40 mM Tris, 1 mM EDTA and 40 mM glacial acetic acid, pH 8.0). Gels were visualized under UV light at a wavelength of 420 nm and the images were captured using a ChemiDoc Imaging System (Bio-Rad ChemiDoc^™ MP Imaging System, California, USA).

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Table 2. Oligonucleotide primer sequences used in the study.

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2.3 Antimicrobial susceptibility testing

The antimicrobial resistance profiles of the isolates were determined by the disc diffusion assay on Mueller Hinton agar following the CLSI (Clinical Laboratory Standards Institute) recommendations [50]. Twelve antibiotics (Mast Diagnostics, UK) belonging to seven groups were used: β-lactam/ β-lactamase inhibitor (Piperacillin-tazobactam, 100 μg /10 μg), Carbapenem (Meropenem, 10 μg), Monobactam (Aztreonam, 30 μg), Aminoglycoside (Gentamicin, 10 μg; Amikacin, 30 μg), Quinolone (Ciprofloxacin), Cephem (Cefepime, 30 μg; Cefotaxime, 30 μg; Ceftriaxone, 30 μg; Cefuroxime, 30 μg and Ceftazidime, 30 μg) and folate pathway inhibitor (Trimethoprim-Sulfamethoxazole, 1.25/ 23.75 μg). These antimicrobial agents were selected based on resistance data from some previous studies conducted in the study area [15, 50]. In addition, third-generation cephalosporins were included in the evaluation due to the fact that they have a wide activity against gram-negative microorganisms and are very useful for the treatment of serious bacterial infections in humans. The resulting growth inhibition zone diameters were used to classify isolates as resistant, intermediate resistant and susceptible based on the CLSI breakpoint values [51]. Isolates presenting intermediate resistant patterns were considered as potential resistant isolates.

2.4 Phenotypic and genotypic detection of AmpC β-lactamases and ESBL

Phenotypic detection of AmpC β-lactamase production was carried out using cefoxitin disc (30 μg) as previously described [22, 30]. E. coli (ATCC 35218), a beta-lactamase-producing strain was used as a positive control. Isolates exhibiting resistance to cefoxitin (inhibition zone ≤ 18 mm) were preliminarily considered positive for AmpC β-lactamase. These potential AmpC beta-lactamase-producing isolates were subjected to a confirmatory phenotypic test using the D96C AmpC detection test based on the manufacturer’s instructions. The D96C AmpC test uses cefpodoxime to screen for chromosomal and plasmid-encoded AmpC [30]. In performing the test, a sterile needle was used to place three discs, A (cefpodoxime and Amp C inducer), B (cefpodoxime, AmpC inducer and ESBL inhibitor) and C (cefpodoxime AmpC inducer, ESBL inhibitor and AmpC inhibitor) at equitable distances on an inoculated bacterial lawn. The plates were incubated aerobically at 37 °C for 24 hours. An isolate was considered AmpC positive if the zone of inhibition of disc C exceeded that of both discs A and B by ≥ 5 mm, while zones differing by ≤ 3 mm were AmpC negative isolates. In a case where the zones of inhibition of both disc B and C exceeded that of disc A by ≥ 5 mm and those of disc B and C had a difference of ≤ 4 mm, the isolate was reported to be negative for AmpC-production but considered to be exhibiting a different resistance mechanism [29]. Molecular detection of AmpC beta-lactamases was achieved through PCR amplification of plasmid-mediated ampC genes encoding the phenotypes MOX, CIT, ACC, EBC, FOX and DHA (Table 2). Each PCR cycling condition comprised an initial denaturation at 95 °C for 2 minutes, followed by 30 cycles of DNA denaturation at 94 °C for 45 seconds, primer annealing at 55.9 °C for 45 seconds, primer extension at 72 °C for 1 minute and a final extension at 72 °C for 5 minutes. All PCR products were resolved by electrophoresis.

Phenotypic identification of ESBL-producing isolates was done using the D64C ceftazidime ESBL identification set (Mast Diagnostics, UK). The test was performed and results interpreted according to the manufacturer’s instructions. Similar to the D63C test, discs containing ceftazidime (30 μg) and ceftazidime/clavulanic acid (30/10 μg) were placed equitably apart on MHA inoculated with the standardised bacterial suspension. Following overnight incubation at 37 °C, the diameter of the zones of inhibition around the discs was recorded. An increase in the inhibition zone diameter of ≥ 5 mm in the presence of clavulanic acid indicated an ESBL-positive strain. ESBL producing isolates were screened for the presence of the bla_TEM gene by PCR assay using specific primers (Table 2).

2.5 Molecular typing of AmpC beta-lactamases producing Escherichia coli using ERIC-PCR

Fifteen (15) PCR confirmed AmpC beta-lactamase-producing E. coli isolates were subjected to ERIC-PCR to generate genetic fingerprints. The primer ERIC 2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) was used based on a previous report [57]. Cycling conditions included an initial denaturation at 94 °C for 2 minutes followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 50 °C for 1 minute and elongation at 65 °C for 8 minutes and a final extension at 65 °C for 8 minutes. The ERIC fingerprints were obtained by resolving PCR products on a 2% (w/v) agarose gel stained with 0.0001 μg/ml ethidium bromide (Bio-Rad Laboratories, Canada). Electrophoresis was performed at 60 V for 120 minutes. The gels were visualized under UV and imaged using a ChemiDoc^™ MP Imaging system (Bio-Rad, Hercules, USA). Fingerprinting patterns were analysed using the BioNumerics software version 7.6 (Applied Maths, Sint-Martens-Latem, Belgium). Salmonella Braenderup H9812 was used as a control and for standardization of the gels. Band similarity was calculated by applying the dice coefficient method with an optimization of 0.5% and a band matching tolerance of 1%. Cluster analysis was performed using the unweighted pair group methods arithmetic average algorithm to construct a dendrogram. The dendrogram was further analysed for associations of isolates in the various clusters originating from the different sources.

2.6 Statistical analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) v. 27. The Fisher Exact test was used to determine relationships between variables and statistical significance was set at p = 0.05.

3. Results

3.1 Occurrence of Enterobacteriaceae in various samples

One hundred and fifty (150) out of the 177 samples screened were positive for Enterobacteriaceae and included all groundwater (n = 40) and cattle faecal (n = 54) samples, 41 (64.1%) clinical swabs, 11 (91.7%) beef and 4 (57.1%) human faecal samples. PCR identification of 203 presumptive Enterobacteriaceae isolates resulted to a higher detection frequency for E. coli (32.0% n = 65), followed by Shigella species (26.1%, n = 36) and Klebsiella pneumoniae 15 (17.7%). The rest (42.9%, n = 87) were considered as other members of Enterobacteriaceae not considered for further analysis in the study. There was a significant difference (p < 0.001) in the distribution of Enterobacteriaceae species with respect to sample source. Fig 1 shows the distribution of various Enterobacteriaceae strains. Representative gels (S1-S3 Figs in S1 File) are documented in the supplementary material.

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Fig 1. Summarized results of PCR-identified isolates from various environments.

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3.2 Antimicrobial resistance profile of isolates

Generally, resistance to the quinolone antibiotic was the most frequent, followed by resistance to the Cephems while high susceptibilities were recorded to Amikacin (Aminoglycoside) and Meropenem (Carbapenem). These patterns were observed in E. coli isolates whereby non-susceptibility was highest to Ciprofloxacin (55.4%, 36/65), followed by the cephems [Cefotaxime (43.1%, 28/65), Cefuroxime and Ceftazidime (33.8%, 22/65)]. Apart from a 29.2% (19/65) resistance recorded to Aztreonam, E. coli isolates revealed < 25% resistance to the rest of the antibiotics tested with a remarkable sensitivity (100%) to Amikacin (Table 3). Similar to the resistance observed in E. coli isolates, Shigella spp. were often resistant to Ciprofloxacin (44.4%, 16/36) and their resistance to Cefuroxime (36.1, 13/36), Cefepime (30.6, 11/36), Cefuroxime and Aztreonam (27.8, 10/36) were higher compared to the rest (susceptibilities of ≥ 75%) of the antibiotics tested. Ciprofloxacin was also the least effective antibiotic (53.3% resistance) against K. pneumoniae. Klebsiella pneumoniae isolates also exhibited a 40% resistance to Aztreonam and Ceftazidime, 33.3% to Cefotaxime, Cefepime and Cefuroxime and 26.7% to Trimethoprim-Sulfamethoxazole. Only 3 (20%) isolates in this group were resistant to Ceftriaxone and Gentamicin, 2 (13.3%) to Amikacin and Meropenem, and 1 (6.7%) to Piperacillin/Tazobactam.

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Table 3. Number and proportion of antibiotic-resistant Enterobacteriaceae strains.

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Generally, the aforementioned differences observed for most of the antibiotics tested were insignificant (p > 0.05), except for the resistance of the various bacterial species/groups obtained against Amikacin (p = 0.024). Table 3 depicts detailed results of the antimicrobial-resistant profiles of the isolates.

3.3 Prevalence of AmpC beta-lactamases/ ESBL among Enterobacteriaceae isolates

Cefoxitin disc revealed about a third of the Enterobacteriaceae isolates (38/116, 32.8%) as cefoxitin-resistant, considered as potential AmpC β-lactamase producers. Nineteen (50%) of these (16 E. coli and 3 Shigella spp.) were confirmed positive using the D69C AmpC detection test comprising 16.4% (19/116) of the overall Enterobacteriaceae population. A large proportion of the AmpC beta-lactamase producers harbored the bla_ACC gene (n = 14; 73.7%), while the bla_CIT, bla_FOX, and bla_DHA were detected in 5 (26%), 3 (16%), and 2 (11%) isolates respectively (Table 4). None of the isolates harboured the bla_ACT and bla_FOX genes. Only one (5.3%) isolate harboured both bla_ACC and bla_DHA genes. S4-S6 Figs in S1 File are representative agarose gels (supplementary material).

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Table 4. Distribution of AmpC beta-lactamase producers and encoding genes among Enterobacteriaceae isolates.

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Out of the 116 Enterobacteriaceae isolates screened for ESBL phenotypes, 34 (29.31%) tested positive which comprised 19 E. coli, 10 Shigella spp. and 5 K. pneumoniae strains (Table 5, Fig 2). Only a small percentage (n = 2; 1.72%) exhibited both AmpC and ESBL phenotypic traits. A large proportion (n = 27; 79.4%) of the phenotypically positive ESBL isolates consisting of 15, 8 and 4 isolates of E. coli, Shigella spp. and K. pneumoniae respectively, harboured the bla_TEM gene (S7 Fig in S1 File).

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Fig 2. Proportion of ESBL-producing Enterobacteriaceae.

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Table 5. Prevalence of ESBL-producing Enterobacteriaceae.

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In summarily, most of the AmpC producers were multidrug-resistant and a Shigella species from cattle faeces, Y10 showed resistance or intermediate resistance to all antibiotics initially tested and doubles as a co-producer of ESBL. All AmpC isolates were susceptible (17/19) or showed intermediate susceptibility (2/19) to the cephem antibiotics. A detailed summary of the characteristics of all AmpC beta-lactamase isolates detected in the study is shown in Table 6.

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Table 6. Summary characteristics of AmpC beta-lactamase positive Enterobacteriaceae.

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3.4 Genetic relatedness of AmpC positive Enterobacteriaceae

The Fifteen AmpC beta-lactamase-producing E. coli strains typed by ERIC-PCR were of beef, clinical environment and cattle faecal origin. Generally, ERIC fingerprints ranged from 3 to 9 fragments per isolate and the relative sizes were between 250 bp to 3000 bp (Fig 3). ERIC fingerprinting profiles (Fig 3) placed the 15 isolates into 6 major clusters indicating genetic diversity with the largest cluster (cluster IV) having four isolates (one each from beef and cattle faeces samples and two from hospital swabs). On the contrary, the smallest cluster (cluster II) possessed only one isolate from a clinic environment in Motlabeng (Fig 4). However, this isolate shared some similarity with those in cluster I that mainly comprised isolates from beef samples collected from Zeerust (Fig 4). Cluster analysis revealed a 45% similarity cut off value regardless of the sampling area or source of the isolate. Fig 4 shows the dendrogram obtained from ERIC-PCR fragments.

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Fig 3. Agarose gel showing ERIC-PCR fingerprints of 15 AmpC- beta-lactamase E. coli isolates.

M = 1Kb DNA molecular weight marker; Lane 1 = BCC; Lane 2 = BCB1; Lane 3 = LPVAW; Lane 4 = 6; Lane 5 = ZM2-B; Lane 6 = ZM1-B; Lane 7 = 22; Lane 8 = 11; Lane 9 = LM1-A; Lane 10 = 27; Lane 11 = ZB1-A; Lane 12 = LM1-C; Lane 13 = MCB2P; Lane 14 = MPCW; Lane 15 = MPCP; Lane 16 = LB1-A.

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Fig 4. Dendrogram generated from ERIC-PCR cluster analysis of AmpC- beta-lactamase-producing Escherichia coli isolates.

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4. Discussion

Enterobacteriaceae pathogens are implicated in intra-abdominal, urinary tract, bloodstream, and various nosocomial infections and may resist antibiotic treatment due to the production of beta-lactamases that hydrolyse beta-lactam antibiotics [19]. The presence of these pathogens in borehole water intended for human use and consumption suggests potential health risks to consumers. Boreholes are usually proved to supply pure water when first drilled with little need for treatment. However, over time, boreholes become vulnerable to pollution with increased microbial contamination. Unfortunately, especially in poor communities, there is a continued water usage without tank cleaning and treatment [58]. Moreover, boreholes are compromised by solid waste dumping sites and general littering by animal droppings from farms [59]. Even in cases where residents are aware of the possible unsafe nature of the water, they are often left with no alternatives as these are usually poverty-stricken communities without access to water purifiers [58].

Meat safety is increasingly becoming an issue of severe concern. Cattle faeces harbouring Enterobacteriaceae such as E. coli is not surprising. However, cross-contamination in slaughterhouses and contamination during processing and distribution due to poor handling practices may occur resulting in the detection of the strains in beef products [60]. The higher isolation frequency of E. coli compared to other Enterobacteriaceae is similar to a report by Gwida et al. [46] in which a large proportion (54%) of the isolates from raw beef were identified as E. coli. The consumption of Enterobacteriaceae-contaminated food or water may lead to illnesses including diarrhoea, urinary and respiratory tract infections, dysentery, and in some cases bacteraemia [61–63]. These bacteria species were also detected on surfaces of clinical environments for which both patients and personnel had been in contact, which serves as potential sources for the development of nosocomial infections. Nosocomial infections are most often associated with invasive medical procedures and present a major challenge to patients’ health [64, 65]. Bloodstream nosocomial infections caused by Gram-negative organisms such as E. coli and Klebsiella species have been previously reported [1, 64] Unlike with other environments, the microbial population in a clinical environment has to be in check, especially as patients are often immunocompromised and unable to combat infections effectively compared to their healthy counterparts. A noticeable percentage of Shigella species were also obtained during the study. Shigellosis, caused by Shigella infection, is a more severe form of gastroenteritis and regularly leads to the death of children under the age of five [62, 66]. Shigella species have earlier been detected in the study area from meat abattoirs [67] and river catchments [68], but not from clinical environments highlighting the immense epidemiological relevance of this data.

The emergence and increased dissemination of beta-lactam resistance, especially in Enterobacteriaceae is an impediment to antibiotic therapy and public health as a whole. In the present study, large proportions of the isolates originating from various sources displayed significant susceptibilities to the aminoglycoside; amikacin and the carbapenem; meropenem. A similar outcome of no resistance against imipenem and two aminoglycosides was obtained in a previous study among young children in the Limpopo Province of South Africa [69]. These findings are of great therapeutic significance particularly because carbapenems especially meropenem are often used as the first-line antibacterial agents for infections caused by AmpC beta-lactamase-producing Enterobacteriaceae. Despite the high susceptibility of AmpC beta-lactamase-producing Enterobacteriaceae to aminoglycosides, these antibiotics are not highly recommended for treating these infections because of their unnecessary increased toxicity [25].

The proportion of resistant isolates against the oxymino-cephalosporins; second (cefepime, cefuroxime) and third-generation cephalosporins (cefotaxime, ceftriaxone and ceftazidime) might have resulted mainly from the expression of plasmid-borne beta-lactamases (ESBL or AmpC) or to a lesser extend hyperproduction of chromosomal-encoded AmpC due to promoter or attenuator mutation [21]. The trend in which isolates from cattle most often displayed resistance to oxymino-cephalosporins and ciprofloxacin was particularly concerning as cattle are food-producing animals. Moreover, given that the projected increase in meat production from 200 million tons to 470 million tons globally by 2050 this may significantly increase the usage of antibiotics for both the prevention and remediation of livestock diseases [70]. This reliance will ultimately increase antimicrobial resistance among bacterial pathogens which in turn will not only affect the quality of food products produced from these animals but also greatly hamper therapeutic processes in humans.

Resistance to cefoxitin is useful in screening for AmpC production in Enterobacteriaceae such as Klebsiella spp. and E. coli especially in areas where ACC-1 and ACC-4 enzymes have never been detected [71]. The large proportion of the isolates positive for AmpC by the use of cefoxitin disc contradicts earlier findings of Polsfuss et al. [15], Helmy and Wasfi [72] and Wassef et al. [73] in which 9.9%, 18.2% and 5.8% of the isolates respectively were resistant to cefoxitin. The presence of transmissible AmpC gene (s) in the majority (15/19) of AmpC positive Enterobacteriaceae is a cause for concern, because these strains or their resistance traits may potentially be dispersed amongst other strains in the environment, a public health threat.

ESBL-producing Enterobacteriaceae have been officially recognized as pathogens of critical priority by the World Health Organization as they have over the years been seen as problematic to global public health [74]. As such, it was of importance to investigate their occurrence especially their co-existence/production with AmpC. A 29.3% (n = 34) ESBL production rate in the current study and co-existence of both enzymes in two of the isolates ranks below the proportion previously obtained in parts of South Africa: Richter et al. [75] obtained a 79.2% ESBL and 41.6% of AmpC in Enterobacteriaceae isolates from vegetables while Founou et al. [76] reported a 19.5% ESKAPE pathogens ESBL producers from clinical samples. Also, the percentage of detection of ESBL in this study was higher than the 9.4% detected from retail foods in China [77] but lower than the proportion (45.2%) detected from clinical strains from Sudan [42]. Although the CTX-M enzymes in ESBL producers are the most frequently detected globally [78], the TEM type enzymes are known to be horizontally transferrable with ease and are degraded by clavulanic acid [78, 79]. The significant number of ESBL producing isolates positive for the bla_TEM gene, do not agree with previous studies by Oduro-Mensah et al. [80] and Bajai et al. [81] who reported lower frequencies of detection; 41.8% and 48.7% respectively although comparable to a very high occurrence rate (95.1%) in penicillin-resistant E. coli isolated from young children in the Limpopo province of South Africa [69]. It is worth mentioning that ESBL producers are clinically significant because associated infections are related to high mortality, lengthened hospital stay, failure of therapy and general health cost. A study for monitoring antimicrobial resistance trends over 10 years, 2002–2011 revealed that ESBL-producing Enterobacteriaceae causing intra-abdominal infections increased from 2003 (about 115) to almost 16% in 2005 in Africa. However, there was a steady decrease in the later years reaching just below 10% by 2010. Unfortunately by 2011, intra-abdominal infections caused by these pathogens was on the rise (almost 12%), especially amongst patients in intensive care units [82]. In neonates and children, ESBL-producing Enterobacteriaceae have been estimated at 15% in Africa [83]. The high incidence of ESBL obtained herein is therefore very worrisome as ESBL-producing Enterobacteriaceae are often associated with various diseases and most infective bacteria are multi-drug resistant.

The ERIC-PCR of 15 isolates revealed low similarity of AmpC beta-lactamase-producing Enterobacteriaceae. Although E. coli have potentially transmissible AmpC beta-lactamase determinants, cross-contamination had unlikely occurred. Despite the genetic diversity and high heterogeneity, the great similarities in the drug resistance genes (identified by the presence of the bla_ACC gene) as shown in clusters I and IV indicate the potential to pose similar public health and/or therapeutic challenges in humans. The data generated through ERIC-PCR proved that there is a need to reduce or eradicate contamination levels in beef products by implementing strict hygiene practices during the handling of beef carcasses.

5. Conclusion

The increased public health threat posed by multidrug-resistant ESBL and AmpC beta-lactamase-producing strains especially among pathogenic Enterobacteriaceae is a cause for concern. The potential of chromosomally encoded ampC genes to be induced and upregulated in most Enterobacteriaceae when exposed to β-lactam antibiotics or other stimuli thus conferring resistance to broad-spectrum Cephalosporins as well as the potential for the rapid dissemination of plasmid-mediated ampC genes in other Enterobacteriaceae strains amplify the need for constant surveillance. In summary, the study revealed the occurrence of AmpC positive E. coli, Shigella species and K. pneumoniae in borehole water, beef, human and cattle faeces as well as clinical environments with high frequencies of occurrence in the latter two. Although AmpC beta-lactamases were confirmed less prevalently than ESBL, their occurrence is a cause for concern because the expression ampC genes in the identified Enterobacteriaceae strains are often carried on transmissible plasmids and may be a source of transmission to other environments and humans, as well as a source of antibiotic resistance in HA- infections, a severe health threat. Thus, strategies to ensure food safety and proper hygienic practices especially with regards to those meant for human consumption are essential. There might be a need to reassess and improve hygienic practices in cattle farms and abattoirs. Moreover, vigorous cleaning methods could be adapted as these pathogens could be thriving within the currently used methods. Continuous surveillance studies are also required for a better understanding of the clinical implications of AmpC infections, which will, in turn, aid in hospital infection control and administration of appropriate antibiotics.

6. Limitation of the study

In this study, Multilocus Sequence Typing (MLST) was not included as a tool to determine the genetic relatedness of isolates from different sources and this is a limitation.

Supporting information

S1 File.

https://doi.org/10.1371/journal.pone.0253647.s001

(PDF)

Acknowledgments

The authors are grateful to the North-West University for providing the space for bench work. The assistance received from the North West Provincial Department of Health, South Africa is also appreciated.

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Evidence of potentially unrelated AmpC beta-lactamase producing Enterobacteriaceae from cattle, cattle products and hospital environments commonly harboring the bla_ACC resistance determinant

Evidence of potentially unrelated AmpC beta-lactamase producing Enterobacteriaceae from cattle, cattle products and hospital environments commonly harboring the bla_ACC resistance determinant

Figures

Abstract

1. Introduction

2. Materials and methods

2.1 Sampling

2.2 Isolation and identification of members of Enterobacteriaceae

2.2.1 Sample processing.

2.2.2 Isolation of members of Enterbacteriaceae.

2.2.3 Molecular identification of members of Enterbacteriaceae.

2.3 Antimicrobial susceptibility testing

2.4 Phenotypic and genotypic detection of AmpC β-lactamases and ESBL

2.5 Molecular typing of AmpC beta-lactamases producing Escherichia coli using ERIC-PCR

2.6 Statistical analysis

3. Results

3.1 Occurrence of Enterobacteriaceae in various samples

3.2 Antimicrobial resistance profile of isolates

3.3 Prevalence of AmpC beta-lactamases/ ESBL among Enterobacteriaceae isolates

3.4 Genetic relatedness of AmpC positive Enterobacteriaceae

4. Discussion

5. Conclusion

6. Limitation of the study

Supporting information

S1 File.

Acknowledgments

References