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Prevalence and Characterization of Extended-Spectrum Beta-Lactamase (ESBL)– and CMY-2–Producing Escherichia coli Isolates from Healthy Food-Producing Animals in Tunisia
Abstract
Abstract The prevalence of extended-spectrum beta-lactamase (ESBL)- and plasmidic AmpC-beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates has been studied in food-producing animals at the farm level in Tunisia, and recovered isolates were characterized for the presence of other resistance genes and integrons. Eighty fecal samples of food-producing animals (23 sheep, 22 chickens, 22 cattle, six horses, five rabbits, and two dromedaries) were obtained from 35 different farms in Tunisia in 2011. Samples were inoculated onto MacConkey agar plates supplemented with cefotaxime (2 mg/L) for cefotaxime-resistant (CTX(R)) E. coli recovery. CTX(R) E. coli isolates were detected in 11 out of 80 samples (13.8%), and one isolate per sample was further characterized (10 from chickens and one from a dromedary). The 11 CTX(R) isolates were distributed into phylogroups: B1 (five isolates), A (two isolates), D (three isolates), and B2 (one isolate). The following beta-lactamase genes were detected: bla(CTX-M-1) (seven isolates), bla(CTX-M-1)+bla(TEM-135) (one isolate), bla(CTX-M-1)+bla(TEM-1b) (one isolate), and bla(CMY-2) (two isolates). All ESBL- and pAmpC-BL-producing E. coli strains showed unrelated pulsed-field gel electrophoresis patterns. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA17-aadA5 (three isolates), dfrA1-aadA1 (two isolates), dfrA15-aadA1 (one isolate), and aadA1 (one isolate). All isolates showed tetracycline resistance and contained the tet(A) +/- tet(B) genes. Virulence genes detected were as follows (number of isolates in parentheses): fimA (10); aer (eight); papC (two); and papGIII, hly, cnf, and bfp (none). Chicken farms constitute a reservoir of ESBL- and pAmpC-BL-producing E. coli isolates of the CTX-M-1 and CMY-2 types that potentially could be transmitted to humans via the food chain or by direct contact.
... In Cameroon, a study found that antibiotics such as oxytetracycline, chloramphenicol, and neomycin were highly abused in poultry (Guetiya Wadoum et al. 2016). A study in Tunisia found ESBL-producing E. coli from food-producing animals, indicating a public health problem as these genes can cause damage to beta-lactam antibiotics (Ben Sallem et al. 2012). ...
... The overuse of antimicrobials in animal husbandry has contributed to the emergence of E. coli resistant to many antimicrobials (Ben Sallem et al. 2012;Guetiya Wadoum et al. 2016;Alonso et al. 2017). In Tanzania, a study found that most E. coli isolates from commercial-layer and free-range chickens were resistant to the most common antibiotics used in poultry (Bernadether et al. 2016). ...
Antimicrobial resistance (AMR) is a global public health challenge exacerbated by the indiscriminate use of antimicrobials. A well-implemented national AMR surveillance system is one of the important strategies for combating AMR. Several bacteria are resistant to antimicrobials, but Escherichia coli (E. coli) and Enterococcus species have been demonstrated to be resistant to most antimicrobials used in veterinary and human medicine, making them valuable indicators for AMR surveillance. The implementation of AMR surveillance in Zambia is still in its infancy. Therefore, this study aimed to evaluate the AMR challenges in layer poultry production and the associated surveillance strategy in the Lusaka and Copperbelt Provinces of Zambia.
This cross-sectional study was conducted in layer farms from September 2020 to April 2021. A total of 365 cloacal samples were collected from 77-layer poultry farms using the National Surveillance AMR protocol for laying hens. E. coli identification and confirmation were done using Analytical Profile Index (API 20E) kits and 16S rRNA sequencing, while Enterococci were identified using API 20 STREP and confirmed by sequencing the 16S rRNA. The expected band size (amplicon size) was 500 bp for E. coli and 600 bp for Enterococci. Antibiotic susceptibility testing (AST) for E. coli was done using a panel of thirteen antibiotics, while that of Enterococci utilised nine antibiotics. Quantitative data were analysed using Stata version 16.1 and WHONET 2020. All statistical tests were conducted at a 95% significance level and a margin error of 5%. Qualitative data were collected using interviews and analysed using the thematic analysis method.
Of the 365 cloacal swab samples, 339 (92.9%) tested positive for E. coli, while 308 (84.4%) tested positive for Enterococci. E. coli isolates were highly resistant to tetracycline (54.6%), ampicillin (54.0%), and cefotaxime (30.4%) but highly susceptible to meropenem 94.7%), chloramphenicol (85.8%), and ceftazidime (85.3%). Additionally, 64.6% of E. coli isolates were multidrug-resistant (MDR). Enterococci isolates were highly resistant to tetracycline (80.5%), erythromycin 53.6%), and quinupristin-dalfopristin (53.2%) but highly susceptible to nitrofurantoin (77.6%), chloramphenicol (71.1%), and ampicillin (63.3%). Further, 86% of Enterococci isolates were MDR. The overall awareness of AMR among layer poultry farmers was 47% (n=36). There was high usage of antibiotics in the layer poultry sector at 86% (n=66), which mainly were accessed from agro-veterinary shops (31%, n=24) and pharmacies (21%, n=16) without prescriptions. Commercial farmers were more likely to be aware of AMR compared to medium-scale farmers (OR = 14.07, 95% CI: 2.09–94.70). Additionally, farmers who used prescriptions to access antibiotics were more likely to be aware of AMR compared to those who did not (OR = 99.66, 95% CI: 7.14–1391.65). Further, farmers who did not treat market-ready birds with antibiotics were more likely to be aware of AMR compared to those who did (OR = 41.92, 95% CI: 1.26–1396.36). Compared to small-scale farmers, commercial farmers were less likely to have AMR problems (AOR=0.20, 95% CI: 0.04-0.99, p=0.049). Similarly, farmers who were aware of AMR were less likely to have AMR problems (AOR=0.26, 95% CI: 0.08-0.86, p=0.027). The AMR surveillance system used in poultry in Zambia effectively identifies indicator microorganisms resistant to antibiotics used in poultry and human health. However, the surveillance system faces challenges, including inadequate human resource and funding shortages, which impact the active surveillance of AMR in layer poultry production.
This study found that E. coli and Enterococci were highly resistant to common antibiotics (tetracyclines and penicillins) used in layer poultry production. The presence of MDR E. coli and Enterococci isolated from laying hens is a public health concern. This study further found that AMR in the sampled farms was due to farmers' low awareness of AMR and its associated factors. To address this problem, there is an urgent need to review and update the current protocol on AMR surveillance in poultry in Zambia and allocate resources efficiently. Additionally, there is a need to strengthen the surveillance and antimicrobial stewardship programmes coupled with poultry farmer educational interventional activities.
... In the last decade, it has been observed that ESBL-producers and multi-drug resistant E. coli isolates are frequently detected in food-producing animals or food products, and thus health authorities are worried about the potential transmission of these resistant microorganisms to humans through the food chain. Likewise, in Tunisia many reports have highlighted a high prevalence of antibiotic resistance and ESBL production in E. coli isolates from food-producing animals or food products, a situation that requires more investigations and vigilance to monitoring large dissemination of such resistant isolates [9][10][11][12]. ...
... Recently, ESBL-producing E. coli isolates have been reported in animal worldwide especially from poultry origin [30][31][32][33]. In Tunisia, the presence of ESBL producers has been previously reported in E. coli from poultry, pets, dromedary, and meat of various animals [9,31,[10][11][12]34]. In those studies, ESBL production was detected by using a selective protocol. ...
Little detailed documentation researched the excessive use of antimicrobials such as tetracycline and sulfonamides in vet-
erinary medicine in Tunisia and more studies are needed. A total of 58 of commensal Escherichia coli isolates recovered
from fecal samples of healthy poultry (n=31) and bovin (n=27) recovered from farms in Tunisia were examinated for 20
antimicrobial as well as, researched the presence of integron, variable regions (VRs), phylogroupes, tetracycline (tetA, tetB et
tetC) and sulfonamides(sul1, sul2, sul3) resistance genes. The most frequently resistance in poultry origin were to tetracycline
(94.3%), sulfonamide (70.69%), nalidixic acid (61.29), amoxicillin (58%), to trimthetoprim-sulfamethoxazole and streptomy-
cin with the same rate (64.51%), ticarcilline (58%). Whereas, the bovine isolates were most resistant to streptomycin (55.5%),
to amoxicillin (18.5), to tetracycline (37%), and have a moderate same rates to kanamycine, to trimthetoprim-sulfamethoxaz-
ole 11.11, to nalidixic acid and to sulfonamide 7.4%, For poultry and bovine class 1 integron were detected in 20, 6 isolates,
respectively as well as class 2 integron were found in 2 and 1 isolates, respectively. Class 1 integrons were significantly asso-
ciated with poultry origin (p=0.001). For poultry sul1, sul2, and sul3 genes were detected in 14 (46.2 %), 7 (23.8 %), and 4
(8.9 %) resistant isolates, respectively. Whereas, for bovine 5 isolates were resistant to sulfonamide and sul1 and sul2 genes
were detected in 4 and 1 isolates with absence of sul3 genes. and tetracycline genes tetA, tetB genes were observed in 27
(84.37 %) and 8 (25%) resistant isolates, respectively while, TetC was not detected amongst our isolates. Seven arrangement
gene cassette were detected; dfrA1-satA1-aadA1 in one identical DNA fragments with approximate size of 2000 bp and six
arrangements of resistance gene cassettes of class 1 integron were detected; dfrA1+aadA1 (5isolates); for dfrA17+aadA1,
dfrA12+ orfF+aadA2 each one two 2isolates; one isolate for aadA1 and dfrA5, respectively. In poultry, 16 isolates were found
to belong to phylogroup A (sub- groupA1: 12, sub- groupA0: 4); 9 to B1, 1 to B2 and 5 to phylogroup D. However, in bovine 9
isolates have the phylogroups A1, 7 isolates B1, 4 isolates B2, and 3 isolates found to phylogroup D. Our results showed that
the prevalence of resistance in E. coli isolates from poultry was much higher than that in bovin.
Transl Med OA, 2023
1. Introduction
Escherichia coli can cause a variety of diarrheal and other ex-
tra-intestinal infections in humans and animals. The emergence
of E. coli isolates with multiple antibiotic resistance phenotypes,
has been previously reported and is considered as a serious
health concern [1,2]. In Enterobacteriaceae and particularly
in E. coli, resistance to beta-lactams due to Extended Spectrum
Beta-Lactamases (ESBL), quinolones, and aminoglycosides
have drawn considerable attention worldwide [3]. ESBL-pro-
ducing isolates are usually resistant to other
... Worryingly, the inadequate biosecurity and poor hygiene and sanitation in poultry production systems in LMICs has resulted in a high reliance on ABU for disease prevention and control (Hedman et al., 2020). Higher incidence of drug-resistant bacteria including ESBLproducing bacteria has been reported in poultry production systems with high levels of ABU (ben Sallem et al., 2012;Donkor et al., 2012). ...
... All three studies conducted in backyard production systems (Ojo et al., 2016;Okpara et al., 2018;Langata et al., 2019) reported low ESBL levels ranging from 0 to 2.9%. Two studies (ben Sallem et al., 2012;Mgaya et al., 2021) assessed ESBL-E. coli in both intensive and extensive systems while one study (Saidani et al., 2019) was carried out on a farm with an intensive production system. ...
Extended-spectrum beta-lactamase (ESBL)-producing bacteria present a unique problem because of their ability to cause infections that are difficult to treat in animals and humans. The presence of ESBL- Escherichia coli ( E. coli ) in poultry raises a major public health concern due to the risk of zoonotic transfer via the food chain and direct contact with birds and the environment. This review aimed to determine the frequency of ESBL-producing E. coli and associated ESBL genes in poultry in Africa. Three databases (PubMed, ScienceDirect, and Web of Science) and predetermined websites were searched to identify scientific and grey literature. Studies (1582) were screened at title, abstract, and full-text levels. This review was registered with PROSPERO (CRD42021259872). Thirty-three studies were deemed eligible for this review. Phenotypic ESBL expression was confirmed in 22 studies (66.7%) with a wide range of colonization noted in sampled poultry (1 – 100%). The bla CTX-M gene was the most commonly isolated with the variants bla CTX-M-1 and bla CTX-M-15 being the most predominant in North and West Africa respectively. ESBL-producing E. coli isolates are frequently detected in poultry in farms and slaughterhouses across Africa thereby posing a potential health risk to humans. The paucity of data however does not allow for inferences to be made about the true extent of ESBLs in poultry in Africa.
... Ngbede et al. (2021) detected Sul2 and Sul3 in 71.42% in camel E. coli isolates from Nigeria. Ben Sallem et al. (2012) isolated Escherichia coli with 100% prevalence of sul2 gene from healthy foodproducing animals in Tunisia. Rawat et al. (2022) observed sulfonamide resistance gene sul3 (44%), sul2 (28%) among the poultry isolates from North India. ...
Background: Escherichia coli is frequently associated with multiple antimicrobial resistances and a major cause of bacterial extraintestinal infections in livestock and humans. Escherichia coli resides in the lower digestive tract as harmless commensals but a subset of E. coli strains has acquired the ability due to acquisition of virulence and antibiotic genes, cause intestinal or extraintestinal diseases. Methods: In this field-laboratory investigation during 2020-2021, samples were taken from different localities of Bikaner district and surrounding are of Rajasthan. A total no. of 70 fecal samples were collected and immediately transferred to the Department of Veterinary Microbiology. In the laboratory, the collected samples were further processed for isolation and identification of E. coli bacteria. Result: Confirmation of E. coli was done using primary and biochemical test which are screened for hemolysin property, biofilm formation, antibiogram study and antibiotic resistance gene. All the isolates of E. coli used to show characteristic metallic sheen on EMB agar plate and excellent identification was done with VITEK 2 system. All the camel isolates shown partial hemolysis on sheep blood agarand 45.71% camel isolates were positive for biofilm formation. All these 70 E. coli isolates from camel were resistant to Penicillin (94.38%) which was followed by amoxicillin+sulbactam (85.71%), erythromycin (71.14%), cefixime+clavulanic acid (71.43%). Highest sensitivity to chloramphenicol (81.28%) followed by sulphadiazine (48.57%) and cotrimoxazole (48.28%). All these 70 isolates were screened for antibiotic resistance genes. On the basis of molecular screening of the antibiotic resistance genes, majority of the isolates carried BlaTEM gene in camel (56/70; 80%), followed by StrA (29/70; 41.40%), Sul-3 in (22/70; 31.42%), Sul-2 (18/70; 25.71%), aadA (28/70; 40%), tet(B) (22/70; 31.42%) isolates.
... The number of recorded antibiotic-resistant isolates (including important human pathogens) is increasing worldwide [2]. Enterobacteriaceae, considered to be normal flora in the intestine of food-producing livestock [3][4][5], make up a large proportion of these resistant isolates. ...
... Abattoir wastes/release often contain several hundred species of bacteria including Aeromonas spp., Enterobacter spp., Klebsiella spp., Salmonella spp., Staphylococcus spp., and Clostridium spp (Sistani et al., 2008;Bello and Oyedemi, 2009;Ben Sallem et al., 2012). These potential pathogens are transmissible via direct contact to humans or indirectly through contaminated water sources. ...
... Similar β-lactamase genes to those from our study have been reported in Southern Thailand [45]. ESBL groups identified from our research (CTX-M-15 and CTX-M-1) also correspond to ESBL groups previously reported in Tunisia [27] from vegetables, irrigation water and soil; and in E. coli from healthy humans, food-producing animals, and food [74,78]. Hence, contamination by ESBL E. coli from farm environments and vegetable samples could have occurred from both animal and human origins in Nigeria. ...
Antimicrobial resistance (AMR) is a major public health concern, especially the extended-spectrum β-lactamase-producing (ESBL) Escherichia coli bacteria are emerging as a global human health hazard. This study characterized extended-spectrum β-lactamase Escherichia coli (ESBL-E. coli) isolates from farm sources and open markets in Edo State, Nigeria. A total of 254 samples were obtained in Edo State and included representatives from agricultural farms (soil, manure, irrigation water) and vegetables from open markets, which included ready-to-eat (RTE) salads and vegetables which could potentially be consumed uncooked. Samples were culturally tested for the ESBL phenotype using ESBL selective media, and isolates were further identified and characterized via polymerase chain reaction (PCR) for β-lactamase and other antibiotic resistance determinants. ESBL E. coli strains isolated from agricultural farms included 68% (17/25) from the soil, 84% (21/25) from manure and 28% (7/25) from irrigation water and 24.4% (19/78) from vegetables. ESBL E. coli were also isolated from RTE salads at 20% (12/60) and vegetables obtained from vendors and open markets at 36.6% (15/41). A total of 64 E. coli isolates were identified using PCR. Upon further characterization, 85.9% (55/64) of the isolates were resistant to ≥ 3 and ≤ 7 antimicrobial classes, which allows for characterizing these as being multidrug-resistant. The MDR isolates from this study harboured ≥1 and ≤5 AMR determinants. The MDR isolates also harboured ≥1 and ≤3 beta-lactamase genes. Findings from this study showed that fresh vegetables and salads could be contaminated with ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Appropriate measures, including improving irrigation water quality and agricultural practices, need to be implemented, and global regulatory guiding principles are crucial to ensure public health and consumer safety.
... Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have been prioritised within the field of novel therapeutic strategy development due to their impact on both human and animal health (WHO, 2017). Beta-lactamase-producing Enterobacteriaceae have been isolated from the gut of both healthy camels and camels experiencing clinical diarrhoea (Ben Sallem et al., 2012;Bessalah et al., 2016;Carvalho et al., 2020) Given the growing camel population within Kenya, the potential for widespread dissemination of these bacterial strains and their beta-lactamase genes represents an important public health threat (Munita et al., 2016;Naseer & Sundsfjord, 2011). Different studies have been conducted in Kenya to determine AMR profiles in food-producing animals reared in different animal production settings (Aliwa et al., 2019;Langata et al., 2019;Mutua et al., 2017;Nyabundi et al., 2017;Ngaywa et al., 2019). ...
Background
Animal husbandry practices in different livestock production systems and increased livestock–wildlife interactions are thought to be primary drivers of antimicrobial resistance (AMR) in Arid and Semi‐Arid Lands (ASALs). Despite a tenfold increase in the camel population within the last decade, paired with widespread use of camel products, there is a lack of comprehensive information concerning beta‐lactamase‐producing Escherichia coli (E. coli) within these production systems.
Objectives
Our study sought to establish an AMR profile and to identify and characterise emerging beta‐lactamase‐producing E. coli isolated from faecal samples obtained from camel herds in Northern Kenya.
Methods
The antimicrobial susceptibility profiles of E. coli isolates were established using the disk diffusion method, with beta‐lactamase (bla) gene PCR product sequencing performed for phylogenetic grouping and genetic diversity assessments.
Results
Here we show, among the recovered E. coli isolates (n = 123), the highest level of resistance was observed for cefaclor at 28.5% of isolates, followed by cefotaxime at 16.3% and ampicillin at 9.7%. Moreover, extended‐spectrum beta‐lactamase (ESBL)‐producing E. coli harbouring the blaCTX‐M‐15 or blaCTX‐M‐27 genes were detected in 3.3% of total samples, and are associated with phylogenetic groups B1, B2 and D. Multiple variants of non‐ESBL blaTEM genes were detected, the majority of which were the blaTEM‐1 and blaTEM‐116 genes.
Conclusions
Findings from this study shed light on the increased occurrence of ESBL‐ and non‐ESBL‐encoding gene variants in E. coli isolates with demonstrated multidrug resistant phenotypes. This study highlights the need for an expanded One Health approach to understanding AMR transmission dynamics, drivers of AMR development, and appropriate practices for antimicrobial stewardship in camel production systems within ASALs.
Background
The World Health Organization (WHO) has classified carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa (P. aeruginosa), and Acinetobacter baumannii (A. baumannii) as high-priority pathogens, and carbapenem-resistant bacteria (CRB) have been reported to spread between humans, animals, and the environment.
Objective
This study aimed to conduct a systematic review of carbapenem resistance in animals, foods, and the environment on the African continent and to provide recommendations and perspectives for better prevention and control of carbapenem resistance in Africa.
Results
A total of 137 research articles collected from 2009 to 2023 were selected for this review, including articles reporting carbapenem-resistant bacteria in animals (81/137; 59.1%), the environment (66/137; 48.2%), and foods (26/137; 19%). Carbapenem-resistant bacterial species belonged to 31 genera and 17 families, including mainly Escherichia spp. (68/127; 53.5%); Klebsiella spp. (45/127; 35.4%); Pseudomonas spp. (20/127; 15.7%), Enterobacter spp. (19/127; 15%) and Acinetobacter spp. (15/127; 11.8%). The prevalence of CRBs by country ranged from 1.1% to 48.5%, and the pooled prevalence of CRBs isolated from animal-environment-food in Africa was 19.1% (2804/14,684; Standard Deviation = 15). Twenty carbapenemase families belonging to A, B, C, and D Ambler classes were reported, including mainly carbapenemase genes from blaOXA (44/84; 52.4%), blaNDM (34/84; 40.5%), blaSHV (23/84; 27.4%), blaKPC (22/84; 26.2%), blaVIM (19/84; 22.6%), and blaIMP (12/84; 14.3%) families. The reported mobile genetic elements (MGE) carrying carbapenemase-encoding genes included plasmids (16/19; 84.2%), integrons (3/19; 15.8%), transposons (3/19; 15.8%), and insertion sequences (2/19; 10.5%). blaOXA-48 was often carried by (60kb-65kb) IncL/M-type pOXA-48 plasmids, while blaNDM-5 was often carried by (45–50kb) IncX-type plasmids. Moreover, 25 articles investigated and reported virulent and hypervirulent CRBs that carried multiple virulence factors.
Conclusion
Animal-environment-food ecosystems would constitute reservoirs of CRBs involved in human infections. The One Health approach and constant collaboration between governments are necessary to drastically reduce the mortality rates linked to antimicrobial resistance.
Çanakkale semt pazarlarında satılan 96 adet yeşil sebze örneğinde “beta laktam” dirençli Enterobacterales suşlarının varlığı araştırılmıştır. Gövde, yaprak kısımlarından alınan örneklerden Violet Red Bile Glucose (VRBG) Agara ekilmiştir. Enterobacterales yükleri <1 log KOB/g ile 6.11 log KOB/g arasında tespit edilmiştir. Kromojenik GSBL Agar ve 2 mg/ml meropenem içeren EMB Agarda üreyen 129 adet izolat MALDI-TOF MS yöntemine göre Serratia liquefaciens (n=35), Serratia plymuthica (n=31), Klebsiella oxytoca (n=25), Klebsiella pneumonia (n=6), Raoultella ornithinolytica (n=14), Raoultella terrigena (n=3), Enterobacter cloacae (n=11), Enterobacter ludwigii (n=4), Enterobacter asburiae (n=1), Lelliottia amnigena (n=16), Escherichia coli (n=2), Escherichia hermannii (n=1), Leclercia adecarboxylata (n=3), Pantoea agglomerans (n=3), Kluyvera intermedia (n=2), Kosakonia cowanii (n=1) ve Hafnia alvei (n=1) olarak tanımlanmıştır. Disk difüzyon yöntemine göre izolatların %11.62’sinin (n=15) Genişlemiş Spektrumlu “Beta Laktamaz” (GSBL), %4.65’inin (n=6) karbapenemaz pozitif olduğu belirlenmiştir. Yeşil sebzelerde GSBL ve karbapenem dirençli izolatların varlığı ısıl işlem görmeden tüketilen bu gıdalardaki halk sağlığı riskini ortaya koymaktadır.
[From Introduction] This guest commentary proposes a set of guidelines for interpreting DNA restriction patterns generated by PFGE. The authors are investigators from the United States who, over the last several years, have correlated epidemiologic data from dozens of outbreaks with strain typing results produced by PFGE. These guidelines are intended to be used by clinical microbiologists in hospital laboratories to examine relatively small sets of isolates (typically, ≤30) related to putative outbreaks of disease. In an effort to make PFGE more easily understood and accessible as a typing method, the use of statistical methods and equipment to digitize patterns has been avoided. Such methods may be appropriate for larger collections of isolates studied in reference laboratories, but they are neither feasible nor necessary for laboratories that will be confronted primarily with short-term outbreaks.
Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 β-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains)
in Spain. Twelve of these strains harbored blaCTX-M genes and showed unrelated pulsed-field gel electrophoresis patterns.
Having shown that the Xeral-Calde Hospital in Lugo (Spain) has been concerned by Escherichia coli clone O25:H4-ST131 producing CTX-M-15 (Nicolas-Chanoine et al. J Antimicrob Chemother 2008; 61: 273-81), the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates and also to molecularly characterize the E. coli isolates producing ESBL other than CTX-M-15.
In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL-producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla(CTX-M-15) gene.
Of the 105 ESBL-producing E. coli isolates, 60 (57.1%) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phylogenetic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15-producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively.
CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 producing CTX-M-15. The triplex PCR developed in this work will be useful for rapid and simple detection of this clone.
Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials
introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes
related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM)
gene variant, blaTEM-135, that may be a precursor in the transitional stage of a traditional blaTEM-1 gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins
in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand.
Molecular analysis of the blaTEM-135 gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different
plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the
blaTEM-135 allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a blaTEM variant (blaTEM-135) that is a possible intermediate precursor for an ESBL, which warrants international awareness.
The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 μg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.
Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla (CTX-M-15) (12 isolates), bla (CTX-M-14a) (one isolate), and bla (CTX-M-14b) (one isolate). The bla (OXA-1) gene was detected in 13 bla (CTX-M)-producing strains and a bla (TEM-1) gene in 6 of them. The ISEcp1 sequence was found upstream of bla (CTX-M) genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17-aadA5 being the most common. One of the strains (bla (CTX-M-14a)-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6')-Ib and cmlA1 genes and it was linked to the bla (CTX-M-14a) gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.
The number and proportion of CTX-M positive Escherichia coli organisms were determined in feces from cattle, chickens, and pigs in the United Kingdom to provide a better understanding
of the risk of the dissemination of extended-spectrum β-lactamase (ESBL) bacteria to humans from food animal sources. Samples
of bovine (n = 35) and swine (n = 20) feces were collected from farms, and chicken cecal contents (n = 32) were collected from abattoirs. There was wide variation in the number of CTX-M-positive E. coli organisms detected; the median (range) CFU/g were 100 (100 × 106 to 1 × 106), 5,350 (100 × 106 to 3.1 × 106), and 2,800 (100 × 105 to 4.7 × 105) for cattle, chickens, and pigs, respectively. The percentages of E. coli isolates that were CTX-M positive also varied widely; median (range) values were 0.013% (0.001 to 1%) for cattle, 0.0197%
(0.00001 to 28.18%) for chickens, and 0.121% (0.0002 to 5.88%) for pigs. The proportion of animals designated high-density
shedders (≥1 × 104 CFU/g) of CTX-M E. coli was 3/35, 15/32, and 8/20 for cattle, chickens, and pigs, respectively. We postulate that high levels of CTX-M E. coli in feces facilitate the dissemination of blaCTX-M genes during the rearing of animals for food, and that the absolute numbers of CTX-M bacteria should be given greater consideration
in epidemiological studies when assessing the risks of food-borne transmission.
Scientific Opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals. EFSA Journal 2011;9(8):2322. [95 pp.] doi:10.2903/j.efsa. ABSTRACT The potential contribution of food-producing animals or foods to public health risks by ESBL and/or AmpC-producing bacteria is related to specific plasmid-mediated ESBL and/or AmpC genes encoded by a number of organisms. The predominant ESBL families encountered are CTX-M, TEM, and SHV; the predominant AmpC-family is CMY. The most common genes associated with this resistance in animals are bla CTX-M-1 (the most commonly identified ESBL), and bla CTX-M-14 , followed by bla TEM-52 and bla SHV-12. Among the genes encoding AmpC-type β-lactamases, bla CMY-2 is the most common.The bacterial species most commonly identified with these genes are Escherichia coli and non-typhoidal Salmonella. ESBL/AmpC transmission is mainly driven by integrons, insertion sequences, transposons and plasmids, some of which are homologous in isolates from both food-production animals and humans. Cefotaxime is used as the drug of choice for optimum detection of bla ESBL and/or bla AmpC genes. The preferred method for isolation of ESBL-and/or AmpC-producers is screening on selective agar preceded by selective enrichment in a broth.The establishment of risk factors for occurrence of ESBL/AmpC-producing bacteria is particularly complicated by the data unavailability or lack of its accuracy. The use of antimicrobials is a risk factor for the selection and spread of resistant clones, resistance genes and plasmids. Since most ESBL-and AmpC-producing strains carry additional resistances to other commonly-used veterinary drugs, generic antimicrobial use is a risk factor for ESBL/AmpC and it is not restricted specifically to the use of cephalosporins. An additional risk factor is extensive trade of animals in EU MS. There are no data on the comparative efficiency of individual control options in reducing public health risks caused by ESBL and/or AmpC-producing bacteria related to food-producing animals. Prioritisation is complex, but it is considered that a highly effective control option would be to stop all uses of cephalosporins/systemically active 3 rd /4 th generation cephalosporins, or to restrict their use (use only allowed under specific circumstances). As co-resistance is an important issue, it is also of high priority to decrease the total antimicrobial use in animal production in the EU.
Clin Microbiol Infect 2011; 17: 873–880
Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as ‘poultry-associated’ (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (blaCTX-M-1, blaCTX-M-2, blaSHV-2, blaSHV-12, blaTEM-20, blaTEM-52): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were blaCTX-M-1 and blaTEM-52 genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.