Antimicrobial resistance of Escherichia Coli in retail chicken meat in Vietnam

K. Sary, J.M. Fairbrother, J. Arsenault And M. Boulianne

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To evaluate the safety of chicken meat in Vietnam whole chicken carcasses, fresh or frozen, were sampled from markets and supermarkets. Primary bacterial cultures were prepared from carcasses rinsed in Vietnam and then sent to the OIE Reference Laboratory for Escherichia coli (EcL) in Canada for further characterization of isolates.

Phenotypic antimicrobial susceptibility was determined by the automated Minimal Inhibitory Concentration (Sensititre ARIS®) system. Antimicrobial resistance of randomly selected Generic E. coli was observed for drugs that are classified as “very high” and “high” importance in human medicine according to Health Canada classification. Resistance was more prevalent in the Possible ExPEC subset, defined by the presence of one or more of the virulence genes iucD, tsh, papC, and cnf. One of the virulence genes, iucD, seemed linked to high resistance to specific antimicrobials. Presence of Extended-Spectrum β-lactamase (ESBL) producing E. coli was confirmed phenotypically by the Sensititre ARIS® system in the Ceftriaxone enriched (1mg/L) colonies and genotypically by the presence of the associated resistance gene family, CTX-M, verified by multiplex PCR. This is the first report of ESBL-producing E. coli in chicken meat from Vietnam. Multi-drug resistance to 5 or 6 classes of antimicrobial was common, being more prevalent in ExPEC isolates. Little difference was observed between retail sources, with the exception that frozen chicken carcasses demonstrated lower prevalence of putative ESBL- producing E. coli, and a higher multi-drug resistance (5 to 6 classes) with greater resistance to specific antimicrobials. Retail sources of fresh carcasses did not differ in antimicrobials patterns. Our results demonstrate that chicken meat in Vietnam represents a potential danger to public health, being a source of ESBL-producing and multi-drug resistant E. coli, including the potentially pathogenic ExPEC.

In Vietnam, chicken carcasses are traditionally bought at urban open-air markets where chickens are slaughtered on-site. Since the economic revolution named Doi moi in late 80’s, Vietnamese have developed more small businesses and a new economic class that possesses an increased purchasing power have emerged. In addition, foreign investments have allowed the implementation of new corporations, such as the introduction of supermarkets. These have restructured retail sale and accessibility to a wide variety of food products as well as other goods. With this revitalized economy and a new class of consumers, their needs and demands have also been modified. They now question quality and food safety, and as such favour supermarkets. Popularity of the traditional markets is explained by a preference for meat from free-range chickens and accessibility. It is often only a short walking distance from home and food is available daily to consumers who cannot afford a fridge. However, the growing young Vietnamese middle-class is turning to another food source, supermarkets. Supermarkets have introduced self-service practices, inexpensive food products such as chicken carcasses from intensive farming, and visibility for other goods. Frozen chicken carcasses are also available in supermarkets. Poultry meat, the second most popular meat consumed in Vietnam, after pork, has been suspected as the main carrier of Extraintestinal pathogen Escherichia coli (ExPEC). Contrary to other Escherichia coli pathotypes, ExPEC is asymptomatic in the digestive tract but is responsible for diseases with elevated morbidity in humans, most commonly urinary tract infections, but also newborn meningitis and septicaemia. ExPEC’s site of infection varies depending on the wide array of virulence genes that it acquires and expresses. This plasticity of virulence and its ability to infect humans at many different extraintestinal sites make ExPEC a major health threat. Due to the increasing prevalence of antimicrobial resistance in clinical cases, fewer options of treatments are available. The objectives of this study are 1) to investigate antimicrobial resistance of Escherichia coli in chicken carcasses in Vietnam; and 2) to compare resistance from different retail sources (fresh carcasses from markets vs fresh and frozen carcasses from supermarkets). Whole chicken carcasses, fresh or frozen, were collected from markets (fresh) and supermarkets (fresh and frozen) in the centermost urban area of Hanoi over a period of six weeks in June and July 2011. Chicken carcasses were processed in Hanoi following a carcass rinse protocol from USDA Food safety and Inspection Service. The primary bacterial cultures inoculated on MacConkey plates were sent to the EcL Laboratory in Saint-Hyacinthe, Canada. Upon reception, a total of 228 samples were recovered: 103 samples from markets (M), 99 samples of fresh carcasses from supermarkets (SM) and 26 samples of frozen carcasses from supermarkets (SMF). High level of contaminants and low presence of Escherichia coli in meat specimens obliged us to use an enrichment protocol inspired by Gill et al. (2012) in order to ensure maximal recuperation of Escherichia coli colonies. Two subsets of E.coli types were collected: 1) Generic, randomly selected colonies on plates and 2) Possible ExPEC positive colonies determined by PCR for selected virulence genes (tsh, papC, iucD, cnf). These were further tested for supplementary virulence genes (afa, sfa and KpsMTII) based on the Johnson definition for Human ExPEC. A third subset of colonies was gathered from randomly selected samples enriched with Ceftriaxone (1mg/L). All isolates were confirmed as E.coli by uidA PCR.

Antimicrobial resistance in Escherichia coli in chicken carcasses in Vietnam was investigated phenotypically by an automated standardized Minimal Inhibitory Concentration system (Sensititre ARIS®) for antimicrobial susceptibility testing. According to auto-read generated results based on the CLSI breakpoints, Generic and Possible ExPEC subsets of 82 and 64 samples, respectively, demonstrated high levels of resistance for antimicrobials classified by Health Canada as of very high importance in human medicine (Category I): Nalidixic acid (85.9%), Ciprofloxacin (37.5%); of high importance in human medicine (Category II): Ampicillin (85.9%), Gentamicin (35.9%), Streptomycin (70.3%), Trimethoprim- Sulfamethoxasole (82.8%); and of moderate importance in human medicine (Category III):

Chloramphenicol (68.8%) and Tetracycline (94%). Chloramphenicol is an antimicrobial banned from use in food animals in Vietnam, as well as in Canada. High resistance to specific antimicrobials in meat could reflect an anarchic use of antimicrobials in poultry farming in Vietnam, which could impact human health if infection occurs with ExPEC.

Interestingly, high resistance of the Possible ExPEC subset could be associated with few virulence genes. In fact, resistance to Ampicillin, Chloramphenicol, Nalidixic acid, Streptomycine, Tetracycline and Trimethoprime-Sulfamethoxasole are more often implicated with virulence gene iucD, which is a frequent virulence gene of ExPEC responsible for its iron-acquiring capacity. Co-selection on a same plasmid for this virulence gene and resistance genes corresponding to those previous antimicrobials could explain enhanced antimicrobial resistance in the possible ExPEC subset. Human ExPEC did not present different resistance patterns. Moreover, the possible ExPEC subset not only demonstrates high levels of resistance to individual antimicrobials but also has a majority of its samples demonstrating multi-drug resistance (MDR) (75%) of 5 to 6 classes of antimicrobials. Definition of multi-drug resistance and extreme drug resistance has been given by Magiorakos et al. (2012). Multi-drug resistance poses an additional threat to human medicine as it considerably reduces clinical treatment options.

The Ceftriaxone resistant subset allowed further investigation of resistance to one of the antimicrobial classes widely used in human medicine, β-lactamases. The resistance pattern obtained from phenotypic testing was compatible with putative Extended-Spectrum β- Lactamase (ESBL): high resistance to Ceftriaxone (≥1mg/L) and susceptible to Cefoxitin. To further confirm the presence of ESBL-producing E.coli, ten isolates were selected from this subset and tested phenotypically using confirmatory test plate with Cephalosporins, Cephalosporins and β-lactamase inhibitors, and Carbapenems. Eight out of ten samples were confirmed as ESBL. This is, to our knowledge, the first report of ESBL in chicken meat in Vietnam. No resistance was found for last resort antimicrobials, 4th generation cephalosporins, or Carbapenems. ESBL profiles were verified genotypically by PCR multiplex for the detection of five β-lactamase resistance genes (SHV, TEM, CMY, OXA, CTX-M). Tested isolates were associated with CTX-M resistance gene alone or incombination; CTX-M is the most widespread resistance gene for 3rd and 4th generation Cephalosporins.

When comparing antimicrobial resistance between retail sources for each subset, frozen carcasses from supermarkets from the generic subset showed a centered distribution to MDR of 5 to 6 classes of antimicrobial. The percentage resistance per antimicrobial reveals the highest levels of resistance for the following antimicrobials: Ampicillin, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, and Tetracycline. This retail source also has the lowest proportion of samples that had growth of E.coli on Ceftriaxone enriched media (27.2% compared to 54.5% for market and 48.6% fresh of supermarket). These findings can lead to our better understanding of the specific use of certain antimicrobials or a combination of selected antimicrobials on the farm, which results in elevated multi-drug resistance without selecting for Ceftriaxone resistance. A common supplier provided most of the sampled frozen chicken carcasses from supermarkets. This could indicate a more harmonized husbandry and antimicrobials management by the provider based on the results in frozen chicken meat.

In conclusion, chicken meat in Vietnam represents health risks to its consumers due to the presence of ESBL-producing E.coli, elevated resistance to antimicrobials of very high importance in human medicine, and multi-drug resistant E.coli. Results by retail sources did not provide any differences, except for frozen chicken carcasses.

From Aust. Poult. Sci. Symp.