Davy Persoons

Diversity of Extended-Spectrum β-Lactamases and Class C β-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms

ABSTRACT A total of 295 ceftiofur-resistant Escherichia coli isolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against β-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different β-lactam resistance phenotypes suggested the presence of an extended-spectrum β-lactamase (ESBL), a class C β-lactamase, or the combination of an ESBL with a class C β-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. coli strains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum β-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC β-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found only in association with blaCMY-2 genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC β-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. coli strains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum β-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampC genes.

Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health.

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard as these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and in animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases - including the exchange of the transposons and plasmids that serve as vehicles for these genes - between different ecosystems (human and animal) are discussed.

Prophylactic and metaphylactic antimicrobial use in Belgian fattening pig herds

The monitoring of antimicrobial use is an essential step to control the selection and spread of antimicrobial resistance. Between January and October 2010 data on prophylactic and metaphylactic antimicrobial use were collected retrospectively on 50 closed or semi-closed pig herds. Ninety-three percent of the group treatments were prophylactic whereas only 7% were methaphylactic. The most frequently used antimicrobials orally applied at group level were colistin (30.7%), amoxicillin (30.0%), trimethoprim-sulfonamides (13.1%), doxycycline (9.9%) and tylosin (8.1%). The most frequently applied injectable antimicrobials were tulathromycin (45.0%), long acting ceftiofur (40.1%) and long acting amoxicillin (8.4%). The treatment incidences (TI) based on the used daily dose pig (UDD(pig) or the actually administered dose per day per kg pig of a drug) for all oral and injectable antimicrobial drugs was on average 200.7 per 1000 pigs at risk per day (min=0, max=699.0), while the TI based on the animal daily dose pig (ADD(pig) or the national defined average maintenance dose per day per kg pig of a drug used for its main indication) was slightly higher (average=235.8, min=0, max=1322.1). This indicates that in reality fewer pigs were treated with the same amount of antimicrobials than theoretically possible. Injectable products were generally overdosed (79.5%), whereas oral treatments were often underdosed (47.3%). In conclusion, this study shows that prophylactic group treatment was applied in 98% of the visited herds and often includes the use of critically important and broad-spectrum antimicrobials. In Belgium, the guidelines for prudent use of antimicrobials are not yet implemented.

Methicillin-resistant Staphylococcus aureus in poultry.

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in several species and animal-derived products. To determine whether MRSA is present in poultry, we sampled 50 laying hens and 75 broiler chickens. MRSA was found in some broiler chickens but no laying hens. In all samples, spa type t1456 was found.

Prospective study on quantitative and qualitative antimicrobial and anti-inflammatory drug use in white veal calves

OBJECTIVES To document and quantify drug use in white veal calves, an intensive livestock production system where multidrug resistance is abundantly present. METHODS Drug consumption data were prospectively collected on 15 white veal production cohorts (n = 5853 calves) in Belgium (2007-09). Treatment incidences (TIs) based on animal defined daily dose (ADD), prescribed daily dose (PDD) and used daily dose (UDD) were calculated. Risk factors were identified by linear regression. RESULTS The average TI(ADD) of antimicrobial treatments was 416.8 ADD per 1000 animals at risk. Predominantly, oral group antimicrobial treatments were used (95.8%). Of the oral group antimicrobial treatments, 12% and 88% were used for prophylactic or metaphylactic indications, respectively. The main indication for group and individual drug use was respiratory disease. The most frequently used antimicrobials (group treatments) were oxytetracycline (23.7%), amoxicillin (18.5%), tylosin (17.2%) and colistin (15.2%). Deviations from the leaflet dosage recommendations were frequently encountered, with 43.7% of the group treatments underdosed (often oxytetracycline and tylosin to treat dysbacteriosis). In 33.3% of the oral antimicrobial group treatments a combination of two antimicrobial preparations was used. Smaller integrations used more antimicrobials in group treatments than larger ones (P < 0.05); an integration is defined as a company that combines all steps of the production chain by having its own feed plant and slaughterhouse and by placing its calves in veal herds owned by producers that fatten these calves for this integration on contract. Producers used higher dosages than prescribed by the veterinarian in cohorts with a single caretaker (P < 0.01). CONCLUSIONS The present study provided detailed information on the intensive antimicrobial use in the white veal industry. Reduction can only be achieved by reducing the number of oral group treatments.

Characterization of Extended-Spectrum β-Lactamases Produced by Escherichia coli Isolated from Hospitalized and Nonhospitalized Patients: Emergence of CTX-M-15-Producing Strains Causing Urinary Tract Infections

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates were obtained from hospitalized and nonhospitalized patients in Belgium between August 2006 and November 2007. The antimicrobial susceptibility of these isolates was determined and their ESBL genes were characterized. Clonal relationships between the CTX-M-producing E. coli isolates causing urinary tract infections were also studied. A total of 90 hospital- and 45 community-acquired cephalosporin-resistant E. coli isolates were obtained. Tetracycline, enrofloxacine, gentamicin, and trimethoprim-sulfamethaxozole resistance rates were significantly different between the community-onset and hospital-acquired isolates. A high diversity of different ESBLs was observed among the hospital-acquired E. coli isolates, whereas CTX-M-15 was dominating among the community-acquired E. coli isolates (n = 28). Thirteen different pulsed-field gel electrophoresis profiles were observed in the community-acquired CTX-M-15-producing E. coli, indicating that multiple clones have acquired the bla(CTX-M-15) gene. All community-acquired CTX-M-15-producing E. coli isolates of phylogroups B2 and D were assigned to the sequence type ST131. The hospital-acquired CTX-M-15-producing E. coli isolates of phylogroups B2, B1, A, and D corresponded to ST131, ST617, ST48, and ST405, respectively. In conclusion, CTX-M-type ESBLs have emerged as the predominant class of ESBLs produced by E. coli isolates in the hospital and community in Belgium. Of particular concern is the predominant presence of the CTX-M-15 enzyme in ST131 community-acquired E. coli.

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration.

Aims:  The behaviour of an Escherichia coli isolate of broiler origin harbouring a blaTEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration.

Risk factors for ceftiofur resistance in Escherichia coli from Belgian broilers.

A cross-sectional study on 32 different Belgian broiler farms was performed in 2007 and 2008 to identify risk factors for ceftiofur resistance in Escherichia coli. On each farm, one E. coli colony was isolated from 30 random birds. Following susceptibility testing of 14 antimicrobials, an on-farm questionnaire was used to obtain information on risk factors. Using a multilevel logistic regression model two factors were identified at the animal level: resistance to amoxicillin and to trimethoprim-sulfonamide. On the farm level, besides antimicrobial use, seven management factors were found to be associated with the occurrence of ceftiofur resistance in E. coli from broilers: poor hygienic condition of the medicinal treatment reservoir, no acidification of drinking water, more than three feed changes during the production cycle, hatchery of origin, breed, litter material used, and treatment with amoxicillin. This study confirms that not only on-farm antimicrobial therapy, but also management- and hatchery-related factors influence the occurrence of antimicrobial resistance.

Prevalence and Persistence of Antimicrobial Resistance in Broiler Indicator Bacteria

This study explored the prevalence and persistence of acquired antimicrobial resistance in Escherichia coli and Enterococcus faecium from healthy broilers. In 32 broiler farms, cloacal samples were taken during two production rounds, with one production round in between. For 10 of the sampled flocks, samples from the carcasses at the slaughterhouse were also collected. For E. coli, high levels of resistance were found for ampicillin, nalidixic acid, streptomycin, tetracycline, and the combination of trimethoprim and sulfonamide. Over 58% of all the isolates showed resistance to four or more antimicrobial agents. Only 4.8% were fully susceptible for all 14 drugs tested. A remarkably high resistance rate (up to 41%) to ceftiofur was found. The enterococci were frequently resistant to macrolides, tetracycline, and the combination quinopristin/dalfopristin. Over 80% displayed acquired resistance to four or more antimicrobial agents, and 3.9% were fully susceptible for the eight agents tested. Resistance was found to persist over consecutive production rounds. There was a good correlation between results obtained with cloacal samples of the live animals and caecal content samples collected in the slaughterhouse for both E. coli and E. faecium. For E. coli but not for E. faecium, the resistance profile of neck skin isolates was different from that of cloacal isolates.

The Importance of Sample Size in the Determination of a Flock-Level Antimicrobial Resistance Profile for Escherichia coli in Broilers

Determining herd- or flock-specific antimicrobial resistance profiles is important to guide therapeutic use of antimicrobials and to assess risk factors for the development and spread of antimicrobial resistance. As such, it is of utmost importance to optimize the sampling strategy for the determination of herd-specific antimicrobial resistance profiles. However, the multitude of prevalences measured at the same time as well as the presence of variation both at the level of the animal and the bacterial population of concern make it impossible to use conventional sample size determination methods. In this article, the use of bootstrapping techniques for sample size determination was explored. In particular, one-stage and two-stage bootstrap samplings were used to determine the optimal number of animals and the optimal number of isolates within one animal. Results show that focus should be on the number of animals sampled rather than on the number of isolates tested within one animal.