Jeroen Dewulf

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.

Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries

OBJECTIVES To evaluate correlations between antimicrobial use and the prevalence of resistance in commensal Escherichia coli isolates from pigs, poultry and cattle, using data from publicly available national or international reports from seven European countries. METHODS The link between the quantities of different classes of antimicrobials administered to food-producing animals per country (expressed in mg/population correction unit) and the prevalence of resistance to the different antimicrobial classes (interpreted by EUCAST epidemiological cut-off values) in E. coli isolates (4831 isolates in total) was assessed by means of polynomial regression analysis and determination of Spearmans rank correlation coefficient. RESULTS A quadratic regression best fitted the antimicrobial use and antimicrobial resistance data. The coefficient of determination was, in decreasing order, 0.99 for fluoroquinolones and amphenicols, 0.94 for third-generation cephalosporins and sulphonamides, 0.93 for aminopenicillins, 0.86 for fluoroquinolones, 0.81 for streptomycin and 0.80 for gentamicin and tetracycline. Spearmans rank correlation coefficient was 1 for amphenicols, 0.96 for sulphonamides, 0.93 for streptomycin and tetracycline, 0.89 for aminopenicillins, 0.71 for gentamicin and 0.70 for third-generation cephalosporins. CONCLUSIONS These remarkably high coefficients indicate that, at a national level, the level of use of specific antimicrobials strongly correlates to the level of resistance towards these agents in commensal E. coli isolates in pigs, poultry and cattle. However, data restraints reveal the need for further detail in collection and harmonization of antimicrobial resistance and use data in Europe.

Antimicrobial Resistance in the Food Chain: A Review

Antimicrobial resistant zoonotic pathogens present on food constitute a direct risk to public health. Antimicrobial resistance genes in commensal or pathogenic strains form an indirect risk to public health, as they increase the gene pool from which pathogenic bacteria can pick up resistance traits. Food can be contaminated with antimicrobial resistant bacteria and/or antimicrobial resistance genes in several ways. A first way is the presence of antibiotic resistant bacteria on food selected by the use of antibiotics during agricultural production. A second route is the possible presence of resistance genes in bacteria that are intentionally added during the processing of food (starter cultures, probiotics, bioconserving microorganisms and bacteriophages). A last way is through cross-contamination with antimicrobial resistant bacteria during food processing. Raw food products can be consumed without having undergone prior processing or preservation and therefore hold a substantial risk for transfer of antimicrobial resistance to humans, as the eventually present resistant bacteria are not killed. As a consequence, transfer of antimicrobial resistance genes between bacteria after ingestion by humans may occur. Under minimal processing or preservation treatment conditions, sublethally damaged or stressed cells can be maintained in the food, inducing antimicrobial resistance build-up and enhancing the risk of resistance transfer. Food processes that kill bacteria in food products, decrease the risk of transmission of antimicrobial resistance.

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.

The impact of different housing systems on egg safety and quality

A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken.

Quantification of gut lesions in a subclinical necrotic enteritis model

Currently Clostridium perfringens-induced necrotic enteritis is a major problem in broiler flocks. In the present study, broilers were inoculated with a combination of Eimeria maxima or overdose coccidial vaccine (one inoculation) with C. perfringens (repeated inoculations). Single C. perfringens, E. maxima or an overdose of live coccidial vaccine inoculations did not result in grossly visible necrotic gut lesions, while combined inoculation resulted in typical necrotic lesions at approximately 4 days after inoculations with C. perfringens in approximately one-half of the inoculated animals. Semi-quantitative histological lesion scoring was done to evaluate gut damage in gut sections of animals in which no gross necrotic lesions were detected. This included scoring of hyperaemia, haemorrhages, the amount of red blood cells and protein precipitate in the lumen, villus fusion and epithelial defects. The villus length and the villus length/crypt depth ratio were also analysed. This approach proved to be discriminative between single E. maxima infection, overdose of coccidial vaccine or C. perfringens inoculations and the non-inoculated control group, and between the double-inoculated and single-inoculated groups. In general, the highest histological scores for gut lesions were observed in the double-inoculated groups, but the single-inoculated groups had higher scores than the control group. It was concluded that oral inoculation of broilers with an overdose of live coccidial vaccine in combination with multiple oral C. perfringens inoculations is a suitable model for necrotic enteritis without inducing mortality of the animals. C. perfringens and Eimeria act synergistically in inducing grossly visible gut damage.

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.

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs

Salmonella Typhimurium infections in pigs are a major source of human foodborne salmonellosis. To reduce the number of infected pigs, acidification of feed or drinking water is a common practice. The aim of the present study was to determine whether some frequently used short- (SCFA) and medium-chain fatty acids (MCFA) are able to alter virulence gene expression and to decrease Salmonella Typhimurium colonization and shedding in pigs using well established and controlled in vitro and in vivo assays. Minimal inhibitory concentrations (MIC) of 4 SCFA (formic acid, acetic acid, propionic acid and butyric acid) and 2 MCFA (caproic and caprylic acid) were determined using 54 porcine Salmonella Typhimurium field strains. MIC values increased at increasing pH-values and were two to eight times lower for MCFA than for SCFA. Expression of virulence gene fimA was significantly lower when bacteria were grown in LB-broth supplemented with sub-MIC concentrations of caproic or caprylic acid (2 mM). Expression of hilA and invasion in porcine intestinal epithelial cells was significantly lower when bacteria were grown in LB-broth containing sub-MIC concentrations of butyric acid or propionic acid (10 mM) and caproic or caprylic acid (2 mM). When given as feed supplement to pigs experimentally infected with Salmonella Typhimurium, coated butyric acid decreased the levels of faecal shedding and intestinal colonization, but had no influence on the colonization of tonsils, spleen and liver. Uncoated fatty acids, however, did not influence fecal shedding, intestinal or tonsillar colonization in pigs. In conclusion, supplementing feed with certain coated fatty acids, such as butyric acid, may help to reduce the Salmonella load in pigs.