Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Technical specifications for a baseline survey on the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in pigs.

The European Commission requested scientific and technical assistance in the preparation of a EU-wide baseline survey protocol for a European Union (EU) coordinated monitoring programme on the prevalence of methicillin-resistant Staphylococcus Aureus (MRSA) in pigs. The objective of the survey is to estimate the MRSA prevalence in batches of fattening pigs at slaughter at both European and national levels, with a 95% level of confidence and a level of precision of 10% considering an expected prevalence of 50%. The survey protocol defines the target population, the sample size for the survey, sample collection requirements, the analytical methods (for isolation, identification, phenotypic susceptibility testing and further genotypic testing of MRSA isolates), the data reporting requirements and the plan of analysis. The samples are to be analysed according to the laboratory protocols available on the European Union Reference Laboratory (EURL-AR) website. Generalised linear models will be used to estimate proportion (with 95% confidence intervals) of batches of slaughter pigs tested positive to MRSA. The necessary data to be reported by the EU Member States to support this baseline survey are presented in three data models. The results of the survey should be reported using the EFSA data collection framework.

Estimation of the wild-type minimum inhibitory concentration value distribution.

Antimicrobial resistance has become one of the main public health burdens of the last decades, and monitoring the development and spread of non-wild-type isolates has therefore gained increased interest. Monitoring is performed based on the minimum inhibitory concentration (MIC) values, which are collected through the application of dilution experiments. In order to account for the unobserved population heterogeneity of wild-type and non-wild-type isolates, mixture models are extremely useful. Instead of estimating the entire mixture globally, it was our major aim to provide an estimate for the wild-type first component only. The characteristics of this first component are not expected to change over time, once the wild-type population has been confidently identified for a given antimicrobial. With this purpose, we developed a new method based on the multinomial distribution, and we carry out a simulation study to study the properties of the new estimator. Because the new approach fits within the likelihood framework, we can compare distinct distributional assumptions in order to determine the most suitable distribution for the wild-type population. We determine the optimal parameters based on the AIC criterion, and attention is also paid to the model-averaged approach using the Akaike weights. The latter is thought to be very suitable to derive specific characteristics of the wild-type distribution and to determine limits for the wild-type MIC range. In this way, the new method provides an elegant means to compare distinct distributional assumptions and to quantify the wild-type MIC distribution of specific antibiotic-bacterium combinations.

Risk factors for Salmonella seroconversion of fattening pigs in farrow-to-finish herds.

We did a prospective observational 9-month long study to quantify risk factors of managerial and hygiene practices, and pig-health status for Salmonella seroconversion of fattening pigs reared in subclinically infected French farrow-to-finish farms. During the fattening phase, 2,649 pigs belonging to the same batch of contemporary pigs, from 89 conventional farrow-to-finish farms were individually followed and regularly blood sampled on a monthly basis. Farm recruitment was based on the farmer's willingness to cooperate. Pig status was assessed using an indirect ELISA test. Evolution of the serological status was studied by means of survival analysis. A Cox proportional-hazards model, taking into account the clustering of animals at the farm level, was used to examine the effects of explanatory variables on the time to Salmonella seroconversion of pigs. Applying group level antibiotic treatment to the pigs during the fattening period (Hazard Ratio (HR) = 2.4; 95% CI: 1.7, 3.4) was identified as a risk factor for Salmonella seroconversion, as the presence of residual Salmonella contamination in the fattening pen before placing the pigs into the pens (HR = 1.9; 95% CI: 1.2, 2.9). Porcine reproductive and respiratory syndrome virus (PRRSV) seropositivity during the fattening period also indicated an increased hazard for seroconversion (HR = 1.6; 95% CI: 1.1, 2.5). The batch size was identified as a risk factor for Salmonella seroconversion: the higher the number of pigs was in the fattening room followed, the higher was the risk (HR(+10 pigs) = 1.05 for a 10-pig increment; 95% CI: 1.03, 1.06). The biosecurity measures of wearing specific clothes before entering the facilities (HR = 0.5; 95% CI: 0.3, 0.9) and enclosing the pig farm facilities were protective (HR = 0.4; 95% CI: 0.2, 0.8).

A pharmaco-epidemiological analysis of factors associated with antimicrobial consumption level in turkey broiler flocks.

An on-farm pharmaco-epidemiological survey of 246 turkey broiler flocks from 131 farms was carried out to assess the homogeneity of antimicrobial use between flocks on the same farm and to explore the possible relationships between farm and farmer characteristics and the level of antimicrobial use. The antimicrobial use in each flock was quantified by an invoice study, expressed as the number of national animal daily doses (ADD)/turkey broiler and characterised as "high", "medium" or "low" according to the tertiles of the resulting distribution. Antimicrobial use was then correlated with variables collected from the farmer by means of an alternating logistic regression method which calculates the pairwise odds ratio (PWOR) for within-farm clustering. Two independent models were fitted: (1) "low" versus "medium" + "high" antimicrobial consumption and (2) "high" versus "medium" + "low" antimicrobial consumption. PWOR from the null models were significant (P < 0.005), but only remained significant in the first final model (P = 0.002). Four explanatory variables were retained for both models. Prophylactic antimicrobial administration and veterinarian antimicrobial prescription attaining the farm technical staff's expectation were associated with a higher antimicrobial consumption level. Administration of competitive exclusion flora and compliance with biosecurity rules of changing clothes and shoes before entering the facilities, were associated with a lower antimicrobial consumption level. In the first model, the number of full-time jobs devoted to the turkey production unit (1 versus more than 1) was also found to be associated with the antimicrobial consumption level. The study tends to confirm the feasibility of the adopted approach to quantify antimicrobial use and to determine the factors likely to influence antimicrobial consumption.

Impact of the Salmonella status of market-age pigs and the pre-slaughter process on Salmonella caecal contamination at slaughter.

The aim of this study was to evaluate the impact of the pre-slaughter process on Salmonella caecal contamination of pigs at slaughter. An observational study was carried out in 2001 on 101 conventional farrow-to-finish pig farms. On each farm, one batch of contemporary pigs was followed from the end of the fattening period until slaughter. The Salmonella bacteriological status of the batches was assessed by environmental samples of faecal material. The serological Salmonella status was obtained on 30 individually identified market-age pigs using an indirect ELISA test. At the slaughterhouse, 25 g of caecal contents were taken from 10 of the identified pigs. Faecal and caecal material were analysed according to a classical bacteriological method. A questionnaire was designed to obtain information about the type of feeding during the fattening period (dry versus wet), the duration of fasting on the farm before leaving for the slaughterhouse, the duration of transport between the farm and the slaughterhouse, the holding time in lairage at the slaughterhouse and loading and unloading conditions on the farm and at the slaughterhouse. To assess the relationships between these factors and the Salmonella caecal status of the pigs and the batches, two logistic models were fitted at the individual and at the batch level, respectively. The first analysis was performed using a random effects logistic regression model. The second analysis was based on a cumulative logit model with a positive caecal rate classified into three classes as the outcome variable. The results showed that the Salmonella status of market-age pigs assessed on the farm either by serological or bacteriological examinations and the time spent in lairage before slaughtering played a crucial role on caecal contamination. In the light of these results, actions should be considered both on the farm and at the slaughterhouse to decrease the risk of Salmonella contamination of the caecal contents.

Listeria monocytogenes contamination of finishing pigs: an exploratory epidemiological survey in France.

Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialised countries. Since L. monocytogenes carriage by pigs at the herd level could be a primary source for carcass contamination, control measures should be designed to reduce the L. monocytogenes load at the pre-harvest stage. For this purpose, an exploratory analytical survey was carried out in 2000-2001 in 93 French farrow-to-finish pig farms concerning L. monocytogenes contamination in pigs before they left for the slaughterhouse. On each farm, the L. monocytogenes status of a batch of contemporary fattening pigs housed in the same room was assessed on faecal material samples taken by means of gauze swabs wiped on the perianal region of the pigs. Fourteen percent of the batches studied had at least one contaminated sample and were therefore classified as L. monocytogenes contaminated batches. Two logistic regression models were used to assess the association between managerial and hygiene practices and the risk of L. monocytogenes contamination of the batch at the end of the finishing period on the whole data set (n = 93) and in the wet feeding farms only (n = 57). Wet feeding during the fattening period was identified as a risk factor for L. monocytogenes contamination. Risk factors related to the introduction of L. monocytogenes in pig facilities were identified for both the general and wet feeding farm data sets. Poor care paid to hygiene on the farms was found to increase the risk of being infected (boots cleaning, change room presence). When the duration of the empty period prior to the introduction of growing pigs was less than one day in the fattening section, the risk of L. monocytogenes contamination was significantly increased. For wet feeding farms, a distribution pipeline cleaning procedure including disinfection was found to be associated with a higher risk of contamination than no cleaning or a procedure consisting of rinsing with water only.

A survey of group-level antibiotic prescriptions in pig production in France.

There is world-wide concern that antimicrobial use in food-producing animals might contribute to antimicrobial resistance both in animals and in humans. The relationship between antimicrobial use and resistance is likely to be related to frequency of prescription of the compound, dose and duration of treatment. Routine collection of that information is not possible today in France. A postal survey of French pig veterinarians therefore was carried out in October 2000. The questionnaire focused on the last antibiotic group-level prescription made; data were collected on the type of animals, presumptive clinical diagnosis and drug prescription. The list frame was defined using a veterinary yearbook. All practitioners with mention of pig in the treated species or with employment in intensive animal production were sent the questionnaire. Out of the 431 selected practitioners, 303 responded to the self-administered questionnaire (overall return proportion 70%). 159 prescriptions were received and analysed (response proportion 37%). Their repartitions according to indications and active compounds were summarised. Mean prescribed daily doses and mean treatment length were calculated for four antibiotics: amoxicillin, colistin, oxytetracycline, tylosin. Prescribed daily dose were in the range of dosages used and recommended in Europe. High variations were encountered in treatment length: from 3 to 21 days.