Characterisation of extended-spectrum ß-lactamase and AmpC ß-lactamase-producing Enterobacteriaceae isolated from companion animals in New Zealand.

AIMS: To assess the occurrence of, and characterise, extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase (AmpC)-producing Enterobacteriaceae isolated by veterinary diagnostic laboratories from infection sites in companion animals in New Zealand. METHODS: Selected Enterobacteriaceae isolates were submitted by seven New Zealand veterinary diagnostic laboratories. They were isolated from infection sites in companion animals between June 2012 and June 2013, and were resistant to amoxicillin-clavulanic acid, fluoroquinolones, or any combination of two or more antimicrobials. Based on disk diffusion test results, the isolates were phenotypically categorised according to production of ESBL and AmpC. Genes for ESBL and AmpC production were amplified by PCR and sequenced. Escherichia coli isolates were also typed by multilocus sequence typing. RESULTS: A total of 115 isolates matching the inclusion criteria were obtained from the participating laboratories, of which 74 (64%) originated from dogs and 29 (25%) from cats. Seven bacterial species were identified, of which E. coli was the most common (87/115, 76%). Of the 115 isolates, 10 (9%) expressed the ESBL phenotype, 43 (37%) the AmpC phenotype, and seven (6%) both ESBL and AmpC phenotypes. Of the 60 ESBL and AmpC-producing isolates, 36 (60%) were E. coli. Amongst these isolates, 27/60 (45%) were classified as multidrug resistant, compared with 15/55 (27%) non-ESBL or AmpC-producing isolates (p<0.01). Ninety five isolates were resistant to amoxicillin-clavulanic acid and 58 (61%) of these were ESBL or AmpC-producing. The predominant ESBL genes were blaCTX-M-14 and blaCTX-M-15, and the dominant plasmid-encoded AmpC gene was blaCMY-2. Thirty-eight E. coli multilocus sequence types (ST) were identified, and the most prevalent were ST12 (12/89, 13%), ST131 (6/89, 7%) and ST648 (6/89, 7%). ESBL and AmpC-producing isolates accounted for 35/1,082 (3.2%) of the Enterobacteriaceae isolated by one laboratory network over the study period. CONCLUSIONS AND CLINICAL RELEVANCE: ESBL and AmpC-producing Enterobacteriaceae were associated with clinical infections in companion animals in New Zealand, and were often multidrug resistant. In this study, these organisms accounted for <5% of all Enterobacteriaceae isolated from infection sites by one laboratory network, but their prevalence among isolates resistant to amoxicillin-clavulanic acid was 61%. Therefore routine secondary testing for ESBL and AmpC production by Enterobacteriaceae that are resistant to amoxicillin-clavulanic acid in primary testing could improve the accuracy of definitive antimicrobial therapy in companion animals in New Zealand.

Molecular-based surveillance of campylobacteriosis in New Zealand--from source attribution to genomic epidemiology.

Molecular-based surveillance of campylobacteriosis in New Zealand contributed to the implementation of interventions that led to a 50% reduction in notified and hospitalised cases of the country's most important zoonosis. From a pre-intervention high of 384 per 100,000 population in 2006, incidence dropped by 50% in 2008; a reduction that has been sustained since. This article illustrates many aspects of the successful use of molecular-based surveillance, including the distinction between control-focused and strategy-focused surveillance and advances in source attribution. We discuss how microbial genetic data can enhance the understanding of epidemiological explanatory and response variables and thereby enrich the epidemiological analysis. Sequence data can be fitted to evolutionary and epidemiological models to gain new insights into pathogen evolution, the nature of associations between strains of pathogens and host species, and aspects of between-host transmission. With the advent of newer sequencing technologies and the availability of rapid, high-coverage genome sequence data, such techniques may be extended and refined within the emerging discipline of genomic epidemiology. The aim of this article is to summarise the experience gained in New Zealand with molecular-based surveillance of campylobacteriosis and to discuss how this experience could be used to further advance the use of molecular tools in surveillance.

Characteristics and comparative performance of direct culture, direct PCR and enumeration methods for detection and quantification of Campylobacter spp. in broiler caeca.

Detection and enumeration of Campylobacter spp. in broiler chicken flocks are key components of research and surveillance studies aimed at reducing Campylobacter infections in people. Direct culture of caecal contents onto selective agar is the typical method used to confirm flock colonisation. Modified charcoal cefoperazone deoxycholate agar (mCCDA) is commonly used for this method, although alternative selective media have been used. Additionally, PCR methods to detect Campylobacter DNA from caecal contents may provide a rapid alternative. However comparative performance data for these methods is limited and therefore required to ensure optimal detection methods for this sample type. In this study, 306 broiler caeca were tested for Campylobacter using direct culture on mCCDA, Skirrows and Preston agars and two real-time PCR methods, one specific for mapA/ceuE regions and another for the flaA gene region. Additionally, the suitability of spread plating and spiral plating methods for enumeration of Campylobacter and the impact of sample storage were assessed. This study confirmed modified CCDA as an optimal media for detection of Campylobacter in broiler caeca. It was significantly more sensitive than Skirrows or Preston agars. This study also demonstrated that the mapA/ceuE PCR had excellent agreement with culture on mCCDA and is a genuine alternative method. Spread plating and spiral plating methods were suitable for enumeration although spiral plating appeared more sensitive for stored samples (72 h). A 1 log reduction in viable Campylobacters was observed in stored samples, therefore storage effects should be considered for quantitative studies with broiler caeca.

Descriptive epidemiological study of the use of antimicrobial drugs by companion animal veterinarians in New Zealand.

AIM: To describe the patterns of use of antimicrobial drugs by veterinary surgeons treating commonly presented bacterial infections in companion animals in New Zealand. METHODS: A postal survey of 800 randomly selected companion animal veterinarians practicing in New Zealand was conducted between August and December 2008. Data were collected regarding the antimicrobials prescribed for recent cases of skin, ear and urinary tract infections; the use of culture and susceptibility testing; and veterinarian characteristics such as proportion of time spent treating companion animals and recent attendance at continuing professional development (CPD) events. Potential associations within the data were explored using extended mosaic plots and multivariable regression models. RESULTS: Completed surveys from 393 respondents were available for analysis, providing data on systemic antimicrobial drug use for 1,799 cases of presumptive bacterial infections. The most frequently prescribed drugs were amoxicillin-clavulanic acid (864 cases, 48%), cephalexin (558, 31%), and fluoroquinolones (198, 11%). Of 359 cases of canine superficial pyoderma, 157 (44%) were treated with amoxicillin-clavulanic acid and 155 (43%) were treated with cephalexin with median reported treatment durations of 7 and 10 days, for these two drugs respectively. Culture and susceptibility tests had been used in 376 of 1,984 (19%) of all reported cases and 160 (43%) of these were suspected urinary tract infections. Practitioners that spent 100% of their time treating companion animals and who had attended a CPD course related to companion animals within the 12 months prior to completing the survey were more likely to submit a sample for culture and susceptibility testing and to prescribe longer courses of antimicrobials for the treatment of canine pyoderma than practitioners who spent less than 100% of their time treating companion animals and had not attended a CPD course in the last 12 months. CONCLUSIONS: Broad-spectrum drugs considered by the World Health Organisation to be critically important for human health, such as fluoroquinolones and amoxicillin-clavulanic acid, are amongst the most frequently prescribed antimicrobials in companion animal medicine, and these drugs are often prescribed without submitting a sample for culture and susceptibility testing. CLINICAL RELEVANCE: Many cases of superficial pyoderma were treated for less than the recommended duration of 21 days, which may contribute to a higher rate of recurrent pyoderma and the development of drug resistance within the causal bacteria. Veterinarians should be aware that the use of fluoroquinolones, in particular, should be based upon the results of a culture and susceptibility test.

Low levels of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand.

AIM: To provide baseline data on the levels and patterns of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand. METHODS: Between July and December 2006, isolates of Escherichia coli (n=407) and Salmonella spp. (n=3) originating from carcass-rinse samples were submitted by testing laboratories affiliated to five major poultry processing plants. Isolates of Campylobacter jejuni (n=193) originating from retail poultry carcasses in 2005-2006 were retrieved from the Massey University archives. All isolates underwent disc diffusion susceptibility testing against panels of 12 (Enterobacteriaceae) and six (Campylobacter spp.) antibacterial drugs. Cephalothin-resistance in isolates of E. coli was confirmed using ETest strips, and confirmation of the resistance phenotypes for a subset of C. jejuni isolates used microbroth dilution assays. Patterns within the resistance phenotypes of the isolates were investigated using hierarchical clustering, and logistic regression modelling. RESULTS: The majority of isolates (71.5% E. coli, 99% C. jejuni, and all three Salmonella spp. isolates) were fully susceptible to the drugs that were tested. Four (1%) E. coli isolates showed resistance to three or more drugs. The proportions of susceptible E. coli differed between the five processing plants. Resistances were detected in E. coli isolates, using disc diffusion to cephalothin (18.2%), ampicillin (4.4%), tetracycline (4.4%) and gentamicin (1.5%). There was an association between cephalothin-resistant isolates of E. coli and decreased susceptibility to gentamicin. Using ETests to ascertain the minimum inhibitory concentrations (MIC) of E. coli for cephalothin gave inconsistent results. One of 193 C. jejuni isolates was resistant to erythromycin, and microbroth dilution assays confirmed that this panel of C. jejuni was generally susceptible to antibacterial drugs. CONCLUSIONS: The levels of resistance shown by Gram-negative bacteria isolated from chicken carcasses in New Zealand are among the lowest reported around the world. No resistance to extended-spectrum cephalosporin drugs was detected in E. coli, suggesting that CTX-M and AmpC beta-lactamases are rare or absent. Salmonella spp. are rarely isolated from poultry carcasses during routine testing in New Zealand, and the isolates identified during this study were fully susceptible to the drugs tested. A panel of C. jejuni isolates originating from retail poultry carcasses were susceptible to first-line and second-line antibacterial drugs. The use of cephalothin as a marker of resistance to first-generation cephalosporins may not be appropriate for non-type-specific E. coli of animal origin.

Sources of variation in the ampicillin-resistant Escherichia coli concentration in the feces of organic broiler chickens.

Currently, there are limited published data for the population dynamics of antimicrobial-resistant commensal bacteria. This study was designed to evaluate both the proportions of the Escherichia coli populations that are resistant to ampicillin at the level of the individual chicken on commercial broiler farms and the feasibility of obtaining repeated measures of fecal E. coli concentrations. Short-term temporal variation in the concentration of fecal E. coli was investigated, and a preliminary assessment was made of potential factors involved in the shedding of high numbers of ampicillin-resistant E. coli by growing birds in the absence of the use of antimicrobial drugs. Multilevel linear regression modeling revealed that the largest component of random variation in log-transformed fecal E. coli concentrations was seen between sampling occasions for individual birds. The incorporation of fixed effects into the model demonstrated that the older, heavier birds in the study were significantly more likely (P = 0.0003) to shed higher numbers of ampicillin-resistant E. coli. This association between increasing weight and high shedding was not seen for the total fecal E. coli population (P = 0.71). This implies that, in the absence of the administration of antimicrobial drugs, the proportion of fecal E. coli that was resistant to ampicillin increased as the birds grew. This study has shown that it is possible to collect quantitative microbiological data on broiler farms and that such data could make valuable contributions to risk assessments concerning the transfer of resistant bacteria between animal and human populations.

Characterization of vancomycin-resistant Enterococcus faecium isolates from broiler poultry and pig farms in England and Wales.

This study aimed to investigate the occurrence and molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) isolates on poultry and pig farms in England and Wales. A total of 217 VREF isolates were obtained from fresh feces and environmental swabs collected from conventional and organic farms. A predominant pulsed-field gel electrophoresis (PFGE) profile was found for each VREF-positive farm, together with less frequent types. All isolates presented the vanA genotype and were esp negative. Seventy-six percent of the VREF isolates were additionally resistant to nine or more antimicrobials, presenting a diverse range of resistance phenotypes. The multiresistance traits did not appear to be specific to individual farms or sample types (i.e., environmental or fecal), nor did they correlate with any specific PFGE type. Ninety-three percent of the isolates were resistant to penicillin, 89% were resistant to tetracycline, 87.5% were resistant to erythromycin, and 50% were resistant to quinupristin-dalfospristin (Synercid). The lack of clonality among these populations may suggest the horizontal transfer of resistance genes and/or a dynamic replacement of clonal lines rather than persistence.