Antimicrobial susceptibility monitoring of mastitis pathogens isolated from acute cases of clinical mastitis in dairy cows across Europe: VetPath results.

VetPath is an ongoing pan-European antimicrobial susceptibility monitoring programme collecting pathogens from diseased cattle, pigs and poultry not recently treated with antibiotics. Non-replicate milk samples were collected from cows with acute clinical mastitis in eight countries. Escherichia coli, Staphylococcus aureus and Streptococcus uberis were isolated by standardised methods. Antimicrobial susceptibility was determined in a central laboratory by CLSI broth microdilution methodology; results were interpreted using clinical breakpoints where available. Among E. coli (n=280), resistance to tetracycline (14.3%) and cefapirin (11.1%) were most common. Resistance to other ß-lactam antibiotics was absent (ceftiofur) or very low (cefalexin, amoxicillin/clavulanic acid). The MIC90 of enrofloxacin and marbofloxacin was 0.03 and 0.06µg/mL, respectively, with 0.7% of strains displaying a deviating high MIC. Staphylococcus aureus (n=250) were susceptible to most antibiotics tested, although 36.0% were resistant to penicillin G. For other ß-lactam antibiotics where a CLSI breakpoint was available, no resistance was detected. Tetracycline resistance was low (5.2%). Streptococcus uberis (n=282) were susceptible to all ß-lactam antibiotics, although 29.8% were intermediately susceptible to penicillin G; 18.8% of strains were resistant to erythromycin and 28.7% to tetracycline. This European study shows that bacteria associated with acute clinical mastitis are susceptible to most antibiotics with the exception of penicillin G against S. aureus, and erythromycin and tetracycline against S. uberis. The results of this study should serve as a reference baseline. This work also highlights the urgent need to set additional clinical breakpoints for antibiotics frequently used to treat mastitis.

Antimicrobial susceptibility monitoring of respiratory tract pathogens isolated from diseased cattle and pigs across Europe: the VetPath study.

VetPath is an ongoing pan-European antibiotic susceptibility monitoring programme collecting pathogens from diseased antimicrobial non-treated cattle, pigs and poultry. In the current study, 1001 isolates from cattle and pig respiratory tract infections were tested for their antimicrobial susceptibilities. Non-replicate lung samples or nasopharyngeal/nasal swabs were collected from animals with acute clinical signs in 11 countries during 2002-2006. Pasteurella multocida and Mannheimia haemolytica from cattle and P. multocida, Actinobacillus pleuropneumoniae and Streptococcus suis from pigs were isolated by standard methods. S. suis was also isolated from meningitis cases. MICs of 16 antibiotics were assessed centrally by broth microdilution following CLSI recommendations. Results were interpreted using CLSI breakpoints where available. P. multocida (231) and M. haemolytica (138) isolates were all susceptible to amoxicillin/clavulanic acid, ceftiofur, enrofloxacin and trimethoprim/sulfamethoxazole. Resistance to florfenicol and spectinomycin was 0.4% and 3.5% in P. multocida, respectively, and absent in M. haemolytica isolates. Tetracycline resistance was 5.7% and 14.6% for P. multocida and M. haemolytica. In pigs, 230 P. multocida, 220 A. pleuropneumoniae and 182 S. suis isolates were recovered. Resistance to amoxicillin/clavulanic acid, ceftiofur, enrofloxacin, florfenicol, tiamulin and tilmicosin was absent or <1%. Trimethoprim/sulfamethoxazole resistance was 3-6% and tetracycline resistance varied from 14.7% in A. pleuropneumoniae to 81.8% in S. suis. In conclusion, low resistance to antibiotics with defined clinical breakpoints, except for tetracycline, was observed among the major respiratory tract pathogens recovered from cattle and pigs. Since for approximately half of the antibiotics in this panel no CLSI-defined breakpoints were available, setting of the missing veterinary breakpoints is important.

Pan-European monitoring of susceptibility to human-use antimicrobial agents in enteric bacteria isolated from healthy food-producing animals.

OBJECTIVES: To determine the antimicrobial susceptibility of Escherichia coli, Salmonella, Campylobacter and Enterococcus from cattle, pigs and chickens across the European Union (EU) using uniform methodology. METHODS: Intestinal samples (1624) were taken at slaughter across five EU countries. Bacteria were isolated in national laboratories, whilst MICs were determined in a central laboratory for key antimicrobials used in human medicine. Clinical resistance was based on CLSI breakpoints and decreased susceptibility based on European Food Safety Authority (EFSA)/EUCAST epidemiological cut-off values. RESULTS: Isolation rates were high for E. coli (n=1540), low for Salmonella (n=201) and intermediate for Campylobacter (n=940) and Enterococcus (n=786). For E. coli and Salmonella, clinical resistance to newer compounds (cefepime, cefotaxime and ciprofloxacin) was absent or low, but decreased susceptibility was apparent, particularly in chicken strains. Resistance to older compounds (except gentamicin) was variable and higher. Colistin resistance was absent for E. coli, but apparent for Salmonella. For Campylobacter jejuni, ciprofloxacin resistance was markedly prevalent for chickens, whereas clinical resistance and decreased susceptibility to erythromycin was absent or very low. For Campylobacter coli, resistance was notably higher. None of the Enterococcus faecium strains was resistant to linezolid, but some were resistant to ampicillin or vancomycin. Resistance to quinupristin/dalfopristin was frequent. CONCLUSIONS: Resistance patterns varied widely depending on bacterial species, antibiotics, hosts and region. Resistance varied among countries, particularly for older antimicrobials, but clinical resistance to newer antibiotics used to treat foodborne disease in humans was generally very low. In the absence of resistance to newer compounds in E. coli and Salmonella, the apparent decreased susceptibility should be monitored.

A pan-European survey of antimicrobial susceptibility towards human-use antimicrobial drugs among zoonotic and commensal enteric bacteria isolated from healthy food-producing animals.

OBJECTIVES: The aim of the study was to study antimicrobial susceptibility in Escherichia coli, Salmonella, Campylobacter and Enterococcus recovered from chickens, pigs and cattle using uniform methodology. METHODS: Intestinal samples were taken at slaughter in five EU countries per host and bacteria isolated in national laboratories. MICs were determined in a central laboratory of key antimicrobials used in human medicine. Clinical resistance was based on CLSI breakpoints and decreased susceptibility on EFSA epidemiological cut-off values. RESULTS: Isolation rates from a total of 1500 samples were high for E. coli (n=1465), low for Salmonella (n=205) and intermediate for Campylobacter (n=785) and Enterococcus (n=718). Resistance prevalence varied among antibiotics, bacteria, hosts and countries. For E. coli and Salmonella, clinical resistance to newer compounds (cefepime, cefotaxime, ciprofloxacin) was absent or low, but a decreased susceptibility was apparent, particularly in chickens. Clinical resistance to older compounds (except colistin and gentamicin) was variable and higher. For Campylobacter jejuni from chickens, ciprofloxacin resistance was markedly higher than in isolates from cattle. Clinical resistance to erythromycin was absent for both hosts; decreased susceptibility very low. Similar trends were determined for Campylobacter coli, but C. jejuni was less resistant. None of the enterococcal strains was resistant to linezolid, but a few displayed resistance to ampicillin or vancomycin. Resistance prevalence to quinupristin/dalfopristin was clearly higher. CONCLUSIONS: Antimicrobial resistance among enteric organisms in food animals varied among countries, particularly for older antimicrobials, but clinical resistance to essential compounds used to treat disease in humans was generally zero or low. In the absence of clinical resistance to newer compounds in E. coli and Salmonella, the apparent decreased susceptibility should be monitored carefully.

Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar typhimurium DT204 acrB mutants selected with fluoroquinolones.

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

A European survey of antimicrobial susceptibility among zoonotic and commensal bacteria isolated from food-producing animals.

OBJECTIVE: To study antimicrobial resistance in zoonotic bacteria isolated from food animals in different countries using uniform methodology. METHODS: Samples were taken at slaughter from chickens, pigs and cattle in four EU countries per host. Escherichia coli (indicator organism; n = 2118), Salmonella spp. (n = 271) and Campylobacter spp. (n = 1325) were isolated in national laboratories and MICs tested in a central laboratory against, where appropriate, ampicillin, cefepime, cefotaxime, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, nalidixic acid, streptomycin, tetracycline and trimethoprim/sulfamethoxazole. RESULTS: Isolation rates were high for E. coli, low for Salmonella and intermediate for Campylobacter. MIC results showed resistance prevalence varied among compounds, hosts and countries. For E. coli and Salmonella, resistance to newer compounds (cefepime, cefotaxime, ciprofloxacin) was absent or low, but to older compounds (except gentamicin), resistance was variable and higher. E. coli isolates from Sweden showed low resistance, whereas among isolates from Spain (pigs), resistance to ampicillin, chloramphenicol, streptomycin, tetracycline and trimethoprim/sulfamethoxazole was higher; the UK, France, the Netherlands, Germany, Italy and Denmark were intermediate. For Campylobacter spp. isolates from chickens, nalidixic acid and ciprofloxacin resistance was >30% in France and the Netherlands, >6% in the UK and zero in Sweden. Nalidixic acid resistance was high in cattle (20%-64%), whereas ciprofloxacin resistance was markedly lower in cattle, variable in pigs (3%-21%) and highest in Sweden. Generally, Campylobacter coli was more resistant than Campylobacter jejuni. CONCLUSION: Antimicrobial resistance among enteric organisms in food animals varied among countries, particularly for older antimicrobials, but resistance to newer compounds used to treat disease in humans was generally low.

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium.

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.