Shabbir Simjee

Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Typhimurium Isolates Obtained from Food Animal Sources

ABSTRACT Molecular characterization (e.g., DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals and from patients with food-borne disease and nosocomial infections. In this study, we compared the abilities of different phenotyping and genotyping methods to distinguish isolates of Salmonella enterica serovar Typhimurium from different food animal sources. One hundred twenty-eight S. enterica serovar Typhimurium strains isolated from cattle, pigs, chickens, and turkeys or derived food products were characterized using pulsed-field gel electrophoresis (PFGE), repetitive element PCR (Rep-PCR), multilocus sequence typing (MLST), plasmid profiling, and antimicrobial susceptibility testing. Among the 128 Salmonella isolates tested, we observed 84 Rep-PCR profiles, 86 PFGE patterns, 89 MLST patterns, 36 plasmid profiles, and 38 susceptibility profiles. The molecular typing methods, i.e., PFGE, MLST, and Rep-PCR, demonstrated the best discriminatory power among Salmonella isolates. However, no apparent correlation was evident between the results of one molecular typing method and those of the others, suggesting that a combination of multiple methods is needed to differentiate S. enterica serovar Typhimurium isolates that genetically cluster according to one particular typing method.

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.

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.

Evaluation of Molecular Typing Methods for Escherichia coli O157:H7 Isolates from Cattle, Food, and Humans

Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

Harmonisation of resistance monitoring programmes in veterinary medicine: an urgent need in the EU?

Antimicrobial surveillance systems in Denmark (DANMAP), The Netherlands (MARAN), Spain (VAV) and Sweden (SVARM) as well as the European Antimicrobial Susceptibility Surveillance in Animals (EASSA) were reviewed. Data have been considered for extended-spectrum cephalosporins, fluoroquinolones and macrolides against food-borne and commensal bacteria. The greatest challenge arises from the lack of agreement between programmes on what is meant by resistance through the use of different interpretive criteria. Indeed, it is shown here that the extent of the differences depends on the antibacterial compound being investigated, the methodology and the interpretive criteria used. This emphasises a need to agree a definition for resistance and for epidemiological cut-off values and to consider harmonising the antimicrobials used in surveillance. This analysis of the data highlights the usefulness of using both epidemiological cut-off values and clinical resistance breakpoints for the purpose of detection of decreased susceptibility and development of clinical resistance, respectively. It is concluded that harmonisation in resistance monitoring programmes is needed since there is potential for data to be appropriately used within risk analysis, providing the opportunity to implement appropriate risk management steps as a response to the public health issues arising from changes in antibiotic resistance in food-borne pathogens and commensal organisms.

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.

Changes in Antimicrobial Susceptibility of Native Enterococcus faecium in Chickens Fed Virginiamycin

ABSTRACT The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.

ANTIMICROBIAL SUSCEPTIBILITY AND DISTRIBUTION OF ANTIMICROBIAL-RESISTANCE GENES AMONG ENTEROCOCCUS AND COAGULASE-NEGATIVE STAPHYLOCOCCUS ISOLATES RECOVERED FROM POULTRY LITTER

Abstract Data on the prevalence of antimicrobial resistant enterococci and staphylococci from the poultry production environment are sparse in the United States. This information is needed for science-based risk assessments of antimicrobial use in animal husbandry and potential public-health consequences. In this study, we assessed the susceptibility of staphylococci and enterococci isolated from poultry litter, recovered from 24 farms across Georgia, to several antimicrobials of veterinary and human health importance. Among the 90 Enterococcus isolates recovered, E. hirae (46%) was the most frequently encountered species, followed by E. faecium (27%), E. gallinarum (12%), and E. faecalis (10%). Antimicrobial resistance was most often observed to tetracycline (96%), followed by clindamycin (90%), quinupristin–dalfopristin (62%), penicillin (53%), erythromycin (50%), nitrofurantoin (49%), and clarithromycin (48%). Among the 110 staphylococci isolates recovered, only coagulase-negative staphylococci (CNS) were identified with the predominant Staphylococcus species being S. sciuri (38%), S. lentus (21%), S. xylosus (14%) and S. simulans (12%). Resistance was less-frequently observed among the Staphylococcus isolates for the majority of antimicrobials tested, as compared with Enterococcus isolates, and was primarily limited to clarithromycin (71%), erythromycin (71%), clindamycin (48%), and tetracycline (38%). Multidrug resistance (MDR) phenotypes were prevalent in both Enterococcus and Staphylococcus; however, Enterococcus exhibited a statistically significant difference in the median number of antimicrobials to which resistance was observed (median  =  5.0) compared with Staphylococcus species (median  =  3.0). Because resistance to several of these antimicrobials in gram-positive bacteria may be attributed to the shuttling of common drug-resistance genes, we also determined which common antimicrobial-resistance genes were present in both enterococci and staphylococci. The antimicrobial resistance genes vat(D) and erm(B) were present in enterococci, vgaB in staphylococci, and mobile genetic elements Tn916 and pheromone-inducible plasmids were only identified in enterococci. These data suggest that the disparity in antimicrobial-resistance phenotypes and genotypes between enterococci and staphylococci isolated from the same environment is, in part, because of barriers preventing exchange of mobile DNA elements.

Monitoring of antimicrobial susceptibility of respiratory tract pathogens isolated from diseased cattle and pigs across Europe, 2009–2012: VetPath results

VetPath is an ongoing pan-European antibiotic susceptibility monitoring programme that collects pathogens from diseased cattle, pigs and poultry. In the current study, 996 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 10 countries during 2009-2012. Pasteurella multocida, Mannheimia haemolytica and Histophilus somni from cattle and P. multocida, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Bordetella bronchiseptica and Streptococcus suis from pigs were isolated by standard methods. S. suis was also isolated from meningitis cases. MIC values of 16 or 17 antibiotics were assessed centrally by broth microdilution following CLSI standards. Results were interpreted using CLSI breakpoints where available. Cattle isolates were generally highly susceptible to most antibiotics, except to tetracycline (3.0-12.0% resistance). Low levels of resistance (0-4.0%) were observed for the macrolide antibiotics. Resistance to spectinomycin varied from 0 to 6.0%. In pig isolates similar observations were made. Resistance to amoxicillin/clavulanic acid, ceftiofur, enrofloxacin, florfenicol, tulathromycin, tiamulin and tilmicosin was absent or <2%. Trimethoprim/sulfamethoxazole resistance varied from 1.9 to 5.3%, but tetracycline resistance varied from 20.4% in P. multocida to 88.1% in S. suis. For most antibiotics and pathogens the percentage resistance remained unchanged or only increased numerically as compared to that of the period 2002-2006. In conclusion, absence or low resistance to antibiotics with defined clinical breakpoints, except for tetracycline, was observed among the major respiratory tract pathogens recovered from livestock. Comparison of all antibiotics and organisms was hampered since for almost half of the antibiotics no CLSI-defined breakpoints were available.

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 β-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 β-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 β-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.