The risk-benefit balance of resistance to ionophores in Enterococcus faecium and Enterococcus faecalis for ionophore coccidiostats in broiler chickens.

In recent years, publications and debate have emerged in the scientific literature that have linked the use of ionophore coccidiostats, which are themselves not medically important and not related to any therapeutic antibiotics used in human and animal medicine, to resistance development to medically important antibiotics in Enterococcus faecium and Enterococcus faecalis, isolated from broilers and broiler meat. This has been based on the discovery of genes, now named NarAB, that appear to result in elevated MICs of the ionophores narasin, salinomycin and maduramycin and that these are linked to genes responsible for resistance to antibiotics that may be clinically relevant in human medicine. This article will seek to review the most significant publications in this regard and will also examine national antimicrobial resistance surveillance programmes in Norway, Sweden, Denmark and the Netherlands, in order to further evaluate this concern. The conclusion of the review is that the risk that enterococci may pass from broilers to humans and that antimicrobial resistance gene transfer may occur is negligible, remains unquantified and is highly unlikely to be of significance to human health. Indeed, to date no human nosocomial infections have been linked to poultry sources. Concurrently a review of the possible impact of a policy that limits access for poultry farmers and poultry veterinarians to ionophore coccidiostats in broilers indicates predictable negative consequences with regard to antibiotic resistance of significance to animal welfare and to human health.

Survey of antimicrobial susceptibility of bacterial pathogens isolated from dogs and cats with respiratory tract infections in Europe: ComPath results.

AIMS: To present antimicrobial susceptibilities for bacteria from dogs and cats with respiratory tract infection (RTI) across Europe in 2013-2014 and compare with data from 2008-2010. METHODS AND RESULTS: Minimal inhibitory concentrations were determined for 464 isolates following Clinical and Laboratory Standards Institute standards using antibiotics approved for RTI treatment. Where possible, susceptibility was calculated using predominantly human-derived breakpoints whilst some antibiotics had no breakpoints. The main pathogen from dogs was Staphylococcus pseudintermedius which was > 90% susceptible to fluoroquinolones and oxacillin (92·5%; six isolates confirmed mecA-positive) and 53·8, 80·0 and 88·8% susceptible to tetracycline, penicillin and trimethoprim/sulfamethoxazole. Streptococci, Escherichia coli, Bordetella bronchiseptica, Staphylococcus aureus and Pseudomonas aeruginosa were also present in dog RTI. Streptococci were fully susceptible to penicillin, ampicillin and pradofloxacin. None were enrofloxacin-resistant but 31·4% had intermediate susceptibility. The least active agent against streptococci was tetracycline (51·4% susceptible). For E. coli, 90·9% were amoxicillin/clavulanic acid-susceptible; susceptibility to other compounds ranged from 63·6 to 81·8%. There are no breakpoints for B. bronchiseptica and Ps. aeruginosa. For Staph. aureus, penicillin susceptibility was low (34·8%); for other compounds 87·0-100%. The main RTI pathogen from cats was Pasteurella multocida, where only pradofloxacin has breakpoints (100% susceptible). Susceptibility of coagulase-negative staphylococci ranged from 66·7% (penicillin) to 97·2% (pradofloxacin). Streptococci from cats were 100% susceptible to all antibiotics except enrofloxacin and tetracycline (both 65·2% susceptible). CONCLUSIONS: Overall, antimicrobial resistance was low to medium in RTI in dogs and cats, although susceptibility varied widely among pathogens studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Responsible use of antibiotics is crucial to maintain susceptibility and continued resistance monitoring is important to support this goal. These findings support the need for the setting of RTI-specific breakpoints for pathogens of dogs and cats.

Present and Future Surveillance of Antimicrobial Resistance in Animals: Principles and Practices.

There is broad consensus internationally that surveillance of the levels of antimicrobial resistance (AMR) occurring in various systems underpins strategies to address the issue. The key reasons for surveillance of resistance are to determine (i) the size of the problem, (ii) whether resistance is increasing, (iii) whether previously unknown types of resistance are emerging, (iv) whether a particular type of resistance is spreading, and (v) whether a particular type of resistance is associated with a particular outbreak. The implications of acquiring and utilizing this information need to be considered in the design of a surveillance system. AMR surveillance provides a foundation for assessing the burden of AMR and for providing the necessary evidence for developing efficient and effective control and prevention strategies. The codevelopment of AMR surveillance programs in humans and animals is essential, but there remain several key elements that make data comparisons between AMR monitoring programs, and between regions, difficult. Currently, AMR surveillance relies on uncomplicated in vitro antimicrobial susceptibility methods. However, the lack of harmonization across programs and the limitation of genetic information of AMR remain the major drawbacks of these phenotypic methods. The future of AMR surveillance is moving toward genotypic detection, and molecular analysis methods are expected to yield a wealth of information. However, the expectation that these molecular techniques will surpass phenotypic susceptibility testing in routine diagnosis and monitoring of AMR remains a distant reality, and phenotypic testing remains necessary in the detection of emerging resistant bacteria, new resistance mechanisms, and trends of AMR.

Pan-European resistance monitoring programmes encompassing food-borne bacteria and target pathogens of food-producing and companion animals.

Antimicrobial resistance is a concern both for animal and human health. Veterinary programmes monitoring resistance of animal and zoonotic pathogens are therefore essential. Various European countries have implemented national surveillance programmes, particularly for zoonotic and commensal bacteria, and the European Food Safety Authority (EFSA) is compiling the data. However, harmonisation is identified as a weakness and an essential need in order to compare data across countries. Comparisons of resistance monitoring data among national programmes are hampered by differences between programmes, such as sampling and testing methodology, and different epidemiological cut-off values or clinical breakpoints. Moreover, only very few valid data are available regarding target pathogens both of farm and companion animals. The European Animal Health Study Centre (CEESA) attempts to fill these gaps. The resistance monitoring programmes of CEESA have been a collaboration of veterinary pharmaceutical companies for over a decade and include two different projects: the European Antimicrobial Susceptibility Surveillance in Animals (EASSA) programme, which collects food-borne bacteria at slaughter from healthy animals, and the pathogen programmes that collect first-intention target pathogens from acutely diseased animals. The latter comprises three subprogrammes: VetPath; MycoPath; and ComPath. All CEESA projects include uniform sample collection and bacterial identification to species level in various European Union (EU) member states. A central laboratory conducts quantitative susceptibility testing to antimicrobial agents either important in human medicine or commonly used in veterinary medicine. This 'methodology harmonisation' allows easy comparisons among EU member states and makes the CEESA programmes invaluable to address food safety and antibiotic efficacy.

Surveillance and monitoring of antimicrobial resistance and antibiotic consumption in humans and animals.

Surveillance and monitoring studies of antimicrobial resistance in bacteria of human and animal origin and antimicrobial consumption in humans and animals have been conducted in various countries throughout the world. In the veterinary field, in particular, programmes have been installed which target bacteria of zoonotic, foodborne and/or veterinary relevance. Each year, the European Surveillance of Veterinary Antimicrobial Consumption project summarises and evaluates antimicrobial consumption in ambulatory and hospital care in many European countries. In contrast, antimicrobial consumption data in veterinary medicine are available from only a few countries and the type of information that is collected or reported varies. To address this challenge, the European Surveillance of Veterinary Antimicrobial Consumption project was launched by the European Medicines Agency in September 2009 and has just published its first report. This comparison of the different studies for surveillance and monitoring of antimicrobial resistance and antimicrobial consumption in humans and animals shows the need to improve harmonisation.

Quinupristin-dalfopristin resistance in Enterococcus faecium isolates from humans, farm animals, and grocery store meat in the United States.

Three hundred sixty-one quinupristin-dalfopristin (Q-D)-resistant Enterococcus faecium (QDREF) isolates were isolated from humans, turkeys, chickens, swine, dairy and beef cattle from farms, chicken carcasses, and ground pork from grocery stores in the United States from 1995 to 2003. These isolates were evaluated by pulsed-field gel electrophoresis (PFGE) to determine possible commonality between QDREF isolates from human and animal sources. PCR was performed to detect the streptogramin resistance genes vatD, vatE, and vgbA and the macrolide resistance gene ermB to determine the genetic mechanism of resistance in these isolates. QDREF from humans did not have PFGE patterns similar to those from animal sources. vatE was found in 35%, 26%, and 2% of QDREF isolates from turkeys, chickens, and humans, respectively, and was not found in QDREF isolates from other sources. ermB was commonly found in QDREF isolates from all sources. Known streptogramin resistance genes were absent in the majority of isolates, suggesting the presence of other, as-yet-undetermined, mechanisms of Q-D resistance.

Prevalence of streptogramin resistance genes among Enterococcus isolates recovered from retail meats in the Greater Washington DC area.

The prevalence of streptogramin resistance genes in enterococci recovered from retail poultry in the Greater Washington DC area was examined. Forty-three chicken and 32 turkey retail samples were analysed. Thirty-one non-Enterococcus faecalis enterococcal strains were isolated that displayed MICs of quinupristin-dalfopristin and virginiamycin of > or = 4 mg/L. These included Enterococcus faecium (turkey n = 4, chicken n = 23), Enterococcus gallinarum (turkey n = 2, chicken n = 1) and Enterococcus hirae (chicken n = 1). The presence of streptogramin resistance genes was examined by PCR in all non-E. faecalis isolates. The vat(E) gene was detected in 10/23 chicken E. faecium and from 2/4 turkey E. faecium. No other streptogramin resistance genes were detected by PCR. In addition, erm(B) was detected in all the E. faecium and E. gallinarum found in turkeys and in 7/23 E. faecium found in chickens. The vat(E) gene was transferable by conjugation from only two of the 12 E. faecium isolates (one from chicken and one from turkey). This study suggests that there is a high prevalence of low-level streptogramin resistance among enterococci found in retail poultry and that other, yet to be identified, mechanisms operate in these isolates that confer streptogramin resistance in enterococci.

Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Identification of vat(E) in Enterococcus faecalis isolates from retail poultry and its transferability to Enterococcus faecium.

Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium.

The food safety perspective of antibiotic resistance.

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens. This issue has been the subject of heated debates for many years, however, there is still no complete consensus on the significance of antimicrobial use in animals, or resistance in bacterial isolates from animals, on the development and dissemination of antibiotic resistance among human bacterial pathogens. In fact, the debate regarding antimicrobial use in animals and subsequent human health implications has been going on for over 30 years, beginning with the release of the Swann report in the United Kingdom. The latest report released by the National Research Council (1998) confirmed that there were substantial information gaps that contribute to the difficulty of assessing potential detrimental effects of antimicrobials in food animals on human health. Regardless of the controversy, bacterial pathogens of animal and human origin are becoming increasingly resistant to most frontline antimicrobials, including expanded-spectrum cephalosporins, aminoglycosides, and even fluoroquinolones. The lion's share of these antimicrobial resistant phenotypes is gained from extra-chromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large, transferable, extra-chromosomal DNA elements, called plasmids, on which may be other DNA mobile elements, such as transposons and integrons. These DNA mobile elements have been shown to transmit genetic determinants for several different antimicrobial resistance mechanisms and may account for the rapid dissemination of resistance genes among different bacteria. The increasing incidence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many aspects of the development of antimicrobial resistance still remain uncertain. The emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms, and the surrounding environments. Although research has linked the use of antibiotics in agriculture to the emergence of antibiotic-resistant foodborne pathogens, debate still continues whether this role is significant enough to merit further regulation or restriction.

Expression of efflux pump gene pmrA in fluoroquinolone-resistant and -susceptible clinical isolates of Streptococcus pneumoniae.

Thirty-four ciprofloxacin-resistant (MIC > or = 2 microg/ml) and 12 ciprofloxacin-susceptible clinical isolates of Streptococcus pneumoniae were divided into four groups based upon susceptibility to norfloxacin and the effect of reserpine (20 microg/ml). The quinolone-resistance-determining regions of parC, parE, gyrA, and gyrB of all ciprofloxacin-resistant clinical isolates were sequenced, and the activities of eight other fluoroquinolones, acriflavine, ethidium bromide, chloramphenicol, and tetracycline in the presence and absence of reserpine were determined. Despite a marked effect of reserpine upon the activity of norfloxacin, there were only a few isolates for which the activity of another fluoroquinolone was enhanced by reserpine. For most isolates the MICs of acriflavine and ethidium bromide were lowered in the presence of reserpine despite the lack of effect of this efflux pump inhibitor on fluoroquinolone activity. The strains that were most resistant to the fluoroquinolones were predominantly those with mutations in three genes. Expression of the gene encoding the efflux pump PmrA was examined by Northern blotting (quantified by quantitative competitive reverse transcriptase PCR) and compared with that of S. pneumoniae R6 and R6N. Within each group there were isolates that had high-, medium-, and low-level expression of this gene; however, increased expression was not exclusively associated with those isolates with a phenotype suggestive of an efflux mutant. These data suggest that there is another reserpine-sensitive efflux pump in S. pneumoniae that extrudes ethidium bromide and acriflavine but not fluoroquinolones.

Variation within the vat(E) allele of Enterococcus faecium isolates from retail poultry samples.

In a survey of retail meat samples, twelve quinupristin-dalfopristin-resistant (MICs, > or =4 mg/liter) Enterococcus faecium isolates that carried a vat(E) gene were recovered. DNA sequence comparison revealed five new variations in the vat(E) allele among 12 isolates, which were designated vat(E-4) through vat(E-8); two isolates had vat(E-1). There was no correlation between the number of base changes and the quinupristin-dalfopristin MIC.

Nature of transposon-mediated high-level gentamicin resistance in Enterococcus faecalis isolated in the United Kingdom.

Forty-two high-level gentamicin-resistant (MIC > 1000 mg/L) strains of Enterococcus faecalis, isolated from diverse geographical locations throughout the UK between 1993 and 1995, were studied to identify the nature of the high-level gentamicin-resistant determinants and the possibility of these determinants being associated with a transposon. High-level gentamicin resistance was attributed to the synthesis of the bifunctional (AAC6'-APH2") aminoglycoside-modifying enzyme. The aac6'-aph2" gene, which was present on a 70 kb plasmid in all 42 isolates, could be transferred by conjugation in association with the 70 kb plasmid in 39 of the isolates studied. In three E. faecalis isolates, however, the high-level gentamicin resistance was transferable independent of the 70 kb plasmid, suggesting the presence of a conjugative transposon. Long-PCR studies showed that all 42 clinical isolates harboured a transposon similar to Tn5281, originally identified in E. faecalis strain HH22 isolated in the USA. Restriction endonuclease and Southern hybridization analysis of the UK transposon showed that it is closely related to the high-level gentamicin resistance-conferring transposon Tn5281. However, the UK transposon lacks the HaeIII site identified in Tn5281. Pulsed-field gel electrophoresis analysis identified seven different patterns. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with nine different plasmid types in E. faecalis.

Plasmid heterogeneity and identification of a Tn5281-like element in clinical isolates of high-level gentamicin-resistant Enterococcus faecium isolated in the UK.

Ten clinical isolates of high-level gentamicin-resistant (HLGR) Enterococcus faecium, collected from six hospitals throughout the UK, were studied to determine whether HLGR was attributed to widespread dissemination of a single plasmid or whether different plasmid types were implicated in the dissemination of this phenotype. HLGR was attributed to the presence of the AAC6'-APH2" bifunctional aminoglycoside modifying enzyme. The aac6'-aph2" gene was present on a 70 kb plasmid in all ten isolates. Conjugation studies indicated that the HLGR marker could transfer with varying frequency, with or without the associated 70 kb plasmid. Detailed molecular genetic analysis suggested that four of the isolates harboured a transposon similar to Tn5281, originally identified in Enterococcus faecalis strain HH22 isolated in the USA. The UK transposon, however, lacked the two symmetrically located HaeIII sites found in Tn5281. The six remaining isolates appeared to have a Tn5281-truncated structure in which the aac6'-aph2" gene is flanked by an IS256 element at the 5' end. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with two different plasmid types in E. faecium. Pulsed-field gel electrophoresis (PFGE) analysis identified three different patterns. The four E. faecium isolates harbouring the Tn5281-like structure were indistinguishable from each other, while the remaining six isolates exhibited two distinct PFGE patterns. This is the first study to indicate that there is heterogeneity among the plasmids that confer the HLGR phenotype in E. faecium isolates in the UK and to report on the presence of a transposon, similar to Tn5281, in E. faecium harbouring the aac6'-aph2" gene.

Gonococcal resistance to beta-lactams and tetracycline involves mutation in loop 3 of the porin encoded at the penB locus.

penB is a chromosomal mutation that confers resistance to beta-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a beta-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101-Ala-102-->Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci.