Development and evaluation of a broth macrodilution method to determine the biocide susceptibility of bacteria.

In comparison to biocide efficacy testing, biocide susceptibility testing of bacteria so far lacks standardized methods for routine use. The aims of the present study were to develop a broth macrodilution method to test bacterial pathogens for their biocide susceptibility and to evaluate this method in an interlaboratory trial. Staphylococcus aureus ATCC®6538 was tested for its susceptibility to benzalkonium chloride, chlorhexidine and isopropanol comparing test strain suspension preparations, test volumes and incubation times. The use of 2 mL volumes for the testing and an incubation time of 24 h were proposed. Ten German laboratories participated in the interlaboratory trial. Four reference strains (S. aureus ATCC®6538, Enterococcus hirae ATCC®10541, Escherichia coli ATCC®10536 and Pseudomonas aeruginosa ATCC®15442) commonly used for biocide activity testing, were included. Strains were tested three times at independent occasions for their susceptibility to benzalkonium chloride, glutardialdehyde and isopropanol. In total, 360 data points were obtained (30 per strain/biocide combination). The modal minimal inhibitory concentration ± one dilution step was defined as acceptable range. For the four reference strains and the three biocides 80-100% of the values were considered as acceptable. The deviations within the laboratories for a strain/biocide combination were rather consistent. In general, the testing was performed without difficulties by the laboratories. Although inoculum plate counts of four laboratories were outside the acceptable range, this did not have a large impact on the results. The proposed method was stable and easy to perform. It may contribute to a harmonization and standardization of biocide susceptibility testing.

Association of farm-related factors with characteristics profiles of extended-spectrum ß-lactamase- / plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolates from German livestock farms.

Resistance to ß-lactam antibiotics, including third-generation cephalosporins, is of major concern for animal and human health. In this study, extended-spectrum ß-lactamase (ESBL) / plasmid-mediated AmpC (pAmpC) ß-lactamase -producing Escherichia coli isolates from German livestock farms were characterised and associations of these isolate characteristics with farm-related factors were investigated across different types of livestock. A total of 469 isolates originating from 150 farms (34 broiler farms, 38 fattening pig farms, 43 dairy cattle farms, 35 beef cattle farms) was included in the analyses. ESBL-gene family, phylogroup and phenotypic antimicrobial susceptibility for several antimicrobial agents were determined. This data was used to define different profiles characterising the isolates. Multivariate analyses using a distance-based non-parametric approach were performed to investigate associations between the profiles of the isolates and farm-related factors (e.g. management, husbandry, and environment of the farms). Co-occurrence of ESBL-gene families were not found in any of the isolates analysed. Sixty-eight percent of the isolates carried blaCTX-M variant genes. The frequency of phylogroups was as follows: A (55%), B1 (35%), D (17%) and B2 (3%). The most frequent phenotypic non-wildtype profile was non-wildtype status of solely cefepime (27%). Profiles of isolates from broilers differed substantially from those of other isolates. Associations between farm-related factors and characteristics profiles differed, depending on the isolate characteristics included in the analyses. Some factors describing the farm environment, like waterfowl in the surrounding of the farm, were associated with all tested profiles. The epidemiological method applied defines distances between isolates on basis of isolate characteristics data and is capable of analysing associations between isolate characteristics and epidemiological factors. As additional data, such as plasmid characteristics, gene type, or sequence information could be included in future studies, the method is suitable to identify points of action to reduce the occurrence of antimicrobial resistant bacteria.

Identification of novel variants of the colistin resistance gene mcr-3 in Aeromonas spp. from the national resistance monitoring programme GERM-Vet and from diagnostic submissions.

Objectives: To investigate Aeromonas spp. isolates for the presence of the novel resistance gene mcr-3 or variants thereof and to characterize the positive isolates by whole genome sequence analysis. Methods: A total of 479 unrelated Aeromonas isolates were investigated by PCR for the genes mcr-1, mcr-2 and mcr-3. Positive isolates were investigated for their colistin MICs. Species assignment was based on sequence analysis of 16s rRNA and gyrB and rpoB genes. The mcr-carrying contigs obtained by WGS were analysed for the genetic environments of the mcr genes. Results: Four (0.84%) Aeromonas isolates were positive in the mcr-3-specific PCR assay, whereas none of the isolates harboured mcr-1 or mcr-2. Each of the four mcr-3 genes encoded a novel variant, which showed amino acid identities of 95.0%-98.0% to the original Mcr-3 protein. These variants were designated Mcr-3.6 [Aeromonas allosaccharophila from golden orfe (Leuciscus idus)], Mcr-3.7 [Aeromonas media from turkey (Meleagris gallopavo)], Mcr-3.8 [Aeromonas jandaei from koi carp (Cyprinus carpio)] and Mcr-3.9 [Aeromonas hydrophila from koi carp]. The isolate harbouring the mcr-3.9 gene carried an additional mcr-3.8 gene and showed a distinctly higher colistin MIC of ≥128 mg/L than all other isolates. The genetic environments of the mcr-3 variant genes in all four isolates differed, but in part resembled the flanking regions of mcr-3.3 from Aeromonas veronii of chicken meat. Conclusions: This study identified four novel Mcr-3 variants. The isolates carrying the respective genes dated back to 2005 suggesting that this gene has existed for more than 12 years.

Analysis of blaSHV-12-carrying Escherichia coli clones and plasmids from human, animal and food sources.

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely. Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing. Results: Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n = 17), IncK ( n = 3), IncF ( n = 1), IncX3 ( n = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to ß-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex. Conclusions: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.

Detection of plasmid-borne extended-spectrum ß-lactamase (ESBL) genes in Escherichia coli isolates from bovine mastitis.

Extended-spectrum ß-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009-2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n=16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n=3), ST117 (n=2), ST361 (n=1), ST362 (n=1), ST540 (n=1), ST1431 (n=2), ST1508 (n=1), and the novel ST5447 (n=1)] and the phylogenetic groups A (n=6), B1 (n=2), B2 (n=1) and D (n=3). ESBL genes blaCTX-M-1 (n=5), blaCTX-M-2 (n=2), blaCTX-M-14 and blaCTX-M-15 (n=4) were found on conjugative plasmids (35-225kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2+P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to ß-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of ß-lactam antibiotics.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates collected from diseased food-producing animals in the GERM-Vet monitoring program 2008-2014.

The aim of this study was to identify extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli collected from diseased food-producing animals in Germany. A total of 6849 E. coli isolates, collected from diseased cattle, pigs and poultry in the German national monitoring program GERM-Vet (2008-2014), were characterized by antimicrobial susceptibility testing and screened for the ESBL phenotype. ESBL genes were identified by PCR and sequencing. The isolates were further characterized by PCR-based phylotyping. The 419/6849 (6.1%) ESBL-producers identified included 324/2896 (11.2%) isolates from cattle, 75/1562 (4.8%) from pigs and 20/2391 (0.8%) from poultry. The ESBL genes detected were: blaCTX-M-1 (69.9%), blaCTX-M-15 (13.6%), blaCTX-M-14 (11.7%), blaTEM-52 (1.9%), blaSHV-12 (1.4%), blaCTX-M-3 (1.0%), and blaCTX-M-2 (0.5%). The phylogroup A was the dominant phylogroup (57.0%) followed by phylogroups D (23.4%), B1 (17.9%), and B2 (1.7%). Bovine isolates belonged predominantly to the phylogroups A and D, whereas the porcine and avian isolates mainly belonged to A and B1. The majority of the ESBL-producing isolates found in each phylogroup were from animals suffering from gastrointestinal infections. In 399/419 isolates (95.2%), additional resistance to non-ß-lactam antibiotics was seen. Multidrug-resistance [resistance to aminoglycosides, fluoro(quinolones), sulphonamides, tetracyclines, and trimethoprim] was seen in 369/419 (88.1%) isolates, which may facilitate the co-selection of ESBL genes, when located on the same mobile genetic element as the others resistance genes, and may compromise the therapeutic options.

Antimicrobial resistance and class 1 integron-associated gene cassettes in Salmonella enterica serovar Typhimurium isolated from pigs at slaughter and abattoir environment.

Forty-five multi-resistant Salmonella enterica subsp. enterica serovar (S.) Typhimurium isolates obtained at five pig abattoirs in Southern Brazil were characterized. Their relatedness was determined by XbaI-macrorestriction analysis. Resistance genes, integrons and plasmid-mediated quinolone resistance genes (PMQR) were investigated by PCR. Amplicons for the variable part of class 1 integrons and the quinolone resistance-determining regions (QRDR) were sequenced. Plasmids were characterized by conjugation assays and replicon typing. Eighteen XbaI-macrorestriction patterns and 19 plasmid profiles were seen. Resistance to ampicillin (blaTEM), chloramphenicol (catA1 and floR), streptomycin (strA-strB), streptomycin/spectinomycin (aadA variants), sulphonamides (sul1, sul2, sul3) and tetracyclines [tet(A) and tet(B)] were commonly found. A trimethoprim resistance gene, dfrA8, was identified on a 100-kb plasmid. Single substitutions in the QRDR of GyrA but no PMQR genes were found. Twenty-five isolates carried class 1 integrons with an aadA23 gene cassette or unusual class 1 integrons with a dfrA12-orfF-aadA27 gene cassette array. Both integrons were found on large conjugative plasmids. Salmonella plasmid-located virulence genes spvR, spvA, spvB, rck and pefA were found on an IncFIB resistance plasmid. Hybrid virulence-resistance plasmids or plasmids harbouring class 1 integrons may play a role in the maintenance and dissemination of antimicrobial resistance among S. Typhimurium in this pig production system.

Circulation of clonal populations of fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany.

Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum ß-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.

Genotypic profile of Listeria monocytogenes isolated in refrigerated chickens in southern Rio Grande do Sul, Brazil / Perfil genotípico de Listeria monocytogenes isolada em frango refrigerados comercializados na região sul do Rio Grande do Sul, Brasil

ABSTRACT: Listeria monocytogenes is of notable concern to the food industry, due to its ubiquitous nature and ability to grow in adverse conditions. This study aimed to determine the genotypic profile of L. monocytogenes strains isolated from refrigerated chickens marketed in the southern part of Rio Grande do Sul, Brazil. The strains of L. monocytogenes isolated were characterized by serotyping and Pulsed Field Gel Electrophoresis (PFGE). Three different serotypes (1/2a, 1/2b and 4e) were evaluated by PFGE, and the macrorestriction patterns utilizing enzymes AscI and ApaI, revealed five different pulsotypes. The presence of such varied genotypic profiles demonstrates the prevalence of L. monocytogenes contamination of chicken processing environments, which combined with ineffective cleaning procedures, allowing the survival, adaptation and proliferation of these pathogens, not only in the processing environment, but also in local grocery stores.

RESUMO: Listeria monocytogenes é uma notável preocupação para a indústria de alimentos, devido à sua natureza ubíqua e a capacidade de se multiplicar em condições adversas. Este estudo objetivou determinar o perfil genotípico de L. monocytogenes isolada a partir de frangos refrigerados comercializados na região sul do Rio Grande do Sul, Brasil. As cepas de L. monocytogenes foram selecionadas e caracterizadas por sorotipagem e Eletroforese em Gel de Campo Pulsado (PFGE). Três sorotipos diferentes (1/2a, 1/2b e 4e) foram avaliados por PFGE, e a combinação dos padrões de macrorestrição utilizando as enzimas AscI e ApaI revelou cinco diferentes pulsotipos. A presença de diferentes perfis genotípicos demonstra a importância da contaminação no ambiente de processamento de frangos, o qual, juntamente com procedimentos de limpeza ineficazes, permitem a sobrevivência, adaptação e proliferação desses patógenos, não somente no ambiente de processamento, mas também no local de comercialização destes produtos.

Emerging issues in antimicrobial resistance of bacteria from food-producing animals.

During the last decade, antimicrobial resistance in bacteria from food-producing animals has become a major research topic. In this review, different emerging resistance properties related to bacteria of food-producing animals are highlighted. These include: extended-spectrum ß-lactamase-producing Enterobacteriaceae; carbapenemase-producing bacteria; bovine respiratory tract pathogens, such as Pasteurella multocida and Mannheimia haemolytica, which harbor the multiresistance mediating integrative and conjugative element ICEPmu1; Gram-positive and Gram-negative bacteria that carry the multiresistance gene cfr; and the occurrence of numerous novel antimicrobial resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus. The emergence of the aforementioned resistance properties is mainly based on the exchange of mobile genetic elements that carry the respective resistance genes.

Analysis and comparative genomics of ICEMh1, a novel integrative and conjugative element (ICE) of Mannheimia haemolytica.

OBJECTIVES: The aim of this study was to identify and analyse the first integrative and conjugative element (ICE) from Mannheimia haemolytica, the major bacterial component of the bovine respiratory disease (BRD) complex. METHODS: The novel ICEMh1 was discovered in the whole-genome sequence of M. haemolytica 42548 by sequence analysis and comparative genomics. Transfer of ICEMh1 was confirmed by conjugation into Pasteurella multocida recipient cells. RESULTS: ICEMh1 has a size of 92,345 bp and harbours 107 genes. It integrates into a chromosomal tRNA(Leu) copy. Within two resistance gene regions of ∼ 7.4 and 3.3 kb, ICEMh1 harbours five genes, which confer resistance to streptomycin (strA and strB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)] and sulphonamides (sul2). ICEMh1 is related to the recently described ICEPmu1 and both ICEs seem to have evolved from a common ancestor. A region of ICEMh1 that is absent in ICEPmu1 was found in putative ICE regions of other M. haemolytica genomes, suggesting a recombination event between two ICEs. ICEMh1 transfers to P. multocida by conjugation, in which it also uses a tRNA(Leu) as the integration site. PCR assays and susceptibility testing confirmed the presence and activity of the ICEMh1-associated resistance genes in the P. multocida recipient. CONCLUSIONS: These findings showed that ICEs, with structurally variable resistance gene regions, are present in BRD-associated Pasteurellaceae, can easily spread across genus borders and enable the acquisition of multidrug resistance via a single horizontal gene transfer event. This poses a threat to efficient antimicrobial chemotherapy of BRD-associated bacterial pathogens.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Identification and characterization of Salmonella enterica subsp. enterica serovar Derby isolates carrying a new aadA26 gene cassette in a class 1 integron obtained at pig slaughterhouses.

Salmonella enterica subsp. enterica (S.) serovar Derby is one of the most prevalent serovars in pigs. Twenty-seven multiresistant S. Derby isolates, obtained at two pig slaughterhouses in Southern Brazil, were investigated for their molecular relationships, genotypic resistance and presence of class 1 and 2 integrons. All isolates shared the same XbaI-macrorestriction pattern and showed a common resistance genotype with resistance to streptomycin/spectinomycin (aadA variant), sulphonamides (sul1) and tetracycline [tet(A)]. They carried chromosome-located class 1 integrons with a new aadA gene variant, designated aadA26, as part of a gene cassette. The sequence of the flanking regions of this integron and the amplification of the merA gene may indicate the location of the class 1 integron into a Tn21-related transposon. The close relationships among these isolates and isolates from an earlier study suggest the persistence of a resistant clone of S. Derby in the pig production chain in Southern Brazil.

Pheno- and genotypic analysis of antimicrobial resistance properties of Yersinia ruckeri from fish.

Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 ± 2°C for 24, 28 and 48 h. Incubation for 24h at 22 ± 2°C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 µg/mL) and nalidixic acid (0.25-0.5 µg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 µg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ∼ 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes.

[Antimicrobial resistance, ESBL and MRSA--definitions and laboratory diagnostics]. / Antimikrobielle Resistenz, ESBL und MRSA--Definitionen und Labordiagnose.

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms "antimicrobial resistance", "ESBL", and "MRSA" are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria--clinical breakpoints and epidemiological cut-off values--including their applications are explained in detail. Furthermore, currently valid diagnostic procedures--as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI)--for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and--as a consequence--for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions.

Biochemical and molecular heterogeneity among isolates of Yersinia ruckeri from rainbow trout (Oncorhynchus mykiss, Walbaum) in North West Germany.

BACKGROUND: Enteric Redmouth Disease (ERM), caused by Yersinia ruckeri, is one of the most important infectious diseases in rainbow trout (Oncorhynchus mykiss) aquaculture in Europe. More recently, non-motile vaccine resistant isolates appear to have evolved and are causing disease problems throughout Europe, including Germany. The aim of this study was to analyse the variation of biochemical and molecular characteristics of Y. ruckeri isolates collected in north west Germany as a basis for strain differentiation. The isolates originated mainly from rainbow trout and were characterised by biochemical profiling, 16S rDNA sequencing, repetitive sequence-based PCRs, including (GTG)5-PCR, BOX-PCR, ERIC-PCR and REP-PCR, and pulsed-field gel electrophoresis (PFGE). RESULTS: In total, 83 isolates were characterised, including 48 isolates collected during a field study in north west Germany. All isolates were confirmed as Y. ruckeri by the API 20E system. Five isolates were additionally confirmed as Y. ruckeri by Y. ruckeri-specific PCR and 16S rDNA sequencing. Only 17 isolates hydrolyzed Tween 80/20. Sixty-six isolates (79.5%) were non-motile. Two different patterns were obtained by REP-PCR, five patterns by ERIC-PCR, four patterns by (GTG)5-PCR and three patterns by BOX-PCR. NotI-directed PFGE resulted in 17 patterns that differed from each other by 25-29 fragments. Isolates from the field study clustered together as PFGE type C. According to the results of API 20E, repetitive sequence-based PCRs and PFGE, these isolates could be subdivided into 27 different groups. CONCLUSIONS: The detailed molecular and phenotypic characterisation scheme developed in this study could be used to help trace the dissemination of Y. ruckeri isolates, and thus may represent part of improved disease monitoring plans in the future.

ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: structure and transfer.

BACKGROUND: Integrative and conjugative elements (ICEs) have not been detected in Pasteurella multocida. In this study the multiresistance ICEPmu1 from bovine P. multocida was analysed for its core genes and its ability to conjugatively transfer into strains of the same and different genera. METHODS: ICEPmu1 was identified during whole genome sequencing. Coding sequences were predicted by bioinformatic tools and manually curated using the annotation software ERGO. Conjugation into P. multocida, Mannheimia haemolytica and Escherichia coli recipients was performed by mating assays. The presence of ICEPmu1 and its circular intermediate in the recipient strains was confirmed by PCR and sequence analysis. Integration sites were sequenced. Susceptibility testing of the ICEPmu1-carrying recipients was conducted by broth microdilution. RESULTS: The 82 214 bp ICEPmu1 harbours 88 genes. The core genes of ICEPmu1, which are involved in excision/integration and conjugative transfer, resemble those found in a 66 641 bp ICE from Histophilus somni. ICEPmu1 integrates into a tRNA(Leu) and is flanked by 13 bp direct repeats. It is able to conjugatively transfer to P. multocida, M. haemolytica and E. coli, where it also uses a tRNA(Leu) for integration and produces closely related 13 bp direct repeats. PCR assays and susceptibility testing confirmed the presence and the functional activity of the ICEPmu1-associated resistance genes in the recipient strains. CONCLUSIONS: The observation that the multiresistance ICEPmu1 is present in a bovine P. multocida and can easily spread across strain and genus boundaries underlines the risk of a rapid dissemination of multiple resistance genes, which will distinctly decrease the therapeutic options.

ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: analysis of the regions that comprise 12 antimicrobial resistance genes.

BACKGROUND: In recent years, multiresistant Pasteurella multocida isolates from bovine respiratory tract infections have been identified. These isolates have exhibited resistance to most classes of antimicrobial agents commonly used in veterinary medicine, the genetic basis of which, however, is largely unknown. METHODS: Genomic DNA of a representative P. multocida isolate was subjected to whole genome sequencing. Genes have been predicted by the YACOP program, compared with the SWISSProt/EMBL databases and manually curated using the annotation software ERGO. Susceptibility testing was performed by broth microdilution according to CLSI recommendations. RESULTS: The analysis of one representative P. multocida isolate identified an 82 kb integrative and conjugative element (ICE) integrated into the chromosomal DNA. This ICE, designated ICEPmu1, harboured 11 resistance genes, which confer resistance to streptomycin/spectinomycin (aadA25), streptomycin (strA and strB), gentamicin (aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or tilmicosin/tulathromycin [msr(E)-mph(E)]. In addition, a complete bla(OXA-2) gene was detected, which, however, appeared to be functionally inactive in P. multocida. These resistance genes were organized in two regions of approximately 15.7 and 9.8 kb. Based on the sequences obtained, it is likely that plasmids, gene cassettes and insertion sequences have played a role in the development of the two resistance gene regions within this ICE. CONCLUSIONS: The observation that 12 resistance genes, organized in two resistance gene regions, represent part of an ICE in P. multocida underlines the risk of simultaneous acquisition of multiple resistance genes via a single horizontal gene transfer event.