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Pasteurella multocida is an important respiratory tract pathogen in intensive livestock farming, especially in pigs. Antimicrobial agents are frequently used to combat infections caused by this pathogen. In a study on antimicrobial resistance among respiratory tract pathogens of pigs from 30 German pig-producing farms, P. multocida isolates (n = 9) with high minimal inhibitory concentration (MIC) values of 16/304 mg/L (n = 2), 32/608 mg/L (n = 3) or ≥64/1216 mg/L (n = 4) for trimethoprim/sulfamethoxazole (1:19) and of ≥512 mg/L (n = 9) for trimethoprim (TMP) were detected in three of these farms. The genetic relatedness of the isolates was investigated via capsule-specific PCR and macrorestriction analyses with ApaI and SmaI. Pulsed-field gel electrophoresis revealed indistinguishable restriction patterns per farm, with slight differences between the three farms. All isolates represented capsular type A. Four representative isolates, that were subjected to whole genome sequencing, shared the multi-locus sequence type (ST) 3. Their plasmids were transformed into E. coli TOP10 with subsequent selection on TMP-containing agar plates. Antimicrobial susceptibility testing and plasmid analysis of the transformants confirmed that they were resistant to sulfonamides and trimethoprim and carried only a single small plasmid. This plasmid was completely sequenced and revealed a size of 6050 bp. Sequence analyses identified the presence of a resistance gene cluster comprising the genes sul2-ΔstrA-dfrA14-ΔstrA-ΔstrB. Further analysis identified a dfrA14 gene cassette being integrated into the strA reading frame. Neither the gene dfrA14 nor this gene cluster have been detected before in P. multocida., (Copyright © 2019. Published by Elsevier B.V.)

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E. coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011-2013, seven putative ESBL-producing E. coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying bla CTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two bla CTX-M-15 - (IncFIB; non-typeable) and the IncN bla CTX-M-65 - and IncHI2 bla CTX-M-125 -carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB bla CTX-M-2 -carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2, streptomycin resistance genes strA and strB, the plasmid-mediated quinolone resistance gene qnrS1 and bla TEM-1 (sul2-strA-strB-IS66-bla TEM-1 -tnpR-ΔtnpA-ISEcp1-bla CTX-M-15 -Δorf477-ΔtnpA-qnrS1) or the fosfomycin resistance gene fosA3 (ΔISEcp1-bla CTX-M-125 -ΔIS903B-fosA3). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E. coli., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Extended-spectrum β-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 bla CTX-M-1 (n=5), bla CTX-M-2 (n=2), bla CTX-M-14 and bla CTX-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 β-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 β-lactam antibiotics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to identify extended-spectrum β-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: bla CTX-M-1 (69.9%), bla CTX-M-15 (13.6%), bla CTX-M-14 (11.7%), bla TEM-52 (1.9%), bla SHV-12 (1.4%), bla CTX-M-3 (1.0%), and bla CTX-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-β-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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes., Methods: A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST., Results: The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons., Conclusions: ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Extended-spectrum-beta-lactamase (ESBL) and plasmid-encoded cephamycinase (pAmpC) producing Escherichia (E.) coli in livestock farms have recently been matter of growing scientific and public concern. This article summarises selected European studies which focus on the prevalence and risk factors associated with the presence of such resistant E. coli isolates in livestock farms. Due to the different methodologies used in these studies, they cannot be compared directly; nonetheless, the overall prevalence found is very high. The prevalence found in broiler farms was higher than 40% and the individual animal prevalence was ca. 30%. The prevalence was more variable in pigs, with reports of pig farms showing prevalence of 1 to 80% and reports of individual animal prevalence of 15 to 100% In studies on cattle farms the production type as well as the age of animals had an influence on the number of positive samples. The highest prevalence was found with calves after birth and in the first weeks, whereas with older cattle the numbers of positive samples were considerably lower. Samples taken from dairy cows were positive more often after calving than before calving. According to the livestock species different risk factors may be assessed for the occurrence of ESBL/pAmpC-producing E. coli isolates. In some studies an association between the occurrence of ESBL-producing E. coli and factors like the use of antimicrobial agents or management factors, as the duration of the fattening period and the acquisition of animals from different origins, were identified. At the moment, there is a lack of systematic and standardised transnational epidemiological investigations on the occurrence of ESBL/pAmpC-producing E. coli in livestock. To control the further spread of ESBL/pAmpC-producing E. coli and the effectiveness of preventive measures, comprehensive monitoring and surveillance systems with harmonised methods are essential. Modern typing methods, in particular the sequence-based methods, can provide more information to clarify transmission pathways.

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla(CTX-M-group-1) dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla(CTX-M-group-1) and bla(OXA-1-like) genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6')-Ib, catB3, bla(OXA-1-like) and bla(CTX-M-group-1). forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Objectives: The aims of this study were (i) to detect extended-spectrum β-lactamase (ESBL) genes among 1378 Escherichia coli isolates from defined disease conditions of companion and farm animals and (ii) to determine the localization and organization of ESBL genes., Methods: E. coli isolates from the German resistance monitoring programme GERM-Vet were included in the study. Plasmids were transferred by conjugation or transformation and typed by PCR-based replicon typing. ESBL genes were detected by PCR; the complete ESBL genes and their flanking regions were sequenced by primer walking. Phylogenetic grouping and multilocus sequence typing (MLST) were performed for all ESBL-producing E. coli isolates., Results: Of the 27 ESBL-producing E. coli isolates detected, 22 carried blaCTX-M-1 genes on IncN (n = 16), IncF (n = 3), IncI1 (n = 2) or multireplicon (n = 1) plasmids. A blaCTX-M-3 gene was located on an IncN plasmid and a blaCTX-M-15 gene was located on an IncF plasmid. A multireplicon plasmid and an IncHI1 plasmid harboured blaCTX-M-2. A blaTEM-52c gene was identified within Tn2 on an IncI1 plasmid. The blaCTX-M genes located within the same or related genetic contexts showed differences due to the integration of insertion sequences. Various MLST types were detected, with ST10 (n = 7), ST167 (n = 4) and ST100 (n = 3) being the most common., Conclusions: This study showed that the blaCTX-M-1 gene is the predominant ESBL gene among E. coli isolates from diseased animals in Germany and a considerable structural heterogeneity was found in the regions flanking the blaCTX-M-1 gene. Insertion sequences, transposons and recombination events are likely to be involved in alterations of the ESBL gene regions.

Objectives: Twenty-five MRSA ST398 isolates from cases of bovine clinical mastitis and two isolates from farm personnel collected from 17 dairy farms in Germany were investigated for genetic relatedness, antimicrobial resistance and virulence properties., Methods: Genomic relationships were determined by ApaI PFGE, spa typing, SCCmec typing and dru typing. Antimicrobial resistance phenotypes were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs., Results: Nine major ApaI PFGE patterns were detected. Three spa types (t011, t034 and t2576) and two SCCmec types (IV and V) were identified. Five different dru types were seen with dt11a being predominant. All isolates were negative for Panton-Valentine leucocidin, enterotoxin and exfoliative toxin genes. Ten resistance patterns were observed with 11 (40.7%) isolates being resistant to only beta-lactam antibiotics and tetracyclines. Several resistance genes were detected: blaZ (penicillin resistance); tet(M), tet(K) and tet(L) (tetracycline resistance); erm(A), erm(B), erm(C) and erm(T) (macrolide/lincosamide/streptogramin B resistance); aacA-aphD, aphA3, aadD and spc (aminoglycoside or aminocyclitol resistance); fexA (phenicol resistance); dfrK (trimethoprim resistance); and vga(A) and vga(C) (pleuromutilin/lincosamide/streptogramin A resistance). The two human isolates were indistinguishable in their genotypic and phenotypic characteristics from the mastitis isolates of the same farm., Conclusions: As previously described for ST398 from swine, isolates of this sequence type from cases of bovine mastitis also demonstrated a high degree of variability when ApaI PFGE profiles and other genotypic and phenotypic characteristics were compared. A uniform virulence gene pattern appeared to be conserved between ST398 isolates from both animal species.

Objectives: Fifty-four methicillin-resistant Staphylococcus aureus (MRSA) ST398 isolates from unrelated diseased swine collected all over Germany were comparatively investigated for their antimicrobial resistance and virulence properties, and for their genomic relatedness., Methods: MICs of 30 antimicrobial agents were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs. The genomic relationships were determined by ApaI-PFGE, spa typing and SCCmec typing., Results: Twenty-two distinct resistance patterns were observed. All 54 isolates were tetracycline resistant, mediated by tet(M), tet(K) and/or tet(L), with 14 isolates being only resistant to beta-lactam antibiotics and tetracyclines. Trimethoprim resistance, seen in 28 isolates, was mostly due to the gene dfrK or dfrG. Among the 24 macrolide/lincosamide-resistant isolates, the genes erm(A), erm(B) and/or erm(C) were detected. The two chloramphenicol/florfenicol-resistant isolates harboured the gene fexA. The eight gentamicin-resistant isolates carried the gene aacA/aphD. Fifty-three isolates harboured SCCmec type V elements while the remaining one carried mecA and ugpQ, but no recombinase genes. All isolates were PVL negative, but one and three isolates, respectively, were positive for the enterotoxin B and enterotoxin K and Q genes. Eight different spa types were identified with t011 being the most predominant. Six ApaI-PFGE clusters with up to nine individual patterns were detected., Conclusions: MRSA ST398 isolates varied slightly in their virulence properties and spa types but differed distinctly in their antimicrobial resistance pheno- and genotypes as well as their ApaI-PFGE patterns. These data underline the ability of ST398 to acquire genetic material that might increase antimicrobial resistance and virulence.

Objectives: In the BfT-GermVet monitoring study, 417 Escherichia coli isolates collected during 2004-06 in Germany from various disease conditions of pigs (n = 87), horses (n = 102) or cats/dogs (n = 228) were investigated for their susceptibility to 24 antimicrobial agents. This study dealt with the identification of integron-associated resistance genes among these isolates., Methods: Class 1 and class 2 integrons were detected by PCR. The variable parts of the integrons were cloned and sequenced. Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons., Results: Class 1 and/or class 2 integrons, alone or in different combinations, were detected in 79 of the 417 E. coli isolates. Four trimethoprim resistance genes (dfrA1/12/14/17), five streptomycin/spectinomycin resistance genes (aadA1/2/4/5/6), two streptothricin resistance genes (estX, sat2), one gentamicin/tobramycin/kanamycin resistance gene (aadB) and one chloramphenicol resistance gene (catB3) were detected. Seven different cassette arrangements were identified within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (18 isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfF + aadA2 (8 isolates), aadB + aadA1 (1 isolate), dfrA14 + recombined aadA6 (1 isolate) and dfrA1 + catB3 + aadA4 (1 isolate). Three different cassette arrangements in class 2 integrons, dfrA1 + sat2 + aadA1 (24 isolates), estX + sat2 + aadA1 (6 isolates) and estX + sat2 + DeltaaadA1 (1 isolate), were identified. The plasmid location of class 1 and/or class 2 integrons was confirmed in 37 isolates., Conclusions: Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 18.9% of the isolates tested. This molecular analysis complements the phenotypic susceptibility testing conducted in the BfT-GermVet monitoring study and helps to explain the persistence of resistance genes even without direct selective pressure.

Objectives: During recent years, methicillin-resistant Staphylococcus aureus (MRSA) strains from animals have become the focus of various studies. In the present study, coagulase-positive staphylococci obtained from pigs, dogs and cats suffering from acute infections, which had been collected in the BfT-GermVet monitoring programme in Germany and phenotypically identified as oxacillin-resistant, were characterized., Methods: The staphylococci were comparatively investigated for their resistance phenotypes and genotypes. Resistance genes were identified by PCR. MRSA strains were further characterized by SmaI macrorestriction analysis and spa typing to assess their genomic relationships., Results: Among the 248 strains tested, 7 strains (5 porcine S. aureus and 2 canine Staphylococcus pseudintermedius) carried the resistance gene mecA. Gentamicin resistance was based on the presence of the gene aacA/aphD while three different tetracycline resistance genes, tet(K), tet(L) and tet(M), alone or in combinations, were detected. The single macrolide/lincosamide-resistant strain carried an erm(A) gene. All MRSA strains proved to be non-typeable by SmaI macrorestriction analysis and exhibited the spa types t011 (four strains) or t034 (one strain)., Conclusions: Based on their spa types and their non-respondence to SmaI digestion, all five porcine MRSA strains resembled MRSA strains of multilocus sequence type ST398, previously detected among pigs in neighbouring countries such as The Netherlands or Denmark. The results of this study indicate that such strains are also involved in defined disease conditions of pigs from various parts of Germany.

MICs for 349 Bordetella bronchiseptica isolates from respiratory tract infections of swine were determined by broth microdilution. The lowest MIC at which 90% of isolates tested are inhibited (MIC90) was that of tetracycline and enrofloxacin (0.5 microg/ml), whereas the highest MIC90s were those of tilmicosin and cephalothin (32 microg/ml) as well as streptomycin (256 microg/ml).