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Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of

Background Recent findings on Antibiotic Resistance (AR) have brought renewed attention to the comparison of data on AR from human and animal sectors. This is however, a major challenge since the data is not harmonized. This study performs a comparative analysis of phenotypical AR data from different routine surveillance and monitoring systems in Germany. Escherichia coli data were used as a model to describe the similarities based on the resistance patterns in human and different animal populations in Germany. Method: Data on E. coli isolates were collected from 2014 to 2017 from human clinical isolates, non-clinical isolates from food-producing animals and food, and clinical isolates from food-producing and companion animals from national routine surveillance and monitoring for AR in Germany. Four antibiotics - ampicillin, cefotaxime, ciprofloxacin and gentamicin - were chosen for the analysis. Resistant isolates were defined according to EUCAST clinical breakpoints for humans. Based on the 16 possible resistance combinations to these four antibiotics, cluster analysis was performed using hierarchical clustering with Euclidian and average distance. All analyses were performed with the software “R”. Result Data of 333,496 E. coli isolates were included in this study. Forty-one different human and animal populations were included in the cluster analysis. Three main clusters were detected. Within these three clusters, all human populations (intensive care unit (ICU), general ward and outpatient care) showed similar relative frequencies of the resistance combinations and clustered together. They demonstrated similarities with clinical isolates from different animal populations and most isolates from pigs from both non-clinical and clinical isolates. Isolates from healthy poultry demonstrated similarities in relative frequencies of resistance combinations and clustered together. However, they clustered separately from the human isolates. All isolates from different animal populations with low relative frequencies of resistance combinations clustered together and likewise separately from the human populations. Conclusion Cluster analysis facilitated the comparison of phenotypical AR data across human and animal sectors. It indicated linkage among human isolates and with isolates from various animal populations based on the resistance combinations in E. coli. Further analyses based on these findings might promote a better one-health approach for AR in Germany.

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.

Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis , one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47 H. parasuis isolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h.

Due to the importance of antimicrobial susceptibility testing (AST) for veterinary diagnostics, a standardised protocol for AST of Rhodococcus equi by broth microdilution has recently been developed and approved by the Clinical and Laboratory Standards Institute (CLSI). The aim of the present study was to test this protocol in an interlaboratory comparative study for its fitness for use in routine laboratory diagnostics. All of the 18 participating laboratories determined the minimum inhibitory concentrations (MIC) of two R. equi strains against 24 antimicrobial agents. The modal MIC values were determined and the acceptable ranges were set as the modal MIC ±1 dilution step. The R. equi field strain Rh110 showed a slightly better performance than the type strain R. equi ATCC® 25729. For the different antimicrobial agents tested, the percentage of MIC values within the acceptable ranges varied from 75.9 to 100% for R. equi ATCC® 25729, and from 85.2 to 100% for R. equi Rh110. The most homogeneous MIC results (i.e. modal MIC ±1 dilution step) were obtained for oxacillin and vancomycin, while the most divergent results were seen with cefotaxime and ceftiofur. Using a success rate of at least 80% of the strain-specific MICs being within the acceptable ranges as an arbitrary cut-off, only one of the participating laboratories failed to reach this cut-off value for one of the two R. equi strains. Thus, we consider the new protocol fit for use in routine AST of R. equi.

Tylosin, a 16-membered macrolide, is - besides other indications - used for the treatment of bovine mastitis. So far, there is only limited information available on the tylosin susceptibility of streptococci isolated from mastitis. The aim of the present study was to comparatively investigate 303 streptococci from bovine mastitis, including 101 Streptococcus agalactiae, 100 Streptococcus dysgalactiae and 102 Streptococcus uberis, for their tylosin and erythromycin susceptibility by broth microdilution and agar disk diffusion. Both tests followed the recommendations of the Clinical and Laboratory Standards Institute (CLSI). For erythromycin, the results were interpreted using the CLSI-approved clinical breakpoints. Moreover, erythromycin-resistant isolates were tested for the presence of macrolide resistance genes and for inducible macrolide resistance. In general, both testing methods showed a good correlation for the three streptococcal species, although for the erythromycin susceptibility testing 11 S. uberis isolates fell into the very major error category. All but one of the erythromycin-resistant isolates harbored at least one macrolide resistance gene, with the erm(B) gene being most common. Moreover, single isolates of S. agalactiae and S. dysgalactiae proved to be inducibly macrolide-resistant. Since inducible macrolide resistance can easily switch to constitutive resistance, tylosin should not be used for the treatment of infections caused by inducibly resistant streptococci.

Background In the European Union, various fluoroquinolones are authorised for the treatment of food producing animals. Each administration poses an increased risk of development and spread of antimicrobial resistance. The aim of this study was to investigate the impact of parenteral administration of enrofloxacin on the prevalence of enrofloxacin and ciprofloxacin susceptibilities in the commensal intestinal E. coli population. Methods E. coli isolates from faeces of twelve healthy pigs were included. Six pigs were administered enrofloxacin on day 1 to 3 and after two weeks for further three days. The other pigs formed the control group. MIC values were determined. Virulence and resistance genes were detected by PCR. Phylogenetic grouping was performed by PCR. Enrofloxacin and ciprofloxacin were analysed in sedimentation samples by HPLC. Results Susceptibility shifts in commensal E. coli isolates were determined in both groups. Non-wildtype E. coli could be cultivated from two animals of the experimental group for the first time one week after the first administration and from one animal of the control group on day 28. The environmental load with enrofloxacin in sedimentation samples showed the highest amount between days one and five. The repeated parenteral administration of enrofloxacin to pigs resulted in rapidly increased MIC values (day 28: MIC up to 4 mg/L, day 35: MIC ≥ 32mg/L). E. coli populations of the control group in the same stable without direct contact to the experimental group were affected. Conclusion The parenteral administration of enrofloxacin to piglets considerably reduced the number of the susceptible intestinal E. coli population which was replaced by E. coli strains with increased MIC values against enrofloxacin. Subsequently also pigs of the control were affected suggesting a transferability of strains from the experimental group through the environment to the control group especially as we could isolate the same PFGE strains from both pig groups and the environment.

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 24 h 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.

Few studies have been conducted on changes in the susceptibility of bacteria due to long-term use of biocides. A total of 375 avian Salmonella isolates collected in Germany from healthy or diseased animals during two time periods, 1979 through 1994 and 2004 through 2010, were included in the present study. The isolates were tested for their MICs of triclosan, acriflavine, benzalkonium chloride, and chlorhexidine by broth macrodilution. MIC50, MIC90, and the distribution of MICs were compared. The MIC ranges were 0.0625 to 0.5 μg/ml for triclosan, 16 to 256 μg/ml for acriflavine, 8 to 128 μg/ml for benzalkonium chloride, and 0.5 to 32 μg/ml for chlorhexidine. MIC50s and MIC90s were equal or differed by not more than one dilution step. For isolates from healthy poultry collected during the two time periods, statistical analysis revealed a significant increase only in MICs for chlorhexidine. Salmonellae from diseased birds were more susceptible to triclosan and benzalkonium chloride but less susceptible to acriflavine and chlorhexidine. Overall, only 25 strains had the highest detected MIC of 0.5 μg/ml triclosan, but an association with multidrug resistance could not be confirmed.

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective blaSHV-12-carrying plasmids and sequencing one of these plasmids completely. Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed blaSHV-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 blaSHV-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 blaSHV-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-blaSHV-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 β-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 blaSHV-12 environment was detected and whole plasmid sequencing revealed a Tn21-derived-blaSHV12-ΔTn1721 resistance complex. Conclusions: Results from this study suggest that the dissemination of blaSHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

We examined 199 group D streptococci isolated from clinically defined and epidemiologically unrelated cases of bovine mastitis. Samples were collected during a 5-month period from 2010 to 2011 from diseased animals in 199 herds (1 isolate per herd) raised in different counties and federal states in Germany. A classical enterococcal species identification procedure started with PYRase and catalase assays, growth on Enterococcoselagar(®) and GCG(®) agar plates and in 6.5% NaCl followed by a biochemical reaction panel. All 199 isolates were also subjected to MALDI-TOF MS diagnostics in which a simple and an extended direct transfer protocol were compared. The latter revealed a much better performance (higher log (score) values) although the same result was obtained in all but three cases. Classical and MALDI TOF MS analyses identified 64 Enterococcus faecalis and 37 Enterococcus faecium isolates which were confirmed by species-specific PCRs. These 101 enterococcal isolates did not display a specific multi-resistance phenotype and resistances to glycopeptides and antibiotics of last resort (linezolid, daptomycin, tigecycline) were absent, resistance to tetracycline was the most frequent resistance feature. Molecular typing of the 64 E. faecalis isolates revealed 3 main PFGE clusters of related strains represented by three MLST types (ST40, ST211, ST268). PFGE and MLST analysis of E. faecium isolates revealed several smaller clusters of only a few related strains and identified a number of previously unknown allele and MLST types (n=6; ST624-ST629) besides known variants (ST22, ST32). One of the 37 E. faecium strains showed properties of hospital-associated E. faecium strains (ampicillin resistance, IS16-positive; MLST CC17).

A standard operating procedure for the determination of minimum inhibitory concentrations (MICs) of antimicrobial agents by the broth microdilution method was developed and evaluated for its fitness for use in an interlaboratory ring trial involving 46 routine diagnostic laboratories. All laboratories tested five strains (one reference strain and four field strains) against a total of 22 different antimicrobial agents. Gram-negative strains were tested against 16 different antimicrobial agents and Gram-positive strains against 14 different antimicrobial agents. Tests were performed once a week for three consecutive weeks. At least 80% of the results determined by 35 of the 46 participating laboratories were within the expected range (mode MIC+/-1 dilution step), with the 18 participating laboratories experienced in MIC determination showing a slightly higher mean percentage of accurate results (89.3% reproducible results) than the 28 non-experienced laboratories (86.7% reproducible results). The most accurate results were obtained for the Escherichia coli field strain, whilst the results for the Streptococcus uberis field strain showed the highest error rate. Among the 22 antimicrobial agents tested, the highest variabilities in the results (mean value for all antimicrobial agents 12.3%) were recorded for ceftiofur (27.8%), penicillin G (20.8%) and cefoperazone (20.6%).

Although the 16-membered macrolide tylosin is commonly used for the treatment of bovine mastitis, little information is currently available about the susceptibility of mastitis pathogens to tylosin. In the present study, 112 Staphylococcus aureus and 110 coagulase-negative Staphylococcus (CoNS) spp. isolates from cases of bovine mastitis were tested by broth microdilution and agar disk diffusion with 30 μg tylosin disks. Susceptibility to erythromycin was tested by broth microdilution and disk diffusion using 15 μg disks. Both test populations showed bimodal distributions of minimal inhibitory concentrations (MICs) and zone diameters with eleven S. aureus and eight CoNS isolates showing tylosin MICs of ≥ 256 μg/ml and no zones of growth inhibition around the tylosin 30 μg disks. All 19 isolates with tylosin MICs of ≥ 256 μg/ml were also resistant to erythromycin. For six additional erythromycin-resistant isolates, tylosin MICs of 1-8 μg/ml were observed. One S. aureus and two CoNS isolates showed inducible macrolide resistance. PCR analysis of the 25 erythromycin-resistant staphylococcal isolates identified the resistance genes erm(A), erm(B), erm(C), erm(T), mph(C) and msr(A) alone or in different combinations. An excellent correlation between the results of the different tylosin susceptibility tests (broth microdilution versus disk diffusion) was seen for S. aureus and CoNS isolates. Since tylosin does not induce the expression of the aforementioned erm genes, isolates with an inducible resistance phenotype may - if only tylosin is tested - be falsely classified as tylosin-susceptible. Thus, erythromycin should be tested in parallel and tylosin should only be used for the treatment of infections caused by erythromycin-susceptible staphylococci.

Sir, Fluoroquinolones are broad-spectrum antimicrobial agents that are effective against Gram-positive and Gram-negative bacteria. Enrofloxacin, the first fluoroquinolone approved for veterinary use, is still commonly used in veterinary practice to treat infections in food-producing, pet and companion animals, caused by a wide variety of bacterial pathogens including methicillinresistant Staphylococcus aureus (MRSA) and methicillinsusceptible S. aureus (MSSA). However, CLSI-approved clinical breakpoints for enrofloxacin applicable to Staphylococcus spp. are restricted to staphylococci from cats and dogs (susceptible, ≤0.5 mg/L; intermediate, 1–2 mg/L; resistant, ≥4 mg/L). No CLSI-approved clinical breakpoints applicable to staphylococci from pigs, cattle or poultry are currently available. DNA gyrase and DNA topoisomerase IV are the target enzymes of fluoroquinolones in S. aureus, and resistance in staphylococci is mainly due to mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA, gyrB, grlA and grlB, which code for the corresponding subunits of these two enzymes. Another mechanism involved in fluoroquinolone resistance of S. aureus is overexpression of the norA gene, which encodes a multidrug efflux pump, by mutations in the norA promoter region. The aim of the present study was to gain insight into target gene mutations and norA regulator mutations present among MRSA and MSSA from diseased pigs, cattle and poultry, as well as food of poultry origin. For this, all MRSA and MSSA isolates that exhibited enrofloxacin MICs of ≥1 mg/L, from five different strain collections, were comparatively analysed. This cut-off was chosen based on the breakpoint for enrofloxacin-non-susceptible canine and feline Staphylococcus spp. isolates. The test population included 8/54 MRSA from diseased pigs and 9/32 MRSA from poultry meat and poultry meat products characterized in two previous studies, 2/11 MRSA from diseased cattle and 1/2 MRSA from diseased poultry collected in the GERM-Vet programme 2008–09, 1/2 MRSA and 12/24 MSSA from diseased poultry collected in the GERM-Vet programme 2006–07, and 3/27 MSSA from diseased pigs collected in the BfT-GermVet study 2006–08. In total, 36 isolates with enrofloxacin MICs of 1 to ≥32 mg/L were identified (Table 1). If not already done in previous studies, isolates were subjected to spa typing (http ://spaserver.ridom.de) as well as to two clonal complex (CC) 398-specific PCRs as described previously. Isolates that were negative in these PCRs were subjected to multilocus sequence typing (MLST; http://saureus.mlst.net). Of the 36 isolates, 24 were assigned to sequence type (ST) 398 or CC398, depending on whether MLST or the CC398-specific PCRs had been conducted. Five isolates had ST9, another five isolates had ST5 and one isolate had ST1791 (both ST5 and ST1791 belong to CC5). One avian isolate had ST2269, which is a novel ST (allelic profile 1-4-1-4-243-250-10) that has been identified for the first time during the course of this study. Nine different spa types (t002, t011, t034, t214, t899, t1197, t1419, t1430 and t2346) were detected (Table 1). The QRDRs of the gyrA, gyrB, grlA and grlB genes and the promoter region of the norA gene of all 36 S. aureus isolates were amplified by PCR using previously described primers. Mutations were identified by sequencing the PCR products, and the results are summarized in Table 1 in relation to the enrofloxacin MICs. Regardless of their origin and their methicillinresistance status, all isolates with enrofloxacin MICs of ≥4 mg/L exhibited mutations in both grlA at codon 80 (resulting in a Ser to Phe exchange) and gyrA at codon 84 (resulting in exchange of Ser to Leu or Ala) (Table 1). The Ser84Ala exchange in GyrA observed in two MRSA isolates has not been reported before in staphylococci. In contrast, isolates with enrofloxacin MICs of 1 or 2 mg/L also exhibited a mutation in grlA at codon 80 (resulting in the exchange of Ser to Phe, Tyr or Leu), but lacked the amino acid substitutions at position 84 in GyrA. Another interesting observation was the presence of the Glu422Asp exchange in GrlB in all isolates assigned to CC398/ ST398, independent of enrofloxacin MIC, the year of isolation

The correct assessment of mastitis pathogens for their susceptibility/resistance to cefoperazone is currently hampered by the lack of harmonized test conditions and interpretive criteria. The aim of this study was to provide a proposal for clinical breakpoints of cefoperazone which are applicable to Staphylococcusaureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis from cases of bovine mastitis and better reflect the situation in the bovine udder than breakpoints adopted from human medicine. For this, pharmacological data and clinical efficacy data of the documents submitted for approval of cefoperazone have been revisited. In addition, 1,086 bacterial pathogens of the aforementioned six species/groups collected in Germany and in the U.S.A. during recent years were tested in parallel for their cefoperazone MICs and the zone diameters using a 75 µg disk. Subsequently, MICs were plotted against zone diameters. Based on the pharmacological data, the clinical efficacy and the microbiological data, a proposal was made for veterinary-specific breakpoints which classify members of the aforementioned species/groups as (a) susceptible to cefoperazone when their MIC is = 2 µg/ml and their zone diameters are = 27 mm (staphylococci or E. coli) or = 21 mm (streptococci), (b) intermediate when their MIC is 4 µg/ml and their zone diameters are 22 to 26 mm (staphylococci or E. coli) or 16 to 20 mm (streptococci), and (c) resistant when their MIC is = 8 µg/ml and their zone diameters are =21 mm (staphylococci or E. coli) or = 15 mm (streptococci).

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. 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. 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. 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

Our understanding of the composition of Escherichia coli populations in wild boars is very limited. In order to obtain insight into the E. coli microflora of wild boars, we studied E. coli isolates from the jejunums, ileums, and colons of 21 wild boars hunted in five geographic locations in Germany. Ten isolates per section were subjected to clonal determination using pulsed-field gel electrophoresis. One representative isolate per clone was further investigated for virulence traits, phylogenetic affiliation, and antimicrobial susceptibility. Macrorestriction analysis of 620 isolates revealed a range of clone diversity among the sections and animals, with up to 9 and 16 different clones per section and animal, respectively. Most of the clones for a given animal were shared between two adjacent intestinal sections. The overall highest clonal diversity was observed within the colon. While the astA gene was present in a large number of clones, other virulence genes and hemolytic ability were detected only sporadically. Clones of all four ECOR groups dominated the intestinal sections. Phylogenetic analysis and the occurrence of virulence genes correlated with the isolation frequencies for clones. All E. coli clones from wild boars were susceptible to all antimicrobial agents tested. In conclusion, though several parameters (including an animal-specific and highly diverse E. coli clone composition, the simultaneous occurrence of single clones in two adjacent intestinal sections of a given animal, and a higher E. coli diversity in the large intestine than in the small intestine) of E. coli populations of wild boars were similar to those of previously described E. coli populations of conventionally reared domestic pigs, our data also indicate possible differences, as seen for the E. coli diversity in the large intestine, the occurrence of certain virulence genes and phylogenetic groups, and antimicrobial susceptibilities.

National Resistance Monitoring of the Federal Office of Consumer Protection and Food Safety (BVL), which was put into service in 2001, has made it possible to implement a valid and representative database on the basis of which the resistance situation, development and spread in animal pathogens can be evaluated. Escherichia coil (E. coli) strains originating from calves and pigs suffering from enteritis were first included in the investigations in the 2004/2005 study. A total of 258 bovine and 492 porcine E. coli strains were tested using the broth microdilution method to determine the in vitro susceptibility (minimum inhibitory concentration) to 23 (fattening pigs) and 28 (calves, piglets, weaners) different antimicrobial substances. Considerable prevalences of resistance were found for some antimicrobials. The strains originating from both animal species displayed high prevalences of resistance for tetracycline, trimethoprim, trimethoprim/sulfamethoxazole, doxycycline and ampicillin. Reduced susceptibility was detected particularly in the E. coli strains from calves. The data reveal that the resistance level of E. coli strains isolated from cases of enteric disease in calves and pigs is altogether higher than has so far been reported in pathogens causing different diseases and in other food-producing animal species. Based on the results presented, it is possible to assess the current resistance situation for E. coli strains in calves and pigs in Germany. This in turn helps to deduce the necessary management measures that can be taken in order to minimise resistance to antibiotics. Furthermore, the data help to decide on adequate therapy of E. coli infections of the intestinal tract in calves and pigs and encourage the responsible use of antibiotics in the interests of animal health and consumer protection.

The National Resistance Monitoring of the Federal Office of Consumer Protection and Food Safety (BVL) is to determine the prevalence of resistance of bacterial pathogens from animals using a valid database. From 2004 to 2006, a total of 1,472 Pasteurella multocida strains isolated from pigs with acute respiratory tract diseases was submitted to the BVL and examined. Of these, 1,11 (75.5 %) were included in the study and tested using 24 different antimicrobial substances. The results showed that the resistance level is generally low, with the exception of the substances tetracycline, trimethoprim, and the combination trimethoprim/sulfamethoxazole. It also became clear that resistance data need to be evaluated separately for each of the animal production categories, so that a realistic figure of the current resistance level can be presented. This knowledge provides information about the resistance situation in Germany, and helps deduce the necessary management measures that must be taken to minimize resistance to antibiotics. Furthermore, it provides valuable information that can form the basis for empirical therapy, so that the National Resistance Monitoring makes an important contribution to the safety of food derived from animals and consequently aids the improvement of consumer protection.

In the study 2004/2005, the current quantitative resistance level of Escherichia coli, Pasteurella multocida, Pseudomonas aeruginosa, Salmonella sp. and Staphylococcus aureus from chickens and turkeys was determined for the first time within the framework of the National Resistance Monitoring of the Federal Office of Consumer Protection and Food Safety (BVL). The objective was to implement a valid database on the basis of which the development and spread of resistance can be evaluated and monitored. During the investigation period from January 2004 to February 2005,927 strains were collected and 857 (92%) bacteria strains which corresponded to the specifications of the study protocol were tested with the broth microdilution method to determine the in vitro susceptibility (minimum inhibitory concentration) to 22 to 28 antimicrobial agents or antibiotic combinations. The results document a prevalence of resistance that exceeds that of bacterial pathogens of other animal species, especially in the case of tetracycline. Apart for S. aureus, clinical resistance to fluoroquinolones can still be considered low in poultry pathogens (E. coli approx. 2%). By applying the MICG of 4 mg/L for enrofloxacin, a susceptibility of approximately 78 % was calculated for S. aureus. A comparison of the prevalence of resistance between chickens and turkeys, showed that a slightly higher prevalence of resistance can be expected in turkeys. Differences between the susceptibility data of chicks and adult animals could only be found in turkeys. In the case of E. coli, the prevalence of resistance of strains isolated from adult turkeys was up to 10% higher than those isolated from chicks for the corresponding antimicrobial agents. It must be pointed out that the number of E. coli strains from adult turkeys was much higher (n = 194) than the number from turkey chicks (n = 21). The results indicate clearly that in a resistance monitoring system it is necessary to categorise poultry by animal species (chicken, turkey) as well as by production stage and type (broiler, laying hen), so that the epidemiology of resistance can be correctly represented and evaluated. This information is the basis for the development of long-term management options for minimizing antibiotic resistance. Furthermore, the knowledge of prevailing resistance levels in Germany is a valuable tool for veterinary practitioners when determining an empirical therapy. The data collected by the BVL make an important contribution to the optimisation of the safety of food from animals, and thus to improving consumer protection.

Bordetella bronchiseptica causes infections of the respiratory tract in swine and other mammals and is a precursor for secondary infections with Pasteurella multocida. Treatment of B. bronchiseptica infections is conducted primarily with antimicrobial agents. Therefore it is essential to get an overview of the susceptibility status of these bacteria. The aim of this study was to comparatively analyse broth microdilution susceptibility testing according to CLSI recommendations with an incubation time of 16 to 20 hours and a longer incubation time of 24 hours, as recently proposed to obtain more homogenous MICs. Susceptibility testing against a panel of 22 antimicrobial agents and two fixed combinations was performed with 107 porcine isolates from different farms and regions in Germany and 43 isolates obtained from companion animals in Germany and other European countries. Isolates with increased MICs were investigated by PCR assays for the presence of resistance genes. For ampicillin, all 107 porcine isolates were classified as resistant, whereas only a single isolate was resistant to florfenicol. All isolates obtained from companion animals showed elevated MICs for β-lactam antibiotics and demonstrated an overall low susceptibility to cephalosporines. Extension of the incubation time resulted in 1–2 dilution steps higher MIC50 values of porcine isolates for seven antimicrobial agents tested, while isolates from companion animals exhibited twofold higher MIC50/90 values only for tetracycline and cefotaxime. For three antimicrobial agents, lower MIC50 and MIC90 values were detected for both, porcine and companion animal isolates. Among the 150 isolates tested, the resistance genes bla BOR-1 (n = 147), bla OXA-2, (n = 4), strA and strB (n = 17), sul1 (n = 10), sul2 (n = 73), dfrA7 (n = 3) and tet(A) (n = 8) were detected and a plasmid localisation was identified for several of the resistance genes.

The objective was to establish and standardise a broth microdilution susceptibility testing method for porcine Bordetella (B.) bronchiseptica. B. bronchiseptica isolates from different geographical regions and farms were genotyped by macrorestriction analysis and subsequent pulsed-field gel electrophoresis. One reference and one type strain plus two field isolates of B. bronchiseptica were chosen to analyse growth curves in four different media: cation-adjusted Mueller-Hinton broth (CAMHB) with and without 2% lysed horse blood, Brain-Heart-Infusion (BHI), and Caso broth. The growth rate of each test strain in each medium was determined by culture enumeration and the suitability of CAMHB was confirmed by comparative statistical analysis. Thereafter, reference and type strain and eight epidemiologically unrelated field isolates of B. bronchiseptica were used to test the suitability of a broth microdilution susceptibility testing method following CLSI-approved performance standards given in document VET01-A4. Susceptibility tests, using 20 antimicrobial agents, were performed in five replicates, and data were collected after 20 and 24 hours incubation and statistically analysed. Due to the low growth rate of B. bronchiseptica, an incubation time of 24 hours resulted in significantly more homogeneous minimum inhibitory concentrations after five replications compared to a 20-hour incubation. An interlaboratory comparison trial including susceptibility testing of 24 antimicrobial agents revealed a high mean level of reproducibility (97.9%) of the modified method. Hence, in a harmonization for broth microdilution susceptibility testing of B. bronchiseptica, an incubation time of 24 hours in CAMHB medium with an incubation temperature of 35°C and an inoculum concentration of approximately 5 x 10(5) cfu/ml was proposed.

The use of antimicrobial substances in human and veterinary medicine inevitably results in a selection pressure for drug resistance in exposed bacteria. Preventive measures, apt to avoid the consequent development of new resistances and selection for existing ones, respectively, have to be elaborated. Moreover, it has to be ensured that neither resistant bacteria nor resistance genes are spread to and consequently via the food chain. Respiratory diseases as well as mastitis in dairy cattle belong to the most frequently occurring diseases in food-producing animals. For the first time in Germany, a comprehensive, cross-sectional study into the antimicrobial susceptibility of bacteria associated with these disease patterns in food-producing animals was conducted by the Federal Office of Consumer Protection and Food Safety (BVL) in 2001. The selection of examined bacterial species comprised Pasteurella multocida and Mannheimia haemolytica associated with respiratory disease in pigs, and Escherichia coli, Streptococcus spp. and Staphylococcus spp. causing mastitis in dairy cattle. Bacterial strains were collected following a representative sampling scheme, taking into account the total number of animals in the individual German federal Lander. In an analogous study conducted in 2002/2003, this selection was extended by the indication respiratory disease in juvenile cattle, caused by P. multocida and M. haemolytica, respectively. In comparison with data from 2001, MIC values determined in 2002/2003 suggested significantly lower or higher degrees of drug susceptibility only for a few antimicrobial agents. Comparison was carried out on the basis of bacterial species and individual federal Lander, respectively. Overall, the data raised in both studies revealed substantially lower resistance rates than published for Germany so far. This is particularly true for results from those Lander, whose animal health services had implemented preventive strategies to control infectious diseases. No correlation could be established between differing animal population densities and differences in the prevalence of resistance in corresponding Lander. However, the geographical distribution of occurrence of resistance against beta-lactam antimicrobial agents suggests different therapeutic strategies employed in different sized animal flocks. In federal Lander marked by large-scale livestock farming, significantly higher resistance values could be measured for cephalosporins than for penicillins, whereas in Lander with rather traditional farming structures, resistance to penicillins was predominant. Assuming otherwise similar factors of influence on the emergence of resistance, this pattern suggests that cephalosporins are preferably used in large enterprises and penicillins in smaller farms, respectively. Currently, mechanisms effecting changes in antimicrobial resistance are being further investigated in a successive study.

The efficacy of enterocoliticin, a phage tail-like bacteriocin, as antimicrobial compound against infections with pathogenic Yersinia enterocolitica serotype O3 strains was assessed. In cell cultures, which were infected with the Y. enterocolitica strains 13 169 or 6471/76, bactericidal activity of enterocoliticin was found for bacteria adhering to the surface of eukaryotic cells, whereas bacteria, which had invaded the eukaryotic cells, were not accessible to the bacteriocin. The interaction of enterocoliticin with Y. enterocolitica was further examined in animals. Female BALB/c mice were experimentally infected with the two Y. enterocolitica strains and enterocoliticin was applied as antimicrobial compound by the oral route. Experimental variations concerning the infectious doses of the Y. enterocolitica strains and the time points of application of the bacteriocin were investigated. The increase of the Yersinia CFU titre in animals was retarded at time points shortly after the application of enterocoliticin indicating that the particles were effective on recently introduced Yersinia . The repeated application of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Yersinia .

The national antimicrobial resistance monitoring determines the current quantitative resistance level of life-stock pathogens, in order to permit the evaluation and surveillance of the distribution of resistances on a valid basis. During the examination period from June 2002 to July 2003, a total of 1849 pathogens was collected, following a representative German-wide pattern in collaboration with 29 laboratories. The selection of examined bacterial strains included different specimen causing respiratory diseases in fattening pigs (Pasteurella multocida, Bordetella bronchiseptica) and cattle (Pasteurella multocida, Mannheimia haemolytica), respectively, as well as strains causing mastitis in dairy cows (Staphylococcus spp., Streptococcus spp., E. coli). Determination of the in-vitro susceptibility (minimal inhibitory concentration) to at least 17 antimicrobial agents was performed centrally by the BVL using the microdilution broth method. The findings approximately match results of an analogous study conducted in 2001, and correspondingly revealed significantly lower resistance-values in comparison to data published for Germany so far. No correlation could be established between the incidence of resistance and differing stock densities. By means of the resistance monitoring data gathered by the BVL, the risk potential of antimicrobials applied in Germany can be reliably defined. This valuable information about the epidemiological situation of resistance in Germany can be helpful to veterinarians as a decision guidance when choosing appropriate means of therapy. Accordingly, the results stated by the BVL are apt to provide an important contribution to improving the safety of food-animal products. The experiences obtained from the monitoring also show that valid data about the antimicrobial susceptibility can only be raised on an interdisciplinary approach (federal agencies, county diagnostic laboratories, universities, industry). Currently, the BVL put into practice a study with an extended spectrum of bacterial species and indications.

Bacteriocins have traditionally been defined as proteinaceous compounds produced by bacteria that inhibit or kill closely related bacteria (16). A special group of bacteriocins are high-molecular-weight particles, which can be sedimented by ultracentrifugation and are resolved by electron microscopy as phage tail-like particles (8, 13). These particles have been regarded as defective bacteriophages, which might have arisen from temperate phages by several successive mutations (8, 13). Bacteriocins of this type have been found in cultures of several gram-negative bacteria, including members of the families Enterobacteriaceae, Vibrionaceae, and Pseudomonadaceae (8, 10). The most thoroughly studied phage tail-like bacteriocins are the F-type and R-type pyocins produced by Pseudomonas aeruginosa. The F-type pyocins resemble flexible but noncontractile tail structures of bacteriophages, whereas the R-type pyocins are similar to contractile but nonflexible tails (26). The bactericidal activity of the R-type pyocins is caused by the depolarization of the cytoplasmic membranes of sensitive bacteria (33). The pyocin biosynthesis genes were chromosomally located, and the gene organization clearly demonstrated that pyocins possess an ancestral origin common with bacteriophages. It was suggested that the pyocins have evolutionarily specialized as phage tails, rather than being just simple defective phages (26). As the increase in bacteria resistant to a wide range of antibiotics has become a major public health problem, alternative strategies are being looked for to counter bacterial infections. In recent years the approach of using bacteriophages for the treatment of bacterial infections has come into focus again (2, 5, 23), as the advantage of phages as therapeutic agents is the high specificity for their target organisms, which would enable the selective elimination of phage-susceptible bacteria from a bacterial community (20). Bacteriocins are comparable to bacteriophages in terms of specificity for target bacteria, and given the morphological similarity, the use of phage tail-like bacteriocins for therapeutic use is conceivable, although bacteriocins lack the ability of phages to multiply during infection of target bacteria. In the genus Yersinia, the production of phage tail-like particles by strains of Y. kristensenii, Y. frederiksenii, and Y. intermedia has been reported (9). These particles were used as diagnostic tools for typing Yersinia strains. Another early report about a phage tail produced by a Y. enterocolitica strain consisted mainly of morphological data (18). By studying a number of Yersinia isolates (19, 25) we found a food-borne strain of Y. enterocolitica which produced a bacteriocin-like substance with an inhibitory activity against serogroups O:3, O:5,27 and O:9 of Y. enterocolitica, which are the dominating pathogenic serogroups for humans (7, 21). The aim of our study was to characterize structural and functional features of this bacteriocin, which was designated enterocoliticin according to the usual nomenclature of bacteriocins (16), by studying its inhibitory activity against a pathogenic Y. enterocolitica O:3 strain.