Jannice Schau Slettemeås

Methicillin-resistant Staphylococcus aureus with the novel mecC gene variant isolated from a cat suffering from chronic conjunctivitis

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) is of growing concern in public and animal health and it is important to ensure efficient and reliable methods for MRSA identification. Standard confirmatory tests include PCR for detection of the mecA gene and protein agglutination for identification of the penicillin-binding protein 2a (PBP2a). In 2011, a novel mecA homologue designated mecC (or mecALGA251) was described in S. aureus. This MRSA variant will not be detected with the usual mecA PCR approaches or with the PBP2a agglutination tests, representing a challenge to MRSA confirmation in diagnostic laboratories. Searching for mecC has recently been performed in several countries. Investigated isolates have either been part of historical collections or originated from current diagnostic submissions. The chosen candidates for mecC screening have typically been isolates with a phenotype corresponding to MRSA but lacking mecA, and/or isolates belonging to clonal lineages already associated with mecC carriage. So far the occurrence of the mecC gene has been confirmed in isolates from the typical hosts, humans and/or cattle, in several countries in Northern Europe: the UK, Denmark, Ireland, Germany, France, Sweden and Norway. – 8 Only two studies examining isolates from hosts other than humans and cattle are known; one of these studies investigated a historical isolate collection and mecC was found in S. aureus from a dog, a seal and a chaffinch (all from the UK), from a rabbit and rats from Belgium, and from sheep in Denmark. The other study, recently published by Walther et al., found mecC-positive MRSA from two dogs, seven cats and a guinea pig. The MRSA isolates investigated originated from samples submitted to a laboratory in Germany during the period 2008–11. Based on current knowledge, mecC seems to have a rather wide geographical and host distribution, but occurs at low frequencies. The Norwegian Veterinary Institute has recently detected mecC in an MRSA isolate from a submission to our diagnostic bacteriological service unit in Bergen. The isolate originated from a cat and represents the first finding from current diagnostic activity performed on samples from companion animals. The mecC-positive MRSA (designated 2012-50-2037) was isolated from an eye swab from a 5-year-old house cat with chronic conjunctivitis and stomatitis. The cat had tested negative for all relevant viruses and Chlamydophila felis. Initial treatment with systemic clavulanate-potentiated amoxicillinand topical fusidicacid was ineffective, but improvement was seen after a change of treatment to systemic enrofloxacin and topical dexamethasone, neomycin and polymyxin B. The cat is currently living in a household with two adults, two children and another cat. The MRSA carrier status of the family members and the other cat is not known. The isolate was routinely tested for susceptibility to antimicrobial agents following the disc diffusion method and breakpoints described by EUCAST (www.eucast.org, 7 November 2012, date last accessed). The isolate showed resistance to b-lactams only. MICs were subsequently determined by the use of a broth dilution method (Trek Diagnostics). The following antimicrobial agents were tested: penicillin, cefoxitin, ciprofloxacin, erythromycin, clindamycin, gentamicin, tetracycline, linezolid, fusidic acid, rifampicin, chloramphenicol, trimethoprim, vancomycin, quinupristin/dalfopristin and mupirocin. The isolate exhibited resistance to the b-lactams only, with a cefoxitin MIC of 16 mg/L. The MIC of oxacillin was 2 mg/L, determined by Etest (bioMérieux). The results were interpreted according to EUCAST. S. aureus ATCC 29213 was included as quality control. DNAwas prepared by the boil lysis method and subsequentlysubjected to PCR for detection of mecA, mecC, nuc and 16S rDNA with primers previously described. In addition to a negative control, positive control strains included S. aureus CCUG 29213 (nuc+), S. aureus CCUG 35603 (nuc+, mecA+), Staphylococcus pseudintermedius CCUG 49543 (nuc, mecA) and S. aureus SVA-AB-773 (nuc+, mecC+). The PCR results confirmed MRSA with mecC. The sequence of the mecC amplicon was determined and showed 100% identity with the previously determined mecC sequence. The mecC gene was probably located on a type XI SCCmec element as PCRs with primers for the mecI, mecR, ccrA, ccrB and blaZ genes related to SCCmecXI produced amplicons of correct sizes. The spa typing showed that the isolate belonged to spa type t6902. This spa type has only one other recording in the Ridom spaserver (http://spaserver.ridom.de/, 7 November 2012, date

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

BackgroundOvine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosu s should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR.MethodsA D. nodosus- specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.ResultsThe developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.ConclusionsThe developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.

Emergence of AmpC-producing Escherichia coli in the broiler production chain in a country with a low antimicrobial usage profile

The aim of this study was to estimate the prevalence of cephalosporin-resistant Escherichia coli at the different levels of the Norwegian broiler production pyramid and identify the mechanisms responsible for the resistance phenotype. Samples from all levels of the broiler production pyramid and retail chicken meat (fillets) were included (n=649). The occurrence of cephalosporin-resistant E. coli at the different production levels ranged from 8 to 43%. All these isolates had an AmpC-phenotype, and the majority carried the blaCMY-2 gene. In addition, a few isolates with up-regulated chromosomal ampC were identified. The results show that Norway has a relatively high prevalence of cephalosporin-resistant E. coli in the broiler production chain in spite of a very low consumption of antimicrobial agents. Cephalosporins have not been used in the Norwegian broiler production, and it has been hypothesised that import of breeding animals and hatching eggs may be the source of these resistant bacteria. We demonstrate that these bacteria are disseminated in the production pyramid despite the lack of selection pressure from antimicrobial agents.

Possible cross-infection of Dichelobacter nodosus between co-grazing sheep and cattle

BackgroundThe aim of this study was to investigate possible cross-infection of Dichelobacter nodosus in Norwegian farms practising co-grazing of sheep and cattle.MethodsThirteen farms practising co-grazing of sheep and cattle were included in this descriptive study: five farms with a history of severe ovine footrot (Group I) and eight farms with free-stall housing of cattle and signs of mild or no footrot in sheep (Group II). Sampling for PCR detection of D. nodosus was performed from animals in all farms, and clinical claw examination of sheep and cattle was performed in Group II. D. nodosus positive samples were analysed by a multiplex PCR method that detects variants of the fim A gene corresponding to D. nodosus serogroups A through I.ResultsD. nodosus serogroup A was identified more frequently in sheep from farms with a history of severe footrot (Group I) versus from Group II, and in most of the farms with a history of severe footrot there was a coexistence of D. nodosus serogroup A in sheep and cattle. In one farm heel horn erosion and dermatitis emerged in cattle after co-grazing with sheep suffering from severe footrot where D. nodosus serogroup A was detected. Six months later heel horn erosion and dermatitis were still diagnosed, and D. nodosus serogroup A was identified. Out of the 16 D. nodosus positive sheep samples from Group II, ten of the samples were positive by the fim A serogrouping PCR. Among these 10 samples all serogroups except G were detected. All the D. nodosus serogroups detected in sheep were also present in the corresponding cattle herds.ConclusionThe clinical findings and the coexistence of the same serogroups in co-grazing sheep and cattle could indicate cross-infection. However, further research including isolation of the bacterial strains, virulence-testing and genetic identification, is needed.

Escherichia coli of animal origin in Norway contains a blaTEM-20-carrying plasmid closely related to blaTEM-20 and blaTEM-52 plasmids from other European countries

Sir, The situation regarding antimicrobial resistance in bacteria from food-producing animals in Norway is, in an international perspective, favourable. The resistance frequencies are moderate and the situation has been stable since the start of the Norwegian monitoring programme in the veterinary sector (NORM-VET) (www. vetinst.no) in the year 2000. We report the first bacterial isolate of animal origin detected in Norway with reduced susceptibility to cephalosporins. The isolate (Escherichia coli 2006-01-1248, hereafter termed E. coli 1248) originated from a broiler domesticated in a cephalosporin-free environment and was included in the NORM-VET 2006 programme. MICs of cefotaxime, ceftiofur and ampicillin were 1, 4 and .32 mg/L, respectively. The cefotaxime MIC of 1 mg/L is close to the clinical breakpoint recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (susceptible 1 mg/L, resistant .2 mg/L). However, this value is considerably higher than those observed for susceptible E. coli strains (wild-type population) having MICs of 0.25 mg/L (www.eucast.org). MICs of cefalotin, ceftazidime and cefepime, determined using Etest (AB Biodisk, Solna, Sweden) with E. coli ATCC 25922 as susceptible control, were 64, 0.5 and 0.25 mg/L, respectively. E. coli 1248 was positive in the double-disc synergy test and in the confirmatory test. The tests were carried out as recommended by the manufacturer using discs containing cefotaxime (30 mg), ceftriaxone (30 mg), ceftazidime (30 mg), cefepime (30 mg), amoxicillin/clavulanic acid (30 mg/15 mg), and ceftazidime and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark) (User’s guide NEO-SENSITABS susceptibility testing 19th Edn, www.rosco.dk). PCR was performed for the detection of blaCTX-M, blaSHV and blaTEM 1,2 using the following control strains: E. coli K4-23 (blaCTX-M-9), E. coli Dak2 (blaSHV) and E. coli 76-33094-7 (blaTEM). Amplicons were produced with the blaTEM-specific primers only. Sequencing showed that a blaTEM-20 gene variant 3 was present. Conjugation showed that blaTEM-20 was located on a self-transferable plasmid. The transconjugant had the following MICs of b-lactams: ampicillin, .32 mg/L; cefotaxime, 0.5 mg/L; and ceftiofur, 1 mg/L. The blaTEM-20 sequence contained three silent mutations at positions 346, 682 and 925 when compared with a previously published sequence (accession number Y17581). The other blaTEM-20 gene variant, conferring high-level resistance to thirdgeneration cephalosporins, showed a 135 bp deletion in the promoter and a G!T mutation at position 162. These mutations were not identified in the sequence from E. coli 1248, and this may explain the lower MICs exhibited. The blaTEM-20 gene has previously been detected in Salmonella Paratyphi B dTþ from poultry in the Netherlands. The strain Salmonella Paratyphi B dTþ 63.48, kindly donated to us for further investigation, was compared with E. coli 1248. The blaTEM-20 gene was also carried by a conjugative plasmid in the Salmonella Paratyphi B dTþ strain, and the two blaTEM-20 sequences were 100% identical. The transconjugant was resistant to b-lactams only with the same MICs as the E. coli 1248 transconjugant. Both blaTEM-20 plasmids were assigned to the incompatibility (Inc) group I1 by PCR-based replicon typing. Plasmid multilocus sequence typing (pMLST) for subtyping IncI1 plasmids was applied to the blaTEM-20 plasmids. 5 The following alleles (accession numbers) were obtained: repI1-1 (EU370458), ardA-2 (EU370453), trbA-pndC-2 (EU40466), sogS-3 (EU70463) and pilL-3 (EU370457). These alleles corresponded to sequence type 5, previously assigned to an IncI1 plasmid carrying blaTEM-52 identified in a Salmonella Infantis strain isolated in Belgium in 2005 (the alleles showed 100% nucleotide identity except for one nucleotide in the ardA locus). This blaTEM-52 plasmid is reported to be widely disseminated among different Salmonella serovars from poultry and humans in Belgium and France. The blaTEM-20 plasmids were large (.100 kb) and showed profiles that seemed to be similar when comparing the PstI and EcoRI restriction patterns with published restrictions patterns of the blaTEM-52 plasmid. 5–7

Methicillin‐resistant Staphylococcus pseudintermedius (MRSP) from healthy dogs in Norway – occurrence, genotypes and comparison to clinical MRSP

The aim of the study was to investigate the occurrence of methicillin‐resistant Staphylococcus pseudintermedius (MRSP) in healthy dogs and further to determine genetic relatedness between carrier isolates and clinical MRSP from dogs in Norway. A total of 189 healthy dogs visiting ten veterinary clinics were screened for MRSP during the period February to April 2013. Carrier isolates were susceptibility tested with disk diffusion and genotyped using multilocus sequence typing (MLST) and pulsed‐field gel electrophoresis (PFGE). Forty‐nine clinical MRSP were characterized for comparison. These isolates were collected from July 2008 to April 2013 and represent all MRSP index isolates from each MRSP‐positive dog detected in Norway until April 2013. Geographical distribution of all MRSP cases was investigated using the ArcGIS 9.3 Software. MRSP was detected from five (2.6%) healthy dogs, sampled at three different clinics. The isolates grouped into three sequence types (STs): ST252 (two isolates), ST71 (two isolates) and ST306 (one isolate). MRSP from dogs sampled at the same animal clinic belonged to the same ST and produced identical PFGE pattern. The 49 clinical MRSP grouped into 15 STs; ST258 (n = 17), ST71 (n = 10), and ST305 (n = 4) were the most prevalent. The MRSP carrier isolates were genetically related to MRSP variants from dogs with infections as ST306 (from a carrier) is related to ST258. MRSP ST252, found in two carriers, was also present among the clinical MRSP isolates. Altogether the MRSP isolates were genetically diverse and MRSP of other lineages than ST71 continues to disseminate in Norway. Susceptibility testing showed that MRSP isolates of the ST71 lineage were the most multiresistant. Our study showed that MRSP could be detected in healthy dogs without infections and with no recent history of antimicrobial therapy stressing the need for future monitoring, infection control and prudent use of antimicrobial agents.

Further diversity of the Staphylococcus intermedius group and heterogeneity in the MboI restriction site used for Staphylococcus pseudintermedius species identification.

Species identification of 200 beta-hemolysin–producing canine staphylococcal isolates was performed using a recently described polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method (based on MboI restriction of a pta gene fragment), supplemented with biochemical testing and sequencing of housekeeping genes. The PCR-RFLP method misclassified a small fraction (approximately 1%) of the Staphylococcus pseudintermedius population as a result of heterogeneity in the MboI restriction site. A potentially novel species within the Staphylococcus intermedius group (SIG) was found, having closest similarity to S. intermedius based on sequence comparison to the genes sodA, pta, hsp60, tuf, and full-length 16S ribosomal DNA, thus demonstrating further species diversity within the SIG.

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing blaCMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.

Integron, Plasmid and Host Strain Characteristics of Escherichia coli from Humans and Food Included in the Norwegian Antimicrobial Resistance Monitoring Programs

Antimicrobial resistant Escherichia coli (n=331) isolates from humans with bloodstream infections were investigated for the presence of class 1 and class 2 integrons. The integron cassettes arrays were characterized and the findings were compared with data from similar investigations on resistant E. coli from meat and meat products (n=241) produced during the same time period. All isolates were obtained from the Norwegian monitoring programs for antimicrobial resistance in human pathogens and in the veterinary sector. Methods used included PCR, sequencing, conjugation experiments, plasmid replicon typing and subtyping, pulsed-field-gel-electrophoresis and serotyping. Integrons of class 1 and 2 occurred significantly more frequently among human isolates; 45.4% (95% CI: 39.9-50.9) than among isolates from meat; 18% (95% CI: 13.2 -23.3), (p<0.01, Chi-square test). Identical cassette arrays including dfrA1-aadA1, aadA1, dfrA12-orfF-aadA2, oxa-30-aadA1 (class 1 integrons) and dfrA1-sat1-aadA1 (class 2 integrons) were detected from both humans and meat. However, the most prevalent cassette array in human isolates, dfrA17-aadA5, did not occur in isolates from meat, suggesting a possible linkage between this class 1 integron and a subpopulation of E. coli adapted to a human host. The drfA1-aadA1 and aadA1 class 1 integrons were found frequently in both human and meat isolates. These isolates were subjected to further studies to investigate similarities with regard to transferability, plasmid and host strain characteristics. We detected incF plasmids with pMLST profile F24:A-:B1 carrying drfA1-aadA1 integrons in isolates from pork and in a more distantly related E. coli strain from a human with septicaemia. Furthermore, we showed that most of the class 1 integrons with aadA1 were located on incF plasmids with pMLST profile F51:A-:B10 in human isolates. The plasmid was present in unrelated as well as closely related host strains, demonstrating that dissemination of this integron also could be attributed to clonal spread. In conclusion, among the systematically collected isolates from two different sources, some significant differences concerning integron prevalence and integron variants were observed. However, closely related plasmids as vehicles for specific class 1 integrons in isolates from meat and from a human with bloodstream infection were found. The occurrence of similar multi-resistance plasmids in bacteria from a food source and from a human clinical sample highlights the possible role of meat as a source of resistance elements for pathogenic bacteria.

Imported food and feed as contributors to the introduction of plasmid-mediated colistin-resistant Enterobacteriaceae to a ‘low prevalence’ country

Sir, In the last year, it has become evident that the plasmid-located colistin resistance gene mcr-1 has a worldwide distribution. Colistin is widely used in veterinary medicine, particularly in pigs and poultry, and the selection pressure in livestock production is a likely driver of the persistence and dissemination of mcr-1. It is not surprising that a large proportion of mcr-1-positive isolates described thus far originate from food-producing animal reservoirs. However, plasmidmediated colistin resistance is also reported from diseased and healthy humans. This is concerning, as colistin is particularly important as one of the last therapeutic options for human infections caused by multiresistant bacteria. Furthermore, plasmid-mediated colistin resistance has been documented for other animal categories such as companion animals, reptiles and birds as well as in environmental samples of sewage, water and vegetables. In Europe, sales of veterinary medicinal products, including the polymyxin colistin, are documented through the European Surveillance of Veterinary Antimicrobial Consumption reports, and wide variations are observed between countries. In Norway, the sale of polymyxin veterinary medicinal products was zero during the years 2010–15. Historical data also show that there has been no sale of polymyxins for use in animals during the years 1993–2009 in Norway (http://www.vetinst.no/overvaking/antibioti karesistens-norm-vet). Retrospective molecular screening of Enterobacteriaceae exhibiting colistin MICs above the breakpoint recommended by EUCAST (.2 mg/L), has demonstrated absence of mcr-1-positive isolates in samples from animals, food and feed originating from Norway for the years 2010–15. However, in 2016 the monitoring programme NORM-VET included samples of imported seafood and imported raw dog food. From two samples, one of each category, mcr-1-positive Escherichia coli were detected. The seafood sample was imported scampi from Bangladesh. The frozen dog food sample originated from the UK and contained turkey meat, fruit and vegetables. These samples were screened selectively for quinolone-resistant E. coli (QREC). Presumptive QREC were susceptibility tested using broth microdilution (Sensititre R ; TREK Diagnostic Systems Ltd, Thermo Scientific, Waltham, MA, USA). The two isolates had colistin MICs of 4 mg/L and were subjected to further investigations. WGS was performed using a MiSeq platform. Data were analysed using online tools available from the Center for Genomic Epidemiology (http://www. genomicepidemiology.org) or manually by search via CLC Main Workbench (CLC bio; QIAGEN, Aarhus, Denmark). WGS data documented mcr-1 in both isolates and the multilocus STs 3014 and 48. In the scampi isolate, a 132 kb contig contained both mcr-1 and the IncHI2 replicon marker, indicating that mcr-1 was located on this plasmid. Both isolates contained genes encoding resistance to several classes of antimicrobial agents (Table 1). Furthermore, a gene responsible for quaternary ammonium compound resistance (qacH) and tellurite resistance operons were present in both isolates. The dog food isolate also contained genes encoding mercury resistance. Conjugation experiments were performed in triplicate, but no transconjugants containing mcr-1 were obtained. Only one finding of plasmid-mediated colistin resistance has been reported in humans in Norway. This was an E. coli with mcr-1 from a traveller returning from India with enteritis. Human travellers have probably contributed to the worldwide spread of antimicrobial resistance. However, international trade in live animals, animal products, feed and food may represent an important route of dissemination, enabling the global spread of important resistance types and their introduction into geographic areas where they may be rare or absent. Humans may acquire antimicrobial-resistant bacteria through handling or consumption of contaminated food. Likewise, companion animals may acquire resistant bacteria via their feed. Sharing of common Enterobacteriaceae clones among family members and their dogs has been described, including mcr-1-positive E. coli, emphasizing the possibility of zoonotic transmission. Interestingly, we found mcr-1-positive isolates via screening for QREC. European studies have described that turkeys and broilers are associated with the highest prevalence of mcr-1. According to European surveillance data these animal species are also associated with high occurrences of QREC. So far, most detections of mcr-1 have been a result of retrospective screening of historical strain collections or WGS databases, whereas fewer reports have dealt with detection in ‘real time’. Our findings show the importance of risk-based screening of relevant samples to uncover possible sources of bacteria resistant to last-resort antimicrobials. Food products imported from areas with a high environmental load of resistant bacteria and with higher usage of antimicrobials should receive special attention. This is exemplified by the findings in the present study, but also by findings from Switzerland where