Showing papers by "Stefan Schwarz published in 2012"

Staphylococcus aureus CC398: Host Adaptation and Emergence of Methicillin Resistance in Livestock

Abstract: Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

First report of the multidrug resistance gene cfr in Enterococcus faecalis of animal origin.

Abstract: The multiresistance gene cfr was identified for the first time in an Enterococcus faecalis isolate of animal origin. The 32,388-bp plasmid pEF-01, which carried the cfr gene, was sequenced completely. Three copies of the insertion sequence IS1216 were identified in pEF-01, and the detection of a cfr- and IS1216-containing amplicon by inverse PCR suggests that IS1216 may play a role in the dissemination of cfr by a recombination process.

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

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

Distribution of the Multidrug Resistance Gene cfr in Staphylococcus Species Isolates from Swine Farms in China

Abstract: A total of 149 porcine Staphylococcus isolates with florfenicol MICs of =16 µg/ml were screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. In total, 125 isolates carried either cfr (16 isolates), fexA (92 isolates), or both genes (17 isolates). The 33 cfr-carrying staphylococci, which included isolates of the species Staphylococcus cohnii, S. arlettae, and S. saprophyticus in which the cfr gene has not been described before, exhibited a wide variety of SmaI pulsed-field gel electrophoresis patterns. In 18 cases, the cfr gene was located on plasmids. Four different types of cfr-carrying plasmids-pSS-01 (n = 2; 40 kb), pSS-02 (n = 3; 35.4 kb), pSS-03 (n = 10; 7.1 kb), and pBS-01 (n = 3; 16.4 kb)-were differentiated on the basis of their sizes, restriction patterns, and additional resistance genes. Sequence analysis revealed that in plasmid pSS-01, the cfr gene was flanked in the upstream part by a complete aacA-aphD-carrying Tn4001-like transposon and in the downstream part by a complete fexA-carrying transposon Tn558. In plasmid pSS-02, an insertion sequence IS21-558 and the cfr gene were integrated into transposon Tn558 and thereby truncated the tnpA and tnpB genes. The smallest cfr-carrying plasmid pSS-03 carried the macrolide-lincosamide-streptogramin B resistance gene erm(C). Plasmid pBS-01, previously described in Bacillus spp., harbored a Tn917-like transposon, including the macrolide-lincosamide-streptogramin B resistance gene erm(B) in the cfr downstream region. Plasmids, which in part carry additional resistance genes, seem to play an important role in the dissemination of the gene cfr among porcine staphylococci.

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

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

Antimicrobial resistance of Staphylococcus pseudintermedius.

Abstract: Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus delphini together comprise the S. intermedius group (SIG). Within the SIG, S. pseudintermedius represents the major pathogenic species and is involved in a wide variety of infections, mainly in dogs, but to a lesser degree also in other animal species and humans. Antimicrobial agents are commonly applied to control S. pseudintermedius infections; however, during recent years S. pseudintermedius isolates have been identified that are meticillin-resistant and have also proved to be resistant to most of the antimicrobial agents approved for veterinary applications. This review deals with the genetic basis of antimicrobial resistance properties in S. pseudintermedius and other SIG members. A summary of the known resistance genes and their association with mobile genetic elements is given, as well as an update of the known resistance-mediating mutations. These data show that, in contrast to other staphylococcal species, S. pseudintermedius seems to prefer transposon-borne resistance genes, which are then incorporated into the chromosomal DNA, over plasmid-located resistance genes.

Erratum: Acquired antibiotic resistance genes: an overview

Abstract: A commentary on http://www.frontiersin.org/Antimicrobials,_Resistance_and_Chemotherapy/10.3389/fmicb.2011.00203/abstract Acquired antibiotic resistance genes: an overview by van Hoek, A. H. A. M., Mevius, D., Guerra, B., Mullany, P., Roberts, A. P., and Aarts, H. J. M. (2011). Front. Microbio. 2:203. doi: 10.3389/fmicb.2011.00203 Dr. Marilyn C. Roberts and Dr. Stefan Schwarz have contacted the authors of the original publication with several comments and suggestions to better harmonize the correct nomenclature of the antibiotic resistance genes, as the gene names were not always correctly presented in the various tables given. Authors often pick their own gene names which in many cases have been approved for use for other genetically distinct genes or give names to determinants which were already given an approved designated name. Therefore, we (Dr. Marilyn C. Roberts and Dr. Stefan Schwarz and Dr. Henk J. M. Aarts on behalf of the authors of the original publication) would like to present here the correct nomenclature and mechanistic features of the antibiotic resistance genes belonging to the following classes: Aminoglycosides (Table ​(Table1),1), Phenicols (Table ​(Table3),3), Macrolides–Lincosamides–Streptogramin B (Table ​(Table4),4), Quinolones (Table ​(Table5),5), Tetracyclines (Table ​(Table6),6), and Trimethoprim (Table ​(Table7).7). In addition some additional information is given on the various classes of antibiotic resistance genes as also a section regarding the antibiotic class Oxazolidinones has been added. Table ​Table22 was correctly displayed by van Hoek et al. (2011) but has been updated. Table 1 Acquired aminoglycoside resistance genes*. Table 2 β-lactamases and ESBLs families. Table 3 Acquired phenicol resistance genes*. Table 4 Acquired macrolide-lincosamide-streptogramin B (MLS) resistance genes*. Table 5 Acquired quinolone resistance genes*. Table 6 Acquired tetracycline resistance genes*. Table 7 Acquired trimethoprim resistance genes*. To the subsection dealing with the “Resistance mechanisms” of the AMINOGLYCOSIDES we would like to add that to date six additional methylases have been reported, i.e., npmA, rmtA, rmtB, rmtC, rmtD, and rmtE (Courvalin, 2008; Doi et al., 2008; Davis et al., 2010). Futhermore, that within the three major classes (AAC, ANT, and APH) an additional subdivision can be made based on the enzymes' target sites within the aminoglycoside molecules: i.e., there are four acetyltransferases: AAC(1), AAC(2′), AAC(3), and AAC(6′); five nucleotidyltransferases: ANT(2″), ANT(3″), ANT(4′), ANT(6), and ANT(9); and seven phosphotransferases: APH(2″), APH(3′), APH(3″), APH(4), APH(6), APH(7″), and APH(9). To the subsection β-LACTAM, Resistance, mechanisms we would like to add that in recent years acquired genes encoding ESBLs have become a major concern (Bradford, 2001). Over time, the genes for the parent enzymes blaTEM−1, blaTEM−2, and blaSHV−1 have undergone point mutations which resulted in amino acid substitutions that changed the substrate spectrum to that of ESBLs, starting with blaTEM−3 and blaSHV−2 (Bradford, 2001). Because chloramphenicol is not an actual antibiotic class the subsection of CHLORAMPHENICOL should be called PHENICOLS. Concerning the history of PHENICOLS, it is worthwhile to know the first antibiotic, chloramphenicol, originally referred to as chloromycetin, was isolated already in 1947 from Streptomyces venezuelae (Ehrlich et al., 1947). Besides the inactivating enzymes (chloramphenicol acetyltransferases), there are also reports on other phenicol resistance systems, such as the inactivation by phosphotransferases, mutations of the target site, permeability barriers, and efflux systems (Schwarz et al., 2004). Of the latter mechanism, cmlA and floR are the most commonly known genes in Gram-negative bacteria (Bissonnette et al., 1991; Briggs and Fratamico, 1999). The macrolides (subsection MACROLIDES–LINCOSAMIDES–STREPTOGRAMIN B) have a similar mode of antibacterial action, comparable antibacterial spectra and in part overlapping binding sites at the ribosome as two other antibiotic classes, i.e., lincosamides and streptogramin antibiotics (comprising streptogramin A and B compounds that act synergistically). Consequently, these antibiotics, although chemically distinct, have been clustered together as MLS antibiotics (Roberts, 1996). Macrolides, lincosamides and streptogramins all inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria (Weisblum, 1995; Roberts, 2002). To Resistance mechanisms of the subsection MACROLIDES–LINCOSAMIDES–STREPTOGRAMIN B. Shortly after the introduction of erythromycin into clinical setting in the 1950s, bacterial resistance to this antibiotic was reported for the first time in staphylococci (Weisblum, 1995). Since then a large number of bacteria have been identified that are resistant to MLS due to the presence of various different genes. The resistance determinants responsible include rRNA methylases that modify the ribosomal target sites, ABC transporters, and efflux proteins of the Major Facilitator Superfamily, as well as genes for inactivating enzymes (Roberts et al., 1999; Roberts, 2008). The latter group can be further subdivided into esterases, lyases, phosphorylases, and transferases (Table ​(Table44). The most common mechanism of MLSB resistance is due to the presence of rRNA methylases, encoded by the erm genes. These enzymes methylate the adenine residue(s) resulting in MLSB resistance. The methylated adenine(s) prevents the drugs from binding to the 50S ribosomal subunit. The other two mechanisms efflux and enzymatic inactivation result in resistance to only 1 or 2 classes of antibiotics belonging to the MLS group. There are currently 77 MLS resistance genes recognized. A new MLS gene must have <79% amino acid identity with all previously characterized MLS genes before receiving a unique name (Roberts et al., 1999; Roberts, 2008). For an actual list of the MLS acquired resistance genes we refer to the website of Dr. Marilyn Roberts, http://faculty.washington.edu/marilynr/. In addition to the subsection of QUINOLONES currently five families of qnr genes have been reported; qnrA (7 subtypes), qnrB (59 subtypes), qnrC (1 subtype), qnrD (1 subtype), and qnrS (8 subtypes) (Jacoby et al., 2008; Cattoir and Nordmann, 2009; Cavaco et al., 2009; Strahilevitz et al., 2009; Torpdahl et al., 2009). Another mechanism of conferring resistance to quinolones is represented by the plasmid-borne gene qepA, which codes for an efflux pump that can export hydrophilic fluoroquinolones, e.g., ciprofloxacin and enrofloxacin (Perichon et al., 2007; Yamane et al., 2007). A variant of this resistance pump, QepA2, was identified in an E. coli isolate from France (Cattoir et al., 2008). Regarding TETRACYCLINE, Resistance mechanisms, currently there are 45 different acquired tetracycline resistance determinants recognized (Roberts, 1996, 2005; Brown et al., 2008) (Table ​(Table6).6). For an up-to-date list of the acquired tetracycline resistance genes, we refer to the website of Dr. Marilyn Roberts, http://faculty.washington.edu/marilynr/. Among these, 26 of the tet genes, 2 of the otr genes and the only tcr determinant code for efflux pumps, whereas 11 tet genes and 1 otr gene code for ribosomal protection proteins (RPPs). The enzymatic inactivation mechanism can be attributed to 3 tet genes. The tet(U) determinant represents an unknown tetracycline resistance mechanism since its sequence does not appear to be related to either efflux or RPPs, nor to the inactivation enzymes. The efflux and RPP encoding genes are found in members of Gram-positive, Gram-negative, aerobic, as well as anaerobic bacteria. In contrast, the enzymatic tetracycline inactivation mechanism has so far only been identified in Gram-negative bacteria. The tet(M) has the broadest host range of all tetracycline resistance genes, whereas tet(B) gene has the widest range among the Gram-negative bacteria. In recent years published data indicate that there are increasing numbers of Gram-negative bacteria that carry tet genes originally identified in Gram-positive bacteria (Roberts, 2002). To the subsection TRIMETHOPRIM, Resistance mechanisms. Initially, the acquired DHFRs fell into two distinct families A and B, encoded by the dfrA and dfrB genes (Howell, 2005). Up to now 6 plasmid-mediated families can be distinguished with relatively few dfr determinants originating from Gram-positive bacteria (Table ​(Table7).7). The dfrK and dfrA28 genes are the newest additions to the trimethoprim resistance determinant family (Kadlec and Schwarz, 2009; Kadlec et al., 2011). In contrast to the latest reported DHFRs, the oldest families, dfrA and dfrB, each contain several members (Roberts, 2002; Levings et al., 2006). For example, the dfrA group accomodates over 30 published genes; however, unpublished, dfrA variants are also present in the public DNA libraries and some genes apparently have changed nomenclature (Table ​(Table77). Furthermore, we suggest an additional section concerning oxazolidinones.

A novel phenicol exporter gene, fexB, found in enterococci of animal origin

Abstract: Objectives: To investigate two porcine Enterococcus isolates for the genetic basis of phenicol resistance and to determine the location and the genetic environment of the novel resistance gene. Methods: A total of 391 isolates with reduced florfenicol susceptibility (MIC =16 mg/L), obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. Isolates that were negative in these PCRs were analysed for their species assignment and antimicrobial susceptibility. Plasmids were extracted and subjected to transformation and conjugation assays. Restriction fragments of the phenicol resistance plasmids were cloned and sequenced. The sequences obtained were analysed and compared with sequences deposited in the databases. Results: The two isolates, Enterococcus faecium EFM-1 and Enterococcus hirae EH-1, exhibited MICs of chloramphenicol and florfenicol of 64 mg/L and carried a new phenicol resistance gene, designated fexB. This gene codes for a phenicol exporter of 469 amino acids organized in 14 transmembrane domains. The fexB gene was located on the 35 kb pEFM-1 from E. faecium and on the 25.3 kb pEH-1 from E. hirae, respectively. Both plasmids were non-conjugative. The fexB gene was found to be embedded in virtually the same genetic environment of 14.8 kb in both plasmids. Conclusion: To the best of our knowledge, this is the first report of the new florfenicol exporter gene fexB. Based on its plasmid location, horizontal transfer from the enterococci to other bacteria is possible.

Detection of the staphylococcal multiresistance gene cfr in Escherichia coli of domestic-animal origin

Abstract: Objectives: To investigate the presence and the genetic environment of the multiresistance gene cfr in Escherichia coli found in domestic animals. Methods: A total of 1230 E. coli isolates, collected from pigs, chickens and ducks, were screened by PCR for the cfr gene. The location of the cfr gene was determined by Southern blotting, the transferability of cfr gene was tested by conjugation and transformation, and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. The location of the cfr promoter sequence was analysed by mapping the cfr transcription start site using rapid amplification of 5' cDNA ends (5' RACE). Results: Only a single strain from the nasal swab of a pig harboured the cfr gene. Southern blotting indicated that the cfr gene was located on a ~110 kb plasmid, designated pEC-01. A cfr-carrying segment of 1545 bp with a sequence identical to that of the cfr-harbouring plasmid pSCFS1 was flanked by two IS26 elements in the same orientation. The IS26 transposition created a new hybrid promoter in which the -35 region was part of the left inverted repeat of IS26 while the -10-like sequence was part of the original cfr upstream region. Conclusions: To the best of our knowledge, this is the first report of the cfr gene in a naturally occurring E. coli strain. Continued surveillance of the presence of the cfr gene in Gram-negative bacteria of domestic-animal origin is warranted.

Rapid Microarray-Based Identification of Different mecA Alleles in Staphylococci

Abstract: To screen isolates and to identify mecA alleles, published mecA sequences were analyzed, and a microarray for the rapid discrimination of mecA alleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated as mecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates of Staphylococcus spp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistant Staphylococcus aureus strains, in S. pseudintermedius, and in coagulase-negative species such as S. epidermidis, S. fleurettii, or S. haemolyticus. There was no correlation between the different mecA hybridization patterns and the SCCmec type. Determination of MICs showed that mecA alleles corresponding to only four of these nine patterns were associated with β-lactam resistance. The mecA alleles that did not confer β-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such as S. sciuri and S. vitulinus. Because of the diversity of sequences and the different impact on β-lactam susceptibility, the existence of different mecA alleles needs to be taken into account when designing diagnostic assays for the detection of mecA.

Detection of the staphylococcal multiresistance gene cfr in Macrococcus caseolyticus and Jeotgalicoccus pinnipedialis

Abstract: Objectives: To investigate the presence and the genetic environment of the multiresistance gene cfr in Jeotgalicoccus pinnipedialis and Macrococcus caseolyticus from pigs. Methods: A total of 391 bacterial isolates with florfenicol MICs =16 mg/L were obtained from nasal swabs of 557 individual pigs; of these, 75 Gram-positive isolates other than staphylococci and enterococci were screened by PCR for the presence of known florfenicol resistance genes. Species assignments of the cfr-carrying isolates were based on the results of biochemical profiling and 16S rDNA sequencing. The locations of the cfr gene were determined by Southern blotting. Regions flanking each cfr gene were sequenced by a modified random primer walking strategy, and the transferability of cfr was assessed by electrotransformation. Results: Two M. caseolyticus isolates and one J. pinnipedialis isolate were cfr positive. The cfr gene was located either on a 7057 bp plasmid, pSS-03, which was widely distributed among staphylococci of pig origin, or on the ~53 kb plasmid pJP1. The region of pJP1 that included the cfr gene and the adjacent IS21-558, showed 99.7% identity to the corresponding region of plasmid pSCFS3. In addition, the genes aadD?+?aacA-aphD, ble and erm(C), coding for aminoglycoside, bleomycin and macrolide-lincosamide-streptogramin B resistance, respectively, were also identified on plasmid pJP1. Conclusions: This study showed that plasmids carrying the multidrug resistance gene cfr are present in two new genera of commensal and environmental bacteria, Macrococcus and Jeotgalicoccus. This observation underlines the role of commensal and environmental flora in the dissemination of clinically important resistance genes, such as cfr.

Increased MICs of gamithromycin and tildipirosin in the presence of the genes erm(42) and msr(E)-mph(E) for bovine Pasteurella multocida and Mannheimia haemolytica

Abstract: Sir, Bovine respiratory disease (BRD) is one of the economically most important diseases in animal production, with global losses of the feedlot industry due to BRD being estimated to be over US$ 3 billion per year. Antimicrobial agents, particularly macrolides, are commonly used to combat bacteria involved in BRD, such as Mannheimia haemolytica, Pasteurella multocida and Histophilus somni. After the approval of the 16-membered macrolide tilmicosin (Micotil) in 1992 and the 15-membered triamilide tulathromycin (Draxxin) in 2005 for use in BRD, two new macrolides were approved during 2011 for the treatment of BRD. These are the 15-membered macrolide gamithromycin (Zactran) and the 16-membered macrolide tildipirosin (Zuprevo). Little is known about the mechanisms of resistance to these two new veterinary antimicrobial agents. Recently, three genes involved in macrolide resistance of P. multocida and M. haemolytica were identified: erm(42), coding for a novel rRNA methylase; msr(E), coding for an ABC transporter; and mph(E), coding for a macrolide phosphotransferase. The last two genes were present in an operon structure. To determine the role of erm(42) and msr(E)-mph(E) in macrolide and lincosamide resistance, these genes were cloned and expressed in P. multocida B130. A closer analysis of these clones for their MICs of selected macrolides and lincosamides revealed that erm(42) conferred resistance to erythromycin, tilmicosin and clindamycin, but not to the triamilide tulathromycin. Although erm(42)-carrying P. multocida B130 showed an 8-fold increase in the tulathromycin MIC to 16 mg/L (Table S1, available as Supplementary data at JAC Online), this MIC classified the P. multocida isolate as susceptible. In contrast, msr(E)-mph(E) conferred resistance to erythromycin, tilmicosin and tulathromycin, but not to the lincosamide clindamycin (Table S1). When the new macrolides became commercially available, we tested the aforementioned P. multocida B130 clones for their MICs of gamithromycin and tildipirosin by broth macrodilution according to CLSI recommendations. The recipient strain P. multocida B130 showed 8-fold lower MICs of 0.25 mg/L for both gamithromycin and tildipirosin, compared with tulathromycin (2 mg/L) (Table S1). In the presence of erm(42), the MIC of tildipirosin increased 128-fold to 32 mg/L, while that of gamithromycin increased only 16-fold to 4 mg/L. In the presence of msr(E)-mph(E), an opposite observation was made: the MIC of tildipirosin increased only 8-fold to 2 mg/L, while that of gamithromycin increased 256-fold to 64 mg/L. Based on these increases in the MICs, it appears that erm(42) has mainly an effect on the tildipirosin MIC, whereas msr(E)-mph(E) increases the gamithromycin MIC for P. multocida B130. To see whether naturally occurring P. multocida and M. haemolytica isolates from BRD cases that carry the genes erm(42) and/or msr(E)-mph(E) show similar MICs of gamithromycin and tildipirosin, a total of 40 P. multocida and 29 M. haemolytica isolates (Table 1), whose macrolide resistance gene status was determined by previously described PCR assays, were tested. These isolates were collected in the Pfizer Animal Health Susceptibility Surveillance Program for BRD between 1999 and 2007 from various states in the USA. The macrolide-susceptible P. multocida isolates (n1⁄48) showed low MICs of 0.25–0.5 mg/L for both gamithromycin and tildipirosin. Slightly higher MICs of 0.5–1 and 0.5–2 mg/L for gamithromycin and tildipirosin, respectively, were detected for the macrolidesusceptible M. haemolytica isolates (n1⁄47). These MICs are in agreement with the gamithromycin MIC50 and MIC90 values for 144 P. multocida and 142 M. haemolytica isolates obtained from animals enrolled in field studies in the USA. Moreover, the observed tildipirosin MICs are in the same range as determined for 105 P. multocida and 88 M. haemolytica isolates. Research letters

High Throughput Multiple Locus Variable Number of Tandem Repeat Analysis (MLVA) of Staphylococcus aureus from Human, Animal and Food Sources

Abstract: Staphylococcus aureus is a major human pathogen, a relevant pathogen in veterinary medicine, and a major cause of food poisoning. Epidemiological investigation tools are needed to establish surveillance of S. aureus strains in humans, animals and food. In this study, we investigated 145 S. aureus isolates recovered from various animal species, disease conditions, food products and food poisoning events. Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis (MLVA), known to be highly efficient for the genotyping of human S. aureus isolates, was used and shown to be equally well suited for the typing of animal S. aureus isolates. MLVA was improved by using sixteen VNTR loci amplified in two multiplex PCRs and analyzed by capillary electrophoresis ensuring a high throughput and high discriminatory power. The isolates were assigned to twelve known clonal complexes (CCs) and –a few singletons. Half of the test collection belonged to four CCs (CC9, CC97, CC133, CC398) previously described as mostly associated with animals. The remaining eight CCs (CC1, CC5, CC8, CC15, CC25, CC30, CC45, CC51), representing 46% of the animal isolates, are common in humans. Interestingly, isolates responsible for food poisoning show a CC distribution signature typical of human isolates and strikingly different from animal isolates, suggesting a predominantly human origin.

Detection of the novel vga(E) gene in methicillin-resistant Staphylococcus aureus CC398 isolates from cattle and poultry

Abstract: Sir, The genes vga(A) and vga(C) and the most recently identified gene, vga(E), as well as the gene cfr, mediate transferable resistance to pleuromutilins in staphylococci. The vga genes encode ABC transporters, which also export streptogramin A antibiotics and lincosamides, and the cfr gene encodes a methyltransferase that confers additional resistance to phenicols, lincosamides, oxazolidinones and streptogramin A antibiotics. All these genes are located on either plasmids or transposons. Point mutations in the domain V of 23S rRNA or in the rplC gene, encoding the ribosomal protein L3, are also known to mediate pleuromutilin resistance in staphylococci. In two previous studies on methicillin-resistant Staphylococcus aureus (MRSA) in dairy cattle (n1⁄425) and in poultry meat and poultry meat products (n1⁄432) and one ongoing study on MRSA from cattle (n1⁄411) and poultry (n1⁄42) collected in the GERM-Vet programme 2008–09, we detected pleuromutilin resistance in a total of nine MRSA isolates, which were negative by PCR for the cfr gene and the staphylococcal genes vga(A), vga(B) and vga(C), but also for the enterococcal gene vga(D) and the streptococcal gene lsa(C). Moreover, point mutations in the 23S rRNA and rplC genes of these isolates were not detected. These nine isolates originated from fresh chicken and turkey meat, turkey meat products, cattle with bovine clinical mastitis and a turkey with an infection of the musculoskeletal system (Table 1). The aim of the present study was to investigate these nine isolates for the presence of the most recently identified gene, vga(E). This gene has so far been found only in MRSA ST398 (where ST stands for sequence type) isolates of porcine origin in Switzerland. If not already done in previous studies, the MRSA isolates were subjected to spa, SCCmec and dru typing as well as two CC398-specific PCRs as described previously. All nine isolates were assigned to the clonal complex (CC) 398, and all but one shared SCCmec type V and showed spa type t034. One isolate from turkey meat had SCCmec type IV and showed spa type t011. Four different dru types were detected, with dru type dt6j found in six isolates and dru types dt10q, dt6m and dt11a in single isolates (Table 1). Despite the different origin of the isolates, susceptibility testing by broth microdilution following the recommendations given in the CLSI documents M31-A3 and M100-S21 revealed rather uniform susceptibility patterns, which included resistance to b-lactams, tetracyclines, trimethoprim, MLSB antibiotics, spectinomycin and tiamulin. The nine isolates differed only slightly in their classification as resistant or intermediate to quinupristin/ dalfopristin (Table 1). All nine isolates carried mecA and the b-lactamase operon blaZ-blaI-blaR. Tetracycline resistance was mediated by the gene tet(M), which was present either alone (n1⁄42) or in combination with tet(K) (n1⁄46) or with tet(K)+tet(L) (n1⁄41). The dihydrofolate reductase gene dfrK was detected in all but one of the trimethoprim-resistant isolates. One isolate carried the gene dfrS1 in addition to dfrK. The rRNA methylase gene erm(A) was detected in all isolates, either alone (n1⁄43) or in combination with erm(B) (n1⁄44) or erm(C) (n1⁄42). The spectinomycin resistance gene spc was also detected in all nine isolates. The simultaneous presence of the genes erm(A) and spc suggested the presence of a Tn554-related transposon. This assumption was supported by the PCR-detected linkage of these two genes. So far, the novel gene vga(E) has been identified in a limited number of porcine MRSA ST398-t034 in Switzerland. In these isolates, vga(E) proved to be part of the Tn554-like multidrug-resistance transposon Tn6133. This transposon consisted of a complete transposon, Tn554, in which a vga(E)containing DNA segment of 4787 bp was integrated between the erm(A) gene and a Tn554-associated reading frame (orf) of unknown function. PCR analysis of whole-cell DNA with primers specific for the vga(E) gene demonstrated that this novel pleuromutilin, lincosamide and streptogramin A resistance gene was present in the nine isolates of cattle and poultry origin collected in Germany. Sequence analysis of the vga(E) amplicons of two randomly chosen isolates (one from cattle and one from poultry) confirmed the specificity of the amplicons. To investigate whether a Tn6133 transposon was also present in the nine isolates of this study, two PCR assays were designed to prove the linkage of the vga(E) gene with its upstream and downstream regions. One primer pair specific for the 5′ end of vga(E) and the 3′ end of the Tn554-associated orf of unknown function (vgaE_fw 5′-GAAATATGGGAAATAGAAGATGG-3′, orf_rv 5′-TAGATTTGGCAAGA TCGAGC-3′; amplicon size 1685 bp; annealing temperature 528C) and the other primer pair specific for the erm(A) regulatory region and the 3′ end of vga(E) (ermA_fw 5′-CTAGCTCTT TGGTAAAATGTCC-3′, vgaE_rv 5′-TGATTCTCTAACCACTCTTC-3′; amplicon size 4977 bp; annealing temperature 508C) yielded fragments of the expected sizes in all nine isolates. These data, in combination with the proved linkage of erm(A) and spc, strongly suggest that the vga(E) gene is also located on a Tn6133 transposon in the bovine and avian MRSA CC398 isolates of this study.

Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Abstract: Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.

Methicillin-resistant Staphylococcus aureus ST9 from a case of bovine mastitis carries the genes cfr and erm(A) on a small plasmid

Abstract: Sir, Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide variety of infections in animals with bovine mastitis being the predominant staphylococcal infection in dairy cattle. MRSA isolates—mainly of multilocus sequence type (ST) 398—have recently been shown to be also involved in bovine mastitis. Although a variety of antimicrobial resistance genes have been identified in MRSA isolates from cases of bovine mastitis, the multidrug resistance gene cfr has not yet been identified in bovine CC398 isolates, although it has been found in a single porcine MRSA ST398 isolate and a single porcine methicillinsusceptible S. aureus ST9 isolate. Although pilot studies have indicated that MRSA ST9 isolates are commonly found among pigs in China, cfr-positive MRSA isolates of animal origin have not yet been reported in China. In 2010, during a routine surveillance study on antimicrobial resistance on dairy farms in China, an MRSA strain (designated SA16) was isolated from fresh raw milk of a cow suffering from mastitis. The MRSA strain was further characterized by multilocus sequence typing (MLST; http://saureus.mlst.net/), spa typing (http://spaserver.ridom.de) and SCCmec typing, as previously described. This strain belonged to ST9 (allelic profile 3-3-1-1-1-1-10), belonged to spa type t899 (allelic profile 07-16-23-02-34), and harboured an SCCmec element of type III. The strain displayed resistance to oxacillin (MIC1⁄4128 mg/L), erythromycin (MIC ≥64 mg/L) and clindamycin (MIC ≥64 mg/L), had a linezolid MIC of 4 mg/L and showed high florfenicol and tiamulin MICs of ≥128 mg/L and 256 mg/L, respectively. The genes mecA (oxacillin resistance), cfr and fexA (phenicol resistance) were detected by PCR and confirmed by sequencing of the respective amplicons. Plasmid preparation using the DNA midi kit (Qiagen, Hilden, Germany) identified an 7 kb plasmid, designated pMSA16. This plasmid was transformed into the recipient strain S. aureus RN4220 by electrotransformation using 0.2 cm cuvettes with a Gene Pulser apparatus at 2.5 kV (Bio-Rad, Munich, Germany). Transformants were selected on BHI agar containing erythromycin (15 mg/L) or florfenicol (10 mg/L). Southern blot analysis using the cfr and fexA amplicons as probes confirmed that the cfr gene was located on pMSA16 in the original strain and its transformants, while the fexA gene was most likely located on the chromosomal DNA. The pMSA16-harbouring transformants showed ≥4-fold increases in the MICs of chloramphenicol, florfenicol, clindamycin, linezolid and tiamulin, which are indicative for the cfr-associated resistance phenotype and demonstrated that the pMSA16-associated cfr gene is functionally active. In addition, the pMSA16-harbouring transformants showed an elevated MIC of erythromycin of ≥64 mg/L, which suggested that a macrolide resistance gene is also located on plasmid pMSA16. To gain insight into the structure and organization of plasmid pMSA16, the sequence of this 7054 bp plasmid was determined by primer walking. Using the open reading frame (ORF) finder software (http://www.ncbi.nlm.nih.gov/gorf/), five reading frames for proteins of ≥100 amino acids (aa) were identified in pMSA16 (Figure 1a). The rep gene codes for a 327 aa plasmid replication protein, which shares 100% and 98.5% aa identity with the same-sized RepU protein of a plasmid from the porcine S. aureus strain 7612628-4 (GenBank accession number JF968539) and the 326 aa RepU protein of the Staphylococcus saprophyticus plasmid pSES22, respectively. A macrolide-lincosamide–streptogramin B resistance gene coding for a 243 aa rRNA methylase was detected, the deduced aa sequence of which was indistinguishable from the same-sized Erm(A) protein of Tn554. Analysis of the erm(A)flanking regions identified a potential recombination site that included the translational stop codon of the erm(A) gene and might have contributed to the replacement of the pSES22-associated erm(C) gene by a Tn554-borne erm(A) gene (Figure 1b). At the other end, homology to Tn554 ended in the translational attenuator 135 bp upstream of the erm(A) translational start codon (Figure 1a and b). The erm(C) and erm(A) translational attenuators are composed of several pairs of inverted repeats, and areas characterized by inverted repeats are considered as preferential areas for illegitimate recombination events. Thus recombination in the erm(A)

In vitro antimicrobial activity of miconazole and polymyxin B against canine meticillin-resistant Staphylococcus aureus and meticillin-resistant Staphylococcus pseudintermedius isolates

Abstract: Background - Meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-resistant Staphylococcus pseudintermedius (MRSP) infections are increasingly reported in dogs, and these bacteria may be isolated from ear infections. Hypothesis/Objectives - The main aim of the present study was to investigate the in vitro antimicrobial activity of miconazole, polymyxin B and a combination of both against 24 canine MRSA and 50 canine MRSP isolates. The minimal inhibitory concentration (MIC) values of 12 other antimicrobial agents were also determined. Methods - All MIC values were determined according to a broth microdilution assay. Results - Acquired resistance was found to all tested agents, except for linezolid, miconazole and polymyxin B. The MIC values for miconazole and polymyxin B against MRSA were in the range of 4-8 and 8-64 µg/mL, respectively, while the MIC values for miconazole and polymyxin B against MRSP were in the range of 1-2 and 0.25-4 µg/mL, respectively. Using a combination of miconazole and polymyxin B, there was no evidence for enhanced in vitro activity of the combination (i.e. synergy) of both products. Nevertheless, MIC(90) values of the combination of these antimicrobial agents and of a commercial product containing both agents were at least 1000 times lower than the concentration present in the commercial product. Conclusions and clinical importance - These results indicate that the topical use of a combination of miconazole and polymyxin B in a 43.5:1 ratio may have potential for the treatment of MRSA-mediated and MRSP-associated otitis externa in dogs.

Detection of Pseudomonas aeruginosa isolates of the international clonal complex 11 carrying the blaPER-1 extended-spectrum β-lactamase gene in Greece

Abstract: Objectives: The extended-spectrum b-lactamase (ESBL) PER-1 initially disseminated among Pseudomonas aeruginosa strains in Turkey. Despite reports from other European countries, such strains have not been detected in Greece until now. We describe the first blaPER-1-positive P. aeruginosa isolates from Greece and their genetic environment. Methods: From January 2008 to December 2009, 287 consecutive non-duplicate P. aeruginosa isolates with reduced susceptibility or resistance to ceftazidime (MIC .8 mg/L) were screened for ESBL production with a modified boronic acid-based double-disc synergy test. Phenotypically ESBL-positive isolates were subjected to agar dilution, PFGE and multilocus sequence typing (MLST). Broad-spectrum bla genes were identified by PCR and sequencing. Plasmid analysis and conjugation experiments were performed. The location of the blaPER-1 gene was detected by Southern blotting and its genetic environment was characterized using inverse PCR. Results: Five isolates were phenotypically positive for ESBL production, exhibited resistance to cefepime, ceftazidime, aztreonam and meropenem, and carried the blaPER-1 gene. MLST showed that they belonged to sequence type (ST) 235, which belongs to the international clonal complex 11. Four isolates had the same PFGE pattern. Southern blotting revealed the chromosomal location of the blaPER-1 gene. Analysis of the blaPER-1 flanking regions showed identity to transposon Tn1213 downstream and 1406 bp upstream of blaPER-1. Further upstream, an orfA gene and ISPa12 were identified; both were truncated by the insertion of IS6100. Conclusions: This study confirmed the presence of PER-1-producing P. aeruginosa strains in Greece. The chromosomal location of blaPER-1, as part of a truncated transposon, suggests clonal expansion rather than horizontal gene transfer.

Unusual small plasmids carrying the novel resistance genes dfrK or apmA isolated from methicillin-resistant or -susceptible staphylococci

Abstract: OBJECTIVES: The aims of this study were to identify small staphylococcal plasmids that carry either the trimethoprim resistance gene dfrK or the apramycin resistance gene apmA and analyse them for their structure and organization with regard to their potential role as precursors of large multiresistance plasmids that carry these genes. METHODS: Trimethoprim- or apramycin-resistant staphylococci from the strain collections of the two participating institutions were investigated for the presence of plasmid-borne dfrK or apmA genes. The dfrK- or apmA-carrying plasmids were sequenced completely and compared with sequences deposited in the databases. RESULTS: Two small plasmids, the 4957 bp dfrK-carrying plasmid pKKS966 from porcine Staphylococcus hyicus subsp. hyicus and the 4809 bp apmA-carrying plasmid pKKS49 from porcine methicillin-resistant Staphylococcus aureus were identified. Structural analysis revealed that both plasmids had a similar organization, comprising a single resistance gene (dfrK or apmA), a plasmid replication gene (rep) and three partly overlapping genes for mobilization proteins (mobA, mobB and mobC). Comparisons showed 71%-82% amino acid identity between the Rep and Mob proteins of these two plasmids; however, distinctly lesser percentages of identity to Rep and Mob proteins of staphylococci and other bacteria deposited in the databases were detected. CONCLUSIONS: Both plasmids, pKKS966 and pKKS49, appeared not to be typical staphylococcal plasmids. The homology to larger plasmids that harbour the genes apmA and/or dfrK was limited to these resistance genes and their immediate upstream and downstream regions and thus suggested that these small plasmids were not integrated into larger plasmids.

Co-location of the multiresistance gene cfr and the novel streptomycin resistance gene aadY on a small plasmid in a porcine Bacillus strain

Abstract: Key Laboratory of Development and Evaluation of Chemical and Herbal Drugs for Animal Use, College of Veterinary Medicine, China Agricultural University, Beijing 100193, P. R. China; Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Holtystr. 10, 31535 Neustadt-Mariensee, Germany; Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA; Shandong Academy of Agricultural Sciences, Institute of Animal Husbandry and Veterinary Medicine, Jinan, 250100, P. R. China

Detection of qnr genes among Escherichia coli isolates of animal origin and complete sequence of the conjugative qnrB19-carrying plasmid pQNR2078

Abstract: Objectives: The aims of this study were to identify qnr genes among quinolone-resistant Escherichia coli isolates from defined disease conditions of companion and farm animals obtained in the BfT-GermVet study, and to gain insight into their localization and the organization of the qnr gene regions. Methods: The qnr genes were detected by PCR and confirmed by sequencing. qnr-positive isolates were checked for mutations in DNA gyrase and topoisomerase IV genes by PCR and sequencing of the quinolone resistance-determining regions. Multilocus sequence typing (MLST) was performed for the qnr-positive E. coli isolates. Plasmids harbouring qnr genes were transferred by conjugation into E. coli recipients, subjected to PCR-based replicon typing and plasmid-MLST, and one qnrB19-carrying plasmid was sequenced completely. Results: Only 2 of 417 E. coli isolates investigated carried qnr genes. Both isolates originated from horses and showed MLST type ST86. They harboured conjugative qnrB19-carrying plasmids, which proved to be indistinguishable by restriction analysis, belonged to incompatibility group IncN, showed plasmid-MLST type ST8 and did not carry other resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence IS26. One of these plasmids, pQNR2078, was sequenced completely and had a size of 42?379 bp. Except for the resistance gene region, plasmid pQNR2078 closely resembled the blaCTX-M-65-carrying plasmid pKC396 from E. coli. Conclusions: qnr genes were rarely detected among E. coli from animals in the BfT-GermVet study. The qnrB19 gene was detected on conjugative plasmids, with IS26 being likely involved in the mobility of qnrB19.

Target gene mutations among methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus with elevated MICs of enrofloxacin obtained from diseased food-producing animals or food of animal origin

Abstract: 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,