Andrea T. Feßler

Characterization of methicillin-resistant Staphylococcus aureus isolates from food and food products of poultry origin in Germany.

ABSTRACT During a survey of fresh chicken and turkey meat as well as chicken and turkey meat products for the presence of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Germany, 32 (37.2%) of 86 samples were MRSA positive. Twenty-eight of these MRSA isolates belonged to clonal complex 398 (CC398), which is widespread among food-producing animals. These CC398 isolates carried SCCmec elements of type IV or V and exhibited spa type t011, t034, t899, t2346 or t6574 and either the known dru types dt2b, dt6j, dt10a, dt10q, dt11a, dt11v, and dt11ab or the novel dru types dt6m, dt10as, and dt10at. In addition, two MRSA sequence type 9 (ST9) isolates with a type IV SCCmec cassette, spa type t1430, and dru type dt10a as well as single MRSA ST5 and ST1791 isolates with a type III SCCmec cassette, spa type t002, and dru type dt9v were identified. All but two isolates were classified as multiresistant. A wide variety of resistance phenotypes and genotypes were detected. All isolates were negative for the major virulence factors, such as Panton-Valentine leukocidin, toxic shock syndrome toxin 1, or exfoliative toxins. In contrast to the MRSA CC398 isolates, the four ST9, ST5, or ST1791 isolates harbored the egc gene cluster for enterotoxin G, I, M, N, O, and U genes. Although the relevance of contamination of fresh poultry meat or poultry products with MRSA is currently unclear, the presence of multiresistant and, in part, enterotoxigenic MRSA emphasizes the need for further studies to elucidate possible health hazards for consumers.

A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin

OBJECTIVES The oxazolidinone-resistant Enterococcus faecalis E349 from a human patient tested negative for the cfr gene and 23S rRNA mutations. Here we report the identification of a novel oxazolidinone resistance gene, optrA, and a first investigation of the extent to which this gene was present in E. faecalis and Enterococcus faecium from humans and food-producing animals. METHODS The resistance gene optrA was identified by whole-plasmid sequencing and subsequent cloning and expression in a susceptible Enterococcus host. Transformation and conjugation assays served to investigate the transferability of optrA. All optrA-positive E. faecalis and E. faecium isolates of human and animal origin were analysed for their MICs and their genotype, as well as the location of optrA. RESULTS The novel plasmid-borne ABC transporter gene optrA from E. faecalis E349 conferred combined resistance or elevated MICs (when no clinical breakpoints were available) to oxazolidinones (linezolid and tedizolid) and phenicols (chloramphenicol and florfenicol). The corresponding conjugative plasmid pE349, on which optrA was located, had a size of 36 331 bp and also carried the phenicol exporter gene fexA. The optrA gene was functionally expressed in E. faecalis, E. faecium and Staphylococcus aureus. It was detected more frequently in E. faecalis and E. faecium from food-producing animals (20.3% and 5.7%, respectively) than from humans (4.2% and 0.6%, respectively). CONCLUSIONS Enterococci with elevated MICs of linezolid and tedizolid should be tested not only for 23S rRNA mutations and the gene cfr, but also for the novel resistance gene optrA.

The diversity of antimicrobial resistance genes among staphylococci of animal origin

Staphylococci of animal origin harbor a wide variety of resistance genes. So far, more than 40 different resistance genes have been identified in staphylococci from animals. This includes genes that confer resistance to virtually all classes of antimicrobial agents approved for use in animals, such as penicillins, cephalosporins, tetracyclines, macrolides, lincosamides, phenicols, aminoglycosides, aminocyclitols, pleuromutilins, and diaminopyrimidines. The gene products of some of these resistance genes confer resistance to only specific members of a class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into three major categories: (i) enzymatic inactivation, (ii) active efflux, or (iii) protection/modification/replacement of the cellular target sites of the antimicrobial agents. Mobile genetic elements, in particular plasmids and transposons, play a major role as carriers of antimicrobial resistance genes in animal staphylococci. They facilitate the exchange of resistance genes with staphylococci of human origin but also with other Gram-positive bacteria.

Characterization of methicillin-resistant Staphylococcus aureus CC398 obtained from humans and animals on dairy farms.

In this study MRSA isolates from dairy farms were investigated for their genetic relationships and antimicrobial susceptibility. In total, 125 MRSA isolates from 26 dairy farms were studied, including isolates from milk samples (n=46), dairy cattle (n=24), calves (n=6), dust samples from pig (n=16) and veal calf sheds (n=1), dogs (n=2), a horse, a sheep and humans (n=28). CC398-specific PCRs, spa typing, SCCmec typing and ApaI macrorestriction analysis were conducted. Susceptibility testing was performed by broth microdilution. All 125 isolates belonged to CC398. Eight spa types (t011, t108, t034, t567, t1184, t1451, t2287 and t3934) were detected. SCCmec elements of types IV (n=48) and V (n=67) were identified with 10 isolates being non-typeable. Six main macrorestriction patterns - with up to 23 sub-patterns - and twelve resistance patterns were identified. Sixty-eight isolates showed a multiresistance phenotype. Farm-by-farm analysis revealed different scenarios: in some farms, the MRSA CC398 isolates from dairy cattle, humans, pig sheds and/or sheep were indistinguishable suggesting an interspecies exchange of the same MRSA CC398 subtype. In other farms, several MRSA CC398 subtypes were detected in different host species/sources with occasionally even more than one MRSA CC398 subtype from the same host species/source. These latter results may suggest that either different MRSA subtypes associated with humans or animals have been imported into the respective farm or that one MRSA CC398 strain has undergone diversification, reflected by more or less expanded changes in PFGE patterns, spa type or resistance pattern, during colonization of different hosts on the same farm.

Co-location of the oxazolidinone resistance genes optrA and cfr on a multiresistance plasmid from Staphylococcus sciuri

OBJECTIVES To identify and characterize the oxazolidinone/phenicol resistance gene optrA in Staphylococcus isolates. METHODS Fifty porcine staphylococci with florfenicol MICs of ≥16 mg/L were screened by PCR for the presence of the optrA gene. Transferability of optrA was examined by transformation and conjugation. Functionality of this gene was confirmed by cloning and expression in a susceptible Staphylococcus host. The optrA-carrying plasmid was completely sequenced and analysed. RESULTS A single Staphylococcus sciuri was optrA positive. This isolate carried the optrA gene on the 60 563 bp multiresistance plasmid pWo28-3, which also harboured the resistance genes, cfr, fexA, aadD, ble and aacA-aphD. Plasmid pWo28-3 is composed of three regions (A, B and C). Region A, which harboured all resistance genes except optrA, showed ≥99.8% nucleotide sequence identity to the corresponding region of plasmids pJP1 and pJP1-like from Jeotgalicoccus pinnipedialis and Staphylococcus lentus, respectively. The optrA gene located in region B differed from the optrA gene of the Enterococcus faecalis plasmid pE349 by four nucleotide substitutions, which also resulted in amino acid substitutions. This optrA variant also conferred resistance to oxazolidinones and phenicols in staphylococci. The 28 genes in region C represent the plasmid backbone and were apparently acquired from staphylococci, enterococci and nosocomiicocci. CONCLUSIONS This is the first report of the optrA gene in staphylococci and of the coexistence of optrA and cfr on the same plasmid. Dissemination of this plasmid will substantially limit the efficacy of oxazolidinones. Surveillance of optrA in staphylococci of both human and animal origin is urgently warranted.

Plasmid‐mediated resistance to protein biosynthesis inhibitors in staphylococci

Protein biosynthesis inhibitors (PBIs) represent powerful antimicrobial agents for the control of bacterial infections. In staphylococci, numerous resistance genes are known to be involved in resistance to PBIs, most of which mediate resistance to a specific class/subclass of PBIs, though a few genes do confer a multidrug resistance phenotype—up to five classes/subclasses of PBIs. Plasmids play a key role in the dissemination of PBI resistance among staphylococci, as they primarily carry plasmid‐borne PBI resistance genes; however, plasmids also can be vectors for transposon‐borne PBI resistance genes. Small plasmids that carry single PBI resistance genes are widespread among staphylococci of human and animal origin. Various mechanisms exist by which they can recombine, form cointegrates, or integrate into chromosomal DNA or larger plasmids. We provide an overview of the current knowledge of plasmid‐mediated PBI resistance in staphylococci, with particular reference to the currently known PBI resistance genes, their association with mobile genetic elements, and the recombination/integration processes that control their mobility.

Novel erm(T)-Carrying Multiresistance Plasmids from Porcine and Human Isolates of Methicillin-Resistant Staphylococcus aureus ST398 That Also Harbor Cadmium and Copper Resistance Determinants

ABSTRACT This study describes three novel erm(T)-carrying multiresistance plasmids that also harbor cadmium and copper resistance determinants. The plasmids, designated pUR1902, pUR2940, and pUR2941, were obtained from porcine and human methicillin-resistant Staphylococcus aureus (MRSA) of the clonal lineage ST398. In addition to the macrolide-lincosamide-streptogramin B (MLSB) resistance gene erm(T), all three plasmids also carry the tetracycline resistance gene tet(L). Furthermore, plasmid pUR2940 harbors the trimethoprim resistance gene dfrK and the MLSB resistance gene erm(C), while plasmids pUR1902 and pUR2941 possess the kanamycin/neomycin resistance gene aadD. Sequence analysis of approximately 18.1 kb of the erm(T)-flanking region from pUR1902, 20.0 kb from pUR2940, and 20.8 kb from pUR2941 revealed the presence of several copies of the recently described insertion sequence ISSau10, which is probably involved in the evolution of the respective plasmids. All plasmids carried a functional cadmium resistance operon with the genes cadD and cadX, in addition to the multicopper oxidase gene mco and the ATPase copper transport gene copA, which are involved in copper resistance. The comparative analysis of S. aureus RN4220 and the three S. aureus RN4220 transformants carrying plasmid pUR1902, pUR2940, or pUR2941 revealed an 8-fold increase in CdSO4 and a 2-fold increase in CuSO4 MICs. The emergence of multidrug resistance plasmids that also carry heavy metal resistance genes is alarming and requires further surveillance. The colocalization of antimicrobial resistance genes and genes that confer resistance to heavy metals may facilitate their persistence, coselection, and dissemination.

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

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.

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

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.

Genetic environment of the transferable oxazolidinone/phenicol resistance gene optrA in Enterococcus faecalis isolates of human and animal origin

OBJECTIVES Aim of this study was to analyse 17 non-related Enterococcus faecalis isolates of human and animal origin for the genetic environment of the novel oxazolidinone/phenicol resistance gene optrA. METHODS WGS and de novo assembly were conducted to analyse the flanking sequences of the optrA gene in the 17 E. faecalis isolates. When optrA was located on a plasmid, conjugation assays were performed to check whether the plasmids are conjugative and to confirm the resistance phenotype associated with these plasmids. RESULTS All nine optrA-carrying plasmids were conjugated into E. faecalis JH2-2 and the transconjugants exhibited the optrA-associated phenotype. In these plasmids, an IS1216E element was detected either upstream and/or downstream of the optrA gene. In eight plasmids, the phenicol exporter gene fexA was found upstream of optrA and in six plasmids, a novel erm(A)-related gene for macrolide-lincosamide-streptogramin B resistance was detected downstream of optrA. When located in the chromosomal DNA, the optrA gene was found downstream of the transcriptional regulator gene araC in four isolates, or downstream of the fexA gene in another four isolates. Integration of the optrA region into a Tn558-Tn554 hybrid, located in the chromosomal radC gene, was seen in two isolates. CONCLUSIONS The findings of the present study extend the current knowledge about the genetic environment of optrA and suggest that IS1216E elements play an important role in the dissemination of optrA among different types of enterococcal plasmids. The mechanism underlying the integration of optrA into the chromosomal DNA requires further investigation.