Tomasz Hauschild

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.

MULTIDRUG RESISTANT ACINETOBACTER BAUMANNII-THE ROLE OF ADEABC (RND FAMILY) EFFLUX PUMP IN RESISTANCE TO ANTIBIOTICS

Acinetobacter baumannii is an opportunistic pathogen which play the more and more greater role in the pathogenicity of the human. It is often attached with the hospital environment, in which is able easily to survive for many days even in adverse conditions. Acinetobacter baumannii is the species responsible for a serious nosocomial infections, especially in the intensive care units. Option of surviving in natural niches, and in the hospital environment could also be associated with the efflux pump mechanisms. Mechanisms of efflux universally appear in all cells (eukaryotic and prokaryotic) and play the physiological important role. In prokaryote, the main functions are evasion of such naturally produced molecules, removal of metabolic products and toxins. These pumps could also be involved in an early stage of infection, such as adhesion to host cells and the colonization. Importantly, they remove commonly used antibiotics from the cell in therapy of infections caused by these bacteria. Efflux pumps exemplify a unique phenomenon in drug resistance: a single mechanism causing resistance against several different classes of antibiotics. In Acinetobacter baumannii, the AdeABC efflux pump, a member of the resistance-nodulation-cell division family (RND), has been well characterized. Aminoglicosides, tetracyclines, erythromycin, chloramphenicol, trimethoprim, fluoroquinolones, some beta-lactams, and also recently tigecycline, were found to be substrates for this pump. Drugs, as substrates for the AdeABC pump, can increase the expression of the AdeABC genes, leading to multidrug resistance (MDR). From this reason, treatment failure and death caused by Acinetobacter baumannii infections or underlying diseases are common. Because the AdeABC pump is widespread in Acinetobacter baumannii, similarly to other pumps in Gram-negative and Gram-positive bacteria, exists a need of searching a new therapeutic solutions. Specific efflux inhibitors of pumps (EPIs), including AdeABC inhibitors, could be suppress the activity of pumps and restore the sensitivity of such important bacteria as Acinetobacter baumannii to commonly used antibiotic.

Identification and Characterization of Clinical Isolates of Members of the Staphylococcus sciuri Group

ABSTRACT A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.

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.

Metallo-beta-lactamases of Pseudomonas aeruginosa - a novel mechanism resistance to beta-lactam antibiotics

Since about twenty years, following the introduction into therapeutic of news beta-lactam antibiotics (broad-spectrum cephalosporins, monobactams and carbapenems), a very significant number of new beta-lactamases appeared. These enzymes confer to the bacteria which put them, the means of resisting new molecules. The genetic events involved in this evolution are of two types: evolution of old enzymes by mutation and especially appearance of new genes coming for some, from bacteria of the environment. Numerous mechanisms of enzymatic resistance to the carbapenems have been described in Pseudomonas aeruginosa. The important mechanism of inactivation carbapenems is production variety of b-lactam hydrolysing enzymes associated to carbapenemases. The metallo-beta-enzymes (IMP, VIM, SPM, GIM types) are the most clinically significant carbapenemases. P. aeruginosa posses MBLs and seem to have acquired them through transmissible genetic elements (plasmids or transposons associated with integron) and can be transmission to other bacteria. They have reported worldwide but mostly from South East Asia and Europe. The enzymes, belonging to the molecular class B family, are the most worrisome of all beta-lactamases because they confer resistance to carbapenems and all the beta-lactams (with the exception of aztreonam) and usually to aminoglycosides and quinolones. The dissemination of MBLs genes is thought to be driven by regional consumption of extended--spectrum antibiotics (e.g. cephalosporins and carbapenems), and therefore care must be taken that these drugs are not used unnecessarily.

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.

The ecological importance of the Staphylococcus sciuri species group as a reservoir for resistance and virulence genes.

The Staphylococcus sciuri species group includes five species that are most often presented as commensal animal-associated bacteria. The species of this group are Staphylococcus sciuri (with three subspecies), Staphylococcus lentus, Staphylococcus vitulinus, Staphylococcus fleurettii and Staphylococcus stepanovicii. Members of these group are commonly found in a broad range of habitats including animals, humans and the environment. However, those species have been isolated also from infections, both in veterinary and human medicine. Members of this group have been shown to be pathogenic, though infections caused by these species are infrequent. Furthermore, members of the S. sciuri species group have also been found to carry multiple virulence and resistance genes. Indeed, genes implicated in biofilm formation or coding for toxins responsible of toxic shock syndrome and multi-resistance, similar to those carried by Staphylococcus aureus, were detected. This group may thereby represent a reservoir for other bacteria. Despite its recognized abundance as commensal bacteria and its possible role as reservoir of virulence and resistance genes for other staphylococci, the S. sciuri species group is often considered harmless and, as such, not as well documented as, for example, S. aureus. More investigation into the role of the S. sciuri species group as commensal and pathogenic bacteria is required to fully assess its medical and veterinary importance.

Isolation and Molecular Characterization of Staphylococcus sciuri in the Hospital Environment

ABSTRACT Staphylococcus sciuri is a principally animal-associated bacterial species, but its clinical relevance for humans is increasing. Our study aimed to provide the first insight into the prevalence of this bacterium in a hospital environment. A 3-month surveillance was conducted in a hospital located in Belgrade, Serbia, and 1,028 samples taken from hands of medical personnel, medical devices, and various hospital surfaces were screened for S. sciuri presence. In total, 108 isolates were obtained, which resulted in a relatively high rate of colonization (10.5%). These isolates, along with 7 S. sciuri strains previously isolated in the same hospital (n = 115), were phenotypically and genotypically characterized. Antimicrobial susceptibility testing revealed that 73% of the strains were resistant to one or more antibiotics, with 4.3% strains displaying multiresistance. Examination of 16S-23S ribosomal DNA intergenic spacer length polymorphism identified the strains at the subspecies level, and 74 (64.3%) strains of S. sciuri subsp. sciuri, 37 (32.2%) strains of S. sciuri subsp. rodentium, and 4 (3.5%) strains of S. sciuri subsp. carnaticus were established. Pulsed-field gel electrophoresis (PFGE) analysis showed 21 distinct pulsotypes, including 17 main types and 4 subtypes. One dominant cluster with 62 strains was found, while 19 (90.5%) of the PFGE types and subtypes identified had 5 or fewer strains. The predominance of small PFGE clusters suggests that the ubiquitous presence of S. sciuri in the outside environment presents the continuous source for colonization of the hospital environment. The presence of one dominant PFGE cluster of strains indicates that some S. sciuri strains may be capable for adaptation to hospital environment conditions and continuous existence in this environment.

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene

ABSTRACT A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

Detection of a new mecC allotype, mecC2, in methicillin-resistant Staphylococcus saprophyticus

Sir, The mecA gene originally identified in methicillin-resistant Staphylococcus aureus (MRSA) encodes penicillin-binding protein 2a (PBP2a), which has low affinity for b-lactam antibiotics. So far, two mecA allotypes, mecA1 and mecA2, have been identified. The genes mecA1 (originally detected in Staphylococcus sciuri) and mecA2 (in Staphylococcus vitulinus) have about 80% and 90% nucleotide sequence identity, respectively, to mecA of S. aureus N315, the first fully sequenced MRSA strain. The allotypes of mecA are located on mobile genetic elements designated staphylococcal cassette chromosome mec (SCCmec). Until recently, 10 different types of SCCmec harbouring mecA (types I–X) as well as numerous subtypes have been described in staphylococci. In 2011, a novel mecA homologue, designated mecC, located in SCCmec type XI, was found in S. aureus strains LGA251 and M10/ 0061. The mecC gene shows only 69% nucleotide sequence identity and the corresponding protein only 63% amino acid sequence identity to mecA and PBP2a of S. aureus N315. Recently, the novel mecC allotype mecC1 (GenBank accession no. HE993884) was detected in oxacillinand cefoxitin-susceptible Staphylococcus xylosus strain S04009 from bovine mastitis and exhibited 93.5% identity at the DNA level to the mecC genes of S. aureus strains LGA251 and M10/0061. The inability of MecC1 from S. xylosus S04009 to confer oxacillin and cefoxitin resistance was due to a frameshift mutation close to the 5′ end of the mecC1 gene, which resulted in a truncated 64 amino acid product. During a survey of oxacillin resistance among staphylococci from wild small mammals (rodents and insectivores) by agar dilution, we detected one isolate (no. 210) with an oxacillin MIC of 32 mg/L and a cefoxitin MIC of 4 mg/L, which was negative by PCR for the mecA gene, from a common shrew (Sorex araneus L.). The isolate was identified by 16S rDNA sequence analysis (GenBank accession no. GQ222241) as Staphylococcus saprophyticus subsp. saprophyticus and further examined for the molecular basis of oxacillin resistance using primers mecC1+2_F and mecC1+2_R for PCR detection of the recently described mecC/mecC1 genes. A single PCR product of the expected size (457 bp) was obtained and sequenced on both strands to confirm the specificity of the amplicon. Sequence analysis showed that this PCR product had only 94.3% nucleotide sequence identity to mecC of S. aureus LGA251 and 94.7% to the new mecC1 allotype of S. xylosus S04009. Based on the assumption that the mecC gene of S. saprophyticus 210 is similar to mecC/mecC1 and is located in a similar genetic context as mecC/mecC1, i.e. bracketed by a blaZ gene in the downstream region and a mecR1 gene in the upstream region, another three primer pairs were designed to obtain the complete mecC gene sequence. The positions of the primers are shown in Figure 1. These primer pairs were: mecC-fw 5′-CTTGGTTATTCAAAGA TGACGA-3′ and mecC-rev 5′-ACGTCTTTAACATTAATCGCCA-3′ (amplicon size 1843 bp; annealing temperature 508C); blaZ-rev 5′-TCTAACACTGGTGAATATGG-3′ and mecC3 5′-GGTATGGAACGTG TAGTGAC-3′ (amplicon size 685 bp; annealing temperature 488C); and mecC4 5′-TCCCAATCTAATTTCCAATG-3′ and mecR1-rev 5′-AAGGCAATTTGYTTAATAGCAG-3′ (amplicon size 873 bp; annealing temperature 488C). Single PCR products of the expected sizes were obtained and cloned into the pGEM-T Easy vector (Promega, Poland). The recombinant plasmids were transformed into Escherichia coli JM109 and sequenced on both strands with the SP6 and T7 promoter primers. For sequencing of the mecC-fw/ mecC-rev amplicon, the primer pair mecC1+2_F/mecC1+2_R, previously used for amplification of the 457 bp mecC fragment, was also employed. Grouping of the sequences obtained revealed that the complete mecC gene of S. saprophyticus 210 (GenBank accession no. KF955540) comprised 1998 bp and encoded a protein of 665 amino acids. The novel mecC gene shared 92.9% identity with the mecC sequences of S. aureus strains LGA251 and M10/ 0061 as well as 94.5% identity with the mecC1 sequence of S. xylosus S04009. These values were below the recommended 95% cut-off value for a new allotype delineation. The deduced amino acid sequence of the MecC protein of S. saprophyticus 210 shared 92.3% identity with the MecC protein sequences of S. aureus LGA251 and M10/0061. These data indicate that the mecC gene of S. saprophyticus 210 represents a new allotype of mecC, herein referred to as mecC2. It should be noted that sequence analysis of the different overlapping amplicons showed that the sequences corresponding to primers mecC3, mecC4 and mecC1+2_R were conserved between the mecC, mecC1 and mecC2 sequences. In contrast, the primer sequences mecC1+2_F, mecC-fw and mecC-rev, as deduced from the mecC/mecC1 sequences deposited in GenBank, revealed one, two and one mismatches, respectively, in comparison with the newly identified mecC2 sequence. For the most specific detection of mecC2, it is suggested to use primers that exactly match the mecC2 sequence, such as mecC1+2_F (5′-AAGTTAATC AAAAATGGGTACAGC-3′), mecC2-fw (5′-CTTGGTTATTCAGAGAT AACGA-3′) and mecC2-rev (5′-ACGTCTTTAACATTAATTGCCA-3 ′). So far, the recently described gene mecC and its mecC1 allotype have been detected in only three coagulase-negative staphylococci: Staphylococcus stepanovicii from Eurasian lynx (Lynx lynx L.), S. xylosus from bovine mastitis and S. sciuri from