Methicillin-resistant Staphylococcus aureus ST398 in Humans and Animals, Central Europe

Isolates found in persons and animals in Germany and Austria show a genetic relationship

Genome-Wide Association Studies for the Detection of Genetic Variants Associated With Daptomycin and Ceftaroline Resistance in Staphylococcus aureus

Background: As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For S. aureus this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical S. aureus isolates. Materials/methods: To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using in silico analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes. Results: GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within mprF and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of mprF. Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome mec (SCCmec) revealed various mecA SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mecA mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in S. aureus. Conclusion: We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in S. aureus. However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.Peer Reviewe

Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of <it>S. aureus </it>infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of <it>S. aureus</it>, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 <it>S. aureus </it>isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods.</p> <p>Results</p> <p>All the <it>S. aureus </it>isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole), respectively (Table <tblr tid="T1">1</tblr>). There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of <it>S. aureus </it>resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 <it>spa </it>types were identified in the study, and the predominant <it>spa </it>type among the methicillin-susceptible <it>S. aureus </it>(MSSA) isolates was t084 (13 isolates). The t037-ST241-SCC<it>mec</it>III type was the only clone identified in Maiduguri (North-East Nigeria) while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCC<it>mec</it>V; t008-ST94-SCC<it>mec</it>IV; t002-ST5-SCC<it>mec</it>V; t064-ST8-SCC<it>mec</it>V) was observed. The toxin genes <it>seh </it>and <it>etd </it>were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and associated with clonal complexes CC1, CC30, CC121 and with sequence type ST152.</p> <tbl id="T1"> <title> <p>Table 1</p> </title> <caption> <p>Antibiotic resistance profile of <it>S. aureu</it><it>s </it>(MSSA and MRSA) from Nigeria</p> </caption> <tblbdy cols="4"> <r> <c> <p/> </c> <c cspan="3" ca="left"> <p><b>Number (%) of resistant isolates among</b>:</p> </c> </r> <r> <c ca="left"> <p><b>Antibiotic</b></p> </c> <c ca="left"> <p><b>MSSA</b></p> <p><b>(n = 57)</b></p> </c> <c ca="left"> <p><b>MRSA</b></p> <p><b>(n = 11)</b></p> </c> <c ca="left"> <p><b>Total</b></p> <p><b>(n = 68)</b></p> </c> </r> <r> <c cspan="4"> <hr/> </c> </r> <r> <c ca="left"> <p>Penicillin</p> </c> <c ca="left"> <p>49 (86)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>60 (88.2)</p> </c> </r> <r> <c ca="left"> <p>Oxacillin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>11 (16.2)</p> </c> </r> <r> <c ca="left"> <p>Teicoplanin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Vancomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Gentamicin</p> </c> <c ca="left"> <p>1 (1.8)</p> </c> <c ca="left"> <p>9 (81.8)</p> </c> <c ca="left"> <p>10 (14.7)</p> </c> </r> <r> <c ca="left"> <p>Tetracycline</p> </c> <c ca="left"> <p>27 (47.4)</p> </c> <c ca="left"> <p>11 (100)</p> </c> <c ca="left"> <p>38 (55.9)</p> </c> </r> <r> <c ca="left"> <p>Ciprofloxacin</p> </c> <c ca="left"> <p>12 (21.1)</p> </c> <c ca="left"> <p>8 (72.7)</p> </c> <c ca="left"> <p>20 (29.4)</p> </c> </r> <r> <c ca="left"> <p>Moxifloxacin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>7 (63.6)</p> </c> <c ca="left"> <p>7 (10.3)</p> </c> </r> <r> <c ca="left"> <p>Trimethoprim/sulfamethoxazole</p> </c> <c ca="left"> <p>39 (68.4)</p> </c> <c ca="left"> <p>10 (90.9)</p> </c> <c ca="left"> <p>49 (72.1)</p> </c> </r> <r> <c ca="left"> <p>Phosphomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Fusidic acid</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Erythromycin</p> </c> <c ca="left"> <p>2 (3.5)</p> </c> <c ca="left"> <p>6 (54.5)</p> </c> <c ca="left"> <p>8 (11.8)</p> </c> </r> <r> <c ca="left"> <p>Clindamycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>6 (54.5)</p> </c> <c ca="left"> <p>6 (8.8)</p> </c> </r> <r> <c ca="left"> <p>Rifampicin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Daptomycin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Mupirocin</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Linezolid</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> <r> <c ca="left"> <p>Tigecycline</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> <c ca="left"> <p>0 (0)</p> </c> </r> </tblbdy> </tbl> <p>Conclusions</p> <p>The use of phenotypic and molecular methods provided useful information on antibiotic resistance and molecular diversity of <it>S. aureus </it>in Nigeria. The high proportion of PVL-positive MSSA isolates affiliated to various clonal complexes and detected in all the health institutions is a major concern, both as a source of severe infections and as a potential reservoir that could lead to the emergence of PVL-positive MRSA. This study presents the first baseline information on the nature of the antibiotic resistance genes from <it>S. aureus </it>isolates in Nigeria. There is the need to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.</p

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches

Mutations in the gdpP gene are a clinically relevant mechanism for β-lactam resistance in meticillin-resistant Staphylococcus aureus lacking mec determinants

In Staphylococcus aureus, resistance to β-lactamase stable β-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus. A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for β-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus), which may lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.Peer Reviewe

Comparison of Different Phenotypic Approaches to Screen and Detect mecC-Harboring Methicillin-Resistant Staphylococcus aureus

Similar to mecA, mecC confers resistance against beta-lactams, leading to the phenotype of methicillin-resistant Staphylococcus aureus (MRSA). However, mecC-harboring MRSA strains pose special difficulties in their detection. The aim of this study was to assess and compare different phenotypic systems for screening, identification, and susceptibility testing of mecC-positive MRSA isolates. A well-characterized collection of mecC-positive S. aureus isolates (n 111) was used for evaluation. Routinely used approaches were studied to determine their suitability to correctly identify mecC-harboring MRSA, including three (semi)automated antimicrobial susceptibility testing (AST) systems and five selective chromogenic agar plates. Additionally, a cefoxitin disk diffusion test and an oxacillin broth microdilution assay were examined. All mecC-harboring MRSA isolates were able to grow on all chromogenic MRSA screening plates tested. Detection of these isolates in AST systems based on cefoxitin and/or oxacillin testing yielded overall positive agreements with the mecC genotype of 97.3% (MicroScan WalkAway; Siemens), 91.9% (Vitek 2; bioMérieux), and 64.9% (Phoenix, BD). The phenotypic resistance pattern most frequently observed by AST devices was “cefoxitin resistance/oxacillin susceptibility,” ranging from 54.1% (Phoenix) and 83.8% (Vitek 2) to 92.8% (WalkAway). The cefoxitin disk diffusion and oxacillin broth microdilution assays categorized 100% and 61.3% of isolates to be MRSA, respectively. The chromogenic media tested confirmed their suitability to reliably screen for mecC-harboring MRSA. The AST systems showed false-negative results with varying numbers, misidentifying mecC-harboring MRSA as methicillin-susceptible S. aureus. This study underlines cefoxitin’s status as the superior surrogate mecC-positive MRSA marker.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I–IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.Peer Reviewe

Single-Nucleotide Polymorphism Genotyping Identifies a Locally Endemic Clone of Methicillin-Resistant Staphylococcus aureus

We developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed ‘t003-X’) within the radiation of methicillin-resistant Staphylococcus aureus (MRSA) ST225. The TaqMan assay achieved 98% typeability and results were fully concordant with DNA sequencing. By applying this assay to 305 ST225 isolates from an international collection, we demonstrate that clade t003-X is endemic in a single acute-care hospital in Germany at least since 2006, where it has caused a substantial proportion of infections. The strain was also detected in another hospital located 16 kilometers away. Strikingly, however, clade t003-X was not found in 62 other hospitals throughout Germany nor among isolates from other countries, and, hence, displayed a very restricted geographical distribution. Consequently, our results show that SNP-typing may be useful to identify and track MRSA clones that are specific to individual healthcare institutions. In contrast, the spatial dissemination pattern observed here had not been resolved by other typing procedures, including multilocus sequence typing (MLST), spa typing, DNA macrorestriction, and multilocus variable-number tandem repeat analysis (MLVA)

Rare Occurrence of Methicillin-Resistant Staphylococcus aureus CC130 with a Novel mecA Homologue in Humans in Germany

MRSA CC130 containing the mecA homologue mecALGA251 were reported from the UK and from Denmark so far from cattle and humans. Here we report on 11 MRSA CC130 among a sample of 12691 isolates of human origin collected from January 2006 until June 2011. MRSA CC130 grew insufficiently on chromogernic agar plates for detection of MRSA; the agglutination test for presence of PBP2a was negative. We designed primers for specific detection of mecALGA251 as well as for concomitant detection of both, mecLGA251 and mecA. As already described, the isolates exhibited spa-types t843, t1736, and t1773. The ccrA homologue indicated the presence SCCmecXI. When subjected to further characterization by means of a commercially available microarray the isolates were negative for sak chp, and scn, and as expected positive for hla, untruncated hlb, and hld. They furthermore contained edinB, aur, slpA, slpB, slpE. From genes coding for surface and cell wall associated products the ica-operon, cap8, clfA, clfF, ebpS, fnbA, fnbB, sdrC were detected but not cna. The isolates were negative for enterotoxin genes and tst, as well as for eta, and etb; agr-type was III

A Timescale for Evolution, Population Expansion, and Spatial Spread of an Emerging Clone of Methicillin- Resistant Staphylococcus aureus

Abstract Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994