Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants.

Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS'fabI' was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense 'fabI' expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.

Bactericidal activity of bacteriophage endolysin HY-133 against Staphylococcus aureus in comparison to other antibiotics as determined by minimum bactericidal concentrations and time-kill analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC50/90 and MBC50/90 values were in the range 0.12-0.5 mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5 mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.

The Energy-Coupling Factor Transporter Module EcfAA'T, a Novel Candidate for the Genetic Basis of Fatty Acid-Auxotrophic Small-Colony Variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA'T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA'T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

In Vitro Susceptibility of Clinical Staphylococcus aureus Small-Colony Variants to ß-Lactam and Non-ß-Lactam Antibiotics.

The Staphylococcus aureus small-colony variant (SCV) phenotype has been associated with relapsing and antibiotic-refractory infections. However, little is known about the activities of antibiotics on clinical SCVs. Here, we demonstrated that SCVs without detectable auxotrophies were at least as susceptible to most ß-lactam and non-ß-lactam antibiotics in vitro as their corresponding clonally identical strains with a normal phenotype. After prolonged incubation, a regrowth phenomenon has been observed in gradient diffusion inhibition zones irrespective of the strains' phenotype.

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.

The pathogenicity and host adaptation of livestock-associated MRSA CC398.

The presence of methicillin-resistant Staphylococcus aureus (MRSA) CC398 in livestock and their transmission to humans followed by their introduction into hospitals led to a significant burden for the human healthcare system, especially in regions with a high density of livestock breeding. The CC398 lineage made two host changes in its evolutionary history: From humans to pigs and other livestock-associated animals and back to the human host. These adaptation processes are mirrored by changes of the equipment with virulence factors necessary for successful host change. Here, we consider these factors and their special role during human colonization and infection. Host adaptation of S. aureus CC398 is accompanied by genetic changes that are mainly driven by exchanges of mobile genetic elements. So far, it is not clear, which virulence or adhesion factors are important for S. aureus CC398 in host interaction. Among human and animal-derived MRSA CC398 virulence factors, e.g. (entero-) toxins, were rarely found. Overall, this review provides a comprehensive overview on the emerging S. aureus lineage CC398 by summarizing current knowledge from microbiological, molecular and cellular interaction studies in relation to clinical and epidemiological perspectives.

Detection of mecA- and mecC-Positive Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates by the New Xpert MRSA Gen 3 PCR Assay.

An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n = 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n = 160, including 10 mecC-positive MRSA) were accurately categorized.

The culturome of the human nose habitats reveals individual bacterial fingerprint patterns.

The complex anatomy of the human nose might offer distinct microbial niches. Microbiota composition may affect nose inflammatory diseases and Staphylococcus aureus carriage. Considering different nasal cavity locations, microbial colonization was analysed across individuals exhibiting chronic nasal inflammatory diseases (n = 18) and those without local inflammation signs (n = 16). Samples were collected systematically during surgery and examined by an extensive culture-based approach and, for a subset, by 16S rRNA gene community profiling. Cultivation yielded 141 taxa with members of Staphylococcus, Corynebacterium and Propionibacterium as most common isolates comprising the nasal core culturome together with Finegoldia magna. Staphylococcus aureus was most frequently found in association with Staphylococcus epidermidis and Propionibacterium acnes, and the posterior vestibules were redefined as S. aureus' principle habitat. Culturome analysis revealed host-specific bacterial 'fingerprints' irrespective of host-driven factors or intranasal sites. Comparisons between cultivable and molecular fingerprints demonstrated that only a small fraction of phylotypes (6.2%) was correlated. While the total number of different phylotypes was higher in the molecular dataset, the total number of identifications down to the species level was higher in the culturomic approach. To determine host-specific microbiomes, the advantages of molecular approaches should be combined with the resolution and reliability of species identification by culturomic analyses.

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge.

Trimethoprim-sulfamethoxazole (SXT) is a possible alternative for the treatment of community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) due to the susceptibility of most MRSA strains to the drug. However, after long-term treatment with SXT, thymidine-dependent (TD) SXT-resistant small-colony variants (SCVs) emerge. In TD-SCVs, mutations of thymidylate synthase ([TS] thyA) occur. Until now, it has never been systematically investigated that SXT is triggering the induction and/or selection of TD-SCVs. In our study, we performed induction, reversion, and competition experiments in vitro and in vivo using a chronic mouse pneumonia model to determine the impact of SXT on the emergence of TD-SCVs. SCVs were characterized by light and transmission electron microscopy (TEM) and auxotrophism testing. Short-term exposure of S. aureus to SXT induced the TD-SCV phenotype in S. aureus SH1000, while selection of TD-SCVs with thyA mutations occurred after long-term exposure. In reversion experiments with clinical and laboratory TD-SCVs, all revertants carried compensating mutations at the initially identified mutation site. Competition experiments in vitro and in vivo revealed a survival and growth advantage of the ΔthyA mutant under low-thymidine availability and SXT exposure although this advantage was less profound in vivo. Our results show that SXT induces the TD-SCV phenotype after short-term exposure, while long-term exposure selects for thyA mutations, which provide an advantage for TD-SCVs under specified conditions. Thus, our results further an understanding of the dynamic processes occurring during SXT exposure with induction and selection of S. aureus TD-SCVs.

Staphylococcus aureus small colony variants show common metabolic features in central metabolism irrespective of the underlying auxotrophism.

In addition to the classical phenotype, Staphylococcus aureus may exhibit the small colony-variant (SCV) phenotype, which has been associated with chronic, persistent and/or relapsing infections. SCVs are characterized by common phenotypic features such as slow growth, altered susceptibility to antibiotic agents and pathogenic traits based on increased internalization and intracellular persistence. They show frequently auxotrophies mainly based on two different mechanisms: (i) deficiencies in electron transport as shown for menadione- and/or hemin-auxotrophs and (ii) thymidylate biosynthetic-defective SCVs. To get a comprehensive overview of the metabolic differences between both phenotypes, we compared sets of clinically derived menadione-, hemin- and thymidine-auxotrophic SCVs and stable site directed mutants exhibiting the SCV phenotype with their corresponding isogenic parental strains displaying the normal phenotype. Isotopologue profiling and transcriptional analysis of central genes involved in carbon metabolism, revealed large differences between both phenotypes. Labeling experiments with [U-(13)C6]glucose showed reduced (13)C incorporation into aspartate and glutamate from all SCVs irrespective of the underlying auxotrophism. More specifically, these SCVs showed decreased fractions of (13)C2-aspartate and glutamate; (13)C3-glutamate was not detected at all in the SCVs. In comparison to the patterns in the corresponding experiment with the classical S. aureus phenotype, this indicated a reduced carbon flux via the citric acid cycle in all SCV phenotypes. Indeed, the aconitase-encoding gene (acnA) was found down-regulated in all SCV phenotypes under study. In conclusion, all SCV phenotypes including clinical isolates and site-directed mutants displaying the SCV phenotype were characterized by down-regulation of citric acid cycle activity. The common metabolic features in central carbon metabolism found in all SCVs may explain similar characteristics of the S. aureus SCVs irrespective of their auxotrophism as well as the specific genetic and/or regulatory backgrounds.

Staphylococcus aureus Small Colony Variants (SCVs): a road map for the metabolic pathways involved in persistent infections.

Persistent and relapsing infections, despite apparently adequate antibiotic therapy, occur frequently with many pathogens, but it is an especially prominent problem with Staphylococcus aureus infections. For the purposes of this review, persistence will encompass both of the concepts of long term survival within the host, including colonization, and the concept of resisting antibiotic therapy even when susceptible in the clinical microbiology laboratory. Over the past two decades, the mechanisms whereby bacteria achieve persistence are slowly being unraveled. S. aureus small colony variants (SCVs) are linked to chronic, recurrent, and antibiotic-resistant infections, and the study of SCVs has contributed significantly to understanding of persistence. In our earlier work, defects in electron transport and thymidylate biosynthesis were linked to the development of the SCV phenotype (reviewed in 2006), thus this work will be discussed only briefly. Since 2006, it has been found that persistent organisms including SCVs are part of the normal life cycle of bacteria, and often they arise in response to harsh conditions, e.g., antibiotics, starvation, host cationic peptides. Many of the changes found in these early SCVs have provided a map for the discovery mechanisms (pathways) for the development of persistent organisms. For example, changes in RNA processing, stringent response, toxin-antitoxin, ribosome protein L6 (RplF), and cold shock protein B (CspB) found in SCVs are also found in other persisters. In addition, many classic persister organisms also show slow growth, hence SCVs. Recent work on S. aureus USA300 has elucidated the impact of aerobic expression of arginine deiminase genes on its ability to chronically colonize the skin and survive in abscesses. S. aureus SCVs also express arginine deiminase genes aerobically as well. Thus, many pathways found activated in electron transport type of SCVs are also increased in persisters that have intact electron transport. Many of these changes in metabolism result in slow growth; hence, small colonies are formed. Another common theme is that slow growth is also associated with reduced expression of virulence factors and enhanced uptake/survival within host cells. These adaptations to survive within the host are rooted in responses that were required for organisms to survive in a harsh environment long before they were mammals on the earth.

Methicillin resistance in Staphylococcus isolates: the "mec alphabet" with specific consideration of mecC, a mec homolog associated with zoonotic S. aureus lineages.

Livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) have globally emerged during the past decade. In Europe, this was particularly due to the occurrence of LA-MRSA strains associated with the clonal complex (CC) 398 as defined by multilocus sequence typing. However, more recently animal-adapted clonal lineages of S. aureus showing phenotypic methicillin resistance have been identified such as CC130, CC599, CC59, CC1943 and CC425. These newly emerging LA-MRSA CCs/STs caused infections in animals and zoonoses in humans. In contrast to other S. aureus clonal lineages, the methicillin resistance of the latter CCs/STs is based on a mecA gene homolog, designated mecC, which is part of a distinct SCCmec type (SCCmec XI). Including mecB found in Macrococcus caseolyticus, henceforth, the "mec alphabet" comprises three major gene types with several allotypes. As known for mecA, the gene homolog mecC is also not restricted to S. aureus, but found in several staphylococcal species including S. sciuri, S. stepanovicii and S. xylosus (mecC1 allotype). First investigations showed a wide geographical distribution of mecC-MRSA in Europe and a broad diversity of host species including livestock, companion and wildlife animals. In particular, wild rodents and insectivores might serve as reservoir for staphylococci harboring mecC. Economic burden may be caused by mastitis of dairy cattle. Livestock animals may likely serve as source for human infections with mecC-MRSA; reported cases comprise skin and soft tissue infections, osteomyelitis and bacteremia. Due to the divergent molecular nature of mecC-MRSA, its diagnostics is hampered by difficulties to verify the methicillin resistance using phenotypic as well as DNA-based procedures, which could have negative consequences for therapy of mecC-MRSA-caused infections.

LA-MRSA CC398 differ from classical community acquired-MRSA and hospital acquired-MRSA lineages: functional analysis of infection and colonization processes.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of the clonal complex (CC) 398 became primarily known as colonizers of livestock animals. In the past few years, they have been increasingly introduced into hospitals with subsequent emergence of human infections. However, the (re-)adaptation to the human host is only incompletely understood. This study aimed to assess virulence properties of LA-MRSA CC398 by functional modeling of infection and colonization processes. A selection of 15 human LA-MRSA CC398 isolates and 11 pig-colonizing isolates were characterized regarding their virulence capacities and compared with human isolates of hospital-acquired (HA)-MRSA (CC5, CC22 and CC45) and community-associated (CA)-MRSA (CC8, CC30 and CC80) clonal lineages. Our investigations demonstrated that LA-MRSA CC398 adhered less efficient to human cells and human/bovine plasma fibronectin than CA-MRSA and HA-MRSA isolates. In contrast, the LA-MRSA CC398 isolates revealed a high cytotoxic potential comparable to certain CA-MRSA. Comparing the most prevalent LA-MRSA CC398 spa types (t011, t034, t108), isolates associated with spa t108 showed an increased adhesive and invasive potential paired with an increased ability to evade phagocytosis. The results underline both the pathogenic potential of LA-MRSA in general and the heterogeneity within the CC398 clade regarding the virulence characteristics of CC398 subpopulations. Assuming an ongoing (re-)adaptation to the human host combined with a huge reservoir of LA-MRSA CC398 in livestock and constant zoonotic transmission, the LA-MRSA CC398 lineage has the potential to pose a serious threat to human health.

Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.

Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

The mecA homolog mecC confers resistance against ß-lactams in Staphylococcus aureus irrespective of the genetic strain background.

In staphylococci, methicillin resistance is mediated by mecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded by mecC, which shares only 70% similarity to mecA. To prove that mecC is the genetic determinant that confers methicillin resistance in Staphylococcus aureus, a mecC knockout strain was generated. The S. aureus ΔmecC strain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 µg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistant S. aureus (MRSA) strain (8 and 16 µg/ml, respectively). Complementing the mutant in trans with wild-type mecC restored the resistance to oxacillin and cefoxitin. By expressing mecC and mecA in different S. aureus clonal lineages, we found that mecC mediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those with mecA. In addition, we showed that mecC expression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosome mec (SCCmec) type XI. In summary, we showed that mecC is inducible by oxacillin and mediates beta-lactam resistance in SCCmec type XI-carrying strains as well as in different S. aureus genetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinical mecC-harboring S. aureus isolates.

Evaluation of a modular multiplex-PCR methicillin-resistant Staphylococcus aureus detection assay adapted for mecC detection.

A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection.

Detection of new methicillin-resistant Staphylococcus aureus strains that carry a novel genetic homologue and important virulence determinants.

In this study, 18 methicillin-resistant Staphylococcus aureus (MRSA) isolates harboring staphylococcal cassette chromosome mec (SCCmec) type XI, recovered in the Dutch-German Euregio, were characterized by DNA microarrays. In contrast to previous data, we found two MRSA strains of different clonal lineages possessing SCCmec XI that carried important virulence determinants. The worrisome emergence of such toxigenic MRSA strains raises concerns that MRSA strains with enhanced virulence potential and impaired detectability by standard molecular assays may spread in Europe.

Population dynamics among methicillin-resistant Staphylococcus aureus isolates in Germany during a 6-year period.

Methicillin-resistant Staphylococcus aureus (MRSA) originated from the health care setting but is now emerging in communities without health care contact (CA-MRSA) or in livestock (LA-MRSA). The impact on the whole MRSA population was assessed in a German prospective multicenter study. Thirty-three laboratories consecutively collected up to 50 MRSA isolates from infection or carriage during two sampling periods in 2004 to 2005 and 2010 to 2011. Patient-related data were collected using a standardized questionnaire. Methicillin resistance was confirmed by the detection of mecA or its homologue mecA(LGA251). The spa type and major virulence factors were analyzed for each isolate. In total, 1,604 (2004 to 2005) and 1,603 (2010 to 2011) MRSA isolates were analyzed; one isolate from each sampling period harbored mecA(LGA251). LA-MRSA increased significantly (odds ratio [OR] = 22.67, 95% confidence interval [CI] = 8.51 to 85.49, P < 0.0005) and spread over Germany, originating from northwestern regions. Panton-Valentine leukocidin-positive CA-MRSA rose significantly, particularly in southern Germany, but the proportion in 2010 to 2011 remained low (2.7%, OR = 2.80, 95% CI = 1.54 to 5.34, P < 0.0005). The emerging MRSA clones changed the MRSA population in Germany during a 6-year period significantly. The ongoing epidemiological shift and changes of MRSA sources create a need for revision of guidelines for MRSA infection control and treatment.

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent.

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.