Britta Ballhausen

Human MRSA Isolates with Novel Genetic Homolog, Germany

To the Editor: Methicillin-resistant Staphylococcus aureus (MRSA) represents a major cause of hospital-, community- and livestock-acquired infections that are increasingly difficult to manage (1–3). Detection and identification of MRSA by culture and nucleic acid–based methods is challenged by heterogeneous penicillin-binding protein 2a (PBP2a) expression and variability of the staphylococcal cassette chromosome (SCCmec) elements. Recently, a new SCCmec element (XI) carried in bovine and human isolates was described (4,5). This SCCmec element contains a novel mecA homolog, designated mecALGA251, that is not detectable by usual mecA-specific PCR approaches and PBP2a agglutination tests. Garcia-Alvarez et al. reported this novel mecA homolog exhibited 70% identity at DNA level to the mecA gene, and suggested these strains were transmitted from livestock to humans (4).

The clinical impact of livestock-associated methicillin-resistant Staphylococcus aureus of the clonal complex 398 for humans.

In the past decade, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains in particular of the clonal complex (CC) 398 have emerged in many parts of the world especially in areas with a high density of pig farming. In those regions, farmworkers and other individuals with professional contact to livestock are very frequently colonized with LA-MRSA. These persons are the presumably source for LA-MRSA transmission to household members and other parts of the human population. Altogether, colonization and/or infection of these individuals lead to the introduction of LA-MRSA into hospitals and other healthcare facilities. Since LA-MRSA CC398 have been found to be specifically adapted to their animal hosts in terms of the equipment with virulence factors, their pathogenicity to human patients is a matter of debate with first reports about clinical cases. Meanwhile, case reports, case series and few studies have demonstrated the capability of LA-MRSA to cause all types of infections attributed to S. aureus in general including fatal courses. Human infections observed comprise e.g. bacteremia, pneumonia, osteomyelitis, endocarditis and many manifestations of skin and soft tissue infections. However, inpatients affected by MRSA CC398 generally show different demographic (e.g. younger, shorter length of hospital stay) and clinical characteristics (e.g. less severe complications) which may explain or at least contribute to a lower disease burden of LA-MRSA compared to other MRSA clonal lineages.

The mecA Homolog mecC Confers Resistance against β-Lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background

ABSTRACT 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.

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.

In the centre of an epidemic: Fifteen years of LA-MRSA CC398 at the University Hospital Munster

Ten years after initial publications on livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in 2005, we report on the course of the LA-MRSA CC398 epidemic among patients of the University Hospital Münster. This tertiary care facility is located in the Dutch-German border region (EUREGIO), which is characterized by a high density of livestock production and is hence a hotspot for the occurrence of LA-MRSA CC398. Taking advantage of the unique opportunity to track the emergence and spread of MRSA CC398 among humans from the very beginning of the epidemic until today, a total of 6555 non-duplicate MRSA isolates from all screenings and clinical specimens cultivated within the period from 2000 to 2014 were included in the analysis. Retrospectively, the first MRSA CC398 isolate (spa type t034) was obtained from a screening specimen of a patient in 2000, which represents one of the first human-associated LA-MRSA CC398 isolates reported in Europe. After sporadic detections between 2000 and 2004, this clonal lineage accounted for 9.6% of all local MRSA in 2005; a proportion which increased to 35% in 2013 and became stable since then. Considering the period from 2000 to 2014, the group of MRSA CC398 isolates comprised a total of 45 different spa types among which t011 (48.3%), t034 (39.3%) and t108 (3.5%) were predominant and so far unreported types were found. Overall, LA-MRSA CC398 emerged rapidly during the past decade, developed enormous sublineage diversity and contributed substantially to the total burden of MRSA colonization and infection at the hospital.

Antimicrobial resistances and virulence markers in Methicillin-resistant Staphylococcus aureus from broiler and turkey: A molecular view from farm to fork.

Little is known about the characteristics of MRSA occurring along the broiler and turkey production chains. The aim of this study was to characterise and compare MRSA of turkey and broiler origin sampled on different production levels using a DNA microarray and antimicrobial susceptibility testing. Main differences could be observed in the prevalence of the resistance genes erm(C), aacA-aphD and tet(K) and the number of non-wild type strains with minimum inhibitory concentration values (MICs) above the epidemiological cut-off values (ECOFFs) for gentamicin and kanamycin. Overall, MRSA with non-wild type phenotype for the macrolide-lincosamide-streptogramin group, tetracycline, and trimethoprim were found in more than 70% of poultry isolates. Non-wild type isolates carrying the qacC gene conferring resistance to quaternary ammonium compound disinfectants were found at all production stages in similar frequencies. Regarding the presence of enterotoxin genes in poultry-derived MRSA the enterotoxin gene cluster (egc) was only found in Non-CC (clonal complex) 398 strains. Three CC398 strains harboured the genes sed (from turkey at slaughter and broiler meat) and sea-N315 (from a chicken carcass). One Non-CC398 strain isolated from turkey meat was found positive for the seb gene and also enterotoxin production. Similarity analysis based on selected resistance and virulence markers revealed a high clonality among Non-CC398 isolates. Isolates of the same clonal complex clustered together according to their common virulence and resistance profiles. Strains of CC9 were grouped in two subclusters due to different resistance genes. Our results underline, that there are other poultry associated clones of MRSA (mainly CC9 and CC5) besides the predominant CC398 which are similar in both poultry species.

Pacemaker lead infection and related bacteraemia caused by normal and small colony variant phenotypes of Bacillus licheniformis.

Here, we report what we believe to be the first case of bacteraemia with small colony variants of Bacillus licheniformis related to a pacemaker lead infection by B. licheniformis displaying the normal phenotype. Arbitrarily primed PCR analysis showed a clonal strain. The infection was cured after the removal of the infected device.

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

ABSTRACT 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.

Evaluation of Multiple-Locus Variable Number of Tandem Repeats Analysis for Typing Livestock-Associated Methicillin-Resistant Staphylococcus aureus

Background The increasing occurrence of livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) associated with the clonal complex (CC) 398 within the past years shows the importance of standardized and comparable typing methods for the purposes of molecular surveillance and outbreak detection. Multiple-locus variable number of tandem repeats analysis (MLVA) has recently been described as an alternative and highly discriminative tool for S. aureus. However, until now the applicability of MLVA for the typing of LA-MRSA isolates from different geographic origin has not been investigated in detail. We therefore compared MLVA and S. aureus protein A (spa) typing for characterizing porcine MRSA from distinct Dutch and German farms. Methodology/Principal Findings Overall, 134 MRSA isolates originating from 21 different pig-farms in the Netherlands and 36 farms in Germany comprising 21 different spa types were subjected to MLVA-typing. Amplification and subsequent automated fragment sizing of the tandem repeat loci on a capillary sequencer differentiated these 134 isolates into 20 distinct MLVA types. Whereas overall MLVA and spa typing showed the same discriminatory power to type LA-MRSA (p  = 0.102), MLVA was more discriminatory than spa typing for isolates associated with the prevalent spa types t011 and t034 (Simpson’s Index of Diversity 0.564 vs. 0.429, respectively; p<0.001). Conclusion Although the applied MLVA scheme was not more discriminatory than spa typing in general, it added valuable information to spa typing results for specific spa types (t011, t034) which are highly prevalent in the study area, i.e. Dutch-German border area. Thus, both methods may complement each other to increase the discriminatory power to resolute highly conserved clones such as CC398 (spa types t011, t034) for the detection of outbreaks and molecular surveillance of zoonotic MRSA.

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.