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.

Prevalence and Genomic Structure of Bacteriophage phi3 in Human-Derived Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for a period of 16 years to investigate the role of phi3 in the adaptation process of LA-MRSA to the human host. For this purpose, 632 MRSA spa-CC011 isolates from patients of a university hospital located in a pig farming-dense area in Germany were analyzed. Livestock-associated acquisition of MRSA spa-CC011 was previously reported as having increased from 1.8% in 2000 to 29.4% in 2014 in MRSA-positive patients admitted to this hospital. However, in this study, the proportion of phi3-carrying isolates rose only from 1.1% (2000 to 2006) to 3.9% (2007 to 2015). Characterization of the phi3 genomes revealed 12 different phage types ranging in size from 40,712 kb up to 44,003 kb, with four hitherto unknown integration sites (genes or intergenic regions) and several modified bacterial attachment (attB) sites. In contrast to the MSSA CC398 clade, phi3 acquisition seems to be no major driver for the readaptation of MRSA spa-CC011 to the human host.

Staphylococcus aureus in two municipal abattoirs in Nigeria: Risk perception, spread and public health implications.

Staphylococcus aureus is a zoonotic pathogen of significant public health concern. Information on the prevalence and risk factors facilitating bacterial colonization and spread under abattoir settings in Nigeria are scarce. This cross-sectional study was designed to determine prevalence of S. aureus as well as risk factors on knowledge and practices facilitating pathogen carriage among workers and slaughter animals in two municipal abattoirs of Ilorin and Ibadan, Nigeria. Swab samples (n = 1671) from nostrils of cattle, goats, pigs and abattoir workers, and from meat tables and abattoir walls were collected for detection of S. aureus. A questionnaire was administered to 275 workers to elucidate risk factors of pathogen carriage applying a logistic regression model. S. aureus prevalence was 6.5%. In total, MSSA and MRSA were detected at a frequency of 5.4% and 1.1%. Molecular analysis of the isolates revealed 19 different spa types, including a novel spa type (t16751). Gender, marital status, occupation and abattoir location were factors influencing worker's practices in relation to pathogen carriage and spread in the abattoir setting. This present study detected not only low MSSA and MRSA prevalence, in both abattoirs but also low risk perception and hygiene practices employed by abattoir workers. Good practices among workers at Nigerian abattoirs are needed to mitigate S. aureus carriage. Further studies expounding the antibiotic resistance and relationships of MSSA and MRSA strains detected in this study are needed to complement understanding of the spread of S. aureus in the abattoir food chain.

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.

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.

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.

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.

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.

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.

The impact of zoonotic MRSA colonization and infection in Germany.

Methicillin-resistant Staphylococcus aureus (MRSA) causes colonization and infection both in animals and humans. In Germany, cases of MRSA colonization among humans, w+hich affect 0.5-1.5% of the general population and 1.0-2.5% of patients at hospital admission, are still mostly associated with previous healthcare contact and defined epidemic clonal lineages. However, MRSA is also distributed in livestock production in Germany, mostly without causing infections in the animals. These MRSA predominantly belong to the clonal complex (CC) 398, but also to CC9 and CC97. Zoonotic transmission of MRSA CC398 from livestock to humans occurs predominantly in people with occupational livestock contact. Spread of MRSA CC398 to household members of these persons is also frequently observed, but dissemination in the general population is limited so far However, especially in areas with intensive livestock husbandry, about 20-38% of MRSA CC398 cases among humans cannot be epidemiologically linked to direct livestock contact, indicating other transmission pathways. MRSA CC398 currently causes about 2% of all human MRSA infections (wound infections, pneumonia, sepsis) in Germany, but up to 10% in regions characterized by a high density of livestock-farming. The burden of MRSA in companion animals was demonstrated to range between 3.6-9.4% within wound samples obtained from dogs, cats and horses, respectively. In contrast to livestock and horses, MRSA distributed in pet animals are mostly associated with MRSA clonal lineages that are also prevalent in human healthcare facilities. Overall, zoonotic exchange of MRSA between humans and animals has relevant impact on the epidemiology of MRSA in Germany.

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.

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.

Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans-2-decenoic acid (SDSF).

In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer-2 (AI-2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell-free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC-MS and proved to be trans-2-decenoic acid. We show that trans-2-decenoic acid does not inhibit AI-2-specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans-2-decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal-specific signature gene of C. albicans, is abolished by trans-2-decenoic acid. trans-2-Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom-signaling molecules and is the first member of this family from a Gram-positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12(T), a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12(T) is able to synthesize the vitamins B(1) and B(12) for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B(12) are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B(12) was confirmed to be functional, and D. shibae DFL12(T) was shown to provide the growth-limiting vitamins B(1) and B(12) to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12(T) is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12(T) has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12(T) shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Constant c10 ring stoichiometry in the Escherichia coli ATP synthase analyzed by cross-linking.

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (cA21C/cM65C). Independent of the carbon source used for growth and independent of the presence of other FoF1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.