Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere ؉ (Ridom GmbH, Münster, Germany). The implemented target genes (n ‫؍‬ 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n ‫؍‬ 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences. Staphylococcus aureus is a Gram-positive facultative pathogenic bacterium that is responsible for a high percentage of hospitaland community-acquired infections worldwide. An infection with S. aureus may manifest itself in a broad variety of diseases, ranging from rather harmless local skin infections to severe bacteremia or intoxications (1). This extensive spectrum of virulence is owed, in part, to the bacterium's individual equipment with virulence factors. Analyzing these virulence factors is difficult because purified staphylococcal toxins do not essentially cause distinctive symptoms when administered in the absence of the bacterium, and the specific knockout of single virulence factors does not necessarily reduce the bacterial virulence (2). Thus, it seems that the combination of different virulence factors, their regulation and transcription, and their allelic variants play a crucial role in determining the eventually expressed virulence phenotype. Therefore, it is important to determine not only the presence or absence of single key factors, such as, e.g., Panton-Valentine leucocidin (PVL) or certain enterotoxins, but to obtain a comprehensive picture of the exact allelic variants of as many virulence-associated genes and their regulatory systems as possible. With regar...

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The term 'neglected tropical diseases' predominantly refers to single-entity, mostly parasitic diseases. However, a considerable morbidity and mortality burden is carried by patients infected with Gram-positive cocci and Gram-negative bacilli that are prevalent all over the world, yet have impact in tropical and developing countries, particularly in children, with much higher incidence rates than those reported from developed countries. Staphylococcus aureus is among these pathogens. The African-German StaphNet consortium uses microbiological characterization of African S. aureus isolates, including identification of virulence factors, alongside the gathering of epidemiological and clinical data in an innovative research network between a European country (Germany) and several African partners. By creating an accessible strain repository and by implementing personnel training and capacity building, this network aims to put staphylococcal disease on the international agenda as a truly neglected condition with a major global impact on public health.

Clonal clusters and gene repertoires of Staphylococcus aureus are essential to understand disease and are well characterized in industrialized countries but poorly analysed in developing regions. The objective of this study was to compare the molecular-epidemiologic profiles of S. aureus isolates from Sub-Saharan Africa and Germany. S. aureus isolates from 600 staphylococcal carriers and 600 patients with community-associated staphylococcal disease were characterized by DNA hybridization, clonal complex (CC) attribution, and principal component (PCA)-based gene repertoire analysis. 73% of all CCs identified representing 77% of the isolates contained in these CCs were predominant in either African or German region. Significant differences between African versus German isolates were found for alleles encoding the accessory gene regulator type, enterotoxins, the Panton-Valentine leukocidin, immune evasion gene cluster, and adhesins. PCA in conjunction with silhouette analysis distinguished nine separable PCA clusters, with five clusters primarily comprising of African and two clusters of German isolates. Significant differences between S. aureus lineages in Africa and Germany may be a clue to explain the apparent difference in disease between tropical/(so-called) developing and temperate/industrialized regions. In low-resource countries further clinical-epidemiologic research is warranted not only for neglected tropical diseases but also for major bacterial infections.

Sperm deposited in the female genital tract receive signals for capacitation. Past work indicates that HCO(3) (-) is the initiating signal that the female reproductive tract contains the HCO(3) (-) -permeant anion channel cystic fibrosis transmembrane conductance regulator (CFTR) and that mutations in CFTR cause subfertility in both sexes. In this study, we examined whether CFTR controls uterine HCO(3) (-) content and sperm responses to it. Both CFTR protein and mRNA were absent in prepubertal murine uterus, but appeared in pubertal and adult tissues. Thus, CFTR is upregulated during development. Uterine CFTR mRNA additionally increased upon induced oestrus, most abundantly in uterus body and distal horns. Uterine fluid of oestrous females contained two-, and nearly fourfold more HCO(3) (-) than that of dioestrous and prepubertal animals, correlating with increased CFTR expression. For sperm incubated in and recovered from prepubertal uteri, flagellar beat frequency was no different from that before incubation. However, for sperm recovered from dioestrous and oestrous uteri, beat frequency was two- and fourfold higher, respectively. Thus, uterine HCO(3) (-) content may have physiological consequences for sperm motility. The male reproductive tract showed no regional distributions or developmental dependence of CFTR expression. Although the sperm flagellum showed CFTR immunoreactivity, CFTR blockers GlyH-101 or CFTR(inh) -172 did neither diminish HCO(3) (-) -evoked increases in sperm motility nor protein tyrosine phosphorylation. Our results indicate that in the uterus, both CFTR expression and the supply of HCO(3) (-) are upregulated hormonally. We propose that these changes coordinate ovulation with increases in sperm motility and promote other components of capacitation by pathways that do not require CFTR in sperm.

As genotyping of S. aureus is important for epidemiologic research and for hygiene management, methods are required for standardized fast and easily applicable evaluation of closely related epidemic strains with high prevalence in hospitals. In this single centre matched control study we compared a new commercially available DNA microarray (IdentiBAC) with standard spa-typing for S. aureus genotyping. Included in the study was a subgroup of 46 MRSA and matched 46 MSSA nasal isolates of the Saarland University Medical Center collected during a state-wide admission prevalence screening. Microarray (MA) and also spa-typing could easily differentiate the genetically diverse MSSA group. However, due to the predominance of CC5/t003 in the MRSA group a sufficient subtyping required analysis of more complex genetic profiles as was shown here by the MA comprising a total number of 334 different hybridization probes. The genetic repertoire of the MRSA group was characterized by more virulence genes as compared to the MSSA group. The standard evaluation of MA results by the original software into CCs, agr-, SCCmec- and capsule-types was substituted in the present study by implementation of multivariate subtyping of closely related CC5 isolates using three different bioinformatic methods (splits graph, cluster dendrogram, and principal component analysis). Each method used was applicable for standardized and highly discriminative subtyping with high concordance. We propose that the identified S. aureus subtypes with characteristic virulence gene profiles are presumably associated also with virulence and pathogenicity in vivo; however, this remains to be analyzed in future studies. MA was superior to spa-typing for epidemiologic and presumably also provide functional respectively virulence associated characterization of S. aureus isolates. This is of specific importance for the hospital setting. In future, MA could become a new standard test for S. aureus typing in combination with multivariate bioinformatic analysis.

Wastewater-based epidemiology (WBE) employs the analysis of wastewater to detect and quantify drug use and discharge within a community. In this work, transformation products (TP) by microbes in the environment were identified after incubations in wastewater and an isolated microbial strain. The microbial strain was isolated from an enrichment culture of wastewater supplemented with 3,4-methylenedioxy-pyrovalerone, and identified by matrix assisted laser desorption - time of flight mass spectrometry as Pseudomonas putida (P. putida). Five pyrrolidinophenone-type psychoactive substances (PPPS) were then incubated in wastewater and in P. putida tryptic soy broth (TSB) growth cultures. TPs were identified using liquid chromatography coupled to mass spectrometry techniques. All TPs observed in P. putida TSB growth cultures were also identified in wastewater incubations. The main TP for all PPPSs in P. putida TSB growth cultures, and two PPPSs incubated in wastewater, were the N-desalkyl-carboxy-TPs. The study showed P. putida TSB growth cultures used for identification of TPs in wastewater, represent parts of the microbial community. With data provided in this type of experiments more information will be available to select targets for monitoring drug use by WBE. Copyright © 2017 John Wiley & Sons, Ltd.

Background: The emergence of community-acquired Staphylococcus aureus infections is increasingly recognized as life threating problem worldwide. In Manhiça district, southern Mozambique, S. aureus is the leading cause of community-acquired bacteremia in neonates.Methods: Eighty-four S. aureus isolates from children less than 5 years admitted to Manhiça District Hospital from 2001 to 2009 were randomly selected and genetically characterized by DNA microarray and spa typing. Antimicrobial susceptibility was determined by VITEK 2.Results: Thirty-eight different spa types and 14 clonal complexes (CC) were identified. Spa-type t084 (n = 10; 12%) was the most predominant while CC8 (n = 18; 21%) and CC15 (n = 14; 16%) were the most frequent CCs. Mortality tended to be higher among children infected with CC45 (33.3%, 1/3) and CC8 (27.8%, 5/18). The majority of isolates possessed the accessory gene regulator I (45%) and belonged to either capsule type 8 (52%) or 5 (47%). Panton valentine leukocidin (PVL) encoding genes were detected in 30%. Antibiotic resistance was high for penicillin (89%), tetracycline (59%) and Trimethoprim Sulfamethoxazole (36%) while MRSA was uncommon (8%).Conclusions: Although MRSA were uncommon, we found high genetic diversity of methicillin susceptible S. aureus causing bacteremia in Mozambican children, associated with high resistance to the most available antibiotics in this community. Some CCs are likely to be more lethal indicating the need for prompt recognition and appropriate treatment.