The hospital epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed in the past few years due to the encroachment of community-associated MRSA (CA-MRSA) strains into health care settings. MRSA strains that were isolated during a 2-year period from patients of the Luzerner Kantonsspital were analyzed to elucidate their epidemiology. Moreover, extended surveillance of individuals who were contacts of those patients was carried out for 6 months to identify the routes of spread and to assess the quality of the infection control measures used in our setting. Patient data were collected to distinguish CA-MRSA strains from health care-associated MRSA (HA-MRSA) strains by epidemiological criteria, as defined by the Centers for Disease Control and Prevention (CDC). On the basis of the CDC definition, the majority of the strains were considered to be HA-MRSA. However, 87% of them belonged to staphylococcal cassette chromosome mec (SCCmec) types IV and V, which are traditionally associated with CA-MRSA. Surprisingly, classical nosocomial SCCmec types I and II represented a minority, whereas SCCmec type III was completely absent. By PFGE analysis, four predominant clonal lineages and 21 highly variable sporadic genotypes were detected. Twentyeight percent of the MRSA strains studied carried the genes encoding the Panton-Valentine leukocidin (PVL), of which 21% and 83% were associated with SCCmec types IV and V, respectively. Among 289 contact individuals screened for MRSA carriage throughout the extended surveillance, a single secondary patient was discovered. The possibility of nosocomial transmission could be excluded. The high proportions of SCCmec type IV and V strains as well as PVL-positive strains suggest strong infiltration of CA-MRSA into our institution. Moreover, the low endemic prevalence of MRSA demonstrates that current infection control measures are sufficient to limit its spreading and the emergence of large epidemic outbreaks.

Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.

Because of the new mechanism of action of bedaquiline-the compound acts via inhibition of mycobacterial ATP synthase (AtpE)-it has been postulated that antimicrobial susceptibility testing (AST) is not needed in patients who have never received bedaquiline (4). However, cross-resistance between bedaquiline and the antimycobacterial drug clofazimine through overproduction of the MmpL5 efflux pump has recently been described (5, 6). Thus, resistance may develop independently of treatment with bedaquiline (2, 7). Delamanid (Deltyba [previously known as OPC-67683]; marketed by Otsuka Novel Products GmbH, Munich, Germany) was approved by the European Medicines Agency (EMA) in April 2014. The mechanism of action of delamanid is incompletely understood; delamanid is suggested to inhibit production of methoxymycolic acid and ketomycolic acid (8). Similarly to the related drug PA-824, delamanid is a prodrug requiring activation by the mycobacterial F420 system, including the nitroreductase Ddn (Rv3547) (8-10). Delamanid resistance is thought to arise from mutations in the mycobacterial F420 genes (ddn, fgd1, fbiA, fbiB, and fbiC) associated with the prodrug's activation (8, 11). The spontaneous rate of delamanid resistance has been reported to be as high as 6.44 ϫ 10 Ϫ6 to 4.19 ϫ 10 Ϫ5 , emphasizing the need to protect delamanid with other active anti-TB drugs during therapy (9).Initially, AST of bedaquiline was reported using radiometric Bactec 460TB (BD, Franklin Lakes, NJ, USA), production of which has since been discontinued (2). Reported MIC 90 s for delamanid range from 0.006 mg/liter to 0.05 mg/liter (depending on the test system) across Mycobacterium tuberculosis isolates (8,9,12). Ten years after the drugs' discoveries, established protocols for automated in vitro AST of bedaquiline and delamanid are still not available. To establish procedures for bedaquiline and delamanid AST, we used well-characterized, fully drug-susceptible clinical M. tuberculosis strains of bedaquiline and delamanid treatment-naive patients, MDR-TB strains, and subsequent isolates of a well-characterized extensively drug-resistant (XDR) strain (6). It has been speculated that the phylogenetic lineage of the M. tuberculosis complex may affect innate drug susceptibility (13). To assess the phylogenetic diversity of the set of strains studied, all M. tuberculosis strains included underwent genotypic characterization by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis using a GenoScreen MIRU-VNTR typing kit (GenoScreen, Paris, France) according to the manufacturer's description. In order to determine the quality control (QC) MIC value, the pan-susceptible M. tuberculosis H37Rv reference strain was used. Details about the resistance patterns of the strains and genotypes are shown in Table S1 and S2 in the supplemental material. With the view to facilitating implementation in the routine laboratories, we used a semiautomated MGIT 960 system and EpiCenter software equipped with a TB eXiST module for q...

Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by grampositive bacteria that might become relevant for the treatment of various infectious diseases. So far, selftoxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.Antibiotic resistance has spread dramatically among pathogens throughout the last two decades. Of particular concern is the increasing emergence of multiresistant pathogens in health care settings. Nowadays, about 70% of nosocomial infections in the United States are resistant to at least one antibiotic (35), and in 2002, the first clinical isolate of Staphylococcus aureus with high-level resistance to vancomycin, a drug of last resort, was isolated (8). As the number of resistant pathogens continues to grow, the number of new antibiotics to fight them is declining steeply (37). Therefore, the development of new antimicrobial agents should be encouraged.Type A lantibiotics are cationic, amphiphilic peptides that are produced by a wide range of gram-positive bacteria and show bactericidal activity against other gram-positive bacteria. They show a dual mode of action at nanomolar concentrations which involves pore formation and inhibition of peptidoglycan biosynth...

cThis study aimed to determine resistant-population cutoffs (RCOFFs) to allow for improved characterization of antimicrobial susceptibility patterns in bacterial populations. RCOFFs can complement epidemiological cutoff (ECOFF)-based settings of clinical breakpoints (CBPs) by systematically describing the correlation between non-wild-type and wild-type populations. We illustrate this concept by describing three paradigmatic examples of wild-type and non-wild-type Escherichia coli populations from our clinical strain database of disk diffusion diameters. The statistical determination of RCOFFs and ECOFFs and their standardized applications in antimicrobial susceptibility testing (AST) facilitates the assignment of isolates to wild-type or non-wildtype populations. This should improve the correlation of in vitro AST data and distinct antibiotic resistance mechanisms with clinical outcome facilitating the setting and validation of CBPs. In antimicrobial susceptibility testing (AST), clinical breakpoints (CBPs) are set with the intent of predicting the clinical outcome (1-3). Epidemiological cutoffs (ECOFFs), as defined by the European Committee for Antimicrobial Susceptibility Testing (EUCAST), separate wild-type from non-wild-type populations, reviving the concept of microbiological breakpoints (4-9). EUCAST uses the ECOFF as one of several tools in the process of CBP setting. Different methods have been suggested to determine ECOFFs (5, 10).For a clinical isolate, prediction of treatment success is usually based on a single laboratory AST result, which is subsequently classified according to the corresponding CBPs. However, both MICs and disk diffusion diameters show significant variations when tested repeatedly (1, 6). For wild-type populations, these variations have been shown to result from measurement imprecision and methodological and biological factors (5, 11, 12). As it is based on a single measurement, the true position of an isolate within a disk diameter or MIC distribution can only be approximated with a certain probability. In addition, it is unclear to what extent the in vitro susceptibility of a clinical isolate reflects the in vivo situation, as in vivo variations within a population are likely to be more pronounced than those observed in vitro in the standardized setting of AST.CBPs should preferably avoid splitting the wild-type population into different interpretative categories as the wild-type is regarded to be a genotypic entity despite population-intrinsic (micro-)variations in drug susceptibility (6). This principle of not splitting wild-type populations by CBPs is widely acknowledged by EUCAST guidelines (13). By logical consequence, these considerations should similarly apply to non-wild-type populations, which represent distinct genotypic/phenotypic entities ("resistotypes"). Thus, CBP setting should not only avoid splitting the wild-type into different interpretative categories but also CBPs should avoid splitting a resistotype into different clinical categories as far as possible in or...