Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements.

Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase gene, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments.

Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB, hsp60, tuf, dnaJ, gap, and sod Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus, the novel strains are pyrrolidonyl arylamidase and ß-glucuronidase positive and ß-galactosidase negative, nitrate is reduced, and acid produced aerobically from d-mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085T (ψSCCmecP5085), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085T (= CCM 8730T = DSM 104441T).IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.

Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETBTY4. Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETBTY4-based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids.

High intraspecies heterogeneity within Staphylococcus sciuri and rejection of its classification into S. sciuri subsp. sciuri, S. sciuri subsp. carnaticus and S. sciuri subsp. rodentium.

A polyphasic taxonomic approach was applied to strains of the species Staphylococcus sciuri in order to clarify the taxonomic legitimacy of the delineation of S. sciuri into S. sciurisubsp.sciuri, S. sciurisubsp.carnaticus and S. sciurisubsp.rodentium. A group of 81 S. sciuri isolates obtained from human (n=62) and veterinary (n=17) clinical materials and foods (n=2) and ten reference and type strains obtained from the Czech Collection of Microorganisms were characterized by extensive biotyping using conventional tests and commercial identification kits (ID 32 Staph, STAPHYtest, Biolog Microbial ID System), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, automated ribotyping with EcoRI restriction enzyme, 16S-23S rRNA gene intergenic transcribed spacer PCR fingerprinting and repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Selected strains representing different ribotypes were further characterized using sequencing of the ß-subunit of RNA polymerase (rpoB) gene. Individual techniques revealed high heterogeneity within the analysed S. sciuri strains but differentiation of the investigated strains into groups corresponding to the aforementioned S. sciuri subspecies and supported by these techniques was not clearly revealed. Based on obtained results and data retrieved from literature we propose rejecting the separation of S. sciuri species into S. sciurisubsp.sciuri, S. sciurisubsp.carnaticus and S. sciurisubsp.rodentium and we suggest reclassification these subspecies as S. sciuri with the type strain W.E. Kloos SC 116T (=ATCC 29062T=BCRC 12927T=CCM 3473T=CCUG 15598T=CNCTC 5683T=DSM 20345T=JCM 2425T=NCTC 12103T).

Staphylococcus petrasii subsp. pragensis subsp. nov., occurring in human clinical material.

Seven coagulase-negative, oxidase-negative and novobiocin-susceptible staphylococci assigned tentatively as Staphylococcus petrasii were investigated in this study in order to elucidate their taxonomic position. All strains were initially shown to form a genetically homogeneous group separated from remaining species of the genus Staphylococcus by using a repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Phylogenetic analysis based on 16S rRNA gene, hsp60, rpoB, dnaJ, gap and tuf sequences showed that the group is closely related to Staphylococcus petrasii but separated from the three hitherto known subspecies, S. petrasii subsp. petrasii, S. petrasii subsp. croceilyticus and S. petrasii subsp. jettensis. Further investigation using automated ribotyping, MALDI-TOF mass spectrometry, fatty acid methyl ester analysis, DNA-DNA hybridization and extensive biotyping confirmed that the analysed group represents a novel subspecies within S. petrasii, for which the name Staphylococcus petrasii subsp. pragensis subsp. nov. is proposed. The type strain is NRL/St 12/356(T) ( = CCM 8529(T) = LMG 28327(T)).

Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

Reclassification of Staphylococcus jettensis De Bel et al. 2013 as Staphylococcus petrasii subsp. jettensis subsp. nov. and emended description of Staphylococcus petrasii Pantucek et al. 2013.

The type and clinical strains of two recently described coagulase-negative species of the genus Staphylococcus, Staphylococcus petrasii and Staphylococcus jettensis, were compared using dnaJ, tuf, gap, hsp60 and rpoB gene sequences, DNA-DNA hybridization, ribotyping, repetitive sequence-based PCR fingerprinting and extensive biochemical characterization. Based on the results, the species description of S. petrasii has been emended and S. jettensis should be reclassified as a novel subspecies within S. petrasii for which the name Staphylococcus petrasii subsp. jettensis subsp. nov. is proposed. The type strain is SEQ110(T) ( = LMG 26879(T) = CCUG 62657(T) = DSM 26618(T) = CCM 8494(T)).

[Corynebacterium imitans isolated from blood culture in a patient with suspected bacteremia - the first isolation from human clinical material in the Czech Republic].

The current view of the clinical importance of nondiphtherial corynebacteria recovered from human clinical material has changed considerably in recent decades; in many cases, a direct etiological role is assumed or has already been demonstrated. Presented is a case of suspected bacteremia in a hospitalized elderly woman with isolation of the very rare species Corynebacterium imitans from blood culture. However, the etiological significance of the isolated microorganism remains unclear. The aim was not to demonstrate the etiological significance of the isolated C. imitans strain but to report the occurrence of this very rare species which is considered to be the first isolation from humans in the Czech Republic.

Staphylococcus brunensis sp. nov. isolated from human clinical specimens with a staphylococcal cassette chromosome-related genomic island outside of the rlmH gene bearing the ccrDE recombinase gene complex.

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

Major clonal lineages in impetigo Staphylococcus aureus strains isolated in Czech and Slovak maternity hospitals.

One hundred and twenty-seven exfoliative toxin-producing (ET-positive) strains of Staphylococcus aureus collected in 23 Czech and one Slovak maternity hospitals from 1998 to 2011 were genotypically characterized by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, and ETA-converting prophage carriage, which resulted in the identification of 21 genotypes grouped into 4 clonal complexes (CC). Ninety-one isolates carried the eta gene alone whilst 12 isolates harboured only the etb gene. Two new, to date not defined, spa types (t6644 and t6645) and 2 novel sequence types (ST2194 and ST2195) were identified in the set of strains under study. The predominant CC121 occurred in 13 Czech hospitals. CC15, CC9, and ST88 (CC88) exclusively included eta gene-positive strains while the strains belonging to ST121 harboured the eta and/or etb genes. This study highlights not only significant genomic diversity among impetigo strains and the distribution of major genotypes disseminated in the Czech and Slovak maternity hospitals, but also reveals their impact in epidermolytic infections.

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains.

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.

Staphylococcus ratti sp. nov. Isolated from a Lab Rat.

Staphylococci from the Staphylococcus intermedius-Staphylococcus hyicus species group include numerous animal pathogens and are an important reservoir of virulence and antimicrobial resistance determinants. Due to their pathogenic potential, they are possible causative agents of zoonoses in humans; therefore, it is important to address the properties of these strains. Here we used a polyphasic taxonomic approach to characterize the coagulase-negative staphylococcal strain NRL/St 03/464T, isolated from the nostrils of a healthy laboratory rat during a microbiological screening of laboratory animals. The 16S rRNA sequence, MALDI-TOF mass spectrometry and positive urea hydrolysis and beta-glucuronidase tests clearly distinguished it from closely related Staphylococcus spp. All analyses have consistently shown that the closest relative is Staphylococcus chromogenes; however, values of digital DNA-DNA hybridization <35.3% and an average nucleotide identity <81.4% confirmed that the analyzed strain is a distinct Staphylococcus species. Whole-genome sequencing and expert annotation of the genome revealed the presence of novel variable genetic elements, including two plasmids named pSR9025A and pSR9025B, prophages, genomic islands and a composite transposon that may confer selective advantages to other bacteria and enhance their survival. Based on phenotypic, phylogenetic and genomic data obtained in this study, the strain NRL/St 03/464T (= CCM 9025T = LMG 31873T = DSM 111348T) represents a novel species with the suggested name Staphylococcus ratti sp. nov.

Correct species identification (reclassification in CNCTC) of strains of Staphylococcus intermedius-group can improve an insight into their evolutionary history.

A group of 59 putative strains of Staphylococcus intermedius/Staphylococcus pseudintermedius deposited in the Czech National Collection of Type Cultures (CNCTC, National Institute for Public Health, Prague, Czech Republic) and the National Reference Laboratory for Staphylococci (NRL for Staphylococci, National Institute for Public Health, Prague, Czech Republic) was reclassified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There the biggest human collection of S. pseudintermedius in Europe was analysed; 44 samples (75%) were of human origin. Twenty-two percent (n = 13) of the strains were isolated from animals, and two staphylococci were of unknown origin. This study revealed the prevalence of Staphylococcus pseudintermedius (94%, n = 53) vs. Staphylococcus intermedius (6%, n = 6) in the collection of human and veterinary staphylococci after reclassification. Results of PCR-RFLP analysis were verified by comparison with a repetitive element sequence-based polymerase chain reaction (Rep-PCR) analysis on 26 (44%) randomly selected strains. Due to a low-resolution ability of PCR-RFLP to separate Staphylococcus intermedius from Staphylococcus delphini, four isolates of Staphylococcus intermedius were biochemically verified further to exclude the presence of Staphylococcus delphini in the collection. Our results indicate that S. intermedius and S. pseudintermedius have occurred independently over an age-long period of their co-evolution.

Lytic and genomic properties of spontaneous host-range Kayvirus mutants prove their suitability for upgrading phage therapeutics against staphylococci.

Lytic bacteriophages are valuable therapeutic agents against bacterial infections. There is continual effort to obtain new phages to increase the effectivity of phage preparations against emerging phage-resistant strains. Here we described the genomic diversity of spontaneous host-range mutants of kayvirus 812. Five mutant phages were isolated as rare plaques on phage-resistant Staphylococcus aureus strains. The host range of phage 812-derived mutants was 42% higher than the wild type, determined on a set of 186 methicillin-resistant S. aureus strains representing the globally circulating human and livestock-associated clones. Comparative genomics revealed that single-nucleotide polymorphisms from the parental phage 812 population were fixed in next-step mutants, mostly in genes for tail and baseplate components, and the acquired point mutations led to diverse receptor binding proteins in the phage mutants. Numerous genome changes associated with rearrangements between direct repeat motifs or intron loss were found. Alterations occurred in host-takeover and terminal genomic regions or the endolysin gene of mutants that exhibited the highest lytic activity, which implied various mechanisms of overcoming bacterial resistance. The genomic data revealed that Kayvirus spontaneous mutants are free from undesirable genes and their lytic properties proved their suitability for rapidly updating phage therapeutics.

Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.

The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Supplementary biochemical tests useful for the differentiation of oxidase positive staphylococci.

Differentiation of the oxidase positive staphylococci, Staphylococcus sciuri, Staphylococcus lentus, Staphylococcus vitulinus and Staphylococcus fleurettii, based on tributyrin, urease, caseinase, gelatinase and DNase activity is described. These tests may be used for preliminary identification of oxidase positive isolates of staphylococci resulting in more accurate identification of these species.

Rapid identification of staphylococci by Raman spectroscopy.

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

Occurance of Staphylococcus nepalensis strains in different sources including human clinical material.

Five isolates of coagulase-negative staphylococci were obtained from human urine, the gastrointestinal tract of squirrel monkeys, pig skin and from the environment. All key biochemical characteristics of the tested strains corresponded with the description of Staphylococcus xylosus species. However, partial 16S rRNA gene sequences obtained from analysed strains corresponded with those of Staphylococcus nepalensis reference strains, except for two strains which differed in one residue. Ribotyping with EcoRI and HindIII restriction enzymes, whole cell protein profile analysis performed by SDS-PAGE and SmaI macrorestriction analysis were used for more precise characterization and identification of the analysed strains. Obtained results showed that EcoRI and HindIII ribotyping and whole cell protein fingerprinting are suitable and reliable methods for the differentiation of S. nepalensis strains from the other novobiocin resistant staphylococci, whereas macrorestriction analysis was found to be a good tool for strain typing. The isolation of S. nepalensis is sporadic, and according to our best knowledge this study is the first report of the occurrence of this species in human clinical material as well as in other sources.

Staphylococcus equorum and Staphylococcus succinus isolated from human clinical specimens.

A polyphasic identification approach was applied to a group of 11 novobiocin-resistant staphylococci isolated from human clinical materials. Phenotypic characteristics obtained by both commercial and conventional tests assigned eight strains as Staphylococcus xylosus and three strains as ambiguous S. xylosus/Staphylococcus equorum. In contrast to biotyping, ribotyping with EcoRI and HindIII restriction endonucleases and whole-cell protein fingerprinting assigned six analysed strains as S. equorum, and five strains as Staphylococcus succinus. Confirmation of the identification was done by partial 16S rRNA gene sequencing and S. equorum isolates were verified by a PCR assay targeting the sodA gene. From the data it has been implied that ribotyping and whole-cell protein analysis can be used to differentiate between the biochemically almost indistinguishable species S. xylosus, S. equorum and S. succinus. The present study confirms what is believed to be the first occurrence of S. equorum in a relevant human clinical material in the Czech Republic and describes what is believed to be the first-ever isolation of S. succinus from human clinical material.

Importance of Multifaceted Approaches in Infection Control: A Practical Experience from an Outbreak Investigation.

BACKGROUND: This study presents the results of a multidisciplinary, nosocomial MRSA outbreak investigation in an 8-bed medical intensive care unit (ICU). The identification of seven MRSA positive patients in the beginning of 2014 led to the closure of the ward for several weeks. A multidisciplinary, retrospective investigation was initiated in order to identify the reason and the source for the outbreak, describe MRSA transmission in the department and identify limitations in infection control. METHODS: The investigation comprised an epidemiological description of MRSA cases from 2012 to 2014 and a characterization of MRSA isolates, including phage-, spa- and PFGE-typing. Additionally, MRSA screening was performed from the hospital staff and the environment. To identify the reason for the outbreak, work-related, psychological and behavioral factors were investigated by impartial audits and staff interviews. RESULTS: Thirty-one MRSA cases were registered during the study period, and 36 isolates were investigated. Molecular typing determined the outbreak strain (phage type 54/812, PFGE type A4, spa type t003) and identified the probable index case. Nasal carriage in one employee and a high environmental contamination with the outbreak strain was documented. Important gaps in nursing procedures and general management were identified. Elevated stress levels and communication problems preceded the outbreak. Compliance with hand hygiene and isolation procedures was evaluated as appropriate. CONCLUSION: This study demonstrates the complexity of controlling hospital-associated infections. The combined use of different typing methods is beneficial for outbreak investigations. Psychological, behavioral and other work-related factors have an important impact on the spread of nosocomial pathogens. These factors should be addressed and integrated in routine infection control practice.