Emergence and dissemination of a linezolid-resistant Staphylococcus capitis clone in Europe.

Objectives: We investigated the epidemiological, clinical, microbiological and genetic characteristics of linezolid-resistant (LZR) Staphylococcus capitis isolates from French ICUs, and compared them with LZR S. capitis isolates from other European countries. Methods: All LZR isolates were subjected to antimicrobial susceptibility testing (AST) and the presence of cfr and optrA genes as well as mutations in the 23S rRNA and ribosomal proteins were investigated using specific PCR with sequencing. The genetic relationship between isolates was investigated using PFGE and WGS. Epidemiological data concerning LZR S. capitis were collected retrospectively in French microbiology laboratories. Results: Twenty-one LZR isolates were studied: 9 from France, 11 from Greece and 1 from Finland. All were resistant to methicillin and aminoglycosides. In addition, this unusual AST profile was identified in S. capitis isolates from seven French hospitals, and represented up to 12% of the S. capitis isolates in one centre. A G2576T mutation in 23S rRNA was identified in all isolates; cfr and optrA genes were absent. All isolates belonged to the same clone on the basis of their PFGE profiles, whatever their geographical origin. WGS found at most 212 SNPs between core genomes of the LZR isolates. Conclusions: We identified and characterized an LZR S. capitis clone disseminated in three European countries, harbouring the same multiple resistance and a G2576T mutation in the 23S rRNA. The possible unrecognized wider distribution of this clone, belonging to a species classically regarded as a low-virulence skin colonizer, is of major concern not least because of the increasing use of oxazolidinones.

In vitro activity of ceftobiprole on 440 Staphylococcus aureus strains isolated from bronchopulmonary infections.

OBJECTIVE: We assessed the in vitro activity of ceftobiprole on 440 Staphylococcus aureus clinical strains isolated from bronchopulmonary infections (2010-2014). METHODS: S. aureus isolates were characterized for methicillin resistance, PVL status, and clonal complex. All isolates were tested for minimal inhibitory concentrations (MIC) determination by broth microdilution method for ceftobiprole, ceftaroline fosamil, and comparator antibiotics (linezolid, tigecycline, vancomycin, and daptomycin). RESULTS: A total of 325 (74%) strains were methicillin-susceptible S. aureus (MSSA) and 115 (26%) were methicillin-resistant S. aureus (MRSA); 105 (24%) S. aureus strains were PVL-positive, including 35.2% (37/105) MRSA and 64.8% (68/105) MSSA. Ceftobiprole was highly active against S. aureus with MIC90 of 1 mg/L, MICs ranging between 0.12 and 4mg/L (only one resistant strain, MIC of 4 mg/L). MIC50 and MIC90 were twice lower in MSSA than MRSA. Moreover, PVL+ MRSA were slightly more susceptible to ceftobiprole (MIC50 of 0.5 mg/L and MIC90 of 1 mg/L) than PVL- MRSA (MIC50 and MIC90 of 1 mg/L). The ceftobiprole-resistant strain was also resistant to ceftaroline fosamil and presented the D239L mutation in PBP2A. The comparator antibiotics were equally active on the strains tested, with MIC90 of 0.5 mg/L for ceftaroline fosamil, tigecycline, and daptomycin; 1 mg/L for vancomycin; and 2 mg/L for linezolid. CONCLUSIONS: Our results suggest that ceftobiprole is highly active against S. aureus and is an effective alternative to vancomycin or linezolid in the management of staphylococcal pneumonia. However, close monitoring of isolates should be maintained to prevent resistant strain diffusion.

Wide geographical dissemination of the multiresistant Staphylococcus capitis NRCS-A clone in neonatal intensive-care units.

Nosocomial late-onset sepsis represents a frequent cause of morbidity and mortality in preterm neonates. The Staphylococcus capitis clone NRCS-A has been previously described as an emerging cause of nosocomial bacteraemia in French neonatal intensive-care units (NICUs). In this study, we aimed to explore the possible unrecognized dissemination of this clone on a larger geographical scale. One hundred methicillin-resistant S. capitis strains isolated from neonates (n = 86) and adult patients (n = 14) between 2000 and 2013 in four different countries (France, Belgium, the UK, and Australia) were analysed with SmaI pulsed-field gel electrophoresis (PFGE) and dru typing. The vast majority of NICU strains showed the NRCS-A pulsotype and the dt11c type (96%). We then randomly selected 14 isolates (from neonates, n = 12, three per country; from adult patients, n = 2), considered to be a subset of representative isolates, and performed further molecular typing (SacII PFGE, SCCmec typing, and multilocus sequence typing-like analysis), confirming the clonality of the S. capitis strains isolated from neonates, despite their distant geographical origin. Whole genome single-nucleotide polymorphism-based phylogenetic analysis of five NICU isolates (from the different countries) attested to high genetic relatedness within the NRCS-A clone. Finally, all of the NRCS-A strains showed multidrug resistance (e.g. methicillin and aminoglycoside resistance, and decreased vancomycin susceptibility), with potential therapeutic implications for infected neonates. In conclusion, this study represents the first report of clonal dissemination of methicillin-resistant coagulase-negative Staphylococcus clone on a large geographical scale. Questions remain regarding the origin and means of international spread, and the reasons for this clone's apparent predilection for neonates.

Community-acquired infections due to Staphylococcus argenteus lineage isolates harbouring the Panton-Valentine leucocidin, France, 2014.

We describe two cases of human infections caused by Staphylococcus aureus clonal complex (CC) 75, also called Staphylococcus argenteus, harbouring the Panton-Valentine leucocidin (PVL). These two sporadic cases were community-acquired, and identified in France in 2014. Both had an epidemiological link with Mayotte, an overseas department of France located in the Indian Ocean off the south-eastern African coast. This report illustrates that, contrary to previous descriptions, S. argenteus can acquire important virulence factors and be responsible for severe infections.

Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains.

Epidemiological data on staphylococcal scalded skin syndromes (SSSS), including bullous impetigo (BI) and generalized exfoliative syndrome (GES), are scarce. To better characterize SSSS and associated Staphylococcus aureus strains, we conducted a retrospective study of 349 cases collected in France between 1997 and 2007 by the National Reference Centre of Staphylococci. Our results showed a stationary evolution of SSSS cases, with a heterogeneous distribution of cases in France. Although notification was not exhaustive, we estimated an incidence of 0.56 cases/year/million inhabitants, in accordance with previous studies conducted in France and Europe, with a median age of 2 years old and sex ratios of 1. A seasonal effect was observed, with a higher GES/BI ratio in autumn compared with other seasons, which could be explained by the impact of viral co-infection. Genetic analysis of S. aureus strains showed that accessory gene regulator (agr) 4, exfoliative toxin A (eta) and B (etb) genes, staphylococcal and enterotoxin-like O (selo) gene and agr4 etb selo profiles were predominantly associated with GES, whereas agr2 eta and agr4 eta selo were more frequently observed with BI. Only one methicillin-resistant strain was found. Protein A (spa) typing identified two main genotypes: spa clonal complex (CC) 159/sequence-type (ST) 121 (75%) and spaCC346/ST15 (18%). spaCC159 was mainly associated with agr4 eta etb selo, agr4 eta selo and agr4 etb selo, and spaCC346 was mainly associated with agr2 eta, suggesting that French SSSS cases are caused by these two main lineages. However, in a multivariate analysis, only etb was independently associated with GES.

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

[Detection of the first strain of glycopeptide intermediary Staphylococcus aureus in Tunis Rabta hospital]. / Détection de la première souche de Staphylococcus aureus de sensibilité diminuée aux glycopeptides à l'hôpital la Rabta de Tunis.

Since the advent of the first glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and its heterogeneous variant hGISA in 1997, debate still ensues as their clinical significance. We report here the first case of GISA in Rabta hospital of Tunisia. Antimicrobial resistance was determined by the disk diffusion method in accordance with CA-SFM (Comity of Antibiogramm of French society of Microbiology). The MIC of vancomycin and teicoplanin was determined by E-test. The detection of mec A gene, virulence factors genes and agr groups (1-4) was performed by multiplex PCR. spa types were determined with the assistance of Ridom of Staph Type software (Ridom GmbH, Wurburg, Germany). The allelic profiles of MRSA were assigned on the basis of their MLST type using the eBURST program. A MRSA bacteraemia patient was treated with teicoplanin for 14 days. S. aureus isolated from patient's blood culture was identified as MRSA and GISA with teicoplanin MIC of 16 mg/l. The molecular study of this strain showed that it belongs to the clonal complex CC8 and is attached to the iberian clone (agr1, enterotoxin A, ST 247, spa type t052). Clinicians and laboratories alike are increasingly aware that patients on long-term vancomycin therapy may signal the presence and potential spread of hGISA/GISA strains. hGISA/GISA strains emerged from lineages with agr types I and II. The multiresitance of the Iberian clone ST247 could be explained by the presence of several resistance genes.

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.

[Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia]. / Comparaison moléculaire de Legionella pneumophila sérogroupe 1 isolées en Tunisie.

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.

Staphylococcal chromosome cassette evolution in Staphylococcus aureus inferred from ccr gene complex sequence typing analysis.

Staphylococcal chromosome cassette mec (SCCmec) elements within major lineages of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) clones were characterised using intra-SCCmec multilocus sequencing. A strong correlation was observed between sequence- and PCR-based typing methods (p <0.001). However, phylogenetic analysis of the SCCmec locus using concatenated sequences evidenced few recombination events. Sequence type (ST)-SCCmec1 was found in SCCmec elements types I and IV, suggesting the evolution of an SCCmecI element into an SCCmecIV element. This coincided with the spread of the clone harbouring this SCCmec element into the community. No correlation was observed between ST-SCCmec lineage and MRSA lineage, confirming multiple acquisitions of SCCmec by S. aureus. This was exemplified by the SCCmecIV ST-SCCmec10 element, which was detected in all of the clonal complexes examined, including healthcare- and community-associated MRSA. The acquisition of this SCCmec element was five- to ten-fold more common than that of others. Models of MRSA clone evolution suggest that this SCCmec was first found in the paediatric clone.

High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study.

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

A rapid and easy PCR-RFLP method for genotyping Serratia marcescens strains isolated in different hospital outbreaks and patient environments in the Lyon area, France.

A new genotyping method for Serratia marcescens is described. This method uses the flagellin gene as target for polymerase chain reaction amplification and Alu I restriction fragment length polymorphism. The strains tested belonged to 13 different hospital clusters of S. marcescens isolated between 1983 and 1988, concerning outbreaks and/or patient environments in different hospital units in Lyon and the Rhone-Alpes region of France. Initially, the classification had been performed by marcescinotyping. These strains were then tested by ribotyping and genotyping of the flagellin gene. Genotyping showed similar classification to ribotyping. The genotyping method is the easiest technique, as reproducible as ribotyping, and with almost the same ability to discriminate different strains. It does not need expensive equipment, is more rapid, and is less labor intensive than ribotyping. With this method, all strains of S. marcescens including sporadic isolates could be amplified and typed. Antibiotic sensitivity determination was found to be a useful complementary and confirmation test for all these typing methods.

Corynebacterium freneyi sp. nov., alpha-glucosidase-positive strains related to Corynebacterium xerosis.

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

Molecular techniques open up new vistas for typing of coagulase-negative staphylococci.

Several methods were used to type 64 clinical isolates of coagulase-negative staphylococci (CNS) derived from hospitals in Morocco. The clinical isolates originated principally from blood cultures and wound sources. These isolates provided the opportunity to substantially compare the proficiency of developing molecular techniques with conventional phenotypic tests for use in the identification of clinical staphylococci. The following molecular methods were examined: Utility ribotyping analysis (Ribotyping); PCR analysis performed with 16S-23S ribosomal-DNA intergenic spacer (ITS-PCR); PCR-based random amplified polymorphic DNA (RAPD). The results obtained by the molecular techniques were contrasted to those of conventional phenotypic tests. Conventional phenotypic tests allowed the outright recognition of the majority of isolates (50/64). These 50 isolates were subdivided into 33 novobiocin-susceptible and 17 novobiocin-resistant strains of CNS. However, 2 other novobiocin-susceptible and 12 other novobiocin-resistant isolates remained unclassified by these tests. There was a good agreement between the conventional phenotypic tests and RAPD for the 33 novobiocin-susceptible isolates. But, the RAPD technique permitted the assignment of the two unidentified novobiocin-susceptible isolates to the Staphylococcus hominis species. A complete correlation was obtained between the three molecular tools for recognition of the 12 novobiocin-resistant isolates that were not identified by phenotypic typing; these were in fact identified as 5 Staphylococcus cohnii and 4 Staphylococcus equorum. Three isolates remained unidentified by all three systems of molecular techniques.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Multicenter evaluation of epidemiological typing of methicillin-resistant Staphylococcus aureus strains by repetitive-element PCR analysis. The European Study Group on Epidemiological Markers of the ESCMID.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Staphylococcus fleurettii sp. nov., isolated from goat's milk cheeses.

A new coagulase-negative and novobiocin-resistant species of the genus Staphylococcus, Staphylococcus fleurettii, isolated from raw-milk cheeses, is described. This species is differentiated from the other novobiocin-resistant staphylococci on the basis of ribotype and intergenic transcribed spacer patterns, DNA-DNA reassociation reactions, cell wall composition and phenotypic characteristics. S. fleurettii could be distinguished by its oxidase activity, by its ability to produce acid aerobically from D-trehalose, D-mannose, D-turanose and maltose and by its inability to produce acid from D-cellobiose. The type strain of S. fleurettii is CIP 106114T (= DSM 13212T).

Improvement of the identification of staphylococci isolated from bovine mammary infections using molecular methods.

Fifty-six Staphylococcus strains isolated from cases of bovine mammary infections were identified by using phenotypic and genotypic methods. Twenty-eight strains (50%) were identified at the species level according to their phenotypic characteristics, whereas the remaining 28 strains presented atypical or unreliable profiles. A combination of phenotypic and genotypic methods allowed the 56 strains studied to be classified. Internal transcribed spacer-polymerase chain reaction (ITS-PCR) based on the polymorphism of the 16S-23S rDNA spacer region appeared as a rapid and reliable method for the classification of bovine staphylococcal isolates at the species and subspecies levels.