Spontaneous Genomic Variation as a Survival Strategy of Nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical device-related infections. However, its mechanisms of evolution and adaptation are still poorly explored. To characterize the strategies of genetic and phenotypic diversity in S. haemolyticus, we analyzed an invasive strain for genetic and phenotypic stability after serial passage in vitro in the absence and presence of beta-lactam antibiotics. We performed pulsed-field gel electrophoresis (PFGE) of the culture and analyzed five colonies at seven time points during stability assays for beta-lactam susceptibility, hemolysis, mannitol fermentation, and biofilm production. We compared their whole genomes and performed phylogenetic analysis based on core single-nucleotide polymorphisms (SNPs). We observed a high instability in the PFGE profiles at the different time points in the absence of antibiotic. Analysis of WGS data for individual colonies showed the occurrence of six large-scale genomic deletions within the oriC environ, smaller deletions in non-oriC environ regions, and nonsynonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes, and insertion sequence (IS) elements. Parallel variation was detected in clinically significant phenotypic traits such as mannitol fermentation, hemolysis, and biofilm formation. In the presence of oxacillin, PFGE profiles were overall stable over time and mainly corresponded to a single genomic variant. Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants. The maintenance of subpopulations in different physiological states may be a strategy to adapt rapidly to stress situations imposed by the host, particularly in the hospital environment. IMPORTANCE The introduction of medical devices and antibiotics into clinical practice have substantially improved patient quality of life and contributed to extended life expectancy. One of its most cumbersome consequences was the emergence of medical device-associated infections caused by multidrug-resistant and opportunistic bacteria such as Staphylococcus haemolyticus. However, the reason for this bacterium's success is still elusive. We found that in the absence of environmental stresses, S. haemolyticus can spontaneously produce subpopulations of genomic and phenotypic variants with deletions/mutations in clinically relevant genes. However, when exposed to selective pressures, such as the presence of antibiotics, a single genomic variant will be recruited and become dominant. We suggest that the maintenance of these cell subpopulations in different physiological states is an extremely effective strategy to adapt to stresses imposed by the host or the infection environment and might contribute for S. haemolyticus survival and persistence in the hospital.

Dissemination of epidemic ST239/ST241-t037-agrI-SCCmecIII methicillin-resistant Staphylococcus aureus in a Tunisian trauma burn intensive care unit.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing health care-infections in the world, especially in burns. The aim of this study was to assess the extent of dissemination of MRSA isolated from burn patients in Burn Intensive Care Unit in Tunisia and to evaluate the frequency of virulence and antibiotics resistance genes. Among the 72 S. aureus isolates analyzed in the study, 54% were MRSA. The majority of MRSA (94.8%) were multidrug resistant and they had a high resistance rates to kanamycin (94.8%), tobramycin (90%), tetracycline (94.8%) and ciprofloxacin and rifampicin (87%, each). The gene aac(6')-Ie-aph(2″)-Ia conferring resistance to kanamycine and tobtamycin were detected in all isolates and the aph(3')-Ia gene conferring resistance to gentamicin were detected in 2.8% of resistant isolates. Tetracycline resistance genes tet(M), tet(K) and tet(L) were detected in 100%, 10.8% and 2.8% of the isolates, respectively. The SCCmec type III and the agr type I were the most predominant (69.2% and 90%, respectively). The 27 SCCmecIII-agrI isolates were clustered into two PFGE types A and B. The two representative isolates of PFGE clusters A and B belonged to ST239-t037 and ST241-t037 respectively. As conclusion, our results showed a high prevalence of MRSA in trauma burn intensive care unit belonging to two multidrug resistant clones ST239/ST241-agrI-t037-SCCmecIII MRSA. We also demonstrated that MRSA was disseminated between burn patients.

Frequent dissemination and carriage of an SCCmec-mecC hybrid in methicillin-resistant Mammaliicoccus sciuri in farm animals from Tunisia.

OBJECTIVES: In this study, we aimed to assess the extent of dissemination of methicillin-resistant Mammaliicoccus sciuri in animal farms in Tunisia and evaluate the distribution of virulence and methicillin resistance genes in the M. sciuri population. METHODS: Staphylococci and mammaliicocci isolated from unhealthy animals and healthy humans from adjacent farms in Tunisia were characterized for antimicrobial susceptibility, biofilm formation, agglutination, and hemolysis abilities. Mammaliicoccus sciuri relatedness and content in antibiotic resistance and virulence genes were analyzed by whole-genome sequencing (WGS). RESULTS: Mammaliicoccus sciuri was the most prevalent species (46.2%), showing the highest resistance rates to fusidic acid (94.6%), oxacillin (73%), penicillin (40.5%), clindamycin (37%), ciprofloxacin (27%), and cefoxitin (24.3%). Some isolates carried genes encoding resistance to nine different antibiotic classes. mecA was found in 35% of M. sciuri and mecC in 16.2%. All isolates carrying mecC were of S. sciuri subspecies carnaticus and carried the hybrid element SCCmec-mecC. Mammaliicoccus sciuri were able to produce strong biofilm (27%) and have clumping ability (16%). Additionally, they carried genes for capsule production (cap8, 100%), iron-regulated surface determinants (isdE, 24%; isdG, 3%), and virulence regulation (clpC and clpP, 100%). Single nucleotide polymorphisms (SNPs) analysis showed that 17 M. sciuri cross-transmission events probably occurred between different animal species and farms. Moreover, SCCmec was estimated to have been acquired five times by S. sciuri subsp. carnaticus. CONCLUSION: Multidrug resistant and pathogenic M. sciuri were frequently disseminated between different animal species within the farm environment. mecA and mecC can be disseminated by both frequent acquisition of the SCCmec element and clonal dissemination.

Staphylococcus saprophyticus Causing Infections in Humans Is Associated with High Resistance to Heavy Metals.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600 mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs ≥ 1,600 mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs ≥ 200 mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

Foodborne Origin and Local and Global Spread of Staphylococcus saprophyticus Causing Human Urinary Tract Infections.

Staphylococcus saprophyticus is a primary cause of community-acquired urinary tract infections (UTIs) in young women. S. saprophyticus colonizes humans and animals but basic features of its molecular epidemiology are undetermined. We conducted a phylogenomic analysis of 321 S. saprophyticus isolates collected from human UTIs worldwide during 1997-2017 and 232 isolates from human UTIs and the pig-processing chain in a confined region during 2016-2017. We found epidemiologic and genomic evidence that the meat-production chain is a major source of S. saprophyticus causing human UTIs; human microbiota is another possible origin. Pathogenic S. saprophyticus belonged to 2 lineages with distinctive genetic features that are globally and locally disseminated. Pangenome-wide approaches identified a strong association between pathogenicity and antimicrobial resistance, phages, platelet binding proteins, and an increased recombination rate. Our study provides insight into the origin, transmission, and population structure of pathogenic S. saprophyticus and identifies putative new virulence factors.

Evidence for the Dissemination to Humans of Methicillin-Resistant Staphylococcus aureus ST398 through the Pork Production Chain: A Study in a Portuguese Slaughterhouse.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), spa, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6-55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCCmec V), but differed in 321-378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.

New Insights into Antibiofilm Effect of a Nanosized ZnO Coating against the Pathogenic Methicillin Resistant Staphylococcus aureus.

ZnO nanoparticles (NPs) are arising as promising novel antibiotics toward device-related infections. The surface functionalization of Zn, a novel resorbable biomaterial, with ZnO NPs could present an effective solution to overcome such a threat. In this sense, the antibacterial and antibiofilm activity of nano- and microsized ZnO coatings was studied against clinically relevant bacteria, methicillin resistant Staphylococcus aureus (MRSA). The bacterial viability of planktonic and biofilm cells together with the corresponding biofilm structures revealed that only the nanosized ZnO coating had an antibiofilm effect. To elucidate this effect, a novel approach was taken: preconditioning of bacteria with this ZnO coating followed by exposure to subinhibitory concentrations of antibiotics with well-known modes of actions. This approached revealed (i) a decreased biofilm formation in combination with gentamycin, targeting protein synthesis, and (ii) an increased biofilm formation in the presence of rifampicin and vancomycin, acting on RNA and cell wall biosynthesis, respectively. The increased bacteria resistance to these two antibiotics gave new insights into the antibiofilm effect of this nanosized ZnO coating. The synergistic effect observed for gentamycin opened new perspectives for the design of effective solutions against implant-related infections. During the in vitro degradation of this nanosized ZnO-coated Zn, a specific corrosion product, hopeite [Zn3(PO4)2], was depicted. Interestingly, the increased deposition of hopeite-derived compounds on MRSA cells surface seemed to be related to unhealthy and dead bacterial cells. This observation suggested that hopeite may well play a key role in this antibiofilm activity. The results obtained herein shed light on the possible antibacterial effect of a nanosized ZnO coating, and strengthened its antimicrobial (antibacterial and antifungal) potential, therefore providing a potentially effective material to overcome the growing trend of implant-related infections.

Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus.

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

Impact of Insertion Sequences and Recombination on the Population Structure of Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most common pathogens associated with medical-device related infections, but its molecular epidemiology is poorly explored. In the current study, we aimed to better understand the genetic mechanisms contributing to S. haemolyticus diversity in the hospital environment and their impact on the population structure and clinical relevant phenotypic traits. The analysis of a representative S. haemolyticus collection by multilocus sequence typing (MLST) has identified a single highly prevalent and diverse genetic lineage of nosocomial S. haemolyticus clonal complex (CC) 29 accounting for 91% of the collection of isolates disseminated worldwide. The examination of the sequence changes at MLST loci during clonal diversification showed that recombination had a higher impact than mutation in shaping the S. haemolyticus population. Also, we ascertained that another mechanism contributing significantly to clonal diversification and adaptation was mediated by insertion sequence (IS) elements. We found that all nosocomial S. haemolyticus, belonging to different STs, were rich in IS1272 copies, as determined by Southern hybridization of macrorestriction patterns. In particular, we observed that the chromosome of a S. haemolyticus strain within CC29 was highly unstable during serial growth in vitro which paralleled with IS1272 transposition events and changes in clinically relevant phenotypic traits namely, mannitol fermentation, susceptibility to beta-lactams, biofilm formation and hemolysis. Our results suggest that recombination and IS transposition might be a strategy of adaptation, evolution and pathogenicity of the major S. haemolyticus prevalent lineage in the hospital environment.

Multilocus sequence typing and further genetic characterization of the enigmatic pathogen, Staphylococcus hominis.

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.

Community-Associated Methicillin-Resistant Staphylococcus aureus Lacking PVL, as a Cause of Severe Invasive Infection Treated with Linezolid.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging public health problem worldwide. Severe invasive infections have been described, mostly associated with the presence of Panton-Valentine leukocidin (PVL). In Portugal limited information exists regarding CA-MRSA infections. In this study we describe the case of a previously healthy 12-year-old female, sport athlete, who presented to the hospital with acetabulofemoral septic arthritis, myositis, fasciitis, acetabulum osteomyelitis, and pneumonia. The MRSA isolated from blood and synovial fluid was PVL negative and staphylococcal enterotoxin type P (SEP) and type L (SEL) positive, with a vancomycin MIC of 1.0 mg/L and resistant to clindamycin and ciprofloxacin. The patient was submitted to multiple surgical drainages and started on vancomycin, rifampicin, and gentamycin. Due to persistence of fever and no microbiological clearance, linezolid was started with improvement. This is one of the few reported cases of severe invasive infection caused by CA-MRSA in Portugal, which was successfully treated with linezolid. In spite of the severity of infection, the MRSA isolate did not produce PVL.

Strong biofilm production but not adhesion virulence factors can discriminate between invasive and commensal Staphylococcus epidermidis strains.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in immunocompromised patients. It originates from the patient's own skin flora, which is subject to severe changes as a result of selective pressure exerted by the hospital environment. This notion led us to compare S. epidermidis isolates from catheter related infections (CRI), non-catheter related bacteremia (NCRB) and catheter hub cultures (commensal isolates). The collection comprised 47 CRI strains from the Bone Marrow Transplant Centre of Tunis, 25 NCRB strains and 25 commensal isolates from patients hospitalized in the same center. Antimicrobial resistance and virulence-associated genes (icaABC, aap, atlE, bhp, fbe, embp, and IS256), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated. The clonal relationship of strains was investigated by pulsed field gel electrophoresis. Whereas bhp, atlE, fbe, embp, and aap were almost ubiquitously amplified, resistance to oxacillin, kanamycin, tobramycin, gentamicin, cotrimoxazole, and fosfomycin, biofilm production, ica genes, and IS256 were significantly more frequent in invasive (CRI and NCRB strains) than in commensal strains. Moreover, strong biofilm production was significantly more frequent among CRI strains than in NCRB strains. In conclusion, when S. epidermidis is isolated from blood cultures, the detection of strong biofilm production may be significant with regard to judging whether the detected strain is an etiologic agent of CRI.

High genetic diversity among community-associated Staphylococcus aureus in Europe: results from a multicenter study.

BACKGROUND: Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent. METHODS AND FINDINGS: A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59% of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83% of MRSA carried Panton-Valentine leukocidin (PVL) and 14% carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson's index of diversity = 0.852 (0.788-0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRSA isolates were highly related, suggesting a probable local acquisition/loss of SCCmec. CONCLUSIONS: Our results imply that CA-MRSA origin, epidemiology and population structure in Europe is very dissimilar from that of USA.

Phenotypic and genotypic characterization of Salmonella enterica recovered from poultry meat in Tunisia and identification of new genetic traits.

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.

Molecular epidemiology of methicillin-resistant Staphylococcus hominis (MRSHo): low clonality and reservoirs of SCCmec structural elements.

BACKGROUND: Methicillin resistant Staphylococcus hominis (MRSHo) are important human pathogens in immunocompromised patients. However, little is known regarding its population structure and staphylococcal chromosomal cassette mec (SCCmec) content. METHODOLOGY/PRINCIPAL FINDINGS: To assess the population structure and the SCCmec content of S. hominis, 34 MRSHo and 11 methicillin-susceptible S. hominis (MSSHo) from neutropenic patients collected over a 3-year period were studied. The genetic backgrounds of S. hominis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and SCCmec types were determined by PCR. Cassette chromosome recombinases (ccr) were characterized by PCR and ccrB sequencing. The 34 S. hominis isolates were classified into as many as 28 types and 32 subtypes (SID = 99.82%); clonal dissemination was occasionally observed. The main SCCmec structures identified were SCCmec type VI (4B) (20%), SCCmec VIII (4A) (15%), and a new SCCmec composed of mec complex A in association with ccrAB1 (38%); 27% of the isolates harbored non-typeable SCCmec. Overall, a high prevalence of mec complex A (73.5%), ccrAB1 (50%) and ccrAB4 (44%) were found. Importantly, ccrB1 and ccrB4 from both MRSHo and MSSHo showed a high nucleotide sequence homology with those found in S. aureus SCCmec I, VI and VIII respectively (>95%). CONCLUSIONS/SIGNIFICANCE: The S. hominis population showed a limited clonality and a low genetic diversity in the allotypes of ccr and classes of mec complex. Moreover, our data suggest that S. hominis might have been a privileged source of mec complex A, ccrB1 and ccrB4, for the assembly of primordial SCCmec types.

Pulsed Field Gel Electrophoresis types of Candida albicans isolates from an intensive care unit in a Tunisian hospital.

Candida albicans is the most important cause of fungal infections in intensive care units. The aim of this work was to compare the profiles of C. albicans in order to specify their genetic polymorphism and to determine the origin of these infections. Thirty-five C. albicans strains were collected from different clinical samples of 12 patients and three health-workers in an intensive care unit (ICU) in Rabta hospital of Tunisia, between August 2007 and April 2008. After digestion with BssHII, the isolates were typed by pulsed field gel electrophoresis (PFGE). The PFGE profiles were analyzed using a visual method, which showed three PFGE types (A, B and C) and the dendrogram generated three clusters (clusters I to III). An average similarity coefficient of 0.83, suggests that isolates are related.

Clonality and occurrence of genes encoding antibiotic resistance and biofilm in methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and bacteremia in neutropenic patients.

Thirty methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and blood cultures from neutropenic patients were studied. They were classified into 17 multidrug-resistance patterns. Polymerase cahin reaction analysis revealed that methicillin resistance was encoded by the mecA gene in all strains, and aminoglycosides resistance was due to aac(6')-Ie-aph(2'')-Ia (23 strains), ant(4')-Ia (13), and aph(3')-IIIa (1) genes. The aac(6')-Ie-aph(2'')-Ia gene was detected concomitantly with aph(3')-IIIa, and ant(4')-Ia genes in one and nine strains, respectively. Erythromycin resistance was encoded by the ermC (11 strains), ermA (6), and msrA (2) genes. The ermC gene was inducibly expressed in five strains, whereas the ermA was exclusively constitutively expressed. The icaA and icaC genes were detected in 19 strains; however, biofilm production was observed in only 16 strains. Most strains harbored multiple plasmids of variable sizes ranging from 2.2 to 70 kb, and two strains were plasmid-free. PFGE identified 15 distinct PFGE types, and five predominant genotypes were found. Our study showed the occurrence of complex genetic phenomenons. In unrelated strains, evidence of horizontal transfer of antibiotic-encoding genes and/or ica operon, and in indistinguishable strains, there is a quite good likelihood of independent steps of loss and/or gain of these genes. This genome dynamicity might have enhanced the invasiveness power of these methicillin-resistant S epidermidis strains.

Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia.

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.