Analysis of Staphylococcal cassette chromosome mec in Staphylococcus haemolyticus and Staphylococcus sciuri: identification of a novel ccr gene complex with a newly identified ccrA allotype (ccrA7)

Methicillin resistance in staphylococci is conferred by the acquisition in its chromosome of the mecA gene, which is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). Genetic type of SCCmec is defined by combination of mec gene complex class and cassette chromosome recombinase gene (ccr) allotype. In this study, we analyzed genetic diversity of the SCCmec in 11 Staphylococcus haemolyticus strains and a Staphylococcus sciuri strain, which were recently isolated from clinical specimens in Bangladesh. Among these strains, only two S. haemolyticus strains were proved to have the known types of SCCmec, that is, SCCmec V (class C2 mec-ccrC) and VII (class C1 mec-ccrC). Five S. haemolyticus strains were assigned two unique mec-ccr gene complexes combination; that is, class C1 mec-ccrA4B4 (four isolates) and class A mec-ccrC (one isolate). In the remaining four S. haemolyticus strains with class C1 mec, no known ccr allotypes could be detected. A single S. sciuri strain with class A mec complex carried a ccrA gene belonging to a novel allotype designated ccrA7, together with ccrB3. The ccrA7 gene in the S. sciuri strain showed 61.7%-82.7% sequence identity to the ccrA gene sequences published so far, and 75.3% identity to ccrA3, which is a component of the type 3 ccr complex (ccrA3-ccrB3) in methicillin-resistant Staphylococcus aureus. The results of the present study indicated that mec gene complex and ccr genes in coagulase-negative staphylococci are highly divergent, and distinct from those of common methicillin-resistant S. aureus. Identification of the novel ccrA7 allotype combined with ccrB3 suggested an occurrence of recombination between different ccr complexes in nature.     

Molecular characterization of Staphylococcus sciuri strains isolated from humans

We previously characterized over 100 Staphylococcus sciuri isolates, mainly of animal origin, and found that they all carried a genetic element (S. sciuri mecA) closely related to the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) strains. We also found a few isolates that carried a second copy of the gene, identical to MRSA mecA. In this work, we analyzed a collection of 28 S. sciuri strains isolated from both healthy and hospitalized individuals. This was a relatively heterogeneous group, as inferred from the different sources, places, and dates of isolation and as confirmed by pulsed-field gel electrophoresis analysis. All strains carried the S. sciuri mecA copy, sustaining our previous proposal that this element belongs to the genetic background of S. sciuri. Moreover, 46% of the strains also carried the MRSA mecA copy. Only these strains showed significant levels of resistance to beta-lactams. Strikingly, the majority of the strains carrying the additional MRSA mecA copy were obtained from healthy individuals in an antibiotic-free environment. Most of the 28 strains were resistant to penicillin, intermediately resistant to clindamycin, and susceptible to tetracycline, erythromycin, and gentamicin. Resistance to these last three antibiotics was found in some strains only. The findings reported in this work confirmed the role of S. sciuri in the evolution of the mechanism of resistance to methicillin in staphylococci and suggested that this species (like the pathogenic staphylococci) may accumulate resistance markers for several classes of antibiotics.     

Prevalence of Staphylococcus aureus carrying Panton-Valentine leukocidin genes among isolates from hospitalised patients in China.

Between January 2005 and January 2006, 25 (12.8%) of 195 Staphylococcus aureus isolates were positive for Panton-Valentine leukocidin (PVL) genes in a teaching hospital in Wenzhou, China. Nineteen (11.9%) of 160 hospital-acquired isolates, and six (17.1%) of 35 community-acquired isolates, harboured lukS/F-PV. Six sequence types (ST88, ST239, ST398, ST25, ST30 and ST59) were found among 18 PVL-positive methicillin-resistant isolates with SCCmec types I, III, IIIA or IV. Only ST88 was found among seven PVL-positive methicillin-susceptible S. aureus isolates. The PVL-positive isolates were associated with lung infection, bloodstream infection and soft-tissue pyogenic infection. Overall, there was a high prevalence of PVL genes in genetically diverse S. aureus isolates.     

First report of infection with community-acquired methicillin-resistant Staphylococcus aureus in South America

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged in the southwestern Pacific, North America, and Europe. These S. aureus isolates frequently shared some genetic characteristics, including the SCCmec type IV and lukS-lukF genes. In this paper we show that typical CA-MRSA isolates have spread to South America (Brazil).     

Partial excision of the chromosomal cassette containing the methicillin resistance determinant results in methicillin-susceptible Staphylococcus aureus

We report a detailed characterization of methicillin-susceptible Staphylococcus aureus isolates from five French hospitals negative for both the mecA and the ccrAB loci but positive for the IS431::pUB110::IS431::dcs structure, present in some Staphylococcus cassette chromosome mec (SCCmec) types. The presence of SCCmec-associated elements suggests that this unusual resistant phenotype is due to a partial excision of SCCmec from epidemic methicillin-resistant S. aureus. The hypothesis of a genetic relatedness is strengthened by common sequence and spa types and similar susceptibility patterns.     

多重PCR对金黄色葡萄球菌杀白细胞素基因的检测

目的应用多重PcR检测含Panton-Valentine杀白细胞素（PVL）基因的金黄色葡萄球菌。方法收集我院2005年1月至2006年1月从临床多种标本中分离的金黄色葡萄球菌，用多重PCR同时检测葡萄球菌16SrRNA基因、mecA基因和lukS/F-PV基因。多重PCR检测MRSA的SCCmec基因型及亚型。结果195株金黄色葡萄球菌经多重PCR检测，121株为耐甲氧西林金黄色葡萄球菌（MRSA），74株为甲氧西林敏感金黄色葡萄球菌（MSSA），共检测到26株金黄色葡萄球菌lukS/F-PV基因阳性，阳性率为13．3％（26，195）。其中19株为MRSA，阳性率为15．7％（19／121）；7株为MSSA，阳性率为12．2％（7／74）。19株lukS/F-PV基因阳性的MRSA的SCCmec基因型分别为SCCmec Ⅲ型10株、SCCmecⅢA型4株、SCCmecⅣ型4株及SCCmecⅠ型1株。26株lukS/F-PV基因阳性的分离株有11株分离自脓液或创面分泌物，10株分离自痰标本，3株分离自血液标本，2株分离自尿液。结论在温州地区分离的MRSA和MSSA中都能检测到Panton-Valentine杀白细胞素基因，含Panton-Valentine杀白细胞素基因MRSA的SCCmec基因型主要为SCCmec Ⅲ，含PVL基因的金黄色葡萄球菌主要引起化脓性感染和肺部感染。     

Distribution of Staphylococcus sciuri subspecies among human clinical specimens, and profile of antibiotic resistance

The distribution of three subspecies comprising Staphylococcus sciuri was determined for a collection of 30 clinical isolates originating from Morocco, the United Kingdom, and France. The sources of these isolates were principally wounds, skin, and soft tissue infections. At the species level, the isolates were identified according to biochemical characteristics and at the subspecies level by the ribotyping technique. PCR analysis performed with the 16S-23S ribosomal DNA intergenic spacer was less powerful for subspecies differentiation. S. sciuri subsp. sciuri was the most frequent subspecies (21 isolates) found in the collection, whereas S. sciuri subsp. rodentium (seven isolates) and S. sciuri subsp. carnaticus (two isolates) were less common. mecA or a mecA-related gene was detected by PCR and Southern blot in all 30 S. sciuri isolates, supporting the suggestion that S. sciuri species are the natural reservoir of the mecA gene. While the linA/linA' gene coding for lincomycin resistance was present in five isolates, an uncharacterized gene for this resistance was suspected in seventeen other isolates.     

Expression of high-level methicillin resistance in Staphylococcus aureus from the Staphylococcus sciuri mec A homologue: role of mutation(s) in the genetic background and in the coding region of mec A

A close homologue of the mec A gene, the primary drug resistance determinant in methicillin resistant Staphylococcus aureus (MRSA), is ubiquitous in the animal commensal species Staphylococcus sciuri, yet most isolates of this staphylococcal species are susceptible to beta-lactam antibiotics including methicillin. Recently, we showed that in a methicillin-resistant mutant of S. sciuri prepared in the laboratory, the mec A homologue is converted to an antibiotic resistance gene by a point mutation introduced into the -10 consensus of the promoter and such promoter-up mutants of the S. sciuri mec A can express a significant degree of methicillin resistance when introduced into an antibiotic-susceptible strain of S. aureus. We now demonstrate that in this system further increase of the drug resistance phenotype may be achieved under antibiotic pressure by at least two different mechanisms. The first one of these involves the introduction of a point mutation at nucleotide Nt 1889 in the coding region of the S. sciuri-derived mec A determinant, resulting in the replacement of an asparagine with a threonine residue downstream of the conserved SXXK motif which causes extensive reduction in the beta-lactam antibiotic binding capacity (affinity) of the penicillin binding protein (PBP) encoded by the S. sciuri mec A homologue. A second, distinct, mechanism causing increased methicillin resistance is associated with mutation(s) of unknown nature in the genetic background of the S. aureus host.     

Investigation of Panton-Valentine leukocidin genes and SCCmec types in clinical Staphylococcus aureus isolates from Turkey

Panton-Valentine leukocidin (PVL) is an important virulence determinant of Staphylococcus aureus. The aim of this study was to investigate the prevalence of PVL genes in clinical S. aureus isolates and to determine the staphylococcal chromosomal cassette mec (SCCmec) types of methicillin-resistant S. aureus (MRSA) strains obtained from inpatients and outpatients of two hospitals in Turkey. Of the 304 S. aureus strains (230 hospital acquired [HA] and 74 community-onset [CO]), 261 were MRSA and 43 were methicillin-sensitive S. aureus (MSSA). PVL positivity was determined in 12 (1 HA and 11 community acquired) strains. Eight were MRSA, and four were MSSA. Seven of the PVL-positive strains were isolated from wound specimens, four from urine, and one from synovial fluid. SCCmec type III (93.78%) was more prevalent among HA-MRSA strains, and SCCmec type IIIB (41.18%) was more prevalent among CO-MRSA strains. Pulsed-field gel electrophoresis patterns of the PVL-positive isolates were different. Our results indicate that PVL-positive strains are able to cause infection in nearly every system without the need for additional risk factors. Our PVL-positive CO-MRSA strains carry SCCmec types other than types IV and V. Due to the presence of PVL-positive strains in the hospitals, it is important to establish appropriate infection control measures to prevent their spread in the community and in hospitals.     

A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I–V

A multiplex PCR with four primer-pairs was designed to identify the five main known SCCmec types. A clear and easily discriminated band pattern was obtained for all five types. The SCCmec type was identified for 98% of 312 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec type IV was by far the most common SCCmec type among both hospital- and community-acquired MRSA isolates in Denmark.     

Detection of diverse SCCmec variants in methicillin-resistant Staphylococcus aureus and comparison of SCCmec typing methods

Non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 436), collected from four hospitals located in three Korean cities between 2001 and 2005, were investigated by SCCmec typing and multilocus sequence typing (MLST). Variations within SCCmec, especially type II, were detected in 165 (37.8%) isolates, and these variants were characterised using four different SCCmec typing methods. The predominant SCCmec type was a type II variant that differed from type II by the absence of a pUB110 insertion. MLST analysis showed that most of the isolates carrying SCCmec variants belonged to ST5.     

Low prevalence of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in Belgian hospitals

Staphylococcus aureus strains with decreased susceptibility to glycopeptides (GISA) have been associated with increased risk of glycopeptide treatment failure. To assess the prevalence of these strains in hospitalised patients in Belgium, 455 methicillin-resistant S. aureus (MRSA) isolates collected in 2001 were screened by two assays: (i) growth on vancomycin agar screen (VAS; brain heart infusion agar (BHI) + vancomycin 6 mg/L); and (ii) a synergy/antagonism test with aztreonam/cefazolin on Mu3 agar (BHI + vancomycin 3 mg/mL). Isolates growing on VAS or Mu3 agar were characterised further by analysis of population susceptibility profiles. MICs of glycopeptides were determined by agar dilution, broth microdilution and Etest (low and high inocula) methods. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and determination of staphylococcal cassette chromosome mec (SCCmec) type. No GISA isolates were found. Three (0.7%) hetero-vancomycin intermediate S. aureus (hVISA) and ten (2.2%) hetero-teicoplanin intermediate S. aureus (hTISA) isolates were identified by population analysis. All but one hetero-GISA isolate belonged to either epidemic PFGE group A/SCCmec type I (69%) or PFGE group D/SCCmec type I (23%), both of which were resistant to gentamicin. The sensitivity and specificity for the detection of hetero-GISA by the two assays were 15.4% and 99.8%, respectively, for VAS, and 84.6% and 95.9%, respectively, for Mu3. The data indicated that hetero-GISA strains were uncommon among Belgian MRSA isolates from hospitalised patients. Use of Mu3 agar was more sensitive, but less specific, than VAS as a screening method.     

Molecular genetics of methicillin-resistant Staphylococcus aureus

A large and growing proportion of Staphylococcus aureus clinical isolates are methicillin resistant, and are resistant to practically all beta-lactam antibiotics. Methicillin-resistant S. aureus (MRSA) strains harbor mecA, which is carried by a unique mobile genetic element, staphylococcal cassette chromosome mec (SCCmec) integrated into the S. aureus chromosome. The mecA gene encodes a methicillin-insensitive transpeptidase, the production of which confers resistance to otherwise inhibitory concentrations of beta-lactam antibiotics. Several distinct clones have been identified among MRSA that apparently have been generated by integration of distinct types of SCCmec. While MRSA are primarily nosocomial pathogens, recent observations indicate that other MRSA clones are colonizing a significant proportion of healthy individuals in the community as well. Community-acquired MRSA (C-MRSA), may become a new threat to humans, and international cooperation of researchers and clinicians will be of cardinal importance in addressing this problem.     

Influence of the incubation atmosphere on the production of biofilm by staphylococci

Biofilm formation by Staphylococcus epidermidis , Staphylococcus hemolyticus , Staphylococcus sciuri and Staphylococcus aureus in aerobic, anaerobic and CO₂ incubation atmospheres was quantified by the modified microtiter plate test. The S. epidermidis and S. aureus strains showed significantly lower biofilm production when grown in a CO₂-rich environment compared to that exhibited in aerobic incubation. The amount of biofilm produced by these strains under anaerobic conditions did not differ significantly from the biofilm formation detected in the aerobic incubation. The incubation atmosphere did not affect S. sciuri biofilm formation. Biofilm production by S. hemolyticus isolates was very low regardless of the experimental conditions used.     

Molecular characterization and antibiotic susceptibility of Staphylococcus aureus from a multidisciplinary hospital in Romania

From 2004 to 2005, 60%-72% of invasive Staphylococcus aureus isolates from Romanian hospitals were resistant to methicillin (methicillin-resistant S. aureus [MRSA]), the highest frequency for any European nation. Few reports, however, have addressed the molecular characteristics of S. aureus in Romania. In this study, we utilized spa typing, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, dru typing, pulsed-field gel electrophoresis, and detection of virulence factors to characterize 146 S. aureus strains isolated from 2004 to 2005 at the Clinic County Hospital in Bra?ov. Antibiotic susceptibility patterns for all MRSA isolates and patient demographic data were also obtained. Fifty-six strains (38.4%) were determined to be MRSA by susceptibility testing and SCCmec typing. All MRSA strains were resistant to beta-lactams and tetracycline, but susceptible to nitrofurans, vancomycin, and clindamycin, with inducible clindamycin resistance in 23/28 clindamycin-sensitive/erythromycin-resistant isolates. Molecular typing identified 15 clonal backgrounds (CC 1, 5, 8, 8/239, 9, 15, 20, 22, 25, 30, 45, 80, 97, 101, and 121), only 4 of which were associated with MRSA (CC 1, 8/239, 30, and 80). Spa types 35 (t127, CC 1) and 351 (t030, CC 8/239) accounted for 27.4% and 21.9% of all S. aureus strains, respectively, and 19.6% and 57.1% of all MRSA strains. Both hospital-associated (SCCmec type III) and community-associated (SCCmec type IV) elements were identified within MRSA strains, whereas Panton-Valentine leukocidin was detected in 10 MRSA and 12 methicillin-sensitive S. aureus strains. These results demonstrate the presence of various endemic S. aureus clones within the Clinic County Hospital in Bra?ov, suggestive of ongoing nosocomial and community transmission.     

Prevalence of SCCmec type IV in nosocomial bloodstream isolates of methicillin-resistant Staphylococcus aureus

Over a period of 7 months, 151 consecutive methicillin-resistant Staphylococcus aureus blood isolates were evaluated. None was community acquired. Twenty (13%) were susceptible to four or more antimicrobials, and 95% of these isolates were identified as SCCmec type IV. Molecular typing demonstrated four patterns, with one predominant pattern. Although usually community acquired, SCCmec type IV in our setting is clearly nosocomial.     

Risk factors and molecular analysis of community methicillin-resistant Staphylococcus aureus carriage

A total of 1,838 subjects from the community and 393 subjects from health care-related facilities in Taiwan were evaluated for the prevalence of nasal Staphylococcus aureus colonization and to identify risk factors associated with S. aureus and methicillin-resistant S. aureus (MRSA) colonization. Among the community subjects, 3.5% had nasal MRSA colonization. Subjects from health care-related facilities had a lower S. aureus colonization rate (19.1%) than community subjects (25.2%) but had a significantly higher rate of colonization with MRSA (7.63%). Age (P < 0.001) was a significant risk factor for S. aureus colonization, with subjects under age 20 years or between 71 and 80 years showing higher rates of colonization. Recent gastrointestinal disease (P = 0.011) and hospital admission (P = 0.026) were risk factors for nasal MRSA colonization. Comparison of hospital MRSA isolates with the colonization strains by staphylococcal cassette chromosome mec (SCCmec) gene typing and pulsed-field gel electrophoresis (PFGE) typing revealed that most MRSA strains carried in the community were SCCmec type IV and that most clinical hospital isolates were type III, while health care facility-related carriage isolates were mainly SCCmec type III and type IV. Two new variant SCCmec types were identified. Six clusters of PFGE patterns were distinguished: two mainly comprised health care facility-related MRSA strains, three mainly comprised community MRSA strains, and one comprised mixed community and health care facility-related MRSA strains. In conclusion, a high prevalence of MRSA colonization was observed among people with no relationship to the hospital setting. The high level of multiple-drug resistance among community MRSA strains in association with the previously reported excessive use of antibiotics in Taiwan highlights the importance of the problem of antibiotic selective pressure. Our results indicate that both the clonal spread of MRSA and the transmission of hospital isolates contribute to the high MRSA burden in the community.     

Molecular pattern and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Northern Taiwan.

BACKGROUND AND PURPOSE: Methicillin-resistant Staphylococcus aureus (MRSA) infection has progressively increased worldwide. Knowledge of the specific epidemiological pattern of isolates at individual hospitals is important. METHODS: MRSA bacteremia was diagnosed in a total of 68 patients from January 2002 through December 2003, stratified for drug susceptibility and molecular pattern (staphylococcal cassette chromosome mec element [SCCmec] typing and genotypes). RESULTS: SCCmec-A-positive isolates were found on polymerase chain reaction in 58 patients. The most frequent SCCmec types were III (40 cases) of which less than 5% were susceptible to other beta-lactam antibiotics and most were health care-associated, followed by SCCmec type IV (15 cases), that were demonstrated to be community-acquired. SCCmec type IV MRSA isolates were more likely to be susceptible to ciprofloxacin (93.3%), gentamicin (46.7%) and trimethoprim-sulfamethoxazole (93.3%) than type III isolates. All MRSA isolates were susceptible to glycopeptides and vancomycin (minimum inhibitory concentrations <2 microg/mL). Pulsed-field gel electrophoresis with SmaI digestion was used to fingerprint these isolates. A total of 9 genotypes with 26 type-subtypes were identified. Genotype A was the most frequent (9 subtypes) indicating that it is epidemic in this hospital. CONCLUSION: After analysis, SCCmec typing could be used to predict drug susceptibility. Specific clones of S. aureus are circulating in hospital and communities in Taiwan.     

Staphylococcal cassette chromosome mec and Panton-Valentine leukocidin characterization of methicillin-resistant Staphylococcus aureus clones

Staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant Staphylococcus aureus MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCCmec type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCCmec type II complex and did not harbor the PVL genes.     

Epidemiologic distribution of the arginine catabolic mobile element among selected methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates

We tested 214 Staphylococcus aureus isolates for the arcA locus of the arginine catabolic mobile element (ACME). All USA300 SCCmec IVa isolates, but no isolates containing other SCCmec subtypes, were arcA positive. arcA was also detected in selected methicillin-susceptible USA300 and methicillin-resistant USA100 isolates. DNA sequence analysis confirmed the integration of ACME in orfX.