Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells

Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.     

Spread of Staphylococcus aureus clinical isolates carrying Panton–Valentine leukocidin genes during a 3-year period in Greece

Three collections of Staphylococcus aureus isolates (n = 1,058) were investigated to assess the spread of Panton-Valentine leukocidin (PVL)-producing strains in Greece and their association with skin and soft-tissue infections (SSTIs). The isolates were collected during 2001-2003 from inpatients and outpatients with invasive infections in two distinct geographical areas. Clonal types were identified according to their ClaI-mecA::ClaI-Tn554::pulsed-field gel electrophoresis patterns, and the presence of the lukS-PV and lukF-PV genes was assessed by PCR. In total, 287 (27%) S. aureus isolates carried the PVL genes: 45% of methicillin-resistant S. aureus (MRSA) and 12% of methicillin-sensitive S. aureus (MSSA). All the PVL-positive MRSA isolates belonged to a single clone that was disseminated in the community and hospitals. The PVL-positive MSSA isolates were polyclonal, with 14 of 65 isolates being associated with hospital-acquired infections. The community-acquired isolates were from SSTIs, while the hospital-acquired isolates were associated with surgical wound infections, especially those involving prosthetic devices. Thus, a unique clone of PVL-positive MRSA has spread in both the community and the hospital setting in Greece, and has replaced older clonal types.     

Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease

Staphylococcus aureus isolates carrying the genes that encode for Panton-Valentine leucocidin (PVL), a highly potent toxin, have been responsible for recent outbreaks of severe invasive disease in previously healthy children and adults in the United States of America and Europe. To determine the frequency of PVL-positive isolates sent to the Staphylococcus Reference Unit (United Kingdom) for epidemiological purposes, we tested 515 isolates of S. aureus, and 8 (1.6%) were positive for the PVL locus. A further 470 isolates were selected to explore the association of PVL-positive S. aureus with clinical disease. Of these, 23 (4.9%) were PVL positive and most were associated with skin and soft tissue infections (especially abscesses). The PVL genes were also detected in isolates responsible for community-acquired pneumonia, burn infections, bacteremia, and scalded skin syndrome. Genotyping by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the PVL-positive isolates were from diverse genetic backgrounds, although one prevalent clone of 12 geographically dispersed methicillin-resistant S. aureus (MRSA) isolates was identified (ST80). All 12 isolates were stapylococcal cassette chromosome mec type IVc, had an agr3 allele, and shared a common toxin gene profile (sea-see, seg-sej, eta, etb, and tst negative but etd positive). ST80 strains with similar genetic characteristics have been responsible for community-acquired infections in France and Switzerland. The remaining PVL-positive isolates were mostly methicillin-sensitive S. aureus and belonged to 12 different sequence types, including ST22 and ST30, which are closely related to the most prevalent MRSA clones in United Kingdom hospitals, EMRSA-15 and EMRSA-16, respectively.     

Accessory gene regulator group polymorphisms in methicillin-resistant Staphylococcus aureus: an association with clinical significance

PURPOSE: Virulent gene expression in Staphylococcus aureus is controlled by regulators such as the accessory gene regulator (agr). Strains can be divided into four major agr groups (agr I-IV) on the basis of agrD and agrC polymorphisms. The purpose of this study was to define the proportion of agr I, II, and III polymorphisms and to compare the clinical characteristics between group I and non-group I polymorphisms of methicillin-resistant Staphylococcus aureus (MRSA) strains in a Korean tertiary care teaching hospital. MATERIALS AND METHODS: A total of 158 clinical isolates were evaluated by RFLPs (restriction fragment length polymorphisms). RESULTS: The mean age of the patients was 50.2 +/- 21.9 years old. There were 74 (49.3%), 66 (44.0%), 10 (6.7%), 7 (4.4%), and 1 (0.6%) strains in agr group I, II, III, I + II, and I + III polymorphisms, respectively. Only ear infections were a statistically significant clinical parameter according to univariate (p=0.001) and multivariate analysis (OR, 4.721 (1.273-17.508), p=0.020). CONCLUSION: This study suggests that agr group I is the most prevalent in Korea, and ear infections are correlated with the group I polymorphism, which is a different clinical trend from western countries. It can also be inferred that community-acquired MRSA correlates with agr group I.     

Increasing incidence and widespread dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in central Europe, with special reference to German hospitals

Objective: to present data on prevalence and interregional spread of methicillin-resistant Staphylococcus aureus (MRSA) in Germany.
Methods: A nationwide collection of MRSA isolates from nosocomial infections in 143 hospitals was established from isolates ( n =4368) sent to a microbiological reference center during 1993–95. As chosen by distinguishable resistance phenotypes at each time of occurrence during the study period, 1830 isolates were subjected to molecular typing by means of Sma l macrorestriction patterns, PCR for RNA gene spacer patterns, and PCR for patterns of DNA stretches flanked by the ERIC-2 sequence and flanked by Tn 976 and ribosomal binding site. In addition, data from a multicenter study on the incidence of antibiotic resistance have been analyzed (32 centers, 637 S. aureus isolates).
Results: In 1995 the prevalence of MRSA among S. aureus isolates was 8.7% overall in central Europe (including Germany), in comparison to 1.7% in 1990. From 1993 until now, a continuous interregional dissemination of six epidemic strains, which were identified by molecular typing, was recorded. Besides these epidemic strains, 15 MRSA strains were identified which could not be allocated to the epidemic MRSA or to the known clonal groups of the species S. aureus. MRSA from three cases of sporadic nosocomial infections exhibited characteristics of the clonal group of S. aureus with the capacity for toxic shock syndrome formation. The pattern of one MRSA corresponded to those of the S. aureus group exhibiting phage pattern 94,96.
Conclusions: The prevalence of MRSA has increased in central Europe (and Germany) during the last 5 years, to 8.7%. The main source of infection with MRSA is obviously interregional dissemination of epidemic strains. At the same time, the mecA gene has been acquired by strains previously sensitive to methicillin.     

Identification of staphylococcal cassette chromosome mec encoding methicillin resistance in Staphylococcus aureus isolates at Charles Nicolle Hospital of Tunis

Staphylococcal cassette chromosome is a mobile element that carries the gene mecA mediating the methicillin resistance in staphylococci. In Staphylococcus aureus five types of SCCmec have been described, which differs in size and genetic composition among strains. SCCmec typing of 34 non redundant methicillin-resistant S. aureus (MRSA) recovered in 2004 at Charles Nicolle Hospital of Tunis was carried out. The isolates were identified by conventional methods. Methicillin resistance was detected by oxacillin and cefoxitin disks and confirmed by mecA PCR. The SCCmec complex types were determined by using PCR which amplify a sequence overlapping the right SCCmec chromosome junction. Strains were recovered mainly from cutaneous pus (61.7%) and blood cultures (17.64%). They were isolated from different wards: medicine (53.1%) especially from dermatology (41.2%); surgery (40.6%) and pediatrics (3.1%). Only two strains were community-acquired MRSA. Two strains (5.9%) were harboring SCCmec type I; five (14.7%) SCCmec type II and 27 (79.4%) SCCmec type III. The two community-acquired MRSA were harboring type II and III SCCmec, usually found in hospital acquired MRSA. Our findings indicate that there are only three SCCmec types at Charles Nicolle Hospital. However, the existence of SCCmec types II and III in community incite us to investigate more community-acquired MRSA.     

Characterization of Staphylococcus aureus nasal carriage from children attending an outpatient clinic in Seoul, Korea

Nasal swabs were collected to isolate S. aureus in 296 children, who visited the pediatrics department with a variety of symptoms. Staphylococcus aureus was isolated from 95 children (32.1%). Of the isolates, 18 were methicillin-resistant S. aureus (MRSA) (18.9%). Antimicrobial susceptibility testing was performed for all S. aureus cultured and the molecular characteristics were investigated. Forty-nine spa types were identified among the S. aureus isolates, and were classified into 13 spa groups (A-L). The most prevalent clone (34 isolates, 35.8%) belonged to the spa group B (spa repeat motif, WG/FKAOMQ), which corresponded to sequence type 30 (ST30) and its variants. Sixteen different spa types, within the spa group B, suggested that this group has evolved over a long period of time. In addition, all S. aureus isolates belonging to the spa group B were methicillin-susceptible, indicating that this group might represent successful adaptation of this clone in the community setting with low antibiotic pressure. The most frequently found clone in the MRSA group was spa group C (spa repeat motif, DMGGM) and SCCmec type IVA, which represented half of the MRSA isolates and corresponded to ST72. ST5-MRSA-II, the most prevalent MRSA clone in Korean hospitals, was found in only two isolates. These findings suggest that strains of S. aureus nasal carriage in Korean children visiting an outpatient pediatric department were different from the strains identified in hospital infections.     

Relationships between Staphylococcus aureus Genetic Background, Virulence Factors, agr Groups (Alleles), and Human Disease

The expression of most Staphylococcus aureus virulence factors is controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide (AIP). A polymorphism in the amino acid sequence of the AIP and of its corresponding receptor divides S. aureus strains into four major groups. Within a given group, each strain produces a peptide that can activate the agr response in the other member strains, whereas the AIPs belonging to different groups are usually mutually inhibitory. We investigated a possible relationship between agr groups and human S. aureus disease by studying 198 S. aureus strains isolated from 14 asymptomatic carriers, 66 patients with suppurative infection, and 114 patients with acute toxemia. The agr group and the distribution of 24 toxin genes were analyzed by PCR, and the genetic background was determined by means of amplified fragment length polymorphism (AFLP) analysis. The isolates were relatively evenly distributed among the four agrgroups, with 61 strains belonging to agr group I, 49 belonging to group II, 43 belonging to group III, and 45 belonging to group IV. Principal coordinate analysis performed on the AFLP distance matrix divided the 198 strains into three main phylogenetic groups, AF1 corresponding to strains of agr group IV, AF2 corresponding to strains of agr groups I and II, and AF3 corresponding to strains of agr group III. This indicated that the agr type was linked to the genetic background. A relationship between genetic background, agr group, and disease type was observed for several toxin-mediated diseases: for instance, agr group IV strains were associated with generalized exfoliative syndromes, and phylogenetic group AF1 strains with bullous impetigo. Among the suppurative infections, endocarditis strains mainly belonged to phylogenetic group AF2 and agr groups I and II. While these results do not show a direct role of the agr type in the type of human disease caused by S. aureus, the agr group may reflect an ancient evolutionary division of S. aureus in terms of this species' fundamental biology.     

Prevalence of methicillin resistant Staphylococcus aureus in Karnataka

This study was undertaken to find out the prevalence of methicillin resistant Staphylococcus aureus (MRSA) infection in our hospital and to compare their antibiotic susceptibility pattern with methicillin sensitive Staphylococcus aureus (MSSA). 100 strains of Staphylococcus aureus isolated from various clinical samples were screened for MRSA by disc diffusion method using 1 gm oxacillin disc. Antibiotic sensitivity testing was done by Kirby-Bauer's disc diffusion method. Out of these, 43% were identified as MRSA and the remaining 57% were MSSA. There was a marked difference in antibiotic sensitivity pattern of these M RSA versus the MSSA isolates. None of the MRSA isolate was found to be sensitive to amoxycillin while 36.8% of MSSA were sensitive to this antibiotic. 9.3%, 18.6%, 34.9% and 95.3% of MRSA were sensitive to cotrimoxazole, cloxacillin, ciprofloxacin and chloramphenicol, while 75.4%, 92.9%, 91.2% and 94.7% of MSSA were sensitive to these antibiotics respectively. Sensitivity to macrolide group of antibiotics like erythromycin and roxithromycin were seen in 7% and 14% of MRSA in comparison to 85.9% and 91.2% of MSSA respectively. Amongst the aminoglycosides like gentamicin and amikacin, the sensitivity of MRSA was found to be 18.6% and 46.5% and that of MSSA was 98.2% and 94.7% respectively. Sensitivity to cephalosporins like cephalexin and cefotaxime was seen in 23% and 25.5% of MRSA, whereas 100% of MSSA were sensitive to these antibiotics. All Staphylococcus aureus isolates were found to be uniformly sensitive to vancomycin. Majority of the isolates belonged to phage group III and the common phage types were 54, 54/75 and 54/75/85.     

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.     

Sporadic "transitional" community-associated methicillin-resistant Staphylococcus aureus strains from health care facilities in the United States

We describe phenotypic and genotypic traits of a group of methicillin-resistant Staphylococcus aureus (MRSA) clones that are either remnants of unsuccessful community-associated MRSA (CA-MRSA) clones or represent a transitional state with some yet-to-be-acquired characteristics of CA-MRSA. These rare strains (n = 20) were identified during a 10-year period (1990-1999) from 13 unrelated health care facilities in Wisconsin. The isolates were recovered from patients in nosocomial or long-term chronic care facilities (60%) and outpatient settings (40%). Sixty percent (n = 12) of the isolates were recovered from skin and soft tissue infections, whereas the remaining isolates (n = 8) were from invasive infections. Ninety percent of isolates were susceptible to all antibiotic classes tested or resistant to erythromycin and clindamycin. Pulsed-field gel electrophoresis, multilocus sequence typing, and spa typing clustered these isolates into 8, 8, and 14 clonal groups, respectively. Eight plasmid profiles were represented in these strains. All four agr types were represented, with type IV being predominant (40%). All strains harbored subtypes of type IV staphylococcal cassette chromosome mec but lacked genes for the virulence factor Panton-Valentine leukocidin (PVL). The strains harbored one or more of the following toxin genes: sea, seb, sec, sed, see, seh, sej, sek, sel, seg, sei, sem, sen, and seo. Individual clonal groups maintained the same set of enterotoxin genes even though they were isolated over extended time periods, suggesting significant genomic stability. The potential role of PVL-carrying phages and plasmids in the success of CA-MRSA clones has been discussed.     

A novel multiplex PCR for molecular characterization of methicillin resistant Staphylococcus aureus recovered from Jeddah, Kingdom of Saudi Arabia

Background: Molecular characterization of staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) is very essential for studying the epidemiology of MRSA. Objectives: This study reports two multiplex PCR for molecular typing of MRSA collected from Jeddah, Kingdom of Saudi Arabia. Materials and Methods: A total of 101 clinical isolates of strains were collected from major hospital laboratories and public health centres, Jeddah, Kingdom of Saudi Arabia during the period from August 2009 to May 2011. All the strains were tested phenotypically by conventional methods and genotypically by a novel multiplex PCR targeting at the same time S. aureus 16S rRNA, Panton - valentine leucocidin (PVL) and mecA resistance genes. All the strains were tested also by multiplex PCR for typing of SCC mec types. Results: All the 101 strains previously identified phenotypically as S. aureus with bacteriological examination were positive for amplification of 756 base pair fragments specific for 16S rRNA of S. aureus. Moreover, all the strains were positive for amplification of 1339 base pair fragments specific for mecA gene, while only 38 strains (37.6%) showed positive amplification of 433 base pair fragments specific for PVL gene. The most predominant SCC mec type among the examined isolates is type V 43 (42.5) followed by SCCmec type III 39 (38.6%). Conclusion: The newly modified multiplex PCR is rapid and sensitive method for detection of MRSA. Moreover, the most predominant SCC mec type among the examined isolates from Jeddah, King Saudi Arabia is type V (42.5%), followed by Type III (38.6%).     

Distribution of major genotypes among methicillin-resistant Staphylococcus aureus clones in Asian countries

To investigate the evolutionary pattern and genotypic characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains in the Asian region, 74 MRSA strains isolated from 12 Asian countries were analyzed by multilocus sequence typing (MLST) and SCCmec typing. Overall, a total of 16 genotypes based on sequence type and SCCmec types were identified among MRSA strains from Asian countries. Data revealed two major genotypes of MRSA strains in Asia, with unique geographic distributions. By MLST analysis, all strains from Korea and Japan except one belonged to clonal complex 5 (CC5) while most MRSA isolates from other Asian countries belonged to CC239. SCCmec typing showed that most isolates from Korea and Japan were SSmec type II whereas SCCmec type III (or IIIA) was the most common type in strains from other Asian countries. Our data documented a unique geographic distribution and evolutionary pattern of MRSA clones in Asia.     

Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis

The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.     

Occurrence of the enterotoxin gene cluster and the toxic shock syndrome toxin 1 gene among clinical isolates of methicillin-resistant Staphylococcus aureus is related to clonal type and agr group

Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group.     

Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains

S. aureus nasal carriage was investigated in 278 healthy humans, determining the antibiotic resistance mechanisms, virulence traits, and genetic lineages of recovered isolates. Nasal samples were cultured in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was detected in 53 of 278 nasal samples (19.1%): MRSA was found in one sample (0.4%) and methicillin-susceptible S. aureus (MSSA) in the remaining 52 samples. The MRSA isolate was typed as ST1649-t701-agrI-SCCmec-IVc and only exhibited resistance to beta-lactams. A high diversity of spa types (n=37) was identified among the 52 MSSA, identifying 5 new spa-types. Multi-locus sequence typing (MLST) typing was performed in 30 selected MSSA, detecting 16 different sequence types, 2 of them being new. MSSA strains presented agr types I (30.2%), II (30.2%), III (34%), and IV (5.6%). Eleven strains showed erythromycin resistance and harbored different combinations of erm(A), erm(B), erm(C), erm(T), and msr(A) genes. Two strains exhibited ciprofloxacin resistance, and one of them presented amino acid changes in GyrA and GrlA proteins. The presence of 28 genes encoding staphylococcal toxins was investigated by PCR in all 53 S. aureus isolates. The toxic shock syndrome toxin 1 (TSST-1) gene was detected in 15 MSSA isolates (11 of them typed within the clonal complex CC30) and the gene of exfoliative toxin A in 2 strains. Different combinations of enterotoxin genes were identified among S. aureus strains. None of the S. aureus isolates harbored the Panton-Valentine leukocidin gene. Two MSSA presented the sequence-type ST398 [harboring erm(T) gene], and 2 additional isolates were typed as ST97. Interestingly, MSSA CC398 and CC97 isolates were detected. These clonal complexes are associated with food-producing animals.     

Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxin 1 gene responsible for hospital- and community-acquired infections in France

Methicillin-resistant Staphylococcus aureus (MRSA) clones harboring the toxic shock syndrome toxin 1 (tst) gene have been detected in France and in Switzerland since 2002. During a passive survey conducted between 2002 and 2003, we collected 103 tst-positive S. aureus isolates from 42 towns in France, of which 27 were resistant to methicillin. The tst-positive MRSA belonged to two clones: a major clone comprising 25 isolates of sequence type (ST) 5 and agr group 2 and a minor clone comprising two isolates of ST30 and agr3. The tst-positive MRSA clones were associated with both hospital-acquired (12 cases) and community-acquired (8 cases) infections. The MRSA clones were mainly isolated from children (overall median age, 3 years). They caused a variety of clinical syndromes, including toxic shock syndrome and suppurative infections. Both clones were found to harbor a type IV staphylococcal chromosomal cassette mec (SCCmec) and to have similar antibiotic resistance profiles (usually resistant to oxacillin, kanamycin, and tobramycin and with intermediate resistance to fusidic acid). The origin of these clones is unclear. The tst-positive agr2 MRSA clone has the same sequence type (ST5) of two pandemic nosocomial MRSA clones, namely, the Pediatric clone and the New York/Japan clone. These findings suggest that all these clones are phylogenetically related. The pulsotype of the tst-positive MRSA clones differed from that of methicillin-sensitive S. aureus (MSSA) clones by a single band involving the SCCmec element. These findings suggest that the tst-positive MRSA clones may have emerged from their respective MSSA counterparts.     

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。     

Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.     

Staphylococcal cassette chromosome mec (SCCmec) analysis and antimicrobial susceptibility patterns of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Tehran, Iran

Oxacillin resistance was present in 99 of 277 (36%) consecutive Staphylococcus aureus isolates collected from hospital patients in Tehran during a 15-month period (January 2004-March 2005). The majority of isolates (77/99 = 78%) had been cultured from wounds or blood. The staphylococcal cassette chromosome mec (SCCmec) types and antimicrobial susceptibility patterns of 99 methicillin-resistant S. aureus (MRSA) strains were determined. Disk diffusion and agar dilution methods were used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. The presence of mecA and SCCmec types was determined by PCR and multiplex PCR. All MRSA isolates were susceptible to vancomycin (MIC90 相似文献