Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.     

Serologic and genetic analysis of group B streptococci isolated from adult patients

Serologic and genetic typing with RAPD (Random Amplified Polymorphic DNA) method was used for epidemiologic analysis of GBS. 125 strains isolated from various clinical samples from adult patients were tested. In serologic typing seven serotypes have been found. Serotypes III and R were the most often encountered, containing 37,6% and 20,8% of samples. There was no dependence between serologic type and disease process. Optimalisation of RAPD reaction parameters was based on the standard strains of GBS. In the group of strains tested with the use of RAPD method, eleven genetic profiles were found, with prevalence of profile B (25,8%). Five other profiles occured with similar frequency (8,8% - 12,8%). Among streptococci isolated from patients with the infection of genitourinary tract, great differentiation in the genetic profiles of the strains has been found. Each serologic type contained various genetic profiles. Genetic variety showed by RAPD method indicates the raised ability of this technique to find differences among isolates of GBS.     

Presence of enterotoxin C and toxic shock syndrome toxin--1 (TSST-1) genes in population of Staphylococcus aureus phage type 187

The aim of this study was to examine whether Staphylococcus aureus of phage type 187 possess the genes of enterotoxins and toxic shock syndrom toxin. Sixteen phage type 187 strains were isolated from the hospital patients (12) and the carriers (4) in twelve medical centres in Poland during 1991 and 2005. Biotyping, phage typing, antibiotic susceptibility, detection of the genes of enterotoxins (sea--sed) and toxic shock syndrome toxin (tst) was tested. The results of this study showed that all staphylococci of phage type 187 belonged to the human biotype (A) and appeared to be sensitive to all of the tested antibiotics, including methicillin (MSSA). Almost all of them (93.8%) had the enterotoxin C gene and TSST-1 gene. This fact allows to consider them the strains of potentially high virulence.     

Multiple-locus variable number of tandem repeats fingerprinting (MLVF) and virulence factor analysis of methicillin resistant Staphylococcus aureus SCCmec type III

Methicillin resistant Staphylococcus aureus (MRSA), particularly strains with type III staphylococcal cassette chromosome mec (SCCmec), represent a serious human pathogen in Tehran, Iran. The disease-causing capability depends on their ability to produce a wide variety of virulent factors. The prevalence of exotoxin genes and multiple-locus variable number of tandem repeats fingerprinting (MLVF) profile among MRSA isolates, from patients in Tehran, was evaluated by PCR and Multiplex-PCR. The MLVF typing of 144 MRSA isolates with type III SCCmec produced 5 different MLVF types. Generally, 97.2% (140/144) of all the isolates were positive for at least one of the tested exotoxin genes. The most prevalent genes were hld, found in 87.5% (126/144) of the isolates followed by lukE-lukD and hla found in 72.9% (105/144) and 70.1% (101/144) of the isolates, respectively. The tst gene, belonging to MLVF types I, IV and V, was found among three of the isolates from blood and wound samples. The sea gene was detected in 58.3% (84/144) of the isolates and the sed and see genes were found in one isolate with MLVF type V. The coexistence of genes was observed in the 87.5% (126/144) of the isolates. The rate of coexistence of hld with lukE-lukD, hla with lukE-lukD and sea with lukE-lukD were 66.7% (96/144), 44.4% (64/144) and 44.4% (64/144), respectively. The present study demonstrated that MRSA strains with type III SCCmec show different MLVF patterns and exotoxin profiles.     

The prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus in milk and dairy products in Riyadh,Saudi Arabia

In terms of life- menaced contagion, methicillin resistant Staphylococcus aureus (MRSA) is known to be one of which and it is truly notable in the contaminated food causing a community health anxiety. However, the occurrence of S. aureus and MRSA in diverse kinds of dairy products have been tested in this study. Samples from: raw milk (unpasteurized) from horse, goat, camel, and cow origins and unpacked cheese were checked for the recovered strains of such bacterium and MRSA. Wholly, MRSA isolates were verified for antimicrobial susceptibility and further characterized by mecA and staphylococcal cassette chromosome mec (SCCmec) typing. Also, Panton-Valentine Leukocidin (PVL), Staphylococcus aureus protein A (spa), and Staphylococcal enterotoxins (SEs) were also tested between all positive MRSA isolates in order to discover the virulence factors. Consequently, 70% of the 100 collected dairy products samples were contaminated by S. aureus bacteria and 72.9% of them were defined as MRSA. 9.8% of MRSA isolates contained mecA genes with SCCmec type II (80%) as the most common SCCmec type. Moreover, large number of MRSA isolates were identified as multidrug resistance and 28.6% of MRSA-mecA positive isolates were also carried vancomycin resistance genes (i.e., vanB). Too, spa gene was detected between 9.8% of MRSA isolates but PVL gene was not spotted at all. Additionally, the existing of SEs was variable between MRSA isolates and the most common type was SEH (51%). In general, our results confirmed that raw milk and unpacked cheese in the Kingdom of Saudi Arabia (Riyadh) is a potential vehicle for multidrug resistant MRSA transmission. It is a critical civic health menace and stresses, thus; the need of applying well cleanliness practices is essential.     

Genotypes of Helicobacter pylori in Polish population

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.     

Application of randomly amplified polymorphic DNA (RAPD) analysis for typing avian Salmonella enterica subsp. enterica

Randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular genetic typing of 30 Salmonella enterica subsp. enterica strains isolated from chickens and ducks in Thailand. Six different primers were tested for their discriminatory ability. While some of the primers could only differentiate between the different serovars, the use of multiple primers showed that the RAPD method could also subdivide within a given serovar. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, we suggest that it may be a valuable new tool for studying the molecular genetic epidemiology of S. enterica ssp. enterica, both inter- and intra-serovars.     

Identification of Escherichia coli Recovered from Milk of Sows with Coliform Mastitis by Random Amplified Polymorphic DNA (RAPD) Using Standardized Reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

Typing of Clostridium perfringens strains by use of Random Amplified Polymorphic DNA (RAPD) system in comparison with zymotyping

The definition of strain clonality postulates that strains showed identical phenotypic and genetic traits are likely to descend from a common ancestor even if they were isolated from different sources and locations. Regarding this definition, non-epidemiologically linked strains might be clonal strains. To overcome this ambiguity, the discriminatory capability of RAPD typing was assessed firstly on eight Clostridium perfringens strains proven to be chromosomally different with one being the mutant of another one. Thirteen primers were tested but only two were able to differentiate seven of the eight strains. With none of the used primers it was possible to differentiate the parental strain and its mutant harboring an insertion of 180 kb. The four most discriminant primers were retained to determine the RAPD fingerprints of a further 20 previously zymotyped strains from which seventeen were unrelated. To compare the two typing systems, the zymotype of the eight chromosomally different strains was determined. Thus, the discriminatory index was calculated on the basis of 25 unrelated C. perfringens strains. This was 0.97 with RAPD typing and 0.99 with zymotyping. From these results we conclude that the RAPD typing which is less fastidious than zymotyping can be used as an epidemiological marker for C. perfringens.     

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Poland: further evidence for the changing epidemiology of MRSA

This study reports the isolation of CA-MRSA strain which was found to colonize the nasal mucosa of a patient undergoing haemodialysis treatment. The MRSA was subjected to molecular analysis by Pulsed Field Gel Electrophoresis (PFGE), multiplex PCR assay for staphylococcal cassette chromosome mec (SCCmec) typing, and PCR detection of the pvl gene encoding for Panton-Valentine leukocidin. The analyzed MRSA harbored the SCCmec type IV and the pvl gene-two unique genetic markers of CA-MRSA. The PFGE pattern of the strain corresponded to the common European CA-MRSA (MLST Type ST80). Moreover, the strain was only resistant to beta-lactam agents and tetracycline. This study adds further evidence for the changing epidemiology of MRSA and indicates the ability of CA-MRSA to affect persons with established risk factors in addition to previously healthy individuals.     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences

Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.     

金黄色葡萄球菌的耐药性分析及基因分型研究

目的通过分析上海地区院内分离金黄色葡萄球菌的药敏谱型及对耐甲氧西林的金黄色葡萄球菌（MRSA）进行基因谱型的研究,了解金黄色葡萄球菌的院内流行状况。方法对临床分离出的43株金黄色葡萄球菌进行药敏试验和SCCmec基因盒的多重PCR检测,并将结果整合后用MEGA3．1软件分析其进化相关关系。结果药敏结果显示43株金葡菌对青霉素和甲氧西林的耐药率最高。甲氧西林的耐药率达到62．8％。MecA阳性菌株SCCmec的分型显示均为Ⅱ型或Ⅲ型,且所占比例相近,未见Ⅰ型和Ⅳ型。进化树分析发现了在同一医院中亲缘关系相近的菌株,为院内感染流行株。结论MecA基因介导的MRSA在分离菌株中所占比例高,存在院内感染爆发性流行。     

SCCmec遗传元件及其在耐甲氧西林金黄色葡萄球菌分子分型中的应用

携带mec基因簇的葡萄球菌盒式染色体(Staphylococcal chromosome cassette mec, SCCmec)遗传元件的获得是耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)耐药的主要原因。SCCmec由一个mec基因簇、一个染色体重组酶(ccr)基因簇及3个J区组成。mec基因簇含有mecA及其调控基因,mecA基因编码的耐药决定簇使MRSA对β-内酰胺类抗生素耐药;ccr基因簇编码的重组酶负责SCCmec元件的整合与切离;J区差异大,导致不同来源MRSA菌株携带SCCmec的大小不一,在组成上也具有多样性。这些特征为利用SCCmec元件进行MRSA分型创造了条件。文章介绍了SCCmec元件的结构和功能,综述了基于SCCmec的MRSA分型研究。     

Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological applications

We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC,, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.     

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India

Aims: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)‐PCR. Methods and Results: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0·95, while ERIC‐PCR showed a DI of 0·94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. Conclusions: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC‐PCR generates useful information in the case of V. parahaemolyticus associated with seafood. Significance and Impact of the Study: The study demonstrates the usefulness of nucleic acid and protein‐based studies in understanding the relationship between various isolates from seafood.     

First Confirmation of Integron-Bearing Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis>

Six methicillin-resistant Staphylococcus aureus MRSA strains from two nosocomial infection cases described in a previous study [15], of which two occurred in March and the other four in May, 2005, were found to possess one copy of class 1 integron with aadA2 gene cassette located on chromosomes by Southern hybridization. Polymerase chain reaction (PCR) detection of mecA and pvl, SCCmec typing, multilocus sequence typing (MLST), spaA typing and coa typing were also performed. The results revealed 6 MRSA fell into the ST239-MRSA-III group (clonal complex 8), with the spaA type GKAOMQ and coa type HIJKL, whereas the pvl locus was not detected. DNA fingerprinting analysis by random amplified polymorphic DNA-PCR using three different assays were also performed, and all strains exhibited identical patterns, indicating that they were clonally related and might be mainly due to a specific clone in the hospital. This was the first time, to our knowledge, that class 1 integron-bearing MRSA (I-MRSA), simultaneously carrying two mobile genetic elements was confirmed: class 1 integron and SCCmec.     

Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37 degrees C, population sizes were already higher than 9.4 x 10(5) UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.