Coagulase Gene Typing Research Articles

Evaluation of aminoglycoside modifying enzymes, SCCmec, coagulase gene and PCR-RFLP coagulase gene typing of Staphylococcus aureus isolates from hospitals in Shiraz, southwest of Iran

Staphylococcus aureus is an important human pathogen that causes various infections. Aminoglycosides are broad-spectrum antibiotics used to treat methicillinresistant S. aureus (MRSA) infections. Typing of S. aureus isolates by coagulase gene typing and PCR-RFLP coa gene is a fast and suitable method for epidemiological studies. Aim of the present study was to evaluate the resistance to aminoglycosides, staphylococcal chromosomal cassette mec (SCCmec) types, coagulation typing and PCR-RFLP coa gene in clinical isolates of S. aureus. 192 S. aureus isolates were collected from Namazi and Shahid Faghihi hospitals. Antibiotic resistance was measured by disk diffusion method and MIC was determined for gentamicin. The presence of genes encoding aminoglycoside modifying enzymes (AME) and mecA gene were assessed by PCR. Also the coagulase typing, PCR-RFLP coa gene, and SCCmec typing were performed. Out of 192 isolated S. aureus isolates, 83 (43.2%) MRSA isolates were identified. In this study, a high resistance to streptomycin and gentamicin (98.7%) were observed. Among the AME genes, the aac (6′)-Ie-aph (2″) gene was the most common. Based on the SCCmec typing, it was determined that the prevalence of SCCmec type III (45.8%) was highest. From the amplification of the coa gene, 5 different types were obtained. Also, in digestion of coa gene products by HaeIII enzyme, 10 different RFLP patterns were observed. According to this study, aminoglycoside resistance is increasing among MRSA isolates. As a result, monitoring and control of aminoglycoside resistance can be effective in the treatment of MRSA isolates. Also, typing of S. aureus isolates based on coagulase gene polymorphism is a suitable method for epidemiological studies.

Investigation of coa Gene Polymorphism in Clinical Isolates of Staphylococcus aureus in North of Iran

Staphylococcus aureus is a common pathogen potentially able to cause a wide range of infectious diseases in human and animals and coagulase enzyme is one of the important virulence factors of this bacterium. Polymorphism of the coagulase encoding gene (coa) is one of the molecular-based typing methods of S. aureus isolates. In this study, the polymorphism of the coagulase gene among MRSA and MSSA isolates were investigated using PCR-RFLP analysis. To perform coagulase gene typing, the repeated units encoding hypervariable regions of coagulase gene of 30 clinical isolates of S. aureus were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In total two amplicons (680 bp and 750 bp) and four distinct RFLP banding patterns (280+400, 340+340, 280+470, and no digested amplicon of 750 bp) were observed. Genotype with PCR-RFLP patterns of 280+400 bp was predominated. The results indicated polymorphism in the investigated regions of coagulase gene. This polymorphism can be used for identification of S. aureus isolates and showing the epidemiological relationship among them.

Detection of tetracycline resistance genes, aminoglycoside modifying enzymes, and coagulase gene typing of clinical isolates of Staphylococcus aureus in the Southwest of Iran.

Objective(s):The aim of the present study was to determine the aminoglycoside modifying enzymes (AMEs) encoded genes, tetracycline resistance genes, and the coa based typing of Staphylococcus aureus isolates in the Southwest of Iran.Materials and Methods:Antimicrobial susceptibility of isolates was carried out by agar disk diffusion methods. Two sets of multiplex PCR mixture were used for detection of AME genes and tet genes. All of the isolates were typed with the coagulase gene typing method. Of the 121 isolates, 29.75% and 47.93% were resistant to at least one aminoglycosides and tetracyclines, respectively.Results:The aac(6’)-Ie-aph(2”) was the most frequent gene (97.22%), and aph (3’)-IIIa and ant (4’)-Ia genes were detected in 61.11% and 11.11% of aminoglycoside resistant isolates, respectively. The tetK and tetM genes were detected in 82.75% and 56.9% of tetracycline resistant isolates, respectively. Overall 31.4% of isolates were MRSA. Totally 17 distinct coa gene RFLP patterns, numbered C1 to C17, were observed. The C5 was the most frequent coa type with 31 isolates.Conclusion:The aac(6’)-Ie-aph(2”) and aph (3’)-IIIa genes were the most important genes contributing to aminoglycosides resistance, while resistance to tetracyclines was mediated by tetK and tetM genes. Interestingly all S. aureus with C5 as the most prevalent coa-type were resistant to at least one of the aminoglycoside antibiotics and tetracycline simultaneously. Moreover, 30 out of 31 isolates with this coa type were MRSA, indicating the importance of the C5 coa-type in MRSA strains and also in isolates that were resistant to aminoglycosides and tetracycline.

Genotyping of coa and aroA Genes of Methicillin-Resistant Staphylococcus aureus Strains Isolated From Nasal Samples in Western Iran

Background:Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen frequently isolated in both hospital and community environments. Methicillin-resistant Staphylococcus aureus is considered a major nosocomial pathogen that causes severe morbidity and mortality.Objectives:The main objective of this study was to determine the genotypes of MRSA strains isolated from the nares of hospitalized and community patients in Kermanshah Hospital, western Iran, by PCR-restriction fragment length polymorphism (PCR-RFLP).Materials and Methods:Of 1387 patients, 1217 patients were screened for more than 48 hours after admission in hospital wards and 170 patients were screened in the hemodialysis unit of Kermanshah Hospital, which is the largest hospital in western Iran. S. aureus was identified by standard biochemical tests, including colonial morphology, production of coagulase, and DNase and the API20 Staph test. Methicillin-resistant Staphylococcus aureus was identified by the Oxacillin strip test.Results:In total, 258 S. aureus isolates were recovered from 1387 samples, of which 96 isolates were MRSA, 82 were hospital acquired, and 14 were community acquired. Digestion of the aro A gene revealed only one distinctive RFLP pattern in the 258 isolates.Conclusions:Methicillin-resistant Staphylococcus aureus is an increasingly common cause of nosocomial infections. Our results are in agreement with those of other studies reporting that a few specialized clones are responsible for most cases of MRSA nasal carriage. In this study, MRSA strains isolated from different wards of hospital were closely related when analyzed by coagulase gene typing. Identifying patients colonized with MRSA during hospitalization and rapidly typing them with these methods may facilitate detection of outbreaks and prevention of the spread of organisms in hospitals.

Molecular typing of Staphylococcus aureus isolated from food samples in Iran

Staphylococcus aureus is the third most common cause of confirmed food poisoning in the world and is the predominant species involved in staphylococcal food poisoning outbreaks. Considerable genetic heterogeneity has been shown in natural populations of S. aureus isolates. Coagulase gene typing is one of the numerous molecular techniques to identify and compare S. aureus genotypes. The present study was conducted to type the coagulase gene in 25 S. aureus isolates isolated from food samples. All isolates were identified by routine biochemical tests and then confirmed by species-specific PCR and yielded products with the expected molecular size of 1.3 kb. PCR amplification of DNA with the primers COAG2 and COAG3 yielded single-banded PCR products in 24 isolates with the molecular size of approximately 500 bp (n = 2, 8 %), 750 bp (n = 1, 4 %), 850 bp (n = 12, 48 %), and 950 bp (n = 9, 36 %), while one isolate produced no band in PCR amplification of coagulase gene. Since human and bovine reservoirs of S. aureus represent two subpopulations that rarely cross-infect, detection of single bands by coagulase PCR in S. aureus isolates suggests that these isolates may be of bovine origin not human one, and contamination of food samples may initiate from the animal source not the food handlers. Digestion of coagulase PCR products with restriction endonuclease enzymes AluI and Hin6I yielded four different restriction profiles that indicate presence of heterogeneity in the coagulase gene of the isolates. This work showed that restriction analysis of the coagulase gene can be considered as a reliable and fast method for determining the origin of S. aureus in food samples.

Genetic analysis of high-level mupirocin resistance in the ST80 clone of community-associated meticillin-resistant Staphylococcus aureus

Four community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) isolates expressing high-level mupirocin resistance (MIC >1024 mg l(-1)) were isolated from four sites of a diabetic patient and characterized for the genetic location of their resistance determinants and typed using PFGE, staphylococcal cassette chromosome mec (SCCmec), the coagulase gene and multilocus sequence typing to ascertain their relatedness. The presence of genes for resistance to high-level mupirocin (mupA), tetracycline (tetK) and fusidic acid (far1), Panton-Valentine leukocidin (PVL), accessory gene regulators (agr) and capsular polysaccharide (cap) were detected in PCR assays. The isolates were resistant to kanamycin, streptomycin, tetracycline, fusidic acid and cadmium acetate, and harboured mupA, tetK, far1, PVL, agr3 and cap8. They had identical PFGE patterns and coagulase gene type, possessed the type IV SCCmec element and belonged to sequence type 80 (ST80). However, they had three different plasmid profiles: (i) 28.0 and 26.0 kb; (ii) 28.0, 21.0 and 4.0 kb; and (iii) 41.0 and 4.0 kb. Genetic studies located the resistance to tetracycline, fusidic acid and cadmium acetate on the 28 kb plasmid and mupA on the related non-conjugative 26 and 21 kb plasmids. One of the 21 kb mupirocin-resistance plasmids was derived from the approximately 41 kb plasmid during transfer experiments. The emergence of high-level mupirocin resistance in the ST80-SCCmec IV MRSA clone demonstrates the increasing capacity of CA-MRSA clones to acquire resistance to multiple antibacterial agents. The presence of different plasmid profiles in genetically identical isolates creates difficulty in the interpretation of typing results and highlights the weakness of using plasmid analysis as the sole method for strain typing.

Molecular Analysis of Coagulase Gene Polymorphism in Clinical Isolates of Methicilin Resistant Staphylococcus aureus by Restriction Fragment Length Polymorphism Based Genotyping

Problem statement: To investigate coagulase gene variants through Restriction Fragment Length Polymorphism (RFLP) amongst clinical isolates of Methicillin-Resistant Staphylococcus Aureus (MRSA) collected from Chennai based hospitals. Approach: A total of 85 MRSA clinical isolates collected from various clinical sources were studied for the molecular typing purpose. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR followed by AluI and HaeIII restriction enzyme digestion of the PCR product and analyzed for RFLP. Results: Cluster analysis performed with Un-weighted Pair Grouping Using Arithmetic Mean Analysis (UPGMA) at >80% similarity level classified all strains into 31 and 21 genotypes based upon AluI and HaeIII RFLP patterns respectively. AluI RFLP patterns gave a high discriminatory index and proved to be better than that of HaeIII in typing of MRSA strains. Conclusion: Method investigated in this study, proved to be quick, reliable and useful for typing large number of MRSA strains from various clinical isolates with high fidelity. Grouping of strains based on RFLP patterns of both enzymes, improved the discriminatory index. Finally, using this methodology group of epidemiological strains can be typed reliably and effectively using both enzymes. Significance and impact of the study: This efficient and reliable typing procedure is beneficial to develop efficient infection control measures in hospitals for staphylococcal infection. This typing procedure could be used to analyze large number of strains with in a short period of time and thus useful for epidemiological investigations.

Combination of capillary electrophoresis, PCR and physiological assays in differentiation of clinical strains ofStaphylococcus aureus

Fast, sensitive and cheap determination of pathogenic bacteria is extremely important in many branches, for example biotechnology, quality control, analysis of samples and antimicrobial therapy. The development and application of analytical techniques in practice could provide new possibilities in this regard. The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality. Rapid and sensitive determination is therefore very important. In the present study, novel methods, based on capillary zone electrophoresis and (as confirmation of these results) molecular analysis of a part of the coag gene, were developed for identification and differentiation of three S. aureus strains. The electrophoretic measurements rely on the differential mobility of bacteria in the fused silica capillary under the direct current electric field. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the S. aureus gene were amplified using the PCR technique followed by restriction enzyme digestion and analysis of restriction fragment length polymorphism patterns as well as sequencing. Finally, the results of electrophoretic measurements with molecular analysis were compared.

Molecular Typing of Methicillin-Resistant Staphylococcus aureusIsolated in a Bahrain Hospital

Objective: To investigate antibacterial resistance patterns and genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) obtained at the Salmaniya Medical Complex in Bahrain. Methods: A total of 53 consecutive MRSA isolates obtained from 53 patients were studied using antibacterial resistance patterns, coagulase gene polymorphism, staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE). Results: There was a high prevalence of resistance to fusidic acid (92.5%), ciprofloxacin (92.5%), erythromycin (90.6%), tetracycline (88.7%), trimethoprim (88.7%), streptomycin (88.7%), kanamycin (83.0%) and gentamicin (73.6%). Coagulase gene typing divided the isolates into five coagulase types comprising coagulase type 36 (86.7%), type 20 (3.8%), type 16 (3.8%), type 38 (1.9%) and type 384 (3.8%). They belonged to SCCmec type III (86.7%) and SCCmec type IV (13.3%). PFGE identified five pulsotypes (types A–E) with PFGE type A and its subtypes comprising 83% of the isolates. PFGE type A isolates were multiresistant and had the SCCmec type III and coagulase type 36 genotype. The SCCmec type IV isolates were nonmultiresistant with different genetic backgrounds. Conclusions: The study identified two types of MRSA in the hospital during the study period. One type consisted of a persistent multiresistant PFGE clone with the SCCmec type III and coagulase type 36 genotypes. The second type consisted of nonmultiresistant isolates that belonged to different genetic backgrounds and were isolated less frequently.

Methicillin‐resistant Staphylococcus aureus in the Australian community: an evolving epidemic

To describe antimicrobial resistance and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in community settings in Australia. Survey of S. aureus isolates collected prospectively Australia-wide between July 2004 and February 2005; results were compared with those of similar surveys conducted in 2000 and 2002. Up to 100 consecutive, unique clinical isolates of S. aureus from outpatient settings were collected at each of 22 teaching hospital and five private laboratories from cities in all Australian states and territories. They were characterised by antimicrobial susceptibilities (by agar dilution methods), coagulase gene typing, pulsed-field gel electrophoresis, multilocus sequence typing, SCCmec typing and polymerase chain reaction tests for Panton-Valentine leukocidin (PVL) gene. 2652 S. aureus isolates were collected, of which 395 (14.9%) were MRSA. The number of community-associated MRSA (CA-MRSA) isolates rose from 4.7% (118/2498) of S. aureus isolates in 2000 to 7.3% (194/2652) in 2004 (P = 0.001). Of the three major CA-MRSA strains, WA-1 constituted 45/257 (18%) of MRSA in 2000 and 64/395 (16%) in 2004 (P = 0.89), while the Queensland (QLD) strain increased from 13/257 (5%) to 58/395 (15%) (P = 0.0004), and the south-west Pacific (SWP) strain decreased from 33/257 (13%) to 26/395 (7%) (P = 0.01). PVL genes were detected in 90/195 (46%) of CA-MRSA strains, including 5/64 (8%) of WA-1, 56/58 (97%) of QLD, and 25/26 (96%) of SWP strains. Among health care-associated MRSA strains, all AUS-2 and AUS-3 isolates were multidrug-resistant, and UK EMRSA-15 isolates were resistant to ciprofloxacin and erythromycin (50%) or to ciprofloxacin alone (44%). Almost all (98%) of CA-MRSA strains were non-multiresistant. Community-onset MRSA continues to spread throughout Australia. The hypervirulence determinant PVL is often found in two of the most common CA-MRSA strains. The rapid changes in prevalence emphasise the importance of ongoing surveillance.

Antimicrobial Susceptibility and Coagulase Gene Typing of Staphylococcus aureus Isolated from Bovine Clinical Mastitis Cases in Turkey

The objectives of this study were to determine antimicrobial resistance patterns of Staphylococcus aureus strains isolated from bovine clinical mastitis cases and to subtype the strains by polymerase chain reaction (PCR) technique based on coagulase gene polymorphism. Two hundred sixty-five S. aureus isolates collected from individual animals in different herds (n = 235) from 1995 to 2004 were tested for susceptibility to penicillin, ampicillin, amoxicillin-clavulanate, oxacillin, oxytetracycline, enrofloxacin, kanamycin-cephalexin, and trimethoprim-sulfamethoxazole using the agar disc diffusion test. Strains were also tested for β-lactamase production. A total of 29.8% of the strains were susceptible to all antimicrobial agents tested. The highest resistance was observed in 63.3% of the strains against β-lactam antibiotics, penicillin and ampicillin. Oxytetracycline resistance was observed in 27.9% of the strains, either alone or in combination with β-lactams. No resistance was detected for amoxicillin-clavulanate, oxacillin, enrofloxacin and kanamycin-cephalexin. β-Lactamase production and resistance to β-lactam antibiotics were usually correlated. Resistance against β-lactams increased from 43.5% in 1995 to 58 to 77% from 1999 to 2004.One hundred twenty-five strains were examined for coagulase gene polymorphism. The isolates were subtyped into 4 types by coagulase gene-based PCR. A predominant 1000-bp PCR product was observed in 60.8% of the isolates typed. The results indicate that a few coagulase gene types of S. aureus are responsible for the majority of bovine clinical mastitis cases in one province of Central Anatolia region, Turkey.

Emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infection in Queensland, Australia

Objectives: To investigate the incidence and epidemiology of non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) infection in south-east Queensland, Australia. Study design: A retrospective survey was done of hospital records of all patients who had non-multiresistant MRSA isolated at Ipswich Hospital (a 250-bed general hospital, 40 km south-west of Brisbane, Queensland, Australia) between March 2000 and June 2001. Laboratory typing of these isolates was done with antibiogram, pulsed-field gel electrophoresis, bacteriophage typing and coagulase gene typing. Results: There were 44 infections caused by nmMRSA. Seventeen infections (39%) occurred in patients from the south-west Pacific Islands (predominantly Samoa, Tonga and New Zealand). Laboratory typing showed that the isolates in Pacific Islanders were Pacific Island strains, and 16/17 of these infections were community acquired. Twenty-three infections (52%) occurred in Caucasians. Eleven of the isolates from Caucasians (48%) were a new predominantly community-acquired strain that we have termed the ‘R’ pulsotype, nine (39%) were Pacific Island strains, and three (13%) were health care institution-associated strains. Four infections occurred in patients who were not Caucasians or Pacific Islanders. Overall, 34 of all 44 infections (77%) were community' acquired. Conclusions: Non-multiresistant MRSA infection, relatively frequently observed in Pacific Islanders in south-east Queensland, is now a risk for Caucasians as well, and is usually community acquired. Clinicians should consider taking microbiological specimens for culture and antimicrobial susceptibility testing in patients with suspected staphylococcal infections who are not responding to empirical therapy with β-lactam antibiotics.

Beef carcass contamination in a slaughterhouse and prevalence of resistance to antimicrobial drugs in isolates of selected microbial species

Meat contaminating bacteria may be the direct cause of foodborne diseases and represent a potential cause for the drug resistance of human pathogenic agents. The prevalence and resistance to 17 antimicrobial drugs of isolates of selected bacterial species were investigated in 70 swabs of beef carcasses and 70 subsequent samples of beef meat. Molecular techniques (coagulase gene typing Staphylococcus aureus and original gene typing Escherichia coli) were used in the differentiation of isolates. Carcasses were already contaminated after evisceration, least frequently with S. aureus strains (7.5% of samples), most frequently with coagulase-negative staphylococci strains (52.2% of samples). During carcass processing, contamination with resistant or polyresistant strains of S. aureus and E. coli significantly increased ( P<0.01). Gene typing isolates of S. aureus and E. coli indicated that the strains probably originated in the processing plant.

Analysis of methicillin-resistant and methicillin-susceptible Staphylococcus aureus by a molecular typing method based on coagulase gene polymorphisms.

A molecular typing method for Staphylococcus aureus based on coagulase gene polymorphisms (coagulase gene typing) was evaluated by examining a total of 240 isolates which comprised 210 methicillin-resistant S. aureus (MRSA) and 30 methicillin-susceptible S. aureus (MSSA) collected from a single hospital. By AluI restriction enzyme digestion of the PCR-amplified 3'-end region of the coagulase gene including 81-bp repeated units, the MRSA and MSSA isolates examined were divided into 6 and 12 restriction fragment length polymorphism (RFLP) patterns, respectively, whereas five patterns were commonly detected in MRSA and MSSA. MRSA isolates that showed a particular RFLP pattern were considered to be predominant in the hospital. Coagulase typing with type-specific antisera was also performed for all S. aureus isolates for comparison. Coagulase types II and VII were most frequently detected and included isolates with four and five different AluI RFLP patterns, respectively, whereas each of the other coagulase types corresponded to a single RFLP pattern. These results indicated that RFLP typing was more discriminatory than serological typing, for typing S. aureus and demonstrated its utility in epidemiologic investigation of S. aureus infection in hospitals.

Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker.

To perform coagulase gene typing, the repeated units encoding hypervariable regions of the Staphylococcus aureus coagulase gene were amplified by the PCR technique; this was followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP) patterns. In order to assess the discriminatory power of this typing method, 30 epidemiologically unrelated S. aureus strains which differed by their pulsed-field gel electrophoresis patterns were examined. Although 18 of the 30 strains had unique and unshared AluI RFLP patterns, there were only four observed patterns in the remaining 12 strains. This finding indicated that unrelated strains may share identical AluI RFLP patterns. To elucidate the degree of genetic variation in the C-terminus-encoding loci within the coagulase genes, the PCR products of these 12 strains were subjected to Taq polymerase-mediated sequencing. Sequence analysis confirmed the AluI recognition sites in each of the four RFLP groups and demonstrated that AluI appears to yield the highest RFLP in restriction enzyme analysis. By their DNA sequences the majority of strains sharing common AluI groups could be clearly differentiated from each other and revealed between 93.2 and 98.5% homology. When we determined the nucleotide sequences of two strains after six subcultivations no significant alterations were observed. Because the discriminatory power of the current coagulase gene typing method is not great enough to be used as the sole method to type S. aureus, additional techniques are necessary. Sequence analysis of the repeated unit-encoding region for the typing of S. aureus may be potentially useful as an alternative to other current molecular typing techniques.