Detection of genes involved in biofilm formation in Staphylococcus aureus isolates.

Staphylococcus aureus is one of the Gram-positive pathogens causing a wide range of nosocomial infections. The present study investigates genotypic and phenotypic aspects involved in biofilm formation in methicillin-resistant Staphylococcus aureus strains isolated from nosocomial infections in Isfahan. A total of 110 S. aureus strains were collected from three major hospitals in Isfahan, the center of Iran. The antibiotic resistance pattern, phenotypes, and biofilm formation genes were studied using Congo red agar (CRA) and multiplex PCR (M-PCR). We found that 103 out of 110 samples (93.6%) were MRSA. The highest frequency of resistance was found to penicillin (89%), ciprofloxacin (87.4%), and erythromycin (86.1%). Phenotypic results showed that 53.5% were high biofilm producers, while 33.3% and 13.2% were intermediate and low biofilm producers, respectively. icaC (69.3%) had the highest frequency in comparison to other intercellular adhesion (ica) genes, icaD (54.8%) was second most common. The results show that the adherence or attachment ability and biofilm production are important for enhancing virulence factors among isolates of S. aureus strains.

Detection of integrons and Staphylococcal Cassette Chromosome (SCCmec) types in Staphylococcus aureus isolated from burn and non-burn patients.

BACKGROUND: Methicillin Resistant Staphylococcus aureus (MRSA) strains have been recognized as an important reason of infections in health care units. Integrons role in antibiotic resistance box gene transfer has been well recognized which are found in Gram positive bacteria. OBJECTIVE: The aim of this study was analyzed of SCCmec typing and determine of integron classes in burn and non-burn specimens. METHODOLOGY: A total of 110 S. aureus strains were isolated from burn and non-burn patients. Antimicrobial susceptibility testing, detection of mecA gene, various SCCmec types and integrons classes were analyzed. RESULTS: In antimicrobial susceptibility test in burn patients, resistant to both gentamicin and oxacilin and in non-burn patients resistance to oxacilin and cefepime showed the highest ratio In PCR molecular test (80%) and (52.7%) of strains harbored the mecA gene. Therefore five different SCCmec types were recognized among our studied strains. Subsequently, integron class I was evaluated as (94.5%) in burn and (12.7%) in non-burn isolates by the multiplex PCR method. CONCLUSION: Albeit MRSA strains have the hospital reservoir so may cause serious treats for hospitalized and non-hospitalized patients, hence clinical decision for prevention and treatment may develop due to, mecA gene, SCCmec elements and integrons detection in health care units.

Evaluation of various staphylococcal cassette chromosome mec (SCCmec) types in Staphylococcus epidermidis invasive strains from hospitalised patients in Iran.

Staphylococcus epidermidis is known to be a major cause of nosocomial infections particularly in catheter-associated bacteraemia, prosthetic valve endocarditis (PVE) and immunocompromised patients in different health care units. The emergence of multidrug-resistant strains, especially to ß-lactam antibiotics such as methicillin, has increased the mortality due to S. epidermidis. A kind of low affinity penicillin-binding protein (PBP2α), which is encoded by the mecA gene that is located in the staphylococcal cassette chromosome mec (SCCmec), mediates the resistance to methicillin. The aim of this study was to investigate the prevalence of SCCmec types and evaluate the antibiotic profile assay in invasive strains isolated from clinical samples. The study focused on invasive strains, determining the antimicrobial resistance profile, designing new primers for detection of the mecA gene and SCCmec typing with the multiplex PCR method. By using the PCR molecular test, 87.1% of all isolates were found to be positive for the mecA gene. In SCCmec typing, different types (I-V) were identified, in which SCCmec type I was detected in 3 isolates, SCCmec type II in 5 isolates, SCCmec type III in 22 isolates, SCCmec type IV in 27 isolates and SCCmec type V was distinguished in 4 isolates. Since coagulase-negative staphylococci are reported as a major cause of hospital infections, molecular typing methods like SCCmec typing would be a helpful method to control and prevent bacterial infections.

RELATIVE EXPRESSION OF EFFLUX PUMPS IN MULTI DRUG RESISTANT PSEUDOMONAS AERUGINOSA.

Pseudomonas aeruginosa is known as an important opportunistic pathogen, resistant to a high number of antibiotics. Efflux pumps are one of the main intrinsic antibiotics resistance mechanisms in P. aeruginosa. MexAB-OprM, MexCD-OprJ, and MexXY-OprM are the main efflux pumps involved in beta-lactam resistant strains which may cause cross resistance to different antimicrobial classes. The aim of this study was to detect relative gene expression in betalactam-resistant clinical P. aeruginosa strains. One hundred fourteen clinical strains of P. aeruginosa were identified by phenotypic and genotypic methods. Antibiotic susceptibility testing was conducted according to CLSI guideline. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor for phenotypic detection of efflux pump mechanism and q-RT PCR was conducted for relative gene expression detection. The highest rate of resistance was observed against cefotaxime and various relative gene expressions levels were observed in all isolates with positive phenotypic test results.

Clinical characteristics of Staphylococcus epidermidis: a systematic review.

Staphylococci are known as clustering Gram-positive cocci, nonmotile, non-spore forming facultatively anaerobic that classified in two main groups, coagulase-positive and coagulase-negative. Staphylococcus epidermidis with the highest percentage has the prominent role among coagulase-negative Staphylococci that is the most important reason of clinical infections. Due to various virulence factors and unique features, this microorganism is respected as a common cause of nosocomial infections. Because of potential ability in biofilm formation and colonization in different surfaces, also using of medical implant devices in immunocompromised and hospitalized patients the related infections have been increased. In recent decades the clinical importance and the emergence of methicillin-resistant Staphylococcus epidermidis strains have created many challenges in the treatment process.

Characterisation of SCCmec elements in methicillin-resistant Staphylococcus aureus isolated from burn patients.

Staphylococcus aureus is an important pathogen, especially in burn units all around the world. Because of the emergence of the ß-lactam antibiotic-resistant strains since 1961, concern about the prevalence of methicillin-resistant S. aureus (MRSA) has increased in these units. Resistance to methicillin is mediated by penicillin-binding proteins (PBPs) that have enough affinity for binding to the ß-lactam ring, but another kind of protein (PBP2α), which is encoded by the mecA gene, has a lower affinity for binding to these antibiotics. The mecA gene is transferred by SCCmec (staphylococcal cassette chromosome mec) as a mobile genetic element, exclusively found in the Staphylococcus genus. Identification of the frequency of the mecA gene, different SCCmec types and also its incidence may have benefit in surveillance prevention and control of MRSA strains in burn units. In this study, 40 S. aureus isolates were collected from patients hospitalised in Motahari burn center of Tehran, during 2012-2013. Conventional microbiological methods were applied and the confirmed isolates were stored at -20°C for molecular polymerase chain reaction (PCR) tests. The antibiotic resistance pattern was performed by disc diffusion method and finally the different SCCmec types were determined by specific primers. During this research, 40 isolates of S. aureus were collected from burn patients, of which (37.5%) of the specimens belonged to female patients and 62.5% to male patients. The aetiology of the burn was classified as follows: open flame (35%), liquid (32.5%), chemical (5%) and other (27.5%). By a disc diffusion method, no resistance pattern was observed to vancomycin and fosfomycin. Based on a multiplex PCR assay, the five different SCCmec types were detected as: 47.5% type III, 25% type IV, 10% type V, 10% type II and 7.5% type I.

Comparison between phenotypic and PCR for detection of OXA-23 type and metallo-beta-lactamases producer Acinetobacter spp.

BACKGROUND: Resistance to carbapenems is developing around the world and can cause many problems for treatment of patients. Production of metallo-beta-lactamase (MBL) is one of the main mechanism for this type of resistance. So, detection of MBL-producer microorganisms can prevent the spread of this type of resistance. MATERIALS AND METHODS: In this study 94 Acinetobacter spp. were investigated. Resistance to imipenem was conducted after purification and identification. Combination disc (CD) and Double Disc Synergy Test (DDST) were performed for phenotypic detection of MBL and the molecular PCR method was done for vim-1, vim-2, imp-1 and OXA-23 genes. RESULTS: According to TSI, SIM and oxidation-fermentation (OF) test and PCR assay 93 Acinetobacter baumannii and one strain Acinetobacter lwoffii were identified. 85% of them were resistant to imipenem. 34% of them have a positive combination disc test (CD) while Double Disc Synergy Test (DDST) was negative for all of them. The vim-1, vim-2 and imp-1 genes were not detected in PCR molecular method, however in 74% of strains with positive results in combination disc, were positive for the OXA-23 gene after PCR test. This study shows that the blaOXA-23 resistance determinant may become an emerging therapeutic problem. DISCUSSION: According to the results, it seems that combination disc does not have enough specificity for detection of MBL-producer Acinetobacter and using Double Disc Synergy Test (DDST) can be more convenient.

Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates.

Staphylococcus aureus is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in men. The ability of microorganisms to produce biofilm facilitates them to withstand the host immune response and is recognized as one factor contributing to chronic or persistent infections. It was demonstrated that the ica-encoded genes lead to the biosynthesis of polysaccharide intercellular adhesion (PIA) molecules, and may be involved in the accumulation phase of biofilm formation. Different studies have shown the decisive role of the ica gene as virulence factors in staphylococcal infections. This study was carried out to demonstrate the relationship between ica gene and production of slime layer in S. aureus strains. Sixty S. aureus strains were isolated from patients. The isolates were identified morphologically and biochemically following standard laboratory methods. After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB), to which 15% glycerol was added, and stored at -20°C. Slime formation and biofilm assay was monitored. A PCR assay was developed to identify the presence of icaD (intercellular adhesion gene) gene in all isolates. Thirty-nine slime producing colonies with CRA plates (65%) formed black colors, the remaining 21 isolates were pink (35%). In the quantitative biofilm assay 35 (58%) produced biofilm while 25 (42%) isolates did not exhibit this property. All isolates were positive for detection of icaD gene by PCR method. The interaction of icaA and icaD in the investigated isolates may be important in slime layer formation and biofilm phenomena. We propose PCR detection of the ica gene locus as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, with implications for therapeutic and preventive measures pertainin to the management of colonized indwelling catheters.

Silver nanoparticles as active ingredient used for alcohol-free mouthwash.

We developed an effective and non-irritant mouthwash that is alcohol-free and has a low concentration of silver nanoparticles (SNP) in order to be used for preventing oral cavity infections in immunocompromised oncologic patients. We studied antimicrobial effects of silver nanoparticles (SNP) in the range of (50-0.024 µg/ml) and 3% of ethanol (30,000 µg/ml) in mouthwash. Antimicrobial effects of two treatments were studied by doing challenge test on microorganisms such as Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and measuring MIC and MBC (MFC) values of SNP toward mentioned microorganisms. These values of SNP respectively were in the range of (0.78-3.12) and (1.56-12.5 µg/ml). Results showed that SNP in the MIC and the lower concentrations killed all of the used microorganisms. No difference was observed between the antimicrobial effect of ethanol-free mouthwash containing SNP and mouthwash containing SNP and ethanol (30,000 µg/ml). SNP has high antimicrobial effects at low concentrations and it can be a good alternative for ethanol (30,000 µg/ml) because ethanol is also irritating, especially to sensitive or inflamed mucosa.