Seyed Asghar Havaei

Genetic Characterization of a Vancomycin-Resistant Staphylococcus aureus Isolate from the Respiratory Tract of a Patient in a University Hospital in Northeastern Iran

ABSTRACT Emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains has led to global concerns about treatments for staphylococcal infections. These strains are currently rare even though there is an upward trend in their reported incidence. Therefore, appropriate screening and epidemiological evaluation of VRSA strains can affect future global health care policies. Isolates of Staphylococcus aureus were obtained from various clinical samples and were then evaluated with agar screening, disk diffusion, and MIC methods to determine resistance to vancomycin and methicillin. After confirmation of the isolated VRSA strain, genetic analysis was performed by evaluating mecA and vanA gene presence, SCCmec, agr, and spa types, and toxin profiles. Multilocus sequence typing (MLST) and plasmid analysis were also performed. The VRSA strain was resistant to oxacillin (MIC of 128 μg/ml) and vancomycin (MIC of 512 μg/ml). Disk diffusion antimicrobial susceptibility tests showed resistance to oxacillin, vancomycin, levofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, clindamycin, rifampin, and tetracycline. The isolate was susceptible to minocycline and gentamicin. PCRs were positive for the mecA and vanA genes. Other genetic characteristics include SCCmec type III, agr I, spa type t037, and sequence type (ST) 1283. The plasmid profile shows five plasmids with a size of ∼1.7 kb to >10 kb. The isolated VRSA strain was obtained from a critically ill hospitalized patient. Genetic analysis of this strain suggested that the strain was a methicillin-resistant S. aureus (MRSA) clone endemic in Asia that underwent some genetic changes, such as mutation in the gmk gene and acquisition of the vanA gene.

Epidemic methicillin-susceptible Staphylococcus aureus lineages are the main cause of infections at an Iranian university hospital.

ABSTRACT The majority of Staphylococcus aureus infections from Isfahan, Iran, were caused by epidemic methicillin-susceptible S. aureus (MSSA) lineages, sequence type 8 (ST8), ST22, ST30, and ST6. The predominant methicillin-resistant S. aureus strain was ST239. We observed a high prevalence of Panton-Valentine leukocidin-positive MSSA strains (19.7%), which is a matter of considerable concern, since these strains have the ability to cause severe infections.

Genetic Characterization of Methicillin Resistant and Sensitive, Vancomycin Intermediate Staphylococcus aureus Strains Isolated from Different Iranian Hospitals

Background. Global concerns have been raised due to upward trend of Vancomycin Intermediate Staphylococcus aureus (VISA) and Vancomycin Resistant Staphylococcus aureus (VRSA) reports which mean casting doubt on the absolute effectiveness of the last line of antibiotic treatment for S. aureus, vancomycin. Hence, epidemiological evaluation can improve global health care policies. Methodology. 171 Isolates of Staphylococcus aureus were collected from different types of clinical samples in selected hospitals in Isfahan, Mashhad, and Tehran, Iran. Then, they were evaluated by agar screening, disk diffusion, and MIC method to determine their resistance to vancomycin and methicillin. The isolated VISA strains were then confirmed with genetic analysis by the evaluation of mecA and vanA genes, SCCmec, agr, and spa type, and also toxin profiles. MLST was also performed. Results and Conclusion. Our data indicated that 67% of isolated S. aureus strains were resistant to methicillin. Furthermore, five isolates (2.9%) had intermediate resistance to vancomycin (VISA). In contrast to usual association of VISA with MRSA strains, we found two isolates of MSSA-VISA. Therefore, our data suggests a probable parallel growing trend of VISA towards MSSA, along with MRSA strains. However, more samples are required to confirm these primarily data. Moreover, genetic analysis of the isolated VISA strains revealed that these strains are endemic Asian clones.

Molecular characterization of intestinal carriage of carbapenem-resistant Enterobacteriaceae among inpatients at two Iranian university hospitals: first report of co-production of bla NDM-7 and bla OXA-48

Gastrointestinal colonization of carbapenem-resistant Enterobacteriaceae (CRE) could serve as a reservoir for the transmission of these pathogens in the clinical setting. The aim of this study was to investigate the intestinal carriage of CRE and to analyze risk factors for CRE carriage. Rectal swabs were collected from 95 patients at two Iranian university hospitals. CRE screening was performed using selective media (CHROMagar and MacConkey agar). Polymerase chain reaction (PCR) was used to detect carbapenemase-encoding genes. Clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE). The rate of carriage of CRE in hospitalized patients was 37.9%. Overall, 54 CRE isolates were identified, of which 47 were carbapenemase-producers. All of the 54 CRE were detected using CHROMagar compared with 52 CRE detected using MacConkey agar. Fifteen patients were colonized by multiple CRE isolates. Three significant risk factors for CRE carriage were detected: intensive care unit (ICU) hospitalization, antibiotic exposure, and mechanical ventilation. blaOXA-48 was the most frequent carbapenemase detected, followed by blaNDM-1 and blaNDM-7. Eleven carbapenemase-producing Enterobacteriaceae (CPE) isolates co-harbored blaNDM-1 and blaOXA-48. Also, six CPE isolates co-harbored blaNDM-7 and blaOXA-48. We did not detect blaKPC, blaGES, blaIMP, or blaVIM. PFGE analysis showed that Escherichia coli clones were diverse, while Klebsiella pneumoniae isolates were divided into four clusters. Cluster I was the major clone carrying blaOXA-48 and blaCTX-M-15 genes. In our study, the carriage rate of CRE was high and the emergence of CPE isolates among patients is alarming. The implementation of adequate preventive measures such as active surveillance is urgently needed to control the spread of CPE in the healthcare setting.

Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran

Background: The rising frequency of methicillin resistant Staphylococcus aureus (MRSA) has led to an increased use of antibiotics such as macrolide, lincosamide, streptogramin B (MLSB) for the treatment of S. aureus infections. Resistance to MLSBin S. aureus is commonly encoded by erm genes, which can be constitutive MLSB (cMLSB) or inducible MLSB (iMLSB). The purpose of this study was to determine the frequency of cMLSB, iMLSB, and MS phenotypes using D-test and polymerase chain reaction (PCR) methods. Materials and Methods: A total of 215 isolates of S. aureus were collected from January 2010 to May 2012 from Al-Zahra Hospital in Isfahan. PCR was performed for detection of mecA gene on all isolates using specific primers. The frequency of MLSB-resistant isolates was determined using D-test, and then a multiplex PCR was performed for detection of ermA, ermB, and ermC genes. Results: Among 215 S. aureus isolates examined, 82 (40.9%) were MRSA, and iMLSB, cMLSB, and MS resistance phenotypes had a frequency of 9 (4.18%), 58 (26.9%), and 11 (5.1%), respectively. Among nine isolates with iMLSBresistance phenotype, four isolates contained ermC gene, two isolates ermB gene, and one isolate ermA gene. Two isolates did not have any erm gene. Conclusion: In the current study, cMLSBwas the most frequent phenotype and ermC was the most common gene in iMLSBresistant phenotypes.

Molecular Typing of Hospital-Acquired Staphylococcus aureus Isolated from Isfahan, Iran

Background. Staphylococcus aureus (S. aureus) is one of the most common pathogens that cause hospital- and community-acquired infections in the world. The use of molecular typing methods is essential for determining the origin of the strains, their clonal relations, and also in epidemiological investigations. The purpose of this study was to determine the prevalence of antibiotic resistant S. aureus isolates and using spa, agr, and SCCmec typing to determine the dominant types in Iran. Material and Method. Fifty isolates of S. aureus were collected from January to May 2010. S. aureus identification was performed by biochemical tests. Disk diffusion method was employed to assess the sensitivity of S. aureus strains to antibiotics and then genetic analysis of bacteria was performed using SCCmec, agr, and spa typing. Results. S. aureus resistance to tetracycline, cefoxitin, clindamycin, ciprofloxacin, gentamicin, Cot: cotrimoxazole, levofloxacin, rifampin, and vancomycin were found to be 36%, 18%, 12%, 12%, 22%, 6%, 6%, and 0%, respectively. The results of this study showed that 16% of the isolates were resistant to methicillin (MRSA) and the majority of isolates were SSC mec type IV. In addition spa and agr typing revealed agr typeI and spa type t7688 to be the most predominant. Conclusion. In this study, spa typing showed 100% reliability and the t7688 spa type had a frequency of 26% compared to the frequency of 0.0% in the Ridom SpaServer. The frequency of t304 spa type was higher than the global average.

Molecular identification of Acinetobacter baumannii isolated from intensive care units and their antimicrobial resistance patterns

Background: Acinetobacter baumannii is one of the most important pathogens in hospital-acquired infections especially in intensive care units (ICUs). This opportunistic pathogen can be easily isolated from water, soil, and hospital facilities. A. baumannii as a nosocomial opportunistic pathogen is resistant to a wide range of antibiotics and responsible for multiple infections, including bacteremia, pneumonia, meningitis, urinary tract infections, and surgical wounds. The aim of this study was to determine frequency and resistance patterns of A. baumannii isolated in ICUs of Isfahan Hospitals. Materials and Methods: During 1 year period (2012-2013), 350 specimens were collected from ICUs of Isfahan hospitals. The isolates were characterized as A. baumannii by conventional phenotypic, biochemical tests and confirmed by PCR for OXA-51-like gene. Susceptibility of isolates was determined by standard disk diffusion method according to CLSI. Results : From total of 350 specimens, 43 isolates were A. baumannii. The antimicrobial patterns of isolates showed that 53.5% of isolates were resistant to amikacin, 83.7% to tetracyclin, 86% to ceftazidime, 90.7% to Trimethoprim sulfametoxazol, 93% to imipenem, cefepime, meropenem, ampicillin-sulbactam. All isolates were resistant to ciprofloxacin. Conclusion: This study showed a high resistance of A. baumannii to a wide range of antimicrobial agent. It is necessary to adopt appropriate strategies to control the spread of the bacteria in care unit centers and wards.

Emergence of carbapenem resistant Escherichia coli isolates producing blaNDM and blaOXA-48-like carried on IncA/C and IncL/M plasmids at two Iranian university hospitals

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was blaOXA-48 (82.8%), followed by blaNDM-1 (31%), blaNDM-7 (6.9%) and blaOXA-181 (3.4%). Seven of the blaNDM positive isolates co-harbored blaOXA-48 carbapenemases. The blaNDM and blaOXA-48 were found in IncA/C and IncL/M conjugative plasmids, respectively. The blaCTX-M-15, qnrA and intI1 genes were also present in most isolates. The PFGE revealed genetic diversity among the 28 E. coli isolates, which belonged to six minor PFGE clusters and 14 isolates were singletons. The 26 isolates were distributed into 18 STs, of which two were dominant (ST648 and ST167). We identified one blaNDM-1-positive ST131 E. coli isolates that harbor the blaCTX-M-15 and blaTEM genes. Horizontal transfer of IncA/C and IncL/M plasmids has likely facilitated the spread of the blaOXA-48 and blaNDM genes among E. coli. Their clonal diversity and the presence of faecal carriers in isolates suggest an endemic spread of OXA-48 and NDM. Therefore, it emphasizes the critical importance of monitoring and controlling the spread of carbapenem resistant E. coli.

Nasal carriage screening of community-associated methicillin resistant Staphylococcus aureus in healthy children of a developing country

Background: The rapid emergence and spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has raised considerable public health concern in both developed and developing countries. The current study aimed to address the extent of this phenomenon in healthy preschool children of a developing country. Materials and Methods: We conducted a prospective study from April 2013 to March 2014 on 410 healthy 2-6 years old preschool children in Isfahan, Iran. Demographic medical data and nasal samples were collected from the participating children. Isolates were identified as S. aureus and MRSA based on microbiological and molecular tests, including the presence of eap and mecA genes. Results: The overall prevalence of S. aureus and CA-MRSA nasal carriage was 28% (115/410) and 6.1% (25/410), respectively. The identity of isolates was confirmed by molecular assay. The factors that were independently associated with nasal carriage of S. aureus were: Children crowding in day-care nurseries and income level of families. A total of 20/90 (22.2%) of methicillin-susceptible S. aureus and all 25 CA-MRSA displayed multiple drug resistance to 3–8 antibiotics. Conclusions: The current report reflects issues and concerns that the high rate of colonization by CA-MRSA in Iranian healthy children provides obliging evidence that MRSA have established a foothold in the community and are emerging as important health threatening pathogens. It is suggested that we need more effective infection control measures to prevent transmission of nasal CA-MRSA in healthy preschool children.

Characterization of Staphylococcus epidermidis isolates from hospitalized patients in Isfahan and Tehran teaching hospitals

Coagulase-negative Staphylococci especially Staphylococcus epidermidis is considered as an important reason of medical implant devices and nosocomial infections. Different virulence factors such as biofilm formation, delta toxin and autolysin increase the pathogenicity of this microorganism. However the ability of biofilm production is one of the main causes of microorganism attachment to various surfaces and may lead to chronic infections. On the other hand resistance to antimicrobial agents containing penicillinase resistant penicillins (i.e. oxacillin, methicillin) has been predominantly emerged in the past several years. PBP2a is encoded by mecA gene that conferred as meticillin resistant main reason. The mentioned gene is carried by the Staphylococcal chromosomal cassette mec (SCCmec) as a mobile genetic element. The aim of this study was evaluating the S. epidermidis isolates biofilm production and determining the various types of SCCmec elements in hospitalized patients. A total 115 Staphylococcus epidermidis strains were studied for the ability of biofilm formation, the frequency of both ica and mecA gene and also the existence of various SCCmec types in hospitalized patients. In CRA (70.4%) of isolates formed black colonies while in the molecular PCR method the prevalence of ica gene among isolates was (82.6%) and 96 were mecA positive. By multiplex PCR method five different types of SCCmec have been identified. Determination of biofilm formation in nosocomial infections, and also evaluation of SCCmec elements may be lead to a significant solution to prevent the infection prevalence in health care units.