Genetic Characterization of blaSHV/VEB/PER Genes in ESBL-producing MDR Klebsiella Pneumonia Strains Isolated from Patients in Isfahan, Iran

This study was conducted to detect three genetical variants of Extended-Spectrum Beta-Lactamase (ESBL, in which142 Klebsiella pneumoniae (K.pneumoniae) isolates were collected from sections of a teaching hospital in Isfahan and were detected using standard IMVIC biochemical tests and urease. These were confirmed by identification of the ureD gene. Antimicrobial susceptibility testing was performed using the standard Kirby-Bauer disk-diffusion method on Mueller-Hinton agar (Merck,Germany) .The performance and interpretation were based on the guidelines from the Clinical Laboratory Standards Institute (CLSI, 2013). Screening and phenotypic identification of ESBLs isolates were performed by DDST. The presence of genes responsible for ESBL resistance, such as SHV, PER and VEB type ESBL genes was identified by PCR and indicator isolates sequencing performed by Macrogen (Seoul, Korea). The nucleotide sequences were analysed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST), the Lahey database, and CromasPro-2 and Mega-4 software to determine the subvarients of the three variants of ESBL (SHV, PER, VEB). These were compared with blaSHV-11 gene from K. pneumonia (accession.no.X98101), blaSHV-5 gene from K. pneumonia (accession no. X55640), blaPER-1 gene from P.aeruginosa transposed on Tn2345 (accession no AY866517.2) and blaVEB-1 gene from K. pneumonia (accession no. AF010416). A total of 120 isolates (84%) were recognized as MDR. The highest rate of resistance was recorded for piperacillin (80%), ceftazidime (76%), and cefotaxime (73%) and the lowest rate was for ertapenem (47.3%), meropenem (50.8%), and imipenem (58.7%) following detection of ESBL isolates of K.pneumoniae (101 isolates; 71%).The ward and the clinical specimen with the most prevalence were ICU with 55(38.7%) and urine with 61(42.9%). The lowest prevalence was related to the neurosurgery ward with 8(5.6%) samples and the clinical specimen with the lowest prevalence was cerebrospinal fluid (CSF) with 2 (1.4%) samples. PCR detection in ESBL-producing K. pneumoniae showed that, of the clinical isolates, 42.2% contained blaSHV (42/101), 2.9% contained blaVEB (3/101) and2% contained blaPER (2/101). Sequencing of 10 selected PCR products of SHV genes showed that 7/10isolates were similar to the strain SHV-11 and 3/7 isolates were similar to the strain SHV-5. The sequencing of two PCR products of the PER genes showed they were similar to the strainPER-1.Sequencing of three PCR products of the VEB genes showed they were similar to the strain VEB-1. The overall prevalence of ESBL-producers was found to vary greatly in different geographical areas; this may be the result of differences in the type and amount of antibiotics consumed and differences in the time of collection of isolates. The present study reflects anincrease in the prevalence of ESBL-producers in Iran. The most common ESBL type found in this study was SHVand that VEB and PER types were rare. In addition, sequence analysis results of our study show the rate of SHV-11, PER-1 and VEB-1was maximum

Genetic Characterization of blaSHV/VEB/PER Genes in ESBL-producing MDR Klebsiella Pneumonia Strains Isolated from Patients in Isfahan, Iran

This study was conducted to detect three genetical variants of Extended-Spectrum Beta-Lactamase (ESBL, in which142 Klebsiella pneumoniae (K.pneumoniae) isolates were collected from sections of a teaching hospital in Isfahan and were detected using standard IMVIC biochemical tests and urease. These were confirmed by identification of the ureD gene. Antimicrobial susceptibility testing was performed using the standard Kirby-Bauer disk-diffusion method on Mueller-Hinton agar (Merck,Germany) .The performance and interpretation were based on the guidelines from the Clinical Laboratory Standards Institute (CLSI, 2013). Screening and phenotypic identification of ESBLs isolates were performed by DDST. The presence of genes responsible for ESBL resistance, such as SHV, PER and VEB type ESBL genes was identified by PCR and indicator isolates sequencing performed by Macrogen (Seoul, Korea). The nucleotide sequences were analysed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST), the Lahey database, and CromasPro-2 and Mega-4 software to determine the subvarients of the three variants of ESBL (SHV, PER, VEB). These were compared with blaSHV-11 gene from K. pneumonia (accession.no.X98101), blaSHV-5 gene from K. pneumonia (accession no. X55640), blaPER-1 gene from P.aeruginosa transposed on Tn2345 (accession no AY866517.2) and blaVEB-1 gene from K. pneumonia (accession no. AF010416). A total of 120 isolates (84%) were recognized as MDR. The highest rate of resistance was recorded for piperacillin (80%), ceftazidime (76%), and cefotaxime (73%) and the lowest rate was for ertapenem (47.3%), meropenem (50.8%), and imipenem (58.7%) following detection of ESBL isolates of K.pneumoniae (101 isolates; 71%).The ward and the clinical specimen with the most prevalence were ICU with 55(38.7%) and urine with 61(42.9%). The lowest prevalence was related to the neurosurgery ward with 8(5.6%) samples and the clinical specimen with the lowest prevalence was cerebrospinal fluid (CSF) with 2 (1.4%) samples. PCR detection in ESBL-producing K. pneumoniae showed that, of the clinical isolates, 42.2% contained blaSHV (42/101), 2.9% contained blaVEB (3/101) and2% contained blaPER (2/101). Sequencing of 10 selected PCR products of SHV genes showed that 7/10isolates were similar to the strain SHV-11 and 3/7 isolates were similar to the strain SHV-5. The sequencing of two PCR products of the PER genes showed they were similar to the strainPER-1.Sequencing of three PCR products of the VEB genes showed they were similar to the strain VEB-1. The overall prevalence of ESBL-producers was found to vary greatly in different geographical areas; this may be the result of differences in the type and amount of antibiotics consumed and differences in the time of collection of isolates. The present study reflects anincrease in the prevalence of ESBL-producers in Iran. The most common ESBL type found in this study was SHVand that VEB and PER types were rare. In addition, sequence analysis results of our study show the rate of SHV-11, PER-1 and VEB-1was maximum

The Effect of Some Physical and Chemical Parameters on Regrowth of Aeromonas Bacterium and Heterotrophic Bacteria in Isfahan Drinking Water System

Aeromonas is one the gram – negative , non spor – formating rod shaping , facultatively anaerobic and opportunistic bactria that can cause systematic infections, leision and diarrhoea in human. Fairly high bactrial population in distribution system is not only of concern because of affecting consumer health but also it makes it difficult to enumerate coliform bacterium indicator. So, the relationships between aeromonas and heterotrophic bacteria growth with pH, temperature, turbidity, free residual cholornie and DO were determined in this study. ADA- V media was used in presumptive stage to count aeromonas bacteria for the first time in Iran on the basis of 1605 EPA (2001) method and used oxidase tests, trehalose fermentation and indol test in confirmative stage. R2A media was used to count HPC bacteria and other factors measured on the basis of standards. The results showed that positive cases of aeromonas bacteria and HPC increase in higher temperature and turbidity and lower pH. In contrast, positive cases of aeromonas bacteria and HPC decrease while free residual cholorine and DO increase. In addition,no positive case of aeromonas was observed in more than 0.2 mg/L concentration of free residual cholorine

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources

Background: Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. Materials and Methods: Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. Results: Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli. PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. Conclusion: The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources

Isolation of toxigenic Clostridium difficile from ready-to-eat salads by multiplex polymerase chain reaction in Isfahan, Iran

Background: Since 2003, the incidence of community associated Clostridium difficile infection (CA-CDI) has increased; different types of food have been supposed to be the vectors of C. difficile strains. The purpose of this study is to investigate the occurrence of C. difficile strains in ready-to-eat salads distributed in food services. Materials and Methods: A total of 106 ready-made salad specimens were sampled from different restaurants and food services located in Isfahan, in the center of Iran. Positive isolates of C. difficile were identified and confirmed for the existence of three genes including tpi, tcdA and tcdB by multiplex PCR. Results: A total of six (5.66%) samples were positive for C. difficile strains. Of which, one strain (16.6%) was positive for A and B toxins. Conclusion: The existence of toxigenic C. difficile in ready-made salads could be a caution for public health. Further investigation is required to assess the relationship between the isolated strains in our study and those from diarrheic patients through molecular typing

Identification of Non Tuberculous Mycobacteria Isolated from Isfahan Different Water Sources Using Phenotypic Characterization Tests

In recent decades, by increasing immunocompromised patients, disease related to nontuberculous mycobacteria, previously known as environmental opportunistic pathogen, has been raised. In this study, 85 water samples from different sources in Isfahan were evaluated for the presence of NTM. Phenotypic tests were used to identify NTM species. Twenty one out of 85 (24.7%) collected water samples had at least one NTM. Of these, 23.8% (5 isolates) and 14.3 % (3 isolates) were M. furtuitum and M. smegmatis, respectively.  Two cases for each isolates (9.5%) were identified as M. chelonae like organisms, M. terrae complex, M. gordonae and M. mucogenicum. One cases for each isolates (4.8%) was determined as M. avium complex, M. phlei, M. xenopi, M. fallax, and M. flavescence. The results showed the incidence of different species of NTM in this geographical region in Iran. Because of increasing immunocompromised disease in communities, and high frequency of NTM in different geographical regions, understanding the NTM distribution in environment would help clinicians to manage proper treatment strategy

Efflux pump regulatory genes mutations in multidrug resistance Pseudomonas aeruginosa isolated from wound infections in Isfahan hospitals

Background: Multidrug resistance Pseudomonas aeruginosa (MDR-P. aeruginosa) is a worldwide threat for public health. Hyperexpression of efflux pump systems (MexAB-OprM and MexCD-OprJ), which is a well-known mechanisms for MDR emerging, is controlled by regulatory genes, mexR and nfxB, respectively. The aim of this study was to evaluate point mutations in mexR and nfxB genes in MDR- P. aeruginosa isolated from wound infections. Materials and Methods: A total of 34 P. aeruginosa cultures obtained from wound infections were analyzed. Among them eight isolates identified as MDR-P. aeruginosa and were subjected to determination of mutations in mexR and nfxB genes. Results: We detected eight-point mutations in mexR and 12-point mutations in nfxB. The most common mutations were common G327-A (eight isolates), G384-A (eight isolates), G411-A (eight isolates). Mutations in A371-C and A372-C were the predominant substitution which was seen in nfxB. Amino acid substitutions were also found at position 124 and 126 for NfxB and MexR, respectively. Conclusions: P. aeruginosa isolates with mutation in efflux pump regulatory genes such as mexR and nfxB could be a main factor contributed to antibiotic resistance and must be considered in antibiotic treatment

Distribution of the Strains of Multidrug-resistant, Extensively Drug-resistant, and Pandrug-resistant Pseudomonas aeruginosa Isolates from Burn Patients

Background: Pseudomonas aeruginosa is an opportunistic and Gram-negative pathogen that is used as the most important factor in burn wound infections, and due to the rapid acquisition of multidrug resistance (MDR), it causes high mortality rates in these sectors. Thus, diagnosis and assessment of antibiotic resistance patterns are very important in these patients. The aim of this study was to evaluate antibiotic resistance pattern and determining P. aeruginosa MDR. Materials and Methods: In this study, phenotypic, biochemical, and polymerase chain reaction tests were used to identify P. aeruginosa from 120 wound burn samples that 96 samples were detected to P. aeruginosa species. In the next step, according to the Clinical and Laboratory Standard Institute standard guidelines, antibiogram test was performed by disk diffusion method for amikacin, ciprofloxacin, norfloxacin, gentamicin, cefepime, aztreonam, meropenem, colistin, ceftazidime, and piperacillin-tazobactam antibiotics. Antibiotic data were analyzed by WHONET software; finally, the rate of antibiotic resistance and MDR strains was determined. Results: The highest antibiotic resistance belonged to amikacin (94.8%) and norfloxacin (90.6%); in contrast, colistin (8.3%) had the lowest and the MDR strains were MDR (95.8%) and extensively drug resistance (XDR) (87.5%). Conclusion: In this study, there was MDR with an alarming rate including MDR (95.8%), XDR (87.5%), and pan-drug resistance (0%). As a result, given antibiotics to patients should be controlled by the antibiogram results to avoid increasing MDR strains