Genomic diversity of ß-lactamase producing Pseudomonas aeruginosa in Iran; the impact of global high-risk clones.

BACKGROUND: Hospital-acquired infections caused by multidrug-resistant Pseudomonas aeruginosa incline hospital stay and costs of treatment that resulted in an increased mortality rate. The frequency of P. aeruginosa high-risk clones producing carbapenemases was investigated in our clinical samples. METHODS: In this cross-sectional study, 155 non-repetitive P. aeruginosa isolates were included from different medical centers of Iran. Antibiotic susceptibility testing was determined, and the presence of ß-lactamases were sought by phenotypic and genotypic methods. The clonal relationship of all isolates was investigated, and multi-locus sequence typing (MLST) was used for finding the sequence types of carbapenemase-producers. RESULTS: The agent with highest percent susceptibility rate was recorded for colistin (94.9%). MOX and FOX were found both as low as 1.95% (3/155). The most frequent narrow spectrum ß-lactamase was SHV with 7.7% (12/155) followed by PER, OXA-1, and TEM with the frequency of 7.1% (11/155), 3.2% (5/155), and 1.3% (2/155), respectively. Carbapenemases were detected in 28 isolates (18%). The most frequent carbapenemase was IMP with 9% (14/155) followed by NDM, 8.4% (13/155). OXA-48 and VIM were also detected both per one isolate (0.65%). MLST of carbapenem resistant P. aeruginosa isolates revealed that ST244, ST664, ST235, and ST357 were spread in subjected clinical settings. REP-PCR uncovered high genomic diversity in our clinical setting. CONCLUSION: Clonal proliferation of ST235 strain plays a key role in the propagation of MDR pattern in P. aeruginosa. Our data showed that high-risk clones has distributed in Iran, and programs are required to limit spreading of these clones.

Occurrence of plasmid-mediated quinolone resistance genes in Pseudomonas aeruginosa strains isolated from clinical specimens in southwest Iran: a multicentral study.

This study aimed to assess the presence of qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6')-Ib-cr determinants as well as quinolone resistance pattern of clinical isolates of P. aeruginosa in Ahvaz, southwest Iran. A total of 185 clinical isolates of P. aeruginosa were collected from 5 university-affiliated hospitals in Ahvaz, southwest Iran. The disk diffusion method was applied to assess the quinolone resistance pattern. The presence of qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6')-Ib-cr genes was investigated by the polymerase chain reaction (PCR) method. Overall, 120 (64.9%) isolates were non-susceptible to quinolones. The most and the less quinolone resistance rates were observed against ciprofloxacin (59.4%) and ofloxacin (45.9%), respectively. The prevalence rates of qnr genes were as follows: qnrA (25.8%), qnrB (29.2%), and qnrS (20.8%). The qnrB gene was the most common type of qnr genes. The qnr genes were occurred in 37.5% (n = 45/120) of quinolne-resistant isolates, simultaneously. The qnrC, qnrD, qepA, and aac(6')-Ib-cr genes were not recognized in any isolates. In conclusion, the ofloxacin was the most effective quinolone. This study was the first to shed light on the prevalence of PMQR genes among P. aeruginosa isolates in southwest Iran.

Molecular characterization of NDM-1-producing Pseudomonas aeruginosa isolates from hospitalized patients in Iran.

BACKGROUND: The emergence of carbapenem-resistant Pseudomonas aeruginosa is one of the most important challenges in a healthcare setting. The aim of this study is double-locus sequence typing (DLST) typing of blaNDM-1 positive P. aeruginosa isolates. METHODS: Twenty-nine blaNDM-1 positive isolates were collected during three years of study from different cities in Iran. Modified hodge test (MHT), double-disk synergy test (DDST) and double-disk potentiation test (DDPT) was performed for detection of carbapenemase and metallo-beta-lactamase (MBL) producing blaNDM-1 positive P. aeruginosa isolates. The antibiotic resistance genes were considered by PCR method. Clonal relationship of blaNDM-1 positive was also characterized using DLST method. RESULTS: Antibiotic susceptibility pattern showed that all isolates were resistant to imipenem and ertapenem. DDST and DDPT revealed that 15/29 (51.8%) and 26 (89.7%) of blaNDM-1 positive isolates were MBL producing isolates, respectively. The presence of blaOXA-10, blaVIM-2, blaIMP-1 and blaSPM genes were detected in 86.2%, 41.4%, 34.5% and 3.5% isolates, respectively. DLST typing results revealed the main cluster were DLST 25-11 with 13 infected or colonized patients. CONCLUSIONS: The presence of blaNDM-1 gene with other MBLs encoding genes in P. aeruginosa is a potential challenge in the treatment of microorganism infections. DLST showed partial diversity among 29 blaNDM-1 positive isolates.

Emerge of NDM-1-Producing Multidrug-Resistant Pseudomonas aeruginosa and Co-Harboring of Carbapenemase Genes in South of Iran.

BACKGROUND: New Delhi metallo-beta-lactamase-1 (NDM-1) is one of the most important emerging antibiotic resistance. Co-harboring three or four carbapenemases is rare and only a few reports exist in the literature. We described the characteristics of the large epidemic outbreaks and reports co-producing blaNDM-1 with the other carbapenemase genes in P. aeruginosa isolates. METHODS: This present cross-sectional research was conducted on 369 P. aeruginosa isolates obtained from burn and general hospitals within years 2013 to 2016. Beta-lactamase classes A, B and D genes were identified by PCR method. Modified hodge test (MHT), double-disk potentiation tests (DDPT) and double disk synergy test (DDST) were performed for detection carbapenemase and metallo beta-lactamase (MBL) production of blaNDM-1 positive P. aeruginos isolates. RESULTS: From 236 carbapenem-resistant P. aeruginosa (CRPA), 116 isolates have had MBL genes and twenty-nine isolates were found positive for blaNDM-1 . In CRPA isolates, blaIMP-1 , blaVIM-2 and blaOXA-10 were identified in 27.5%, 21.1% and 32.2% of isolates respectively, while co-producing blaNDM-1 , blaIMP-1 , blaOXA-10 , co-producing blaNDM-1 , blaVIM-2 , blaOXA-10 and co-producing blaIMP-1 , blaVIM-2 were determined in 11 (4.6%), 8 (3.4%) and 27 (11.4%) of isolates respectively. CONCLUSION: The finding of this co-existence of multiple carbapenemase resistance genes is threating for public health. Dipicolinic acid is a superior MBL inhibitor in DDPT antique than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P. aeruginosa.

Distribution of Genes Encoding Virulence Factors and the Genetic Diversity of Enteroinvasive Escherichia coli (EIEC) Isolates from Patients with Diarrhea in Ahvaz, Iran.

BACKGROUND: Entero-invasive E. coli (EIEC) is one of the causes of bacillary dysentery in adults and children. The ability of EIEC to invade and colonize the surface of epithelial cells is influenced by many virulence factors. This study aimed to investigate the distribution of virulence factor genes in EIEC strains isolated from patients with diarrhea in Ahvaz, Iran, as well as the genetic diversity between these isolates by Multilocus variable-number tandem repeat analysis (MLVA). MATERIALS AND METHODS: A total of 581 diarrheic stool samples were collected from patients with diarrhea attending two hospitals, in Ahvaz, Iran. The E. coli strains were identified by biochemical methods. Subsequently, all E. coli isolates were identified as EIEC by polymerase chain reaction (PCR) for the ipaH gene. The EIEC isolates evaluated by PCR for the presence of 8 virulence genes (ial, sen, virF, invE, sat, sigA, pic, and sepA). All EIEC strains were genotyped by the MLVA typing method. RESULTS: A total of 13 EIEC isolates were identified. The presence of ial, virF, invE, sen, sigA, pic, and sat genes was confirmed among 92.3%, 84.6%, 84.6%, 76.9%, 69.2%, and 15.3% of EIEC isolates, respectively. On the other hand, none of the isolates were positive for the sepA gene. The EIEC isolates were divided into 11 MLVA types. CONCLUSION: Our results showed a high distribution of virulence genes among EIEC isolates in our region. This study showed that MLVA is a promising typing technique for epidemiological studies. MLVA can supply data in the form of codes that can be saved in the database and easily shared among laboratories, research institutes, and even hospitals.

First investigation of the presence of SPATE genes in Shigella species isolated from children with diarrhea infection in Ahvaz, southwest Iran.

Background: SPATE (serine protease autotransporters of enterobacteriaceae) genes are considered as a group of the main virulence factors of Shigella species This study aimed to investigate for the first time the distribution of SPATE genes among Shigella spp. isolated from children with diarrhea infection in Ahvaz, Iran. Methodology: In this study, a total of 74 Shigella isolates were collected between August 2016 and June 2017 from feces of children with diarrhea and identified by biochemical and molecular methods for Shigella species. The frequency distribution of the SPATE genes, including pic, pet, sat, sigA and sepA, was evaluated using PCR. The genetic relationship of all isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR. Results: The most common species of Shigella was S. flexneri, followed by S. sonnei and S. boydii. In total, 95.94% of Shigella isolates had at least one of the SPATE genes. The presence of pic, pet, sat, sigA and sepA genes was confirmed among 35.13%, 27%, 47.29%, 58.1% and 39.18% of Shigella isolates, respectively. Of these SPATE genes, the sat and sigA genes were recognized as the most common autotransporters among S. flexneri and S. sonnei isolates, respectively. Also, either S. flexneri or S. sonnei isolates belonging to a same clone type had similar SPATE genes profile. Conclusion: Our results revealed that the high distribution of SPATE genes among Shigella isolates in our region. Hence, this study highlights a need for epidemiological programs to monitor the distribution of SPATE genes locally for prevention from further dissemination of the Shigella isolates harboring them.

Molecular epidemiology of colistin-resistant Pseudomonas aeruginosa producing NDM-1 from hospitalized patients in Iran.

OBJECTIVES: Resistance to carbapenems is the principal reason for the continuing utilization of colistin as a last resort choice for treating the infections resulted from multidrug carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates. The assessment of antimicrobial resistance pattern, the prevalence of carbapenem-resistance determinants, and molecular epidemiology of colistin-resistant isolates among CRPA strains were the aims of the present research. MATERIALS AND METHODS: The current cross-sectional research was conducted on 269 CRPA isolates collected from various clinical samples from 2013 to 2016. After performing identification tests, disk diffusion as well as MIC methods were used for testing sensitivity to the antibiotics. Modified Hodge Test (MHT) was utilized to produce carbapenemase. PCR technique identified beta-lactamase classes A, B, and D genes. RESULTS: In total, from 269 CRPA, five isolates (1.3%) were resistant to colistin. It was found that blaNDM-1, blaIMP-1, blaVIM-2, and blaOXA-10 genes were present in 40%, 40%, 20%, and 100% of colistin-resistant isolates, respectively. DLST type 25-11 is a significant cluster of colistin-resistant P. aeruginosa isolates. CONCLUSION: The appearance of colistin-resistant isolates in CRPA carrying blaNDM-1 with multiple carbapenem-resistant genes shows the great problem in the treatment of P. aeruginosa infections.

Frequency of quinolone resistance genes among extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli strains isolated from urinary tract infections.

BACKGROUND: As an opportunistic pathogen, Escherichia coli (E. coli) is widely recognized as the main cause of nosocomial infections as well as some disorders especially those associated with urinary tract infections (UTIs). This study, therefore, sets out to determine the extent of antibiotic resistance to quinolones and to measure the frequency of qnr genes (A, B, and S) within extended-spectrum beta-lactamase (ESBL) and non-ESBL-producing strains of E. coli isolated from UTI-diagnosed patients as well as to investigate their antimicrobial susceptibility patterns for some selected antibiotics in southwest Iran. METHODS: Two hundred E. coli strains were isolated from UTI-diagnosed patients, hospitalized in nine different wards of Ahvaz Golestan Hospital between November 2015 and March 2016. The isolates were confirmed through well-practiced phenotypical methods. Moreover, the antimicrobial susceptibility test was successfully performed using a disk diffusion method. ESBL production among the isolates was screened by double disk synergism test (DDST), and the qnr genes were identified using a multiplex PCR. RESULTS: Out of the 200 samples collected, 167 isolates were confirmed to be E. coli strains. Maximum and minimum resistance were reported against nalidixic acid and chloramphenicol with 65.3% and 17.4%, respectively. Most of the isolates were resistant to all three types of quinolones studied in this research. Using multiplex PCR, the qnr genes were found in 100 (59.88%) strains (qnrA = 10, qnrB = 21, qnrS = 41, qnrB-S = 21, qnrB-A = 1, qnrA-S = 3, qnrA-B-S = 3), 58% of which was found among ESBL-producing isolates. CONCLUSIONS: Resistance to quinolones antibiotics was highest among ESBL-producing isolates harboring, especially qnrS among other determinants of the qnr gene. There is a need for sensitive antibiotic stewardship especially in hospitals of Ahvaz, Khuzestan province. Further research is needed to ascertain the gravity of quinolones resistance in Iran and to quickly act against its spread among other nosocomial pathogens.

Evaluation of Antibiotic Susceptibility Pattern and Distribution of coa Genes in Coagulase-negative S. aureus from Ahvaz, Iran.

BACKGROUND: Staphylococcus aureus is one of the important causes of clinical infections that can be more destructive by its antibiotic resistant strains. OBJECTIVE: This study aimed to determine the antibiotic susceptibility pattern and distribution of mecA and coa genes in clinical isolates of S. aureus. METHODS: Two hundred seventy-three specimens suspected to S. aureus were taken from hospitals of Ahvaz, southwest of Iran. Isolates were identified by standard microbiologic tests and confirmed by the molecular method. Antimicrobial susceptibility testing was carried out by disk diffusion method. The presence of mecA and coa genes was determined by PCR method. RESULTS: Of a total of 200 isolates which were tested for coagulase tube test, 143 (71.5%) showed coagulase positive, and 57 (28.5%) showed a coagulase-negative reaction. Antibacterial susceptibility pattern of 200 S. aureus isolates showed the highest and lowest susceptibility rate to linezolid (98%) and ciprofloxacin (42%), respectively. The prevalence of methicillin-resistant S. aureus (MRSA) by detection of mecA gene was estimated as 47.5 % (95/200), of which the rate of MRSA in coagulase positive and negative isolates was 35% (50/143), and 65% (45/57), respectively. Meanwhile, coa gene was detected in 100% of coagulase positive and 28.1% of coagulasenegative isolates. CONCLUSION: The results of this study showed that the number of atypical CNSA in our area is high. Since the coagulase test is an essential test for diagnosis of S. aureus, our findings regarding the emergence of CNSA are a warning about the misdiagnosis and selection of appropriate treatment approach for S. aureus isolates.

Prevalence of class 1 integrons and plasmid-mediated qnr-genes among Enterobacter isolates obtained from hospitalized patients in Ahvaz, Iran.

Quinolones are frequently used classes of antimicrobials in hospitals, crucial for the treatment of infections caused by Gram-negative bacteria. The inappropriate use of quinolones and other antimicrobial agents for the treatment of bacterial infections leads to a significant increase of resistant isolates. The acquisition of antimicrobial resistance may be related to achievement of resistance determinant genes mediated by plasmids, transposons and gene cassettes in integrons. The objective of this cross-sectional study, conducted from December 2015 to July 2016 at two teaching hospitals in Ahvaz, southern Iran, was to screen for the presence of class 1 integrons and quinolone resistance genes in clinical isolates of Enterobacter spp. In all, 152 non-duplicated Enterobacter isolates were collected from clinical specimens and identified as Enterobacter spp. using standard microbiological methods. Antimicrobial susceptibility test was determined using the disc diffusion method according to the CLSI recommendation. Determination of class 1 integrons and PMQR genes was assessed by PCR. Analysis of antibiotic susceptibility tests showed that the highest antibiotic resistance was toward ciprofloxacin (55.3%), while the lowest level was observed against meropenem (34.9%). Moreover, 47.4% (72/152) and 29% (44/152) of isolates were positive for class 1 integron and quinolone resistance genes, respectively. The relative frequencies of antibiotic resistance were significantly higher among class 1 integron-positive isolates. In summary, our results highlight the importance of PMQR genes in the emergence of quinolone-resistant Enterobacter isolates. Moreover, it seems that class 1 integrons have a widespread distribution among Enterobacter isolates and have clinical relevance to multiple-drug-resistant isolates.

Molecular characteristics and antibiotic resistance pattern of Staphylococcus aureus nasal carriage in tertiary care hospitals of Isfahan, Iran.

Carriage of S. aureus in the anterior nares seems to play a significant role in the pathogenesis of infection. This study aimed to determine the molecular characteristics and antibiotic susceptibility pattern of S. aureus isolates obtained from the nasal carriage of health care workers (HCWs). This study was performed during July 2014 to July 2015 at three tertiary care hospitals. Nasal samples were collected from the nasal cavity of HCWs. Standard microbiological methods were used for identification of S. aureus isolates. Antibiotic susceptibility pattern was determined by the disc diffusion method. Determination of SCCmec typing and virulence genes was performed by the PCR method. From the isolates of 340 nasal swab samples of HCWs, 65 S. aureus strains (19%) including 22 (33.8%) MRSA were isolated. The highest sensitivity for MRSA isolates was towards vancomycin and rifampicin, each with 90.9%. Overall, 17% (11/65) and 92.3% (60/65) of S. aureus isolates were positive for pvl and hla genes, respectively. The rates of SCCmec types II, III, IV, V and I among MRSA isolates were 36.4 %, 22.7 %, 22.7 %, 9.1% and 4.5% respectively. The results of the present study indicate that S. aureus nasal carriage with potential virulence ability still remains a significant healthcare problem, especially in hospital environments.

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 MLSB in 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 iMLSB resistance 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, cMLSB was the most frequent phenotype and ermC was the most common gene in iMLSB resistant 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.