Microbial Profile and Antibiotic Susceptibility Pattern in Diabetic Patients with Mild, Moderate, and Severe Foot Infections in Tehran.

It is estimated that 10-25% of diabetic patients will encounter diabetic foot ulcers (DFU) during their lifetime. This study evaluated the microbiology of DFUs and determined the antibiotic resistance pattern of bacterial isolates based on the severity of wounds and infections in different grades of ulcer. The specimens were collected from115 diabetic foot infections (DFI) deep tissue by needle aspiration and biopsy. The aerobic and anaerobic cultures and antimicrobial susceptibility testing were carried out. The presence of resistance genes including metallo-beta-lactamases (MBL), extended-spectrum ß-lactamase (ESBL), ermA, ermC, and mecA was also determined. A total of 222 microorganisms were isolated. The prevalence of poly-microbial infections was 69.6%. Bacterial isolates comprised 64.2% Gram-positive bacteria (GPB), 33.5% Gram-negative bacteria (GNB), and five isolates of anaerobic bacteria were also detected. The most prevalent GPB and GNB were Staphylococcus spp. (52.2%) and Escherichia coli (33.3%), respectively. The prevalence of poly-microbial infections and GNB was positively associated with increased grades of Wagner and IDSA classifications. Among Staphylococcus aureus isolates, resistance to clindamycin (73.5%), ciprofloxacin (70.6%), and erythromycin (70.6%) were noticeable. GNB was also highly resistant to cephalosporins and ciprofloxacin. ESBL genes were detected in approximately 40% of isolates of Enterobacteriaceae. The prevalence of ermA, ermC, and mecA genes in S. aureus isolates were 8.8%, 32.3%, and 14.7%, respectively. In conclusion, our data suggest that GPBs are the most common isolates from DFIs. Furthermore, with the development of wounds and infection, the prevalence of GNB in DFIs are increased.

Identification of Non-Tuberculosis Mycobacteria by Line Probe Assay and Determination of Drug Resistance Patterns of Isolates in Iranian Patients.

The potentially pathogenic Non-Tuberculosis Mycobacteria (NTM) are emerging nowadays which result in pulmonary and non-pulmonary infections in human. This group of bacteria consists of at least 200 different species. While the pulmonary disease is the most common form of NTM infections, NTM can cause diffused infections as well as extrapulmonary infections in every organ, such as bone marrow, skin, eye, and brain. The NTM cause tuberculosis-like infections, therefore, correct identification of these Mycobacteria is necessary to avoid faulty treatment. Different species of NTM isolates were identified from clinical specimens using phenotypic methods and Line Probe Assay. Minimum Inhibitory Concentration for selected antibiotics was obtained by the broth micro-dilution method. Totally, 42 NTM isolates were identified in this study. Moreover, the frequency of NTM between all positive mycobacterium cultures was estimated at 12%. The most common Rapidly Growing Mycobacteria included Mycolicibacterium fortuitum (30.9%), Mycobacterium abscessus (7.1%), and Mycobacterium chelonae (2.3%), whereas Mycobacterium simiae (40.4%), Mycobacterium kansasii (16.6%), and Mycobacterium avium complex (2.3%) were the most recurring among the Slowly Growing Mycobacteria. Amikacin, clarithromycin, and ciprofloxacin were the most effective antibiotics against isolated NTM. The NTM isolates are frequently being separated from Iranian patients, and are mostly resistant to the wide spectrum of antibiotics. Correct identification and determination of antibiotic susceptibility can be helpful in the healing process of the patients who suffer from non-tuberculosis mycobacterial infections.

Mycobacterium simiae pulmonary disease in Iran: systematic review and meta-analysis.

Mycobacterium simiae is one of the most common nontuberculous mycobacteria (NTM) microorganisms causing lung disease in many countries in the world. A reliable estimate of the extent of M. simiae pulmonary disease has not been well investigated in Iran. We systematically searched multiple databases to identify relative studies. Studies were excluded if they did not use the American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) diagnostic criteria for NTM diseases. Data were extracted independently and in duplicate. We assessed pooled estimate by using a random model effect, and sources of heterogeneity were assessed by using Cochran's Q and the I 2 statistic. The potential for publication bias was explored by using Begg's and Egger's tests. All analyses were conducted with Stata 14.0 (StataCorp, College Station, TX, USA). Of 172 articles identified, seven met the inclusion criteria. Of 355 patients who were culture positive for NTM, 82 had M. simiae pulmonary disease according to the ATS/IDSA diagnostic criteria. The pooled frequency of M. simiae pulmonary disease among patients with NTM was 25.0% (95% confidence interval, 16.8-33.2). No evidence of publication bias was observed among the included studies (p >0.05 for Begg's and Egger's tests). Clinical isolates of M. simiae are increasingly being recognized as a cause of pulmonary disease in Iran and need further attention by health authorities.

The reliability of rifampicin resistance as a proxy for multidrug-resistant tuberculosis: a systematic review of studies from Iran.

In Iran, patients showing rifampicin (RIF) resistance detected by the Xpert® MTB/RIF assay are considered as candidates for multidrug-resistant tuberculosis (MDR-TB) treatment. Despite the fact that RIF resistance has been used as a proxy for MDR-TB, little is known about the proportion of isoniazid (INH) resistance patterns in RIF-resistant TB. We systematically searched MEDLINE, Embase, and other databases up to March 2017 for studies addressing the proportion of INH resistance patterns in RIF-resistant TB in Iran. The data were pooled using a random effects model. Heterogeneity was assessed using Cochran's Q and I2 statistics. A total of 11 articles met the eligibility criteria. Data analysis demonstrated that 33.3% of RIF-resistant isolates from new TB cases and 14.8% of RIF-resistant isolates from previously treated cases did not display resistance to INH. The relatively high proportion of INH susceptibility among isolates with RIF resistance indicated that RIF resistance may no longer predict MDR-TB in Iran. Therefore, the detection of RIF resistance by the Xpert MTB/RIF assay will require complementary detection of INH resistance by other drug susceptibility testing (DST) methods in order to establish the diagnosis of MDR-TB.

High rates of nontuberculous mycobacteria isolation from patients with presumptive tuberculosis in Iran.

Nontuberculous mycobacteria (NTM) can cause disease which can be indistinguishable from tuberculosis (TB), posing a diagnostic and therapeutic challenge, particularly in low- and middle-income settings. We aimed to investigate the mycobacterial agents associated with presumptive clinical pulmonary TB in Iran. A total of 410 mycobacterial isolates, obtained between March 2014 and January 2016, from 7600 clinical samples taken from consecutive cases of presumptive diagnosis of TB were identified. Phenotypic and molecular tests were used to identify the isolated organisms to the species level. Single-locus and multilocus sequence analysis based on 16S rRNA, rpoB, hsp65 and ITS locus were used to confirm the results. Of 410 consecutive strains isolated from suspected TB subjects, 62 isolates (15.1%) were identified as NTM. Patients with positive NTM cultures met American Thoracic Society diagnostic criteria for NTM disease. Mycobacterium simiae was the most frequently encountered (38.7%), followed by Mycobacterium fortuitum (19.3%), M. kansasii (17.7%) and M. avium complex (8.0%). Isolation of NTM, including M. simiae, from suspected TB cases is a serious public health problem and merits further attention by health authorities, physicians and microbiologists.

Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis.

BACKGROUND: Acinetobacter baumannii is usually multi-drug resistant (MDR), including third generation cephalosporins, amino glycosides and fluoroquinolone. Resistance to these antibiotics is mediated by multiple factors such as: lactamases, efflux pumps and other mechanisms of resistance. Pulsed-field gel electrophoresis (PFGE) was then used to investigate the genetic relationships among the MDR isolates. AIM: The aim of this study was to determine MDR isolates and the existence of OXAs genes among MDR isolates of A. baumannii collected from Kermanshah hospitals in west of Iran. MATERIALS AND METHODS: Forty-two MDR A. baumannii were collected from patients at Kermanshah hospitals. The isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics by disk diffusion method was determined. Polymerase chain reaction (PCR) was performed for detection of blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like betalactamase genes in isolates and clonal relatedness was done by PFGE (with the restriction enzyme ApaI) and patterns analyzed by Bionumeric software. RESULTS: This study showed high resistant to ciprofloxacin, piperacillin, ceftazidime and also resistant to other anti-microbial agents and more spread blaOXA-23-like gene (93%) in MDR isolate. The PFGE method obtained six clones: A (10), B (9), C (5), D (4), E (11) and F (3) that clone E was outbreak and dominant in different wards of hospitals studied. CONCLUSION: An isolate from the emergency ward of these hospitals had indistinguishable isolates PFGE profile and similar resistance profile to isolates from intensive care unit (ICU), suggesting likely transmission from ICU to emergency via patient or hospital staff contact.

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis.

BACKGROUND: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. MATERIALS AND METHODS: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. RESULTS: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. CONCLUSION: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.

Mycobacterium arupense infection in HIV-infected patients from Iran.

Here we report two cases of infection caused by Mycobacterium arupense in HIV-infected patients who had received Mycobacterium avium complex medication after primary treatment with antituberculous drugs. The causative agents were isolated from the respiratory and blood specimens of the patients. The identification was based on conventional and molecular tests. Our study provides further evidence on the role of this microorganism in clinical cases.

Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae.

AIMS: To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae. METHODS AND RESULTS: Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 µmol 1(-1) primer, 4 mmol 1(-1) MgCl(2), 0·4 mmol 1(-1) dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 µl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson's diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson's similarity coefficient ≈ 100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates. CONCLUSIONS: Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae.

Antimicrobial susceptibility patterns and distribution of blaOXA genes among Acinetobacter spp. Isolated from patients at Tehran hospitals.

Multiple drug-resistant strains of Acinetobacter have created therapeutic problems worldwide. This study was conducted to determine the antimicrobial susceptibility patterns and prevalence of bla(OXA-type) carbapenemases among isolates of Acinetobacter spp. obtained from Iranian patients. Here, 128 Acinetobacter isolates were identified at the species level, and their susceptibilities to different antibiotics were determined using disk agar diffusion testing. Isolates were then subjected to multiplex-PCR targeting bla(OXA) genes. More than 50% of the isolates showed multidrug resistance to different antibiotics. The rates of susceptibility to imipenem, meropenem, piperacillin-tazobactam, and amikacin were 50.7, 50, 42.1, and 38.2%, respectively. The MICs of carbapenems for the resistant isolates ranged from 64 to > or = 256 microg/ml. All strains of Acinetobacter baumannii possessed a bla(OXA-51-like) gene. The co-existence of bla(OXA-51-like)/bla(OXA-23-like) and bla(OXA-51-like)/bla(OXA-24-like) was detected in 25% (n=32) and 17.9% (n=23) of the isolates, respectively. Over 70% of carbapenem-resistant strains contained at least two genes encoding OXA-type carbapenemase. Resistance to carbapenems in the population of Acinetobacter strains in Iran is high, with the majority of isolates showing multidrug resistance. A wide diversity of OXA genes exists among the strains of A. baumannii in Iran. Detection of bla(OXA-51-like) can be used as a simple and reliable method to differentiate A. baumannii strains from other species.

Pulsed field gel electrophoresis & plasmid profile of Pseudomonas aeruginosa at two hospitals in Tehran, Iran.

BACKGROUND & OBJECTIVE: Pseudomonas aeruginosa is a common cause of nosocomial infections and exhibits innate resistance to a wide range of antibiotics. This study was undertaken to determine the resistance patterns of P. aeruginosa isolates recovered from patients at two hospitals in Tehran, to investigate the presence of plasmids and to genetically characterize them by pulsed field gel electrophoresis (PFGE). METHODS: The susceptibility of 104 isolates of P. aeruginosa to 13 different antibiotics was determined by agar disk diffusion method. The alkaline lysis method was used for plasmid extraction. PFGE technique was optimized for DNA fingerprinting of isolates. RESULTS: The isolates showed resistance to 13 different antibiotics ceftizoxime (99%), lomefloxacin (94.3%), ceftazidime (59.6%), ticarcillin (50%), ceftriaxone (44.3%), cefoperazone (37.5%), tobramycin (34.6%), piperacillin and gentamicin (33.7%), carbenicillin (25%), amikacin (22%), ciprofloxacin (15.4%) and imipenem (2.9%). Plasmids were detected in 31 isolates (29.8%) that produced 15 different patterns. In total, 84 DNA banding patterns were detected by PFGE. The dominant PFGE type, Pattern A with 14 isolates was found at both hospitals. The remaining isolates were grouped in B, C, D and PF1-PF80. The majority of isolates with the identical plasmid profiles and resistance patterns produced closely related DNA fingerprints by PFGE. INTERPRETATION & CONCLUSION: Isolates in pattern A were distributed widely at both hospitals and the environment. Absence of plasmids in majority of isolates indicated low typeability and discriminatory power of this technique.

Serovar determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (IRAN).

The serovars and drug susceptibility patterns of 265 isolates of Pseudomonas aeruginosa cultured from burn patients during 2001-2002 at Motahari and Tohid Hospital were determined. Distribution of serovars was different at two hospitals. Most of the isolates at Tohid Hospital belonged to serovar 0:1, but 21 and 13% of them were untypeable or polyagglutinable, respectively. Serovar 0:11 was the most prevalent serovar at Motahari Hospital. All the strains were multi resistant to tetracyclin, carbenicillin, amikacin, ceftazidime, sulfamethoxazol, ciprofloxacin, tobramycin, kanamycin, cefotaxime and gentamicin. Further analysis of the strains by plasmid profiling demonstrated that 95% of the isolates carried two megaplasmids. However, there was not any correlation between the serotyping and presence of plasmids. Changes in the drug susceptibility patterns and beta-lactamase production of some cured derivatives were observed after the strains lost their plasmids. The emergence of multi-drug resistant strains of P. aeruginosa is a serious concern in burn patients who are hospitalized in Tehran.

Multidrug-resistant strains of Mycobacterium tuberculosis isolated from patients in Tehran belong to a genetically distinct cluster.

Restriction fragment length polymorphism (RFLP) analysis was used to study the molecular epidemiology of tuberculosis (TB) in certain areas of Tehran. 120 isolates of Mycobacterium tuberculosis, including drug-resistant strains (n = 23), were analysed using polymorphic GC-rich sequence (PGRS) and IS6110 probes. There was considerable diversity among the strains cultured from patients from certain areas. The results of RFLP showed that multidrug resistant (MDR) isolates of M. tuberculosis in Tehran belong to a group of strains with low copies of IS6110 and PGRS. The degree of clustering was higher for the drug-resistant strains than for the susceptible ones (65% vs 20%). Based on the demographic data and results of RFLP, it appears that recent transmissions of TB from old patients have occurred in Tehran. However, drug-resistant TB in the city is mainly caused by strains that look different from those cultured from such patients. The majority of MDR isolates (85%) in this study contained a low copy number of IS6110 and PGRS in RFLP, and were mostly recovered from immigrants and refugees.

Genetic differentiation of Australian isolates of Mycobacterium tuberculosis by pulsed-field gel electrophoresis.

As part of an epidemiological study of tuberculosis in Australia, 84 isolates of Mycobacterium tuberculosis from patients were analysed by pulsed-field gel electrophoresis (PFGE). The isolates were genetically heterogeneous, with 66 different DNA banding patterns obtained following digestion of genomic DNA with Dra1 and 53 patterns with Xba1. When the results were compared with those previously obtained in restriction fragment length polymorphism analysis (RFLP), in 87% of cases the results with Dra1 were consistent with those obtained with insertion sequence IS6110 as a probe in RFLP. However, PFGE was able to differentiate four of eight isolates which were identical with IS6110 typing. The high polymorphism amongst strains and the high average age of the patients (51 years) suggested that most organisms were cultured from patients who had reactivation of existing infections. Isolates with identical DNA patterns were found in different states of Australia, but no one strain predominated in any area. This suggests that tuberculosis has been introduced into Australia from various sources.

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis.

OBJECTIVES: To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis. DESIGN: Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates. PROCEDURE: DNAs from 35 Australian isolates of M paratuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared. RESULTS: The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales. CONCLUSION: Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis, and could be used to study the transmission of strains in Australia.

Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis.

Initially, multilocus enzyme electrophoresis was used to examine genetic relationships among 63 isolates of Mycobacterium bovis and 13 other members of the M. tuberculosis complex. The isolates were divided into five electrophoretic types, with a mean genetic diversity of 0.1. The strains were genetically homogenous, indicating that members of the complex were closely related. This supported the suggestion that they should be considered as subspecies of a single species. Pulsed-field gel electrophoresis (PFGE) was then used to differentiate these isolates, as well as 59 additional isolates of M. bovis from different parts of the world. PFGE differentiated these strains into 63 patterns (53 patterns for M. bovis). Isolates of M. bovis from Western Australia (n = 46) were more homogenous than isolates from other regions. Eight strains were identified in that state, and one predominantly bovine strain was isolated from two human beings and a feral pig. Although M. bovis isolates from different parts of the world had distinct DNA patterns, some were very similar. PFGE is a highly discriminatory technique for epidemiological studies of bovine tuberculosis. For example, it allowed differentiation between isolates of M. bovis cultured from animals in separate outbreaks of tuberculosis, it suggested the transmission of infection between certain properties, and it demonstrated the existence of multiple infections with different strains at certain farms.

Genetic characterization of Mycobacterium avium isolates recovered from humans and animals in Australia.

Genetic relationships amongst 115 mainly Australian isolates of Mycobacterium avium were assessed using multilocus enzyme electrophoresis (MEE). The isolates were divided into 58 electrophoretic types (ETs), with a mean genetic diversity of 0.29. Isolates from humans were closely related to but distinct from those cultured from birds, whilst some porcine isolates belonged to the same ETs as certain human isolates. Pulsed field gel electrophoresis (PFGE) was used to differentiate related isolates, and those from birds and some from other animals, including pigs, were distinguished from the human isolates. The results of MEE and PFGE suggested that certain strains of M. avium may be transmitted between birds and pigs, but there was no clear evidence of transmission to humans. The serovar of the M. avium isolates was not obviously related to their ET assignment or their PFGE type.