Iranian Journal of Microbiology Tehran University of Medical Sciences en-US Iranian Journal of Microbiology 2008-3289 Rapid detection of multidrug resistant tuberculosis in respiratory specimens at a tertiary care centre in south coastal Karnataka using Genotype MTBDR plus assay <p><strong>Background and Objectives:</strong> Despite high prevalence of MDR-TB in India very limited information about MDR-TB and mutation patterns in <em>rpoB, inhA</em> and <em>katG</em> genes among MDR-isolates of <em>Mycobacterium tuberculosis</em> in south coastal Karnataka region is available; thus present study is an attempt to explore the extent of MDR-TB and mutation patterns prevalent among clinical isolates in this region using GenoType MTBDR <em>plus</em> assay.</p> <p><br><strong>Materials and Methods:</strong> A total of 256 sputum samples from Pulmonary TB patients suspected of MDR-TB were tested by GenoType MTBDR <em>plus</em> as per manufacturer’s guidelines for detection of mutations conferring resistance to rifampicin and isoniazid. The results of GenoType MTBDR <em>plus</em> were recorded and analysed using SPSS version 22. For all analyses, a <em>p</em> value &lt;0.05 was considered statistically significant.</p> <p><br><strong>Results:</strong> Fifty (19.53%) isolates were found MDR, 32 (12.50%) isolates were found mono-resistant to isoniazid and 15 (5.86%) isolates were found mono-resistant to rifampicin. Eleven isolates (4.3%) were found NTM. Mutation in codon S531L, S315T1 and C15T were most common in <em>rpoB, katG</em> and <em>inhA</em> genes respectively. Unknown mutations were found in 50.77% (33/65), 3.66% (3/82) and 26.83% (22/82) isolates for <em>rpoB, katG</em> and <em>inhA</em> genes respectively. Hetero-resistance in MDR, rifampicin monoresistant and isoniazid monoresistant isolates was found to be 26% (13/50), 20% (3/15) and 34.37% (11/32) respectively.</p> <p><br><strong>Conclusion:</strong> Mutation in codon S531L, S315T1 and C15T were most common mutations associated with MDR-TB. Further high number of isolates showed mutations in unknown regions and hetero-resistance thus more elaborate studies based on sequencing are desirable in this region.</p> Vishnu Prasad Shenoy Ajay Kumar Kiran Chawla ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 275 280 Evaluation of a modified protocol that decreases the turnaround time of positive blood cultures <p><strong>Background and Objectives:</strong> Neonatal septicemia can be rapidly fatal if not treated promptly. A speedy laboratory diagnosis would improve the outcome. The BacT/ALERT 3D system (bioMerieux, Durham, North Carolina) is currently being used for laboratory diagnosis of blood stream infections. In the present study, a modified protocol was employed in which the broth was subcultured into two nutrient broth tubes and these tubes were used for biochemical tests and antimicrobial susceptibility testing to decrease the turnaround time.</p> <p><br><strong>Materials and Methods:</strong> A prospective study was conducted in the Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad from October 2010 to July 2012 after receiving clearance from the institutional ethics committee. Automated blood cultures of 250 neonates admitted to the Neonatal Intensive Care Unit (NICU), clinically diagnosed to have septicemia, were performed using BacT/ALERT 3D. Bottles flagged positive within 72 hours of loading were processed for identification and antibiotic susceptibility testing using a modified protocol. The results were assessed for time saved in reporting in comparison with standard protocol. Student’s t test was used for statistical analysis.</p> <p><br><strong>Results:</strong> Of the 250 cases studied, 117 cases yielded a positive blood culture giving a yield of 46.8%. The number of cases yielding monomicrobial growth were 73, which were included for further analysis. Of the remaining samples, 133 did not show growth, 11 were polymicrobial while 33 samples were flagged positive after 72 hours. <em>Candida</em> spp. grew in 34 cases, Gram negative bacilli grew in 28 cases and Gram positive cocci grew in 11 cases. In four cases, 66 hours were saved, 60 and 54 hours were saved in 18 cases each, 48 hours were saved in 27 cases, and 24 hours were saved in 6 cases. Methicillin resistant<em> Staphylococcus aureus</em> and <em>Klebsiella pneumoniae</em> were the most common isolates among Gram positive cocci and Gram negative bacilli, respectively, while <em>C. guilliermondii</em> was the most common <em>Candida</em> isolate. All Gram positive isolates were susceptible to vancomycin and linezolid. Most of the Gram negative isolates were susceptible to imipenem.</p> <p><br><strong>Conclusion:</strong> This method can be employed in peripheral laboratory settings where there is no complete automation. Modification in processing blood culture can provide speedy identification and sensitivity report in blood stream infections. Time saved in reporting would play a crucial role in improving morbidity and mortality rates in neonatal septicemia.</p> Renuka Anegundi Raghavendra Dheerendra Kulkarni Satish S Patil Vidyavathi B Chitharagi Ganavalli Subramanya Ajantha ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 281 286 Diagnosis of Clostridium difficile infection by toxigenic culture and PCR assay <p><strong>Background and Objectives:</strong> <em>Clostridium difficile</em> is responsible for 15-25% of nosocomial antibiotic associated diarrhea (AAD) cases and all cases of pseudomembranous colitis. <em>C. difficile</em> has two major virulence factors, toxin A (enterotoxin) and toxin B (cytotoxin). The aim of this study was to determine the frequency of <em>C. difficile</em> strains in patients with diarrhea in Babol' hospitals with toxigenic culture and PCR assay.</p> <p><br><strong>Materials and Methods:</strong> One hundred stool specimens were taken from diarrheal patients in hospitals of the city of Babol. All patients had a history of antibiotic use. The samples were cultured on CCFA medium. In the next stage, toxigenic culture was performed for isolated <em>C. difficile</em> strains. Then, PCR assay was used to identify <em>gdh, tcdA</em> and <em>tcdB</em> genes among isolated <em>C. difficile</em> strains.</p> <p><br><strong>Results:</strong> From the 100 stool samples, eight (8%) samples were positive in <em>C. difficile</em> culture. In toxigenic culture, two (2%) of these strains had cytopathic effects on Vero cells. All eight strains had the <em>gdh</em> gene. This gene is specific for <em>C. difficile</em>. Two strains that had cytopathic effects on toxigenic culture were positive for toxin genes.</p> <p><br><strong>Conclusion:</strong> The frequency of toxigenic strains in different parts of the world is variable, and needs to be continually investigated. In the present study, the PCR method had a good correlation with toxigenic culture. Thus, it can replace the laborious and costly cell culture method.</p> Elnaze Zare Mirzaei Mahdi Rajabnia Farzin Sadeghi Elaheh Ferdosi-Shahandashti Mahmoud Sadeghi-Haddad-Zavareh Soraya Khafri Abolfazl Davoodabadi ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 287 293 Distribution of extended-spectrum β-lactamases (ESBLs) among Salmonella serogroups isolated from pediatric patients <p style="margin: 0cm 0cm 0pt; text-align: justify; line-height: 115%; unicode-bidi: embed; direction: ltr;"><strong>Background and Objectives:</strong> Extended-spectrum β-lactamases (ESBLs) and fluoroquinolones are generally used to treat invasive <em>Salmonella</em> infections, but emergence of antibiotic-resistant strains are increasing worldwide. This study was aimed to investigate the distribution of ESBLs among <em>Salmonella</em> serogroups isolated from pediatric patients in Tehran, Iran.</p> <p style="margin: 0cm 0cm 0pt; text-align: justify; line-height: 115%; unicode-bidi: embed; direction: ltr;"><br><strong>Materials and Methods:</strong> The study included all <em>Salmonella</em> isolates recovered from pediatric patients admitted to Children’s Medical Center, Tehran, Iran during 2015-2016. Bacterial isolation and identification were performed by standard biochemical and agglutination tests. Antimicrobial susceptibility testing was done according to the Clinical and Laboratory Standards Institute (CLSI). Polymerase chain reaction was used to identify the genetic determinants responsible for ESBL phenotypes.</p> <p style="margin: 0cm 0cm 0pt; text-align: justify; line-height: 115%; unicode-bidi: embed; direction: ltr;"><br><strong>Results:</strong> A total of 138 <em>S. enterica</em> serovars were isolated from stool specimens, including serogroup A (1), serogroup B (18), serogroup C (41) and serogroup D (78). Forty isolates out of 138 <em>Salmonella</em> strains had shown ESBL-positive phenotype. All ESBL-positive isolates showed multiple resistant phenotype. Resistance to more than 3 antimicrobial agents was observed among ESBL-positive strains. The frequency of <em>Salmonella</em> strains carrying the <em>bla</em><sub>CTX</sub>, <em>bla</em><sub>TEM</sub> and <em>bla</em><sub>SHV</sub> genes was 17 (12.3%), 40 (29.9%) and 4 (2.89%) respectively.</p> <p style="margin: 0cm 0cm 0pt; text-align: justify; line-height: 115%; unicode-bidi: embed; direction: ltr;"><br><strong>Conclusion:</strong> The high rates of ESBLs positive-<em>Salmonella</em> strains recovered from pediatric patients is alarming and indicates a necessity to substitute the cephalosporins with a proper alternative.</p> Reza Ranjbar Mehrdad Ardashiri Sakineh Samadi Davoud Afshar ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 294 299 Prevalence of class 1, 2 and 3 integrons among multidrug-resistant Pseudomonas aeruginosa in Yazd, Iran <p><strong>Background and Objectives:</strong> Antibiotic resistance in <em>Pseudomonas aeruginosa</em> is an increasing health problem. Integrons are associated with a variety of gene cassettes, which confer resistance to multiple classes of antibiotics. This study aimed at screening the presence of class 1, 2 and 3 integrons in <em>P. aeruginosa</em> in Yazd, Iran.</p> <p><br><strong>Materials and Methods:</strong> This study was carried out on <em>P. aeruginosa</em> strains from March 2016 to March 2017. Clinical specimens were initially identified by the standard biochemical methods and their resistance patterns to antibiotics were studied using the disc diffusion method. PCR was carried out for the detection of class 1, 2 and 3 integrons using <em>intI1, intI2</em> and <em>intI3</em> gene primers, respectively.</p> <p><br><strong>Results:</strong> Antimicrobial susceptibility test showed that 75% of isolates were detected as multi-drug resistant (MDR), and lowest resistance was observed in ciprofloxacin (48.6%) and most resistance was in gentamicin (63.2%). Moreover, PCR results showed that 22 (15.3%) and 119 (82.6%) of <em>P. aeruginosa</em> isolates carried <em>intI2</em> and <em>intI1</em> genes, but <em>intI3</em> gene was not found.</p> <p><br><strong>Conclusion:</strong> Since it is customary to observe Class I integrons in <em>P. aeruginosa</em> isolated from clinical samples, they are often responsible for antibiotic resistance gene transfer, which calls for evaluation of integrons as contributing factors in antibiotic resistance.</p> Mohadeseh Zarei-Yazdeli Gilda Eslami Hengameh Zandi Masoumeh Kiani Kazem Barzegar Hanieh Alipanah Seyed Morteza Mousavi Marzieh Shukohifar ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 300 306 Prevalence, antimicrobial susceptibility, serotyping and virulence genes screening of Listeria monocytogenes strains at a tertiary care hospital in Tehran, Iran <p><strong>Background and Objectives:</strong> <em>Listeria monocytogenes</em> is the etiological agent of listeriosis, a highly fatal infection which causes miscarriage or stillbirth in pregnant women. The objective of this study was to detect the prevalence, serotypes, antimicrobial susceptibility and virulence factors of <em>L. monocytogenes</em> isolated from pregnant women with vaginitis at a tertiary care hospital in Tehran, Iran.</p> <p><br><strong>Materials and Methods:</strong> During September 2015 to February 2017, a total of 400 vaginal swabs were collected from pregnant women. The presumptive isolates were characterized biochemically. All<em> L. monocytogenes</em> isolates were further analyzed by serotyping and antimicrobial susceptibility tests. All positive samples for <em>L. monocytogenes</em> were analyzed for presence of virulence genes (<em>hlyA, actA, inlA, inlC, inlJ</em> and <em>prfA</em>).</p> <p><br><strong>Results:</strong> Twenty-two (5.5%) of the samples were found positive for presence of <em>L. monocytogenes</em>. Most isolates are resistant to trimethoprim/sulfamethoxazole (81.82%) and chloramphenicol (54.55%). The majority of tested isolates (59.10%) belonged to serotype 4b, followed by 1/2a (22.73%), 1/2b (13.63%), and 3c (4.54%). The <em>hlyA, actA</em> and <em>inlA</em> were detected in all of the 22 <em>L. monocytogenes</em> isolates, but two, three and five isolates were found to lack <em>inlC, inlJ</em> and <em>prfA</em>, respectively. Only one isolate lacked three<em> inlC, inlJ</em> and <em>prfA</em> genes, and two isolates simultaneously lacked both<em> inlJ</em> and <em>prfA</em> genes.</p> <p><br><strong>Conclusion:</strong> Evaluation of virulence factors and antimicrobial susceptibility can be highly helpful to develop effective treatment strategies against <em>L. monocytogenes</em> infections. This study is noteworthy in that it documents prevalence, virulence characteristics, and antimicrobial resistance of <em>L. monocytogenes</em>.</p> Siamak Heidarzadeh Mohammad Mehdi Soltan Dallal Mohammad Reza Pourmand Reihaneh Pirjani Abbas Rahimi Foroushani Matina Noori Aida Babazadeh Naseri ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 307 313 Diversity of lactic acid bacterial in inasua fermentation <p><strong>Background and Objectives</strong>: Inasua is one of the traditional fermented fish products in Maluku, Indonesia. There are twotypes of inasua, i.e. with and without sap. The research aimed to study the succession of lactic acid bacteria (LAB) duringfermentation and microbial composition in inasua.<br><strong>Materials and Methods</strong>: The sample of inasua was taken from two traditional producers in Layeni village, Ceram Island.The diversity of lactic acid bacteria was analyzed based on the 16S rRNA gene sequence.<br><strong>Results</strong>: The succession of lactic acid bacteria was strongly influenced by the physicochemical characteristics during fermentation.Lactobacillus plantarum was found dominant in both inasuas fermentation processes. At end of fermentation,L. plantarum was still found dominant in inasua with sap while inasua without sap was dominated by Leuconostoc mesenteroides.In addition, Lactobacillus paracasei (LAB) was found only in inasua with sap. The result of Denaturing Gradient Gel Electrophoresis (DGGE) revealed that Lactobacillus was the dominant bacteria in inasua with sap while Staphylococcuswas dominant in inasua without sap.<br><strong>Conclusion</strong>: Inasua with sap was found with higher bacterial diversity index and lower evenness and dominance indices, aswell as more complex LAB succession pattern during fermentation and bacterial composition, as opposed to inasua withoutsap.</p> Ferymon Mahulette Nisa Rachmania Mubarik Antonius Suwanto Widanarni Widanarni ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 314 323 Studies on agarolytic bacterial isolates from agricultural and industrial soil <p><strong>Background and Objectives:</strong> Soil is rich in microbes which can be used for a variety of purposes starting from decomposition to antibiotic production. Agar-agar, extracted from the marine environment, is an important polysaccharide that has multiple uses after degradation by microbes. The aim of this study was to isolate bacteria that produced agarase enzyme, from a variety of soil sources and study their morphological and biochemical characterization. The enzyme activity of the isolates was also studied at 3 different pH, temperature and agar concentration.</p> <p><br><strong>Materials and Methods:</strong> Agarolytic isolates, were identified from industrial and agar- enriched agriculture soil by serial dilution method using MSA media that contains agar as the only source of carbon. Qualitative analysis of the isolates was determined by iodine assay while for quantitative analysis of enzyme activity, at standard and variable conditions, DNSA method was used. Genus of SELA 4 was identified.</p> <p><br><strong>Results:</strong> 4 isolates were obtained from industrial soil and 6 were obtained from agriculture soil enriched with laboratory agar. Isolate ‘SELA 4’ showed maximum relative activity (OD 0.92) followed by ‘CCIL 2 (OD 0.91) under standard culture conditions. Isolate ‘SELA 1' showed maximum activity between 37°C- 40°C, pH 5-7 with 1.5% agar concentration. “CGIPL 1” showed maximum activity at pH 9 while “SELA 2” and “SELA 4” showed maximum activity at pH 5. SELA 4 belonged to genus <em>Microbacterium</em> (Accession no. MG203882.1).</p> <p><br><strong>Conclusion:</strong> The results showed that agar degrading bacteria can also be isolated from soil sources other than the usual marine sources and can be used for the industrial production of agarase enzyme.</p> Shomini Parashar Narendra Kumar ##submission.copyrightStatement## 2018-11-23 2018-11-23 10 5 324 333 Comparison of antibacterial effects of a carrier produced in microemulsion system from aqueous extract of Aloe vera with selected antibiotics on Enterobacteriacea <p><strong>Background and Objectives:</strong> Antibiotics resistance has recently increased. The aim of this study was the evaluation of antibacterial efficacy of Aloe vera carrier produced in microemulsion system in comparison with ordinary antibiotics against some Enterobacteriacea.</p> <p><br><strong>Materials and Methods:</strong> The aquatic extract of Aleo vera was produced by the Soxhlet method and a nonocarrier in the microemulsion system was prepared by two emulsifiers. The clinical isolates of <em>Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Shigella dysenteriae, Salmonella Typhimurium, Salmonella Paratyphi, Serratia marcescens, Proteus mirabilis, Enterobacter aerogenes, Citrobacter freundii</em> and <em>Morganella morganii</em> were obtained from patients and were identified by microbiological methods. Diffusion disk was used for evaluation of antibacterial properties in comparison with selected ordinary antibiotics. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for tested materials were determined using MTT in the Micro Broth dilution method.</p> <p><br><strong>Results:</strong> The results proved that effect of carrier on studied isolates is dependent on concentration level. The inhibitory effect of carrier in concentration of 15 µg/ml by 18 mm zone of inhibition for <em>Klebsiella pneumoniae</em> was comparable to Ceftazidime and Cefalothin. The lowest MIC and MBC determined by the Microbroth dilution method with MTT belonged to <em>Klebsiella pneumoniae</em> as 0.1 and 3 µg/ml and higher concentrations belonged to <em>Enterobacter aerogenes</em> at 7 and 15 µg/ml. The greatest effect of carrier of Aleo vera aquatic extract was observed for <em>Klebsiella pneumoniae</em> and the lowest effect belonged to <em>Enterobacter aerogenes, Citrobacter freundii</em> and <em>Morganella morganii</em>.</p> <p><br><strong>Conclusion:</strong> It was concluded that the carrier of Aloe vera produced in microemulsion system was most effective and had equal effects in comparison with ordinary antibiotics against Enterobacteriacea.</p> Ghasem Habibi Mohammad Arjomandzadegan Maryam Tayeboon Farshideh Didgar Hossein Sarmadian Maryam Sadrnia Farid Mirhosseini Somayeh Geravand Mahboobeh Abdoli ##submission.copyrightStatement## 2018-11-24 2018-11-24 10 5 334 341 Prevalence of cytomegalovirus infection in patients with ulcerative colitis: a prospective cross-sectional study in Tehran, Iran <p><strong>Background and Objectives:</strong> Cytomegalovirus (CMV) infection has been reported in ulcerative colitis (UC), but limited data are available on its prevalence in Iran. The aim of this study was to evaluate the prevalence of CMV infection in patients with UC.</p> <p><br><strong>Materials and Methods:</strong> A prospective, cross-sectional study was conducted in 86 consecutive patients with UC. Prevalence of CMV infection was determined by rectal biopsies for hematoxylin and eosin staining and PCR. CMV-positive specimens was measured for CMV loads by real-time PCR assay.</p> <p><br><strong>Results:</strong> In six out of 86 (7%) patients with UC, CMV was diagnosed. These patients had detectable CMV DNA in their biopsies as indicated by PCR. In all CMV-positive patients, viral load was more than 250 copy/mg. Histochemical staining did not show any CMV inclusion bodies. No significant demographic and clinical differences existed between patients with and without a CMV infection.</p> <p><br><strong>Conclusion:</strong> UC and its treatment may put patients at risk of CMV infection. Real-time PCR test for the detection of CMV in UC patients may enable diagnosis of CMV infection with a high sensitivity and allow effective treatment to be administered in these patients. The impact of antiviral therapy on the clinical outcome of the UC patients with CMV remains to be elucidated.</p> Davood Yadegarynia Shabnam Tehrani Manijeh Roohi Latif Gachkar Seyed Alireza Nadji Mohammad Hashemi Saadat Molanaei ##submission.copyrightStatement## 2018-11-24 2018-11-24 10 5 342 347