Jundishapur Journal of Microbiology
Volume:8 Issue: 10, Oct 2015

Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients. The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients. This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing. In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%). The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing nosocomial infections..

Pseudomonas aeruginosa possesses a variety of virulence factors and infections caused by multidrug-resistant P. aeruginosa (MDRPA) in burn patients are a public health problem. The aim of this study was to determine the antibiotic resistance pattern, the biofilm formation, the prevalence of MDRPA and two virulence genes (nan1 and exoA) among P. aeruginosa isolated from burn patients.Patients and A total of 144 isolates of P. aeruginosa were collected from burn patient at the Burn Centre of Tehran, Iran, between March 2013 and July 2013. Antibiotic susceptibility test was performed via agar disk diffusion method. The ability of producing biofilm was examined by crystal violet microtiter plate assay and the prevalence of the exoA and nan1 genes among the isolates was determined by polymerase chain reaction (PCR). A high rate of resistance was seen against ciprofloxacin (93.7%), aztreonam (86.8%), piperacillin (85.4%), ceftazidime (82.6%), amikacin (82%) and imipenem (79.2%). In total, 93.1% of the isolates were characterized as MDRPA. Biofilm formation was seen in 92.4% of the isolates. The prevalence of the exoA and nan1 genes were 75% and 11.8% among the isolates, respectively. The high rate of MDRPA and its ability to produce biofilm is an alarm for public health. The statistical analysis showed that biofilm production in the MDRPA isolates was significantly higher than that in the non–MDRPA isolates (P < 0.001).

Leptospirosis is recognized as a re-emerging infectious disease; therefore, understanding the epidemiology of the disease is vital for designing intervention programs and diminishing its transmission. Recently, Multilocus variable number tandem repeat analysis (MLVA) is used for segregating and identifying Leptospira serovars. The method has potential application in investigating the molecular epidemiology of Leptospira. The propose of this study was genomic identification of pathogenic Leptospires in Iran by MLVA. Leptospira serovars were obtained from National Reference Laboratory of Leptospira at Razi Vaccine and Serum Research Institute, Karaj, Iran. Serovars were cultured into the liquid EMJH medium and incubated at 28˚C for 7 days. DNA of serovars was extracted using the phenol-chloroform method. PCR was performed with 5 selected variable number tandem repeat analysis (VNTR) loci. The amplified products were analyzed by agarose gel electrophoresis. The size of the amplified products was estimated by 100 bp ladder and sequencing analysis. The saprophytic serovar showed no amplified fragments. PCR products in all pathogenic serovars were observed. The 12 reference serovars used for the development of technique displayed distinct patterns. Results showed that MLVA technique with its range of polymorphism is a good marker for identification of pathogenic serovars. Some VNTR loci are more powerful than the other ones with regard to differentiation. Serovars from the same geographical area have more genetic similarity than same serovars from different places. MLVA is a suitable technique for epidemiological survey.

Staphylococcus aureus is a significant pathogen that can colonize the nares of different animals, causing a wide range of infections in various hosts. We intended to determine the prevalence of S. aureus in the nasal cavity of healthy ruminants and also to investigate the presence of antibiotic resistance genes. In the present study, healthy cattle (n = 79), sheep (n = 78) and goats (n = 44) were screened for nasal carriage of S. aureus by the Polymerase Chain Reaction (PCR). Staphylococcus aureus isolates were further assessed for the presence of blaZ (encoding penicillin resistance), mecA (encoding methicillin resistance), tetK and tetM (encoding tetracycline resistance), and ermA and ermC (encoding macrolide-lincosamide-streptogramin B resistance) genes. The proportion of S. aureus-positive nasal swabs from cattle, sheep and goats were four (5.06%), 11 (14.1%) and 11 isolates (25%), respectively. The blaZ gene was detected in 20 out of 26 S. aureus isolates (76.9%), including four cattle (100%), nine sheep (81.8%) and seven goats (63.6%). Two of the four cattle isolates possessing the blaZ gene also had the tetK gene. Of the nine sheep isolates harboring the blaZ gene, one possessed the mecA and tetK genes together. Of the seven goat isolates with blaZ gene, one harbored the tetM gene. None of the S. aureus isolates were positive for the ermA and ermC genes. In contrast to cattle, S. aureus is frequently present in the nose of sheep and goats, which may represent the primary reservoir of S. aureus in small ruminant flocks. This study also showed that nasal isolates of S. aureus from healthy ruminants might be a potential reservoir of antimicrobial-resistance.

Cervical cancer is regarded as the second highest cause of cancer deaths in Nigeria, with an overall prevalence similar to most developing countries. Screening for cervical cancer is primarily performed using papanicolaou (PAP) staining procedure, in Nigeria. This study aimed to use human papillomavirus (HPV) DNA typing, as a means of ascertaining the presence of high risk HPV in cytology samples, which are positive for the presence of cervical intraepithelial neoplasia (CIN), using the PAP screening procedure.Patients and Amplification of DNA was done using polymerase chain reaction. Gene sequencing was carried out to determine the presence of high risk HPV from cervical smears that were positive for abnormal cytology, from a cross-sectional study involving women between the ages of 16 - 65 years, screened for CIN and cervical cancer, in Lokoja, Nigeria. Result showed a 100% presence of high risk HPV in all the samples with abnormal cytology. The HPV genotype 35 accounted for the highest percentage of the HPVs cases, with a 40% incidence. The HPV genotype 31 accounted for 30% of samples, while HPV genotype 16 and 18 accounted for 20% and 10% of samples, respectively. The high prevalence of HPV in abnormal cytology underlines to the fact that the presence of HPV is a critical factor in the development of cervical cancer. The use of HPV DNA techniques could actually become an effective and fast means of ascertaining the presence of HPV in abnormal cytology.

Demodex species are ectoparasites living in the hair follicles and sebaceous glands in human. Only two species, Demodex folliculorum and D. brevis were identified in human. While the D. folliculorum is settling in infundibular part of the hair follicles mostly, D. brevis settles into the sebaceous glands and ducts, which are deeper. These parasites live preferentially in hair follicles on the face and in the sebaceous glands, although they have also been reported to reside in seborrheic parts of the human body. The Demodex species have the highest rate on the face which has thesignificant number of sebaceous glands and sebum production in the skin. However, the rate of infestation increases with age in healthy skin. The aim of this study was to determine the prevalence of Demodex species in healthy women and the relationship between the incidence of Demodex and metabolic syndrome (MetS).Patients and This study consisted of 151,498 women aged ≥ 20 years who reside in the central district of Malatya province, Turkey. In 5% confidence interval of sample size, while the design effect was 1.5 it was calculated as 552 individuals and while the design effect was 2 it was calculated as 736 individuals. The World Health Organization 30 cluster sampling method was used to select the samples. Women aged ≥ 20 years who were not pregnant or lactating were included in the study. From a total of 669 subjects included in this study, 90.89% of the largest sample was accessed. Parasites were detected in 263 (39.3%) of 669 subjects and 3 of them were D. brevis. In chi-square analysis, nosignificant relationship was found between the incidence of the parasite, age, education level, occupation, marital status, family type, and MetS. However, a significant relationship was found between the diastolic pressure and those who fed with fatty foods and the incidence of parasite’s occurrence. According to the results of this study, MetS has no effect on the frequency of occurrence of the parasite; however, weight, fatty foods, and high diastolic pressure are effective in the frequency of occurrence of the parasite. The effects of these factors on the incidence of parasites should be supported by further study designs.

Bacterial parapneumonic pleural effusions (PPEs) have high morbidity. The accurate identification of pathogens is vital for initiating the appropriate treatment. A previous study suggested that the use of blood culture bottles might improve the bacterial yield in PPEs. The aim of this study was to compare the culture positivity rate by the blood culture bottles and the standard culture bottles in bacterial PPEs.Patients and Patients diagnosed with PPEs at the Khon Kaen Hospital, Khon Kaen, Thailand, which is an endemic area of melioidosis, were enrolled consecutively and prospectively. The study period was from June first, 2012 to December 31st, 2013. The inclusion criteria were adult patients aged > 18 years, with exudative, neutrophilic parapneumonic effusion. Of the pleural fluid samples, 5 mL from all the eligible patients were collected in both blood culture bottles and the standard culture bottles. Patient baseline characteristics, laboratory results, and culture results were collected and analyzed. During the study period, 129 patients met the study criteria. The bacteria-positive rate of pleural fluid culture using the standard culture bottle was 14.0%, whereas the positive rate using blood culture bottles was 24.0% (P < 0.001). The blood culture bottle method is more effective than the standard culture bottle method for the detection of bacterial pathogens in PPE.

Exploring the rate of naturally occurring NS3 protease mutants in HCV infected population is influential in the future therapeutic approaches. This study explored naturally occurring resistant mutations to protease inhibitors in a pilot study.Patients and We analyzed NS3 gene sequences in 7 HCV infected patients, referred to the central liver center, south of Iran. The protease domain was amplified by PCR followed by product extraction. Amplified NS3 genes were cloned by TA/cloning system. For each patient, clonal-sequencing was performed to improve mutation detection sensitivity. Then, the obtained sequences were compared with the reference sequences and final phylogenic tree was constructed. Afterwards, the sequences were studied to investigate point mutations. Phylogenetic analysis between reference and amplified sequences demonstrated high similarity of all sequences with genotype 1. Interestingly, crucial protease resistant mutations were detected in V36 and R155 positions in one patient’s sequence. Checking different clones of this patient confirmed V36L, as the dominant mutation while R155K was detected only in a few cases. As revealed, naturally occurring resistant mutations, especially R155K in protease sequence were identified in 1 out of the 7 patients, so the rate of such mutations is estimated to be high. It seems that checking HCV patients before protease inhibitor treatment are necessary in the region.

Blood stream infections (BSIs) are major causes of morbidity and mortality in burn patients. Microorganisms responsible for BSI are generally bacteria; however, Candida spp. are the infection agents in as many as 8% of all cases. Burn wound colonization and infections are generally the first steps to systemic infection. Candidemia in burn patients has been associated with high mortality and a prolonged hospital stay. Candidemia in burn patients has been defined as a preterminal event, leading to high morbidity and mortality rates among these patients. The aim of this study was to establish the incidence of candidemia in burn patients in Iran.Patients and We consecutively collected 405 blood samples from 113 burn patients. The yeast isolates were identified to the species level using conventional procedures. In vitro antifungal susceptibility of the Candida isolates to amphotericin B, fluconazole, voriconazole and caspofungin was performed using the Etest. Twenty-seven samples (6.7%) of the blood cultures from 13 patients (12%) were positive for Candida species. Candida parapsilosis (38%) and C. tropicalis (38%) were the most commonly found Candida species, followed by C. albicans (15%) and C. guilliermondii (15%) in the patients. The incidence of candidemia was significantly correlated with increased duration of hospitalization, increased time of stay in the intensive care unit, and higher mortality. The antifungal susceptibility tests demonstrated that amphotericin B and voriconazole had the lowest minimum inhibitory concentrations (MICs) against Candida spp. Non-albicans Candida should be considered as significant pathogens in burned patients with candidemia.

Chitosan, an important biodegradable and biocompatible polymer, has demonstrated wound-healing and antimicrobial properties. This study aimed to evaluate the antimicrobial properties of mafenide acetate-loaded nanofibrous films, prepared by the electrospinning technique, using chitosan and polyvinyl alcohol (PVA). A 32 full factorial design was used for formulating electrospinning solutions. The chitosan percentage in chitosan/PVA solutions (0%, 10%, and 30%) and the drug content (0%, 20%, and 40%) were chosen as independent variables. The release rate of mafenide acetate from nanofibrous films and their microbial penetration were evaluated. The antimicrobial activity of different nanofibrous film formulations against Staphylococcus aureus and Pseudomonas aeruginosa was studied. The results indicated that all nanofibrous films, with and without drug, can prevent bacterial penetration. Incorporation of mafenide acetate into chitosan/PVA nanofibers enhanced their antimicrobial activity against P. aeruginosa and S. aureus. Overall, the results showed that chitosan/polyvinyl alcohol (PVA) nanofibrous films are applicable for use as a wound dressing with protective, healing, and antimicrobial effects.

Enterococci are opportunistic pathogens and are a major factor in nosocomial infections. They may contain ebp operon, which upon expression makes them highly prone to biofilm formation on biotic and abiotic surfaces. The aim of the current study was to detect the polymorphism of ebp genes in Enterococcus faecalis. Samples were isolated from patients (n = 58) and hospital environments (n = 32) of two hospitals in Tehran, Iran. All enterococcal species were identified by species-specific polymerase chain reaction (PCR); the antibiotic resistance pattern against nine antibiotics was determined. The ebp A, ebp B, ebp C and srt C genes were detected by PCR and the biofilm formation by the isolates was evaluated using the microtiter plate method. The genetic diversity of ebp genes was analyzed by restriction fragment length polymorphism (RFLP). The results indicated that, 86% of patient and 29% of environmental isolates carried ebp genes. The ability of the isolates to strongly attach was 62% and 71% for patient and environmental samples, respectively. The RFLP of the ebp showed no genetic variations amongst the isolates. The results of the antibiotic resistance and other data suggest that there is a possible common clone of E. faecalis, which could rapidly disseminate in patients and the environment.

Thrombolytic therapy has become a conventional treatment for acute myocardial infarction (AMI), yet currently, clinically prescribed thrombolytic drugs have problems such as delayed action and other side effects. Fibrinolytic enzymes have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process, including plasmin activation. Nattokinase (NK) is a potent fibrinolytic agent for thrombosis therapy. The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement. In the present study, a potent NK-producing strain was isolated from cow milk and identified. To enhance the yield of NK, effect of various parameters such as pH, temperature, carbon source, nitrogen source and inoculum size were optimized. Strain improvement of P. aeruginosa CMSS was done by random UV-mutagenesis. Nattokinase was partially purified and the activity was determined by the casein digestion method, blood clot lysis and fibrin degradation assay. Based on morphological, biochemical and molecular characterization, the strain was confirmed as P. aeruginosa (GenBank accession number: JX112657), designated as P. aeruginosa CMSS. The optimum condition at pH 7 and temperature at 25˚C showed activity of NK as 1514 U mL-1 and 1532 U mL-1, respectively. Sucrose as the carbon source and shrimp shell powder (SSP) as the nitrogen source expressed NK activity of 1721 U mL-1 and 2524 U mL-1, respectively. At 1% inoculum size, the maximum rate of enzyme production was achieved with 2581 U mL-1. The NK activity of the mutant strain UV60 was 4263 U mL-1, indicating a two-fold increase in activity compared to the wild strain (2581 UmL-1). Nattokinase produced from mutant strain P. aeruginosa CMSS UV60 showed 94% blood clot lysis at ten minutes. The degradation of fibrin clot by the produced NK was observed after two hours of incubation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the molecular mass of CMSS UV60 NK to be 21kDa. The current study demonstrated the enhanced production of NK by P. aeruginosa CMSS. This study is unique and the findings are the first report on the production of NK from Pseudomonas aeruginosa CMSS isolated from cow milk.

Tympanosclerosis is a condition caused by calcification of tissues in the middle ear mucosa that sometimes results hearing loss. Helicobacter pylori is one of the pathological and etiologic factors in the development of tympanosclerosis. The purpose of this study was to show the role of H. pylori in the different aspects of chronic suppurative otitis media using the polymerase chain reaction (PCR) technique.Patients and This case-control and cross-sectional study was performed on all patients with chronic otitis media, candidates for surgical operations, in 2013. They were allocated into the case group with tympanosclerosis and the control group without tympanosclerosis. During the surgical operation, biopsy was done from middle ear and the samples were studied to see if they contained H. pylori using the PCR method. From a total of 19 patients with tympanosclerosis, 16 cases (84.2%) were H. pylori positive, while in the control group 15 (45.4%) cases out of the 37 cases were H. pylori positive, which showed a significant difference (P = 0.002). Age and gender of the patients, ear dryness and perforation size were not correlated with the presence or absence of H. pylori. There is a significant correlation between tympanosclerosis and H. pylori (P = 0.002). This correlation can single out H. pylori as a pathological factor in the development of tympanosclerosis; however, further studies are needed to prove this correlation.

Blastocystis hominis is a common globally distributed parasite. The prevalence of this parasite has been shown to vary among different countries. Molecular studies have also shown that there is a high level of genetic diversity among Blastocystis spp. isolated from humans and animals. Extensive information on parasitic genotypes will aid in devising more effective strategies for the identification and potential control of these pathogenic parasites. This study aimed to gain information on the prevalence and abundance of Blastocystis subtypes in Iran. Over a period of 3 months, 1,410 stool samples were collected and examined by microscopy. Samples found to be positive for B. hominis were concentrated and phylogenetic analysis was subsequently performed. A questionnaire was completed by all study participants. Blastocystis hominis was found to have a prevalence of 3.33% in the study population. There was no significant association of Blastocystis infection with age (P = 0.3) or gender (P = 0.57). The Blastocystis subtypes (ST) identified in this study were ST3, ST4, ST5, and ST7 with the most prevalent being ST4 (40.9%). The prevalence of B. hominis in the study area was lower than that reported for most developed countries, and unlike in other countries in the Middle East, ST4 was the most prevalent subtype.

Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly. In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared. Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles. The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set. Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.