Evaluation of Biofilm Formation and Frequency of Multidrug-resistant and Extended Drug-resistant Strain in Pseudomonas aeruginosa Isolated from Burn Patients in Isfahan.

BACKGROUND: Pseudomonas aeruginosa is a biofilm-forming bacterium which can result in serious health problems, particularly in burn patients. Biofilm has been assumed to protect the bacteria from environmental fluctuations such as antimicrobial agent. Mucoid strains generate extensive levels of the alginate exopolysaccharide, which is an important factor of its biofilm. MATERIALS AND METHODS: Totally, 100 isolates of P. aeruginosa has been gathered from wound infections of burn patients. Polymerase chain reaction of exoA gene has been carried out to confirm the bacteriologic identification of isolates. The biofilm-forming capacity has been specified by capsule staining and microtiter plate test as qualitative and quantitative determination, respectively. Antimicrobial susceptibility of the isolates has been specified by disk diffusion method. RESULTS: All the isolates carried the exoA gene. The antibiotic resistance was imipenem (90%); levofloxacin (93%); aztreonam (87%); piperacillin-tazobactam (85%); tobramycin (92%); polymyxin b (PB) (2%); and ceftazidime (CAZ) (32%). Totally, multidrug-resistant (MDR) and extended drug-resistant (XDR) isolates were 19% and 75%, respectively. Fortunately, pan drug-resistant (PDR) strain has not been observed. The assessment of biofilm formation has shown that 7% of the isolates were nonbiofilm (N), weak (W) 67%, moderate (M) 22%, and strong (S) 4%. CONCLUSIONS: As a result, the findings of this survey indicated that PB and CAZ were the most effective antibiotics against P. aeruginosa, which of course indicate a serious problem about the emergence of the PDR strains. There was no relationship between the patterns of biofilm production and antibiotic susceptibility, but high frequency of MDR/XDR and biofilm producer strains has been detected.

Overexpression of spoT gene in coccoid forms of clinical Helicobacter pylori isolates.

Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.

Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction.

BACKGROUND: Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. MATERIALS AND METHODS: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. RESULTS: The CTAB method showed more positive results at 1:10-1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. CONCLUSIONS: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

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

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

The Bioinformatics Report of Mutation Outcome on NADPH Flavin Oxidoreductase Protein Sequence in Clinical Isolates of H. pylori.

frxA gene has been implicated in the metronidazole nitro reduction by H. pylori. Alternatively, frxA is expected to contribute to the protection of urease and to the in vivo survival of H. pylori. The aim of present study is to report the mutation effects on the frxA protein sequence in clinical isolates of H. pylori in our community. Metronidazole resistance was proven in 27 of 48 isolates. glmM and frxA genes were used for molecular confirmation of H. pylori isolates. The primer set for detection of whole sequence of frxA gene for the effect of mutation on protein sequence was used. DNA and protein sequence evaluation and analysis were done by blast, Clustal Omega, and T COFFEE programs. Then, FrxA protein sequences from six metronidazole-resistant clinical isolates were analyzed by web-based bioinformatics tools. The result of six metronidazole-resistant clinical isolates in comparison with strain 26695 showed ten missense mutations. The result with the STRING program revealed that no change was seen after alterations in these sequences. According to consensus data involving four methods, residue substitutions at 40, 13, and 141 increase the stability of protein sequence after mutation, while other alterations decrease. Residue substitutions at 40, 43, 141, 138, 169, and 179 are deleterious, while, V7I, Q10R, V34I, and V96I alterations are neutral. As FrxA contribute to survival of bacterium and in regard to the effect of mutations on protein function, it might affect the survival and bacterium phenotype and it need to be studied more. Also, none of the stability prediction tool is perfect; iStable is the best predictor method among all methods.

Study on Prevalence, Antibiotic Susceptibility, and tuf Gene Sequence-Based Genotyping of Species-Level of Coagulase-Negative Staphylococcus Isolated From Keratitis Caused by Using Soft Contact Lenses.

OBJECTIVE: To study on antibiotic susceptibility and identify coagulase-negative Staphylococcus (CoNS) species based on tuf gene sequencing from keratitis followed by using soft contact lenses in Isfahan, Iran, 2013. METHODS: This study examined 77 keratitis cases. The samples were cultured and the isolation of CoNS was done by phenotypic tests, and in vitro sensitivity testing was done by Kirby-Bauer disk diffusion susceptibility method. RESULTS: Thirty-eight of isolates were conveniently identified as CoNS. In this study, 27 (71.1%), 21 (55.3%), and 16 (42.1%) were resistant to penicillin, erythromycin, and tetracycline, respectively. One hundred percent of isolates were sensitive to gentamicin, and 36 (94.7%) and 33 (86.8%) of isolates were sensitive to chloramphenicol and ciprofloxacin, respectively. Also, resistances to cefoxitin were 7 (18.4%). Analysis of tuf gene proved to be discriminative and sensitive in which all the isolates were identified with 99.0% similarity to reference strains, and Staphylococcus epidermidis had the highest prevalence among other species. CONCLUSIONS: Results of this study showed that CoNS are the most common agents causing contact lens-associated microbial keratitis, and the tuf gene sequencing analysis is a reliable method for distinguishing CoNS species. Also gentamycin, chloramphenicol, and ciprofloxacin are more effective than the other antibacterial agents against these types of bacteria.

The influence of impact delivery mode, lactation time, infant gender, maternal age and rural or urban life on total number of Lactobacillus in breast milk Isfahan - Iran.

BACKGROUND: Breast milk is known as the most crucial postpartum issue in metabolic and immunologic programming of neonatal health. Human milk microbial changes over Lactation. The factors influencing the milk microbiome as well as potential impact of microbes on infant health have not yet been discovered. The objective was to identify pre- and post-natal factors that can potentially influence the bacterial communities inhabiting human milk. MATERIALS AND METHODS: Breast milk samples (n = 40) with all full-term breastfed infants were collected from lactating randomized. Information on personal characteristics, dietary habits, information about infants were collected after birth. The samples were plated with serial dilutions on three selective culture media man rogosa sharp and then colonies were counted. Colonies tested for catalase reaction, Gram-staining and microscopic examination. RESULTS: The result of this study showed that the overall incidence of positive Lactobacillus in mother's milk was 87.5%. The results based on (infant gender, mode of delivery, rural or urban and lactation time) rural or urban and lactation time were significant (P < 0.05). The results showed that all of the variables were significant in this regression model (P < 0.001). The median of log10 Lactobacillus counts in rural mothers, vaginal delivery, infant male gender and Lactation time for first 3-month were meaningfully high. CONCLUSIONS: The findings of this study about the breast milk Lactobacillus potential probiotic bacteria of healthy Iranian mothers, suggested that the breast milk microbiome is significantly influenced by several factors, mode of delivery, rural or urban and lactation time.

Helicobacter pylori in Iran: A systematic review on the antibiotic resistance.

OBJECTIVES: Helicobacter pylori (H. pylori) is a pathogenic bacterium that colonizes the stomachs of approximately 50% of the world's population. Resistance of H. pylori to antibiotics is considered as the main reason for the failure to eradicate this bacterium. The aim of this study was to determine the rate of resistant H. pylori strains to various antimicrobial agents in different areas of Iran. MATERIALS AND METHODS: A systematic review of literatures on H. pylori antibiotic resistance in Iran was performed within the time span of 1997 to 2013. Data obtained from various studies were tabulated as following, 1) year of research and number strains tested, 2) number of H. pylori positive patients, 3) study place, 4) resistance of H. pylori to various antibiotics as percentage, and 5) methods used for evaluation of antibiotic resistance. RESULTS: Over the period, a total of 21 studies on H. pylori antibiotic resistance have been conducted in different parts of Iran. In these studies, H. pylori resistance to various antibiotics, including metronidazole, clarithromycin, amoxicillin, tetracycline, ciprofloxacin, levofloxacin and furazolidone were 61.6%, 22.4%, 16.0%, 12.2%, 21.0%, 5.3% and 21.6%, respectively. We found no study on H. pylori resistance to rifabutin in Iran. CONCLUSION: Compared to the global average, we noted that the prevalence of H. pylori resistance to metronidazole, clarithromycin, amoxicillin, and tetracycline has been rapidly growing in Iran. This study showed that in order to determine an appropriate drug regimen against H. pylori, information on antibiotic susceptibility of the bacterium within different geographical areas of Iran is required.

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

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

The study of mutation in 23S rRNA resistance gene of Helicobacter pylori to clarithromycin in patients with gastrointestinal disorders in Isfahan - Iran.

BACKGROUND: Helicobacter pylori antimicrobial resistance is an important factor responsible for treatment failure. The purpose of this study was evaluating the prevalence of point mutations in clarithromycin-resistant clinical isolates of H. pylori in Isfahan city of Iran. MATERIALS AND METHODS: Thirty isolates of H. pylori from 130 biopsy specimens were isolated by culture and confirmed by biochemical and PCR tests. The MIC of clarithromycin antibiotic for 30 clinical isolates of H. pylori was determined by E-test method. The point mutations in the 288 bp of 23S rRNA gene of H. pylori were investigated in four clarithromycin-resistant clinical isolates by PCR followed by sequencing. RESULTS: Among 30 isolates of H. pylori, 4 cases were resistant to clarithromycin. One point mutation was found at position T2243C in the 23S rRNA gene in all resistance isolates. CONCLUSIONS: In our study, H. pylori resistance to clarithromycin associated with point mutation at position 2243 (T2243C).

The mutation of the rdxA gene in metronidazole-resistant Helicobacter pylori clinical isolates.

BACKGROUNDS: Antibiotic resistance is an increasing problem throughout the developed world, and knowledge about different resistance mechanisms is consequential for efficient treatment of bacterial infections. Although metronidazole has been frequently used in treatment regimens for H. pylori infection, but antibiotic resistance is now a major contributing factor in treatment failure. Nevertheless metronidazole has been greatly used as a critical component of combination therapies for H. pylori infection. OBJECTIVE: This study is trying to describe the mutational mechanisms of metronidazole resistance in H. pylori in our clinical isolates in Isfahanian patients, Iran and compare with the findings of previous studies in world. MATERIALS AND METHODS: MIC values of metronidazole for H. pylori strains were determined by E- test. Both rdxA and glmM genes used for confirmation of isolates as H. pylori and then amplification of another rdxA oligonucleotide pair was done. Finally, the six resistant strains were sent to sequencing for other processing and further analysis was done by software. RESULTS: The result of six clinical isolates in comparison with 26695, J99 and 69A as a sensitive and resistant reference strains showed plenty of mutations. No frame shift and nonsense mutation was seen in our clinical isolates. CONCLUSION: An interesting finding in metronidazole-resistant strains in our study was the detection of one mutation not previously described in the literature in the rdxA gene and this W(209)R substitution presumably plays a role in inducing metronidazole resistance.

Assessment of cagE and babA mRNA expression during morphological conversion of Helicobacter pylori from spiral to coccoid.

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90-99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.

Resistance pattern of Helicobacter pylori strains to clarithromycin, metronidazole, and amoxicillin in Isfahan, Iran.

BACKGROUND: Helicobacter pylori (H. pylori) resistance to antibiotics has become a global problem and is an important factor in determining the outcome of treatment of infected patients. The purpose of this study was to determine the H. pylori resistance to clarithromycin, metronidazole, and amoxicillin in gastrointestinal disorders patients. MATERIALS AND METHODS: In this study, a total of 260 gastric antrum biopsy specimens were collected from patients with gastrointestinal disorders who referred to Endoscopy Section of the Isfahan Hospitals. The E-test and Modified Disk Diffusion Method (MDDM) were used to verify the prevalence of antibiotic resistance in 78 H. pylori isolates to the clarithromycin, metronidazole, and amoxicillin. RESULTS: H. pylori resistance to clarithromycin, metronidazole, and amoxicillin were 15.3, 55.1, and 6.4%, respectively. In this study, we had one multidrug resistance (MDR) isolates from patient with gastritis and peptic ulcer disease. CONCLUSION: Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases. According to the results obtained in this study, H. pylori resistance to clarithromycin and metronidazole was relatively high. MDR strains are emerging and will have an effect on the combination therapy.

Helicobacter pylori in Iran: A systematic review on the association of genotypes and gastroduodenal diseases.

BACKGROUND: Helicobacter pylori (H. pylori) infection is known as a major etiologic factor for a variety of gastroduodenal diseases. In Iran, with a high rate of H. pylori infection close to 90%, numerous studies have revealed many aspects of interaction between the bacterium, mucosal surface and induction of disease outcome. The organism is genetically diverse and several virulence factors are attributed to the more virulent strains. The well-characterized virulence factors of H. pylori are cytotoxin associated gene A and vacuolating cytotoxin gene A. The distribution pattern of H. pylori genotypes and its association with disease status varies geographically. The present review focused on the virulence factors and genotyping of H. pylori in relation to gastroduodenal disorders in different regions of Iran. METHODS: In total, 398 studies were reported on different aspects related to H. pylori in our electronic search from 1995-2011. H. pylori infection and its virulence factors in association with disease status were investigated in 159 reports. Looking specifically at the gastrointestinal tract disorders, the most relevant reports including 37 papers were selected. RESULTS: We found no correlation of cagA genotype and disease status in the majority of studies, whereas vacA was demonstrated as a useful marker in predicting the disease outcome. The results of reports on other virulence factors of H. pylori such as blood group antigen-binding adhesion gene A, the induced by contact with epithelium gene A, the outer inflammatory protein A, the duodenal ulcer promoting gene A, and Helicobacter outer membrane gene and their relation with disease status were contradictory. CONCLUSIONS: Although different markers of H. pylori were emphasized as useful when predicting disease outcomes in some studies, the inconsistent researches and the scarcity of data made any conclusion or even comparison impossible. Considering the gap of information observed during our search relating to genotyping and other aspects of H. pylori infection, further investigations are suggested.

Development of PCR-based method for detection of Enterobacteriaceae in septicemia.

OBJECTIVE: Sepsis is a systemic inflammatory response associated with high mortality rates in the clinical setting. A multiplex endpoint polymerase chain reaction (PCR) based assay for rapid detection of enterobacteriaceae involved in septicemia, which included Internal Control (IC) and 16S rDNA, is presented here. To develop a panel of primers for DNA fragments of 16S rDNA, enterobacteriaceae, IC, and evaluate analytical sensitivity and specificity of the test. MATERIALS AND METHODS: Primers for amplification of enterobacteriaceae, IC, and16S rDNA were designed, and then PCR was performed. Minimal analytical sensitivity was determined by cloning and colony PCR, and specificity was tested on the basis of their respective standard strains. This study is a cross-sectional Model. RESULTS: Our results showed the rpoB gene as the most promising target for detection of enterobacteriaceae by PCR amplification. Specificity and sensitivity of endpoint PCR were 100%, 100%, and 100%, and 10, 1, and 100 copies/reaction for enterobacteriaceae, IC, and 16S rDNA, respectively. CONCLUSION: The molecular panel presented offers the advantage of an easy, reliable, and cost-effective system when compared to other molecular detection methods. However, further evaluation is needed. Our assay holds promising for more rapid pathogens related in clinical sepsis.

Helicobacter pylori as a zoonotic infection: the detection of H. pylori antigens in the milk and faeces of cows.

BACKGROUND: The prevalence of Helicobacter pylori infection, which may increase the risk of gastritis, peptic ulcers, and cancer, has increased worldwide. This number is estimated to be around 70-90% in developing countries and 25-50% in developed countries. It is possible that the bacterium can be transmitted via food and water as well as zoonotically and iatrogenically. Because of high prevalence of this infection in Iran, the aim of this study is to examine whether H. pylori infection might be transmitted from cow's milk and faeces. METHODS: The existence of the H. pylori antibody and antigen was investigated in samples of serum, milk, and faeces from 92 lactating Holstein cows in Shahrekord, Iran. The H. pylori antigen and antibody were detected using ELISA and were confirmed by PCR. RESULTS: It was found that out of 92 serum specimens, 25 (27%) of the cows were positive for the H. pylori antibody and 67 specimens were negative. From these 25 seropositive cows, 10 (40%) faeces samples and four (16%) milk samples were antigen positive for H. pylori. Four of the antigen-positive milk specimens were also antigen positive for faeces. The existence of the UreC gene was also confirmed in positive samples of milk and faeces. CONCLUSIONS: There is a possibility that cow's milk is a transmission mode in H. pylori infection and faecal contamination and inappropriate management processes could transfer H. pylori to humans. The awareness of the H. pylori epidemiology and its method of distribution are necessary for public health measures and controlling the spread of this bacterium. Further investigation with a greater sample number is necessary to verify the ability of H. pylori transmission via milk consumption.