Enhanced antibacterial properties of polyvinyl alcohol/starch/chitosan films with NiO-CuO nanoparticles for food packaging.

Packaging is very important to maintain the quality of food and prevent the growth of microbes. Therefore, the use of food packaging with antimicrobial properties protects the food from the growth of microorganisms. In this study, antibacterial nanocomposite films of polyvinyl alcohol/starch/chitosan (PVA/ST/CS) together with nickel oxide-copper oxide nanoparticles (NiO-CuONPs) are prepared for food packaging. NiO-CuONPs were synthesized by the co-precipitation method, and structural characterization of nanoparticles (NPs) was carried out by XRD, FTIR, and SEM techniques. Composites of PVA/ST/CS, containing different percentages of NPs, were prepared by casting and characterized by FTIR and FESEM. The mechanical properties, diffusion barrier, and thermal stability were determined. The nanoparticles have a round structure with an average size of 6.7 ± 1.2 nm. The cross-section of PVA/ST/CS film is dense, uniform, and without cracks. In the mechanical tests, the addition of NPs up to 1% improved the mechanical properties (TS = 31.94 MPa), while 2% of NPs lowered TS to 14.76 MPa. The fibroblast cells toxicity and the films antibacterial activity were also examined. The films displayed stronger antibacterial effects against Gram-positive bacteria (Staphylococcus aureus) compared to Gram-negative bacteria (Escherichia coli). Furthermore, these films have no toxicity to fibroblast cells and the survival rate of these cells in contact with the films is more than 84%. Therefore, this film is recommended for food packaging due to its excellent mechanical and barrier properties, good antibacterial activity, and non-toxicity.

Preparation and characterization of gelatin/chitosan nanocomposite reinforced by NiO nanoparticles as an active food packaging.

Food packaging with antibacterial properties has attracted much attention recently. In this study, nickel oxide nanoparticles (NiONPs) were synthesized by co-precipitation and then gelatin/chitosan polymer films (GEL/CS) with different percentages of NiONPs, bio-nanocomposites, were prepared by casting. Morphology, crystal microstructure, molecular interactions and thermal stabilities of the NPs and the composite films were characterized by FESEM, XRD, FTIR and TGA, respectively. The bio-nanocomposite films exhibited excellent barrier, thermal and mechanical properties by addition of an optimized content of NPs. For example, the tensile strength (TS) of the GEL/CS film without NPs was 23.83 MPa and increased to 30.13 MPa by incorporation of 1% NPs. The antibacterial properties and toxicity of the films were investigated. These films show good antibacterial behavior against Gram-positive (Staphylococcus aureus) bacteria compared to Gram-negative (Escherichia coli) bacteria. Furthermore, the films were found to be non-toxic to fibroblast cells that came into contact with the films, with a survival rate of more than 88%. Therefore, these films can be applied for food packaging due to their excellent mechanical, barrier, and antibacterial properties.

Detection of Genes Related to Linezolid Resistance (poxtA, cfr, and optrA) in Clinical Isolates of Enterococcus spp. from Humans: A First Report from Iran.

Background: Enterococci may develop resistance to linezolid through chromosomal mutations that involve specific linezolid resistance genes, such as cfr, optrA, and poxtA. The objective of this study was to evaluate the antibiotic susceptibility of enterococcal isolates and identify cfr, optrA, and poxtA genes in MDR isolates. Materials and Methods: Enterococcal isolates were collected from various clinical specimens at Al-Zahra, Amin, and Khorshid Hospitals in Isfahan. The Enterococcus isolates were identified as belonging to the E. faecalis and E. faecium species by using specific gene (D alanine D alanine ligase ddl) sets in PCR. To detect cfr, optrA, and poxtA genes among the species, a multiplex-PCR assay was performed. Results: Out of 175 isolates, E. faecalis predominated 129/175 (73.7%). Furthermore, the prevalence of vancomycin-resistant Enterococci (VRE) and linezolid-resistant Enterococci (LRE) was 29.7% and 4%, respectively. The overall prevalence of MDR was 91.1%, 68.9%, and 66.6% of E. faecium, E. faecalis, and other Enterococcus spp., respectively. Interestingly, the frequency of optrA (71.4%) in E. faecium and poxtA and crf (42.8%) in E. faecalis were detected among LRE species. A statistically significant relationship (P < 0.05) was found between the presence of the three genes and the occurrence of LRE. Conclusion: This is the first study to report the detection of linezolid resistance genes (cfr, optrA, and poxtA) in clinical Enterococcus spp. isolates from Iran, conducted at Isfahan University of Medical Sciences hospitals. The emergence of enterococcal strains that resist linezolid is concerning as it can lead to the spread of resistant strains among patients, resulting in treatment failure.

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.

Biofilm Formation in Nonmultidrug-resistant Escherichia coli Isolated from Patients with Urinary Tract Infection in Isfahan, Iran.

BACKGROUND: Escherichia coli is a Gram-negative, opportunistic human pathogen in which increasing antibiotic resistance is a great concern for continued human survival. Although biofilm formation is a mechanism that helps E. coli to survive in unfavorable conditions, according to the importance of biofilm formation in developing the antibiotic resistance here, we studied the relation between antibiotic resistance and in vitro qualitative rating method biofilm formation in E. coli isolated from patients with urinary tract infection (UTI). MATERIALS AND METHODS: The clinical isolates of E. coli (n = 100) were collected from urine of patients with UTI attending Isfahan Alzahra hospital. The strains were confirmed as E. coli using biochemical tests and molecular method. The Kirby-Bauer disk diffusion tests were done according to the Clinical and Laboratory Standards Institute protocol, and the biofilm synthesis was performed by microplate method. The binary logistic test was applied and P < 0.05 was considered statistically significant. RESULTS: Our results showed a high outbreak of multidrug-resistant (MDR) E. coli strains (73%) and the highest resistance was observed toward ampicillin. The prevalence of biofilm producer isolates was 80% that 29% produced strong biofilm. The distribution of non-MDR isolates was high among strong biofilm producers, which shows a significant negative correlation between biofilm production and MDR pattern (P < 0.001). CONCLUSIONS: We found a negative correlation between MDR phenotype and biofilm formation capacity. This transmits the concept that more antibiotic susceptibility of strong biofilm producers may be due to the reduced exposure to multiple antibiotics.

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.

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources.

BACKGROUND: Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of ß-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. MATERIALS AND METHODS: Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. RESULTS: Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli. PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. CONCLUSION: The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources.

The Effect of Er:YAG Laser on Entroccocus faecalis Bacterium in the Pulpectomy of Anterior Primary Teeth.

Introduction: Successful root canal therapy depends on the complete elimination of microorganisms such as Entroccocus faecalis, which is impossible to achieve with the traditional methods. Lasers are recently introduced as a new method to solve the problem. The present study is planned and performed to examining the antibacterial effect of Er: YAG laser. Methods: Sixty extracted anterior primary teeth were prepared and sterilized. E. faecalis bacterium was cultured in canals. Samples were randomly divided into two groups. The first group was disinfected by NaOCl 5/25% and Er: YAG laser and the second group just by NaOCl 5/25%. Samples of canal contents were cultured and colony counts were calculated. The results were analyzed statistically by SPSS software and Mann Whitney test. Results: There was no significant difference between colony counts in both groups (P=0.142). But the number of colonies in the first group was lower than in the second group. Conclusion: Although, Er: YAG laser cannot completely eliminate E. faecalis bacterium, its simultaneous use with NaOCl decreases E. faecalis.

Detection of Panton-valentine Leukocidin Gene Isoforms of Staphylococcus aureus Isolates in Al-Zahra Hospital, Isfahan-Iran.

BACKGROUND: Panton-Valentine leukocidin (PVL) is a gamma-toxin produced by Staphylococcus aureus encoded by genes lukS/lukF-PV with several single-nucleotide polymorphisms. A mutation at nucleotide position 527 results in substitution of histidine (H) to arginine (R) at amino acid 176. The groups defined based on the amino acid change, the "R isoform" group and the "H isoform" group. The purpose of this study was to determine the frequency of PVL gene isoforms in S. aureus strains isolated from patients at Al-Zahra Hospital Isfahan and molecular characterization of PVL-positive methicillin-resistant S. aureus (MRSA) strains including the detection of mecA gene and staphylococcal chromosomal cassette mec (SCCmec) typing. MATERIALS AND METHODS: In this study, 130 isolates of S. aureus were collected from Al-Zahra Hospital. The PVL gene identified using polymerase chain reaction (PCR); PCR products were sequenced to identify the type of isoform. The molecular characterization of isolates of PVL-positive MRSA including detection of mecA gene by PCR and also SCCmec typing was performed by multiplex PCR. RESULTS: Out of 130 isolates, 23% were positive for the presence of PVL genes. The PVL positive isolates were comprised 37% (11/30) of methicillin-resistant isolates and 63% (19/30) of methicillin-susceptible S. aureus (MSSA) isolates. The results showed that 17 isolated carrying isoform H and 13 isolated carrying the R isoform. CONCLUSION: The PVL gene was predominantly found in MSSA isolates. There was no relation between PVL isoforms and the presence of mecA and SCCmec types.

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 study of H. pylori putative candidate factors for single- and multi-component vaccine development.

Helicobacter pylori has grown to colonize inside the stomach of nearly half of the world's population, turning into the most prevalent infections in the universe. Medical care failures noticeably confirm the need for a vaccine to hinder or deal with H. pylori. This review is planned to discuss the most known factors as a vaccine candidate, including single (AhpC, BG, CagA, KatA, Fla, Hsp, HWC, Lpp, LPS, NAP, OMP, OMV, SOD, Tpx, Urease, VacA) and multi-component vaccines. Many promising results in the field of single and multivalent vaccine can be seen, but there is no satisfactory outcome and neither a prophylactic nor a therapeutic vaccine to treat or eradicate the infection in human has been acquired. Hence, selecting suitable antigen is an important factor as an appropriate adjuvant. Taken all together, the development of efficient anti-H. pylori vaccines relies on the fully understanding of the interactions between H. pylori and its host immune system. Therefore, more work should be done on epitope mapping, analysis of molecular structure, and determination of the antigen determinant region as well due to design a vaccine, preferably a multi-component vaccine to elicit specific CD4 T-cell responses that are required for H. pylori vaccine efficacy.

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.

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.

Morphological and Bactericidal Effects of Amikacin, Meropenem and Imipenem on Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa might be converted to coccoid bacteria under antibiotic stress. Bacterial conversion would increase resistance to antibiotics due to changes in cell wall crosslink or decreased metabolic activity. Morphology of P. aeruginosa under stress conditions (presence of antibiotics) can be changed to elongated bacilli, U shape and finally coccoid bacteria. Results of several researches showed that coccoid bacteria are one of the most important aspects of drug resistance. It would be the major reason for treatment failure. OBJECTIVES: The aim of this study was to determine in vitro morphological and bactericidal effects of amikacin, meropenem and imipenem on P. aeruginosa isolated from clinical specimens. MATERIALS AND METHODS: Eight P. aeruginosa isolates obtained from clinical samples of burned patients and standard strain ATCC 27853 were used in this study. Isolates were identified by biochemical tests and confirmed by PCR method using ITS specific primer. Minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. Bacteria were exposed to antibiotics at different concentrations. Bacterial morphology in different days was examined by specific microscope and viability of isolates was examined by flow cytometry. RESULTS: All used antibiotics at sub MIC concentration had capability to induce coccoid bacteria. The highest rate of induced coccoid bacteria was 98.2% after 8 days, with contribution of imipenem and meropenem at 2 µg/mL concentration. Amikacin at 4 µg/mL concentration induced lower rate of coccoid bacteria (55.05%). Amikacin had a strong bactericidal effect on coccoid bacteria at 8 µg/mL concentration. Imipenem and meropenem showed very weak bactericidal effect on coccoid bacteria. CONCLUSIONS: Induction of coccoid form of P. aeruginosa may be one of the important reasons for antibiotic treatment failure; therefore, prescribed dose of antibiotics should be carefully managed to prevent increasing antibiotic resistance and coccoid bacteria induction.

Morphological and Bactericidal Effects of Different Antibiotics on Helicobacter pylori.

BACKGROUND: Helicobacter pylori (H. pylori) is a spiral Gram negative bacteria that can transform to the coccoid form in adverse conditions. OBJECTIVES: The aim of this study was to determine the in vitro morphological and bactericidal effects of metronidazole, amoxicillin and clarithromycin on H. pylori. MATERIALS AND METHODS: The standard strain 26695 of H. pylori was cultured on Brucella agar (BA) and the minimum inhibitory concentrations (MICs) of three antibiotics were determined by E-test method. The bacteria were exposed to antibiotics at 1/2 MIC, MIC and 2X MIC concentrations in Brucella broth (BB). Induced coccoid forms were confirmed by Gram staining and light microscopy. The viability of cells as well as the susceptibility of viable coccoids to antibiotics were examined using the flow cytometry method. RESULTS: All of the three antibiotics at sub-MIC induced coccoid forms. The highest rates of coccoids (> 90%) were induced at 0.008 µg/mL concentration (1/2 MIC) of amoxicillin, 72 hours postexposure. Metronidazole and clarithromycin with 1/2 MIC (0.5 and 0.125 µg/mL respectively) induced lower rates of coccoid forms (60% and 40% respectively). Potent bactericidal effects on coccoids were observed with Metronidazole at 2X MIC and clarithromycin at MIC (0.25 µg/mL) (80 - 90%). Amoxicillin with MIC and 2X MIC had no bactericidal effect on coccoid forms. CONCLUSIONS: Despite the good in vitro bactericidal effect of amoxicillin on spiral forms of H. pylori, this antibiotic has little effect on induced coccoids that may develop after the inappropriate in vivo antibacterial treatment. Hence, for successful therapy, it is essential not only to eradicate the spiral forms, but to eliminate the viable coccoids.

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.

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.

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.