Microbial susceptibility, virulence factors, and plasmid profiles of uropathogenic Escherichia coli strains isolated from children in Jahrom, Iran

Background: Urinary tract infections [UTIs], including cystitis and pyelonephritis, are the most common infectious diseases in childhood. Escherichia coli [E. coli] accounts for as much as 90% of the community-acquired and 50% of nosocomial UTIs. Therefore, identification of E. coli strains is important for both clinical and epidemiological implications. Understanding antibiotic resistance patterns and molecular characterization of plasmids and other genetic elements is also epidemiologically useful

Methods: To characterize uropathogenic strains of E. coli, we studied 96 E. coli strains recovered from urine samples of children aged 1 month to 14 years with community-acquired UTIs in Jahrom, Iran. We assessed virulence factors [VFs], drug sensitivities, and plasmid profiles

Results: Drug sensitivities of the isolates were: 19.8% [ampicillin], 24% [trimethoprim-sulfamethoxazole], 29.2% [tetracycline], 75.5% [nalidixic acid], 80.4% [cefixime], 84.6% [gentamicin], 91.4% [ciproAoxacin], 96.8% [nitrofurantoin], 96.8% [amikacin] and 100% [imipenem]. Totally, 76 isolates harbored plasmids with an average of 5.5 plasmids [range: 1-10] in each strain. Plasmid profiling distinguished 22 different E. coli genotypes in all isolates that ranged in similarity from 50% to 100%. PCR showed that the prevalence of virulence genes ranged from 15.62% forhly to 30.2% for pap

Conclusion: These data mandate local monitoring of drug resistance and its consideration in empirical therapy of E. coli infections. Plasmid analysis of representative E. coli isolates also demonstrates the presence of a wide range of plasmid sizes, with no consistent relationship between plasmid profiles and resistance phenotypes. Plasmid profiles distinguished more strains than did the antimicrobial susceptibility pattern

Antifungal susceptibility of the Aspergillus species by Etest and CLSI reference methods

Because resistance to antifungal drugs is seen in patients, susceptibility testing of these drugs aids in choosing the appropriate drug and respective epidemiology. This study has investigated and compared susceptibility patterns of the Aspergillus species sisolated from patients by the Clinical and Laboratory Standards Institute [CLSI] reference broth microdilution [MD] assay and Etest method. The minimum inhibitory concentrations [MICs] of various antifungal agents [amphotericin B, ketoconazole, itraconazole, and voriconazole] for 108 Aspergillus species isolated from patients were determined by CLSI M38-A broth MD and Etest. The isolates were obtained from clinical samples that included tissues, sputum, bronchoalveolar lavage, abdominal tap, and cerebrospinal fluid. As revealed by the MD method, 63.9% of the isolates were sensitive to amphotericin B and 36.1% were resistant. Etest revealed that 61.1% were sensitive to amphotericin B and 38.9% were resistant. As for ketoconazole, 108 isolates [100%] were shown to be sensitive through the MD method; while the Etest revealedan 88.9% sensitivity and 11.1% were resistant. All species were susceptible to voriconazole, according to both methods. The measure of agreement [Kappa Index] for these three drugs was satisfactory [>/= 0.6]. According to the MD method, 69.4% of the species were susceptible to itraconazole, whereas 30.6% were not. For this drug, the Etest showed 86.1% susceptible and 13.9% resistant. Voriconazole was the most effective agent against isolates. Using RPMI agar, we found the Etest to be helpful, readily available, and easy to use for determining invitro susceptibilities of Aspergillus species to voriconazole, amphotericin B, ketoconazole, and itraconazole in the region of this study

Characterization of Pseudomonas aeruginosa strains isolated from burned patients hospitalized in a major burn center in Tehran, Iran

Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen and plays a prominent role in serious infections in burned patients. The current study was undertaken to characterize P. aeruginosa strains isolated from burned patients in Tehran, Iran. The study was conducted in a major burn center in Tehran, Iran in 2007. A total of seventy specimens obtained from different clinical origin with positive culture results for P. aeruginosa were included in the study. Antimicrobial susceptibility test was performed according to the standard CLSI guideline. The relationship between the strains was also determined using antimicrobial drug resistance pattern analysis and plasmid profiling. All strains were multi drug resistant. The percentage of resistance to tested antibiotics was: imipenem 97.5%, amikacin 90%, piperacillin 87.5%, ceftizoxime 72.7%, gentamicin 67.5%, ciprofloxacin 65%, ceftriaxone 60%, and ceftazidime 57.5%. Thirteen resistant phenotypes were recognized, R3 [TET, IPM, AMK, CIP, PIP, GM, CAZ, CRO, CT] was the predominant resistance pattern seen in 27.5% of isolates. Results obtained from Etest showed that 100% of P. aeruginosa strains were resistant to cefoxitin, 97% to cefotetan, 93% to ticarcillin, 89% to ticarcillin/clav, 76% to gentamicin and imipenem, 63% to piperacillin, 49% to tetracycline, and 20% to meropenem. Nine different plasmid profiles were observed among the strains. The current study showed an increase rate of resistance for some antibiotics tested among P. aeruginosa strains isolated from burned patients in Tehran. A combination of antibiotic susceptibility testing and profile plasmid analysis, which are relatively cheap and available methods, showed to be useful to characterize the clinical strains of P. aeruginosa isolated from burned patients in Iran

cholera outbreak associated with drinking contaminated well water

Cholera has been a significant public health challenge in many communities. An outbreak of acute diarrheal illness occurred among participants in a wedding ceremony in a village in Qazvin, Iran, in 2008. We conducted an epidemiological, environmental and microbiological investigation to determine the causative agent, source and extent of this outbreak. Clinical and environmental samples were collected and analyzed for the presence of diarrhea-causing bacterial organisms, which included Vibrio cholera. The relationship between the strains was determined using enterobacterial repetitive intergenic consensus poly-merase chain reaction [ERIC-PCR]. The attack rate was 21.8%. Clinical and environmental samples were positive for V. cholerae serotype Inaba. All tested isolates had a similar ERIC-PCR pattern, which indicated that a single clone of V. cholerae was responsible for this outbreak. Our findings demonstrated that well water was the source of this outbreak

Relationship between O serotype and virulent genes in Escherichia coli causing urinary tract infections

Escherichia coli are the most frequent pathogens in acute urinary infections. They are classified based on various types of O antigen. Escherichia coli strains that cause urinary tract infections possess several genes encoding urovirulent factors. To assay the relation of virulent factors of E coli in acute urinary infections, the serotypes and virulence factor genotypes were determined. We studied 96 E coli isolates from children with acute urinary infections. Four urovirulence determinants were analyzed by DNA colony hybridization, including the genes for type 1 fimbriae [pil], P fimbriae [pap], S fimbriae [sfa], hemolysin [hly], and cytotoxic necrotizing factor 1 [cnfl]. O serotypes were also determined. The most frequently found virulence factor-encoding gene in the E coli strains studied was the gene for type 1 fimbriae [27.4%]. The prevalence of pap, sfa, hly, and cnf1 were higher in serotypes causing pyelonephritis than cystitis. The most common type of O antigen was O1 [12.2%]. There was a significant correlation between serotype and genotype in uropathogenic E coli. The high prevalence of O6 serotypes in children urinary tract infections and the high percentage of virulent genes in serotype O6 suggested a close relation between serotype and genotypes of uropathogen E coli

Occurrence of class 2 integrons among multi-drug resistant Shigella sonnei isolated from Tehran, Iran in 2005

Shigellosis is one of the major causes of morbidity in children with diarrhea in Iran. Integrons play an important role in the evolution and dissemination of multidrug resistance in gram-negative bacteria. The occurrence of integrons among Shigella spp. is frequently reported throughout the world. The aim of this study was to assess the occurrence of class 2 integrons among the multi drug resistant S. sonnei isolated from Iranian children in 2005. The study was conducted in two major pediatric hospitals in Tehran, Iran. Fecal specimens and rectal swab collected from patients were cultured and identified as Shigella by the conventional methods. Antimicrobial susceptibility test was performed according to the standard CLSI guideline. Multi-drug resistant isolates of S. sonnei were further examined for the presence of class 2 integron by PCR using specific primers. Amplicons were confirmed by restriction endonuclease analysis. A total of 83 multi-drug resistant S. sonnei strains were isolated. Of these, 45 [54%] exhibited a class 2 integron of 2.1 kbp, and 34 [41%] a class 2 integron of 1.3 kbp. Class 2 integrons were not detected in four isolates. The results showed an increased occurrence of class 2 integron carrying S. sonnei isolated from children in Tehran in 2005

Il-1 beta [+3953 C/T] and IL-8 [-251 A/T] gene polymorphisms in H.pylori mediated gastric disorders

Previous studies imply that IL-1 and IL-8 gene variations may play a crucial role in the genetic predisposition to different gastric disorders upon H. pylori infection. The aim of this study was to determine the potential association between the prevalence of certain polymorphic sites and the risk of gastric disorders in Iranian population. One hundred and forty three unrelated individuals with different gastric disorders and 374 normal individuals with no gastric disorders and with a negative serology test for H. pylori [control group] were studied for the association between IL-1 beta [+ 3953 C/T] and IL-8 [-251 A/T] gene polymorphisms and H. pylori - mediated gastritis and gastric ulcer. An analysis of genotype frequency for these genes was performed using RFLP- PCR. Based on the data obtained from culture and pathologic findings, the patients were classified into three subpopulations: H pylori [+] non-ulcerative gastritis [+], H. pylori [+] ulcerative gastritis [+] and H. pylori[-] non-ulcerative gastritis [+]. A significantly higher frequency of TT genotype [p=0.02] in IL-1 beta +3953 in H.pylor[+] ulcerative gastritis [+] was revealed compared to the control group. There were no significant differences among other subpopulations. No significant differences in allele and genotype frequencies of IL-8 [-251A/T] were found among the patients. The data suggest that TT genotype in IL- 1 beta +3953 may be a major contributing genetic risk factor for H. pylori induced gastric ulcer. Moreover, the role of other bacterial and host response factors, such as bacterial adherence peptides, host chemokines, and genes involved in gastric acid secretion, must be further investigated in different ethnic populations

Fatality due to shigellosis with special reference to molecular analysis vi Shigella sonnet strains isolated from the fatal cases

Shigellosis as a global human health problem is more severe than other forms of gastroenteritis and causes over a million deaths in developing countries worldwide annually. Fatality due to shigellosis is usually due to dehydration and two-third of fatalities are seen among children. The aim of current study was to describe fatal cases of shigellosis due to infection with Shigella sonnei and S.flexneri. We investigated the fatal cases of shigellosis among all children with acute diarrhea admitted to Children's Medical Center, Tehran, Iran. Bacterial isolation and identification was achieved according to standard bacteriological methods. Antibiotic susceptibility tests, plasmid profiling and ribotyping were performed to investigate the clonal relationship among the isolates. Among 1200 children with acute diarrhea, 140 [12.7%] cases had shigellosis. Of these, three patients died. No signs of severe dehydration were observed among the fatal cases. The symptoms were not improved following antibiotic therapy and all three cases died after 24 h of hospitalization despite receiving intensive treatments. Stool cultures yielded S.flexneri and S. sonnei for one and two cases, respectively. The isolates were resistant to streptomycin, ampicillin, and sulfamethoxazole-trimethoprim. 5. sonnei strains were further studied and showed a single pattern of antibiotic susceptibility and ribotyping. Mortality due to species other than 5. dysenteriae is rare, however, in current study we found S. sonnei and S.flexneri as the cause of fatality among pediatric patients during the study

Changing prevalence of Helicobacter pylori in south of Iran

The prevalence of Helicobacter pylori has declined rapidly in Asia. This has been shown in both seroprevalence-based and endoscopy-based studies. The present study was conducted to determine the prevalence of gastric infection caused byH. pylori in an Iranian population residing in south of Iran. A total of 522 patients [266 females and 256 males with the mean age of 44.3 +/- 13.0, range 18-83 years] underwent endoscopy in Shiraz, southern Iran. The diagnosis of H. pylori infection was established by rapid urease test, culture and gram staining and the gastric disease was confirmed by an expert pathologist. From ulcerative [n=296] and non-ulcerative [n=226] patients, 156 [52.7%] and 94 [41.6%] H. pylori strains were isolated by culture, respectively. The prevalence of H. pylori infection was significantly higher in patients aged 21-30 and >50 years [66.66% and 62.12%, respectively]. However, H. pylori was not detected in 22 patients aged < 20 years The present study revealed a significant decline in the prevalence of H pylori infection in the studied population. It seems that in parallel with better therapeutic approaches and elimination of bacteria, an improvement in the personal hygiene and living conditions of the Iranian population contribute to lower prevalence of H. pylori

Evaluation of a PCR based approach to study the relatedness among Shigella sonnei strains

Infections caused by Shigella re a major cause of diarrheal disease in the developing and developed countries. The present study was conducted to apply and evaluate arbitrarily primed PCR [AP-PCR] for investigation of genetic relatedness among the strains of Shigella sonnei isolated from cases of acute diarrhea in Tehran. Totally, 60 S. sonnei strains isolated from children hospitalized due to enteritis at five hospitals in Tehran during 2003 and two sporadic isolates recovered in 1984 were investigated. Molecular typing was performed by AP-PCR. Depending on the number and size of amplified DNA bands, the strains were clustered into AP-PCR profiles. All strains of S. sonnei were typeable with this approach. AP-PCR generated nine indistinguishable bands ranged from 0.35 to 2.5 kbp in all studied strains. Only a single AP-PCR pattern was observed among the S. sonnei strains recovered in 2003. Two sporadic isolates recovered in 1984 showed different AP-PCR patterns compared to recent clinical isolates. Results suggest that a very homogeneous AP-PCR cluster types might be responsible for shigellosis caused by S. sonnei in Tehran in 2003. Further molecular analysis conducted on a larger selection of isolates could confirm our findings

Comparison of arbitrarily primed-polymerase chain reaction and plasmid profiles typing of Pseudomonas aeruginosa strains from burn patients and hospital environment

To identify the strengths and weakness of arbitrary primed-polymerase chain reaction [AP-PCR] and plasmid profiles for typing of Pseudomonas aeruginosa [P. aeruginosa] and tracking of source of infections. Seventy-four strains of P. aeruginosa were isolated from burn patients and hospital environment between January to April 2003 in Ghotbadden Burn Hospital, Shiraz, Iran. The strains were classified by photo Capt Mw program, similarity and clustering of strains were assessed using NTSYS-PC version 2.02K software. Based on 50% and 64.7% and 67.5% similarity on the plotted dendrogram, 38 plasmid profiles were classified into: 2, 3 and 5 clusters, respectively. Photo Capt Mw program categorized AP-PCR products to 47 different types of 6 to 12 bands between 0.376 to 3.7 kb. Based on dendrogram pattern 3 levels [62%, 81% and 84.6%] of similarity were selected. Using these criteria 2, 5and 11 clusters were obtained, respectively. As compared with plasmid profiles, AP-PCR analysis protocol is rapid, reproducible and differentiated the isolates with higher discrimination power. These results suggest that during admission of patients in burn center a limited number of common strains cross-contaminate burn victims. However, transmissions of infection from hospital environment to patients also occur in the minority of the victims. To control cross-contamination of the patient wounds with antibiotics resistant isolates, strong disinfection of patients' bathroom after scrubbing of each patient wounds is mandatory

Effect of cyclophosphamide on the course of candida albicans infection in normal and vaccimated mice

To evaluate the immunomodulating effect of cyclophosphamide [Cy] on the course of Candida albicans [C. albicans]. We performed this study in the Shiraz Medical School, Shiraz, Iran during April to November 2003. Five groups of 10 mice [vaccinated group] were immunized by 5 equal injections of 2x105, 2.5x105 and 3x105 of the organism intraperitoneally. Then, the group received Cy on day zero and was challenged with lethal doses of C. albicans [7.74x105 colony forming unit] on days zero, one, 3, 6 and 12 post-Cy injection. Another 5 equal groups of 10 mice [non-vaccinated group] received Cy on day zero and similar to vaccinated ones were challenged with lethal doses of the organism too. The control groups received just Cy on day zero and were sacrificed on days zero, one, 3, 6 and 12 days post-Cy injection. We performed the hemogram and the spleen and studied the renal tissues microscopically and macroscopically. In vaccinated group, we observed an increase in survival time and in spleen and renal weights were visible while in non-vaccinated ones, a significant decrease was also observed on days one and 3 and an increased on days 6 and 12 post-Cy injection. We observed atrophy and necrosis in the spleen while inflammation and necrosis were also observed in the kidneys on days one and 3. We noticed a significant hyperplasia in the white pulp on days 6 and 12 post-Cy injection. We conclude that hyperplasia in the white pulp of spleen and the increase in peripheral polymorphonuclears due to selective effects of Cy could effectively protect the animal against C. albicans infection

Immunodominant antigens of helicobacter pylori strains isolated from patients with different gastroduodenal diseases

To detect the immunogenic proteins in Helicobacter pylori [H. pylori] strains isolated from patients with different gastric diseases. We performed this study in the Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, during July 2003 to September 2004. Total proteins of H. pylori strains isolated from the gastric biopsies of 3 groups of patients were separated by 1D-SDS-PAGE and then blotted with the sera of their respective hosts. In SDS-PAGE the members of each group showed high correlation according to similarity in their patterns, resulting in considering them in the same cluster. The patterns of immunoblots differed from that of Coomassie Brilliant Blue stained gels. The blotting method did not recognize some of the protein bands in the SDS-PAGE. Only the bands of 106 and 45 kDa from H. pylori strains isolated from patients with gastric cancer were significantly [p<0.05] recognized specifically with the sera of their respective patients, and the band of 13 kDa was recognized specifically [p<0.05] with the sera of nonulceric patients. With the exception of these bands, in the patterns of blotting of the sera from all patients no significant differences were observed. By using 1D blotting methods we could find 2 antigenic protein bands [106 and 45 kDa] for H. pylori strains isolated from cancerous patients, and one [13 kDa] for the strains isolated from nonulceric patients, which were specifically recognized with their respective host

Discrimination of dominant helicobacter pylori strains isolated from patients with different gastroduodenal pathologies by protein profiling

It is not clear what factors determine divergent outcomes of infections caused by H. pylori. In the present study, the protein profiles of different strains of H. pylori, isolated from three groups of patients with ulcerative disease, non-ulcerative gastritis and cancer disease, were analyzed using 1D-SDS-PAGE. The patterns of different H. pylori strains were highly divergent. About 30.76% [7 bands] of the 26 observed protein bands were common in all strains isolated from 3 groups of the patients. While the similarity for the strains inside each group were 75% [15 from 20], 76.47% [13 from 17] and 78.57% [11 from 14] for cancerous, ulcerative and nonulcerative group, respectively. Some of the observed bands were significantly specific for each group. Therefore, we speculated that some H. pylori strains might be more associated with a specific disease than others, giving the clustering of some, but not all, strains within each disease group. In conclusion, this study showed that protein profile can be a characteristic in discrimination of dominant strains in different gastric clinical status. Specific and dominant proteins of different strains isolated from three groups of patients under study were candidates for further exploration for laboratory tests, which analyze disease-specific H. pylori strains, and for diagnosis of the different diseases and outcomes associated with this widespread bacterium

Distribution patterns of methicillin resistance genes [mecA] in staphylococcus arureus isolated from clinical specimens

There is a growing concern about the application of molecular methods in epidemiological studies of infectious diseases. The objective of this study was to determine the genetic stability of methicillin resistance genes in Staphylococcus aureus for the evaluation of resistance strain distribution. One hundred and fifteen S. aureus isolates from patients with staphylococcal infection were collected. The isolates were screened for methicillin resistance by minimum inhibitory concentration [MIC] examination. The stability of methcillin resistance genes was examined by physical curing and PCR screening methods. The results showed that methicillin resistance Staphylococcus aureus [MRSA] had risen up to 43% in Nemazi Hospital [Shiraz, Iran]. Indeed, the incidence of MRSA in our hospital was 10% during the last four years. The ability to lose [curability] of methicillin resistance genes [mecA] was examined by physical curing method in 49 isolates with MIC >/= 16 micro g ml -1. No sign of curability of mecA gene was observed where 500 colonies from each strain have been studied and exhibited by the same MIC values before and after curing test. Positive PCR results for isolates with MIC >/= 16 micro g ml -1 before and after curing experiment have been achieved. These data confirm the results of curing method, indicating that stable genetic determinants confer methicillin resistance. These results support the hypothesis that resistance isolates may be selected due to clonal selection under antibiotic pressure used in clinics rather than transmission of mobile genetic determinant. The high prevalence of MRSA immerged in our Hospital could be originated due to antibiotic pressure and poor control measures

Simultaneous detection of helicobacter genus and helicobacter pylori species using a multiplex PCR method

In order to improve simultaneous detection and identification of Helicobacter genus in general and Helicobacter pylori specifically and reduce the number of amplifications needed, we established a multiplex PCR. In this study, two pairs of primers: Hcom1 and Hcom2 specific for Helicobacter genus, Hicd1 and Hicd2 specific for Helicobacter pylori species were used. To determine the sensitivity of our multiplex PCR, the lower limits of DNA detection from pure culture were established using phenol-chloroform method. To evaluate the specificity of our protocol, we tested DNA extracted from various Gram-negative and -positive bacteria. A study was subsequently undertaken on stomach tissue samples from 18 patients to evaluate this protocol for detection of Helicobacter in tissue samples. In our optimized PCR, two fragments of 389- and 1200-bp were produced using Hcom1-Hcom2 and Hicd1-Hicd2 primers, respectively. Amplification of Helicobacter pylori genomic DNA was achieved at concentration as low as 0.03 pg equivalent to 150 bacteria. No DNA amplification of other Gram- positive and -negative bacteria was seen. Twelve specimens, positive in culture and rapid urease test for Helicobacter pylori were positive for DNA of this organism using this multiplex PCR. Our results demonstrate that this protocol represents a specific and sensitive assay for simultaneous detection of Helicobacter genus members, in general, and Helicobacter pylori species, specifically, in clinical samples yielding no false-positive

Production and characterization of monoclonal antibodies against brucella S [99] surface antigens

By immunizing mice with killed whole bacterial cells of Brucella abortus S [99], a panel of six hybridomas producing monoclonal antibodies [mAb] specific for the surface antigens of this bacterium were produced. ELISA was used to screen the hybridoma supernatants. Immunoblots of the cell extract indicated that three mAb were specific for S-LPS [Ba-1, Ba-2, Ba-3] and three others were reactive with major outer membrane proteins [OMP] [Ba-4, Ba-5, Ba-6]. The OMP recognized by these antibodies were the proteins with molecular masses of 25-27 kDa [Ba-4, Ba-5] and 36-38 kDa [Ba-6]. None of the four mAb including Ba-3, Ba-4, Ba-5 and Ba-6 cross reacted with any other bacteria close to Brucella abortus, but Ba-1 and Ba-2 cross reacted with B. melitensis 16M and B. suis. By using cell extract and killed whole cell Ag in ELISA, it was indicated that all mAb except Ba-6 have better reactivity with cell extract Ag, but Ba-6 mAb reacted with killed whole cell Ag better than cell extract Ag