Distribution of genes encoding virulence factors and molecular analysis of Shigella spp. isolated from patients with diarrhea in Kerman, Iran

Shigella is one of the important causes of diarrhea worldwide. Shigella has several virulence factors contributing in colonization and invasion of epithelial cells and eventually death of host cells. The present study was performed in order to investigate the distribution of virulence factors genes in Shigella spp. isolated from patients with acute diarrhea in Kerman, Iran as well as the genetic relationship of these isolates. A total of 56 isolates including 31 S. flexneri, 18 S. sonnei and 7 S. boydii were evaluated by polymerase chain reaction (PCR) for the presence of 11 virulence genes (ipaH, ial, set1A, set1B, sen, virF, invE, sat, sigA, pic and sepA). Then, the clonal relationship of these strains was analyzed by multilocus variable-number tandem repeat analysis (MLVA) method. All isolates were positive for ipaH gene. The other genes include ial, invE and virF were found in 80.4%, 60.7% and 67.9% of the isolates, respectively. Both set1A and set1B were detected in 32.3% of S. flexneri isolates, whereas 66.1% of the isolates belonging to different serogroup carried sen gene. The sat gene was present in all S. flexneri isolates, but not in the S. sonnei and S. boydii isolates. The result showed, 30.4% of isolates were simultaneously positive and the rest of the isolates were negative for sepA and pic genes. The Shigella isolates were divided into 29 MLVA types. This study, for the first time, investigated distribution of 11 virulence genes in Shigella spp. Our results revealed heterogeneity of virulence genes in different Shigella serogroups. Furthermore, the strains belonging to the same species had little diversity

Molecular diagnosis and anti-microbial resistance patterns among Shigella spp. isolated from patients with diarrhea

Aim: This study aims to determine the serogroup distribution and molecular diagnosis, as well as antimicrobial resistance profiles among Shigella spp. isolated from patients with diarrhea in Kerman, southeast of Iran. Background: Shigella species are frequent cause of bacterial dysentery worldwide. Previous studies have been reported that S. sonnei and S. flexneri are the most prevalent serogroups in various parts of Iran. Patients and methods: A total of 624 stool samples were randomly collected from patients with diarrhea from June 2013 to August 2014. Biochemical and serological characterizations were performed for identifying Shigella spp. In addition, the multiplex PCR assay was carried out for the detection and differentiation of three pathogenic Shigella spp. Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Results: Fifty six (9%) Shigella strains were isolated from stool samples. The most common species were S. flexneri 31(55.4%), followed by S. sonnei 18(32.1%) and S. boydii 7(12.5%). S. dysentery was not detected in the present study. All the isolates that identified by serological test as Shigella spp. were confirmed by the multiplex PCR method. The highest rate of resistance was observed for ampicillin and trimethoprim-sulphamethoxazole antibiotics with 52(92.9%) resistant, followed by tetracycline 44(78.6%) and cefotaxime 33(58.9%). All Shigella isolates were susceptible to ciprofloxacin. A significant relationship was found between the Shigella species and cefotaxime resistance (p<0.05). Conclusion: S. flexneri was found as the most prevalent serogroup causing shigellosis. The high rate of resistance to third-generation cephalosporins limits the treatment options available for the management of shigellosis in Kerman, Iran

Virulence Gene Profile and Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) of Enteroinvasive Escherichia coli (EIEC) Isolates From Patients With Diarrhea in Kerman, Iran

Background: Enteroinvasive Escherichia coli (EIEC) isolates cause dysentery in humans. Several virulence factors associated with EIEC pathogenesis have been characterized. Multilocus variable-number tandem-repeat analysis (MLVA) is a PCR-based method that has been used for genotyping bacterial pathogens. Objectives: The aim of this study was to investigate the distribution of virulence factor genes in EIEC isolates from patients with diarrhea in Kerman, Iran, as well as the genetic relationships between these isolates. Patients and Methods: A total of 620 diarrheic stool samples were collected from patients attending two hospitals in Kerman from June 2013 to August 2014. All isolates were confirmed as EIEC by PCR for the ipaH gene. The EIEC isolates were evaluated by PCR for the presence of nine virulence genes (ial, set1A, sen, virF, invE, sat, sigA, pic, and sepA). MLVA was performed for all EIEC isolates. Results: A total of 11 EIEC isolates were identified, and all were positive for the ial gene. The invE and virF genes were observed in 81.8% of the isolates, while sen, sigA, and pic were detected in 72.7%, 63.6%, and 27.3% of the isolates, respectively. None of the isolates were positive for the sat, set, and sepA genes. Using MLVA, the 11 total isolates were divided into five types. Conclusions: By studying the profiles of virulence genes and MLVA, it can be concluded that EIEC isolates do not have high heterogeneity and are derived from a limited number of clones. Keywords: EIEC, MLVA, Virulence Factors, Diarrhe

Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii

Background: Acinetobacter baumanniiis commonly resistant to nearly all antibiotics due to presence of antibiotic resistance genes and biofilm formation. In this study we determined the presence of certain antibiotic-resistance genes associated with biofilm production and the influence of low iron concentration on expression of the biofilmassociated protein gene (bap) in development of biofilm among multi-drug-resistant A. baumannii (MDRAB). Methods: Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR). Biofilm formation was assayed by the microtiter method. Results: The sequence of bap was determined and deposited in the GenBank database (accession no. KR080550.1). Expression of bap in the presence of low iron was analyzed by relative quantitative real time PCR (rqRT-PCR). Nearly half of the isolates belonged to RAPD-types A and B remaining were either small clusters or singleton. The results of biofilm formation revealed that 23 (35.4%), 18 (27.7%), 13 (20%), and 11 (16.9%) of the isolates had strong, moderate, weak, and no biofilm activities, respectively. ompA and csuE genes were detected in all, while bap and blaPER-1 were detected in 43 (66%) and 42 (64%) of the isolates that showed strong and moderate biofilm activities (p ≤ 0.05), respectively. Analysis ofbapexpression by rqRT-PCR revealed five isolates with fourfold bap overexpression in the presence of low iron concentration (20 µM). Conclusion: The results suggest thatbapoverexpression may influence biofilm formation in presence of low iron concentration

Iron limitation enhances acyl homoserine lactone (AHL) production and biofilm formation in clinical isolates of Acinetobacter baumannii

Acinetobacter baumannii is an important source of infections in intensive care units (ICUs) of our hospitals in Kerman, Iran and the most frequently isolated strains produce biofilm. There is a little information about role of iron (Fe) levels on acyl homoserine lactone (AHL) production and biofilm formation in this microorganism. In the present study, we investigated the influence of iron-III limitation on AHL, siderophore, catechol and virulence factors in the biofilm forming clinical strains of A. baumannii. A total of 65 non-duplicated multidrug resistance (MDR) strains of A. baumannii were isolated from patients in ICUs of 2 hospitals in Kerman, Iran. Antibiotic susceptibility, siderophore and other iron chelators, hemolysis, cell twitching motility, capsule, gelatinase and DNase were studied. Presence of quorum sensing, LuxI and LuxR genes was detected by multiplex-PCR. AHL activity quantified by colorimetric method and the functional groups were determined by Fourier Transform Infra-Red Spectroscopy (FT-IR). Biofilm formation was detected by microtiter plate technique. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin, levofloxacin, tetracycline, whereas, 78% and 81% were resistant to amikacin and carbapenems, respectively. The siderophore activity was highest at 20 mM Fe3C (70%); however, it decreased to 45% as concentration of Fe3C increased to 80 mM. Furthermore, screening of the isolates for LuxI and LuxR genes showed that presence of both genes required in the isolates with high AHL activity. FT-IR analysis indicated CDO bond of the lactone ring and primary amides. Significantly, a higher amount of AHL (70%) was detected in the presence of low concentration of iron-III (20 mM); as iron concentration increased to 80 mM, the AHL activity was reduced to 40% (P � 0.05). All the isolates exhibited twitching motility and had a capsule. No any gelatinase or DNase activity was detected. Quantification of the biofilm formation introduced 23 isolates with efficient attachment to microplate wells and strong biofilm. We found that both the AHL production and biofilm formation were regulated by iron concentration in a dose dependent manner. These findings provide evidence that iron limitation plays an important regulatory role in AHL and siderophore production resulting in strong or weak biofilm, thereby helping the organism to persist in less available micronutrient environment

Prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) and Antibiotic Resistance Patterns of the Isolates from the Nose of Training Soldiers in Kerman in 2012

Background and Aim: The increases in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) from outside healthcare settings are recently reported. This study was performed to determine the prevalence of community-acquired MRSA and antibiotic resistance of the isolates from nose of military training soldiers in Kerman city- 2012. Materials and Methods: Nasal samples were collected from 567 training soldiers. S. aureus was identified using standard Methods. MRSA phenotype was screened by oxacillin and cefoxitin disc. The MRSA was genetically confirmed by detection of mecA gene by PCR methods. Antibiotic susceptibility to six antibacterial agents was determined by standard agar disc diffusion method. Induction of resistance to clindamycin  was performed using the D-test. Results: samples were isolated from the nose of 39.8% of training soldiers. By the methods used 7.6% of the isolates were identified as MRSA all of them harbored mecA gene and were sensitive to vancomycin. The highest rate of resistance was detected against erythromycin (23.3%). Resistance to, clindamycin, gentamicin, trimethoprime sulfamethoxazole and ciprofloxacin were 14%, 16.3%, 14% and 9.3% respectively. Induced of Clindamycin resistance among MRSA isolates was not observed. Conclusions: The high frequency of isolation of&nbsp;S aureus from nose in military population studied is remarkable. The prevalence of MRSA in this group was %7.8.Although resistance to other antibacterial agents was not high in these isolates, but due to the increasing importance of community acquired MRSA frequent surveillance of this and similar type of population are required in intervals, and physicians should be informed about the presence of these bacteria to choose appropriate drug for therapy

Clonal relationships, antimicrobial susceptibilities, and molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates from urinary tract infections and fecal samples in Southeast Iran

Abstract INTRODUCTION: Multidrug-resistant (MDR) Escherichia coli, a species that is a leading cause of urinary tract infections (UTIs) and is a major global public health concern. This study was designed to detect the differences in antibiotic resistance patterns, the production and type of extended spectrum β-lactamases (ESBLs), and the clonal relationships among E. coli isolates from UTIs and fecal samples. METHODS: Antibacterial resistance was determined by the disk diffusion method. ESBL, carbapenemase, and AmpC-producing isolates were detected phenotypically. Then, the ESBL genes were sequenced to detect the type. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed on the ESBL-positive isolates. RESULTS: The most common effective antibacterial agents were colistin, imipenem, and amikacin. Among the isolates, 204 (56.6%) were MDR. Of the 163 ESBL-positive isolates, 11 (6.7%) produced AmpC, and the frequencies of beta-lactamase-positive genes were as follows: bla CTX-Mgroup1, 76%; bla TEM1, 74.8%; bla SHV12, 1.2%; and bla OXA1, 12.88%. ERIC PCR showed a diverse pattern, suggesting that clonal spread of E. coli in this area is uncommon, and that most of the infecting strains are endogenous. CONCLUSIONS: The high rates of antibacterial-resistant and MDR isolates are quite important since these strains can act as source of resistant bacteria that can be spread in the community. Controlling antibiotic use, against inappropriate use and abuse, in the community and continuous surveillance of emerging resistance traits are critical to controlling the spread of resistance

The frequency of adherence, biofilm-associated, Arginine Catabolic Mobile element genes, and biofilm formation in clinical and healthcare worker coagulase-negative staphylococci isolates

Abstract Background Healthcare workers may pave the way for increased infections in hospitalized patients by coagulase-negative staphylococci (CoNS). Biofilm formation and antibiotic resistance are the major problems posed by CoNS in nosocomial infections. In this study, we determined biofilm production level and the distribution of biofilm-associated and virulence genes, including icaADBC, aap, bhp, atlE, embp, and fbe, as well as IS256, IS257, mecA, and ACME clusters (arc-A, opp-3AB) among 114 clinical (n = 57) and healthcare workers (n = 57) CoNS isolates in Kerman, Iran. Results In this study, more than 80% (n = 96) of isolates were methicillin-resistant CoNS (MR-CoNS). Out of 114 isolates, 33% (n = 38) were strong biofilm producers. Strong biofilm formation was found to be significantly different between clinical and healthcare workers’ isolates (P < 0.050). In addition, 28% (n = 32) of isolates were positive for icaADBC simultaneously, and all were strong biofilm producers. The prevalence of icaADBC, mecA, bhp, fbe, and IS256 in clinical isolates was higher than that in healthcare workers’ isolates (P < 0.050). A significant relationship was observed between clinical isolates and the presence of icaADBC, mecA, bhp, and IS256. Although these elements were detected in healthcare workers’ isolates, they were more frequent in clinical isolates compared to those of healthcare workers. Conclusions The high prevalence of ACME clusters in healthcare workers’ isolates and biofilm formation of these isolates partially confirms the bacterial colonization in the skin of healthcare workers. Isolating MR-CoNS from healthcare workers’ skin through similar genetic elements to clinical isolates, such as icaADBC, mecA, and IS256, calls for appropriate strategies to control and prevent hospital infections

Novel Combinations of Synthesized ZnO NPs and Ceftazidime: Evaluation of their Activity against Standards and New Clinically Isolated Pseudomonas aeruginosa

Abstract Background: Antibiotic resistant bacteria can be considered as a main problem in infection management. Zinc oxide nanoparticles (ZnO NPs), individually or in combination with antibiotics, can be considered as good candidates for struggling against drug resistant bacteria. Methods: In this study, Zinc oxide nanoparticles were synthesized using sol-gel method in low temperature as a cost effective procedure and characterized by X-ray diffraction and Scanning Electron Microscopy. Antibacterial activity of 9 new combinations of Zinc oxide nanoparticles and ceftazidime was assessed against standards and new clinically isolated multi drug resistant Pseudomonas aeruginosa (P. aeruginosa), in order to evaluate enhancement effect of synthesized Zinc oxide nanoparticles on antibacterial activity of ceftazidime. Results: The results indicated that desirable effects can be seen at 6 and 7 mM of Zinc oxide nanoparticles (60 to 100% inhibition). Moreover, after evaluation of 9 new combinations with various concentrations of both components, it was demonstrated that Zinc oxide nanoparticles can enhance the antibacterial activity of ceftazidime, against some bacterial strains of P. aeruginosa. The highest activity was observed with the concentration of 20 μg/ml ceftazidime in the presence of 5, 6 or 7 mM of Zinc oxide nanoparticles. Conclusion: Zinc oxide nanoparticles in appropriate concentrations can be proposed as new and promising candidates for overcoming bacterial resistance

Development and immunereactivity evaluation of a chimeric recombinant protein encoding <i>Brucella</i> antigen: <i>In silico </i> to <i>in vitro</i>

30-36Brucellosis is an important health problem in developing countries and no vaccine is available for the prevention of infection in humans. Because of clinically infectious disease and its economic consequences in human and animals, designing a proper vaccine against Brucella is desirable. In the present study, we evaluated the immune responses induced by a designed recombinant chimera protein and investigated the immunogenic potential of some immune reactive antigens of Brucella. Three immune dominant antigens of Brucella including trigger factor (TF), Omp31 and Bp26 (have been characterized as potential immunogenic and protective antigens) were fused together by EAAAK linkers to produce a chimera. Recombinant chimeric protein was synthesized, cloned and expressed in Escherichia coli BL21 (structure were designed in silico). The purification of recombinant protein was performed by using Ni-NTA agarose, and anti-His antibody was used for confirmation (Western blot). The recombinant chimeric protein could be a new potential antigen candidate for the development of a subunit vaccine against Brucella. These results demonstrate the role of the Bioinformatics in vaccine design, assisted by experimental procedures