Particular distribution of enterobacter cloacae strains isolated from urinary tract infection within clonal complexes

Background: Based on biochemical properties, Enterobacter cloacae represents a large complex of at least 13 variant species, subspecies, and genotypes that progressively identified as the most species causing hospital-acquired infections. The aim of this study was to determine the relevance between phylogenetically related strains within the E. cloacae complex and the frequency of urinary tract infection caused by them

Methods: A 268-bp fragment was obtained from hsp60 gene for 50 clinical E. cloacae isolates from urine cultures of inpatients that admitted to six hospitals in Tehran, Iran during December 2012 to November 2013. The 107 nucleo de sequences were analyzed and the evolutionary distances of sequences were computed and neighbor-joining tree was calculated

Results: It showed that all of the genetic clusters have not an equal involvement in pathogenesis of urinary tract infections. Three superior clusters were found, together representing more than two third [80%] of the isolates [cluster VI with 25 members; clusters III and VIII with 9 and 6 members, respec vely] andsome genetic clusters were absent [IV, X, XII, and xiii], some of which are supposed to be associated with plants and no human infection has been reported

Conclusions: This study, for the first time, reports the unequal contribution of E. cloacae complex subspecies and clusters in urinary tract infections in Iran and together with studies from other countries suggest that the subspecies of E.hormaechei subsp. Oharae is the most prevalent E. cloacae complex subspecies regardless of country under study

Comparison of biofilm formation between methicillin-resistant and methicillin-susceptible isolates of Staphylococcus aureus

Background: The aim of this study was to compare the biofilm formation and the prevalence of biofilm-associated genes between the isolates of methicillin-resistant [MRSA] and methicillin-susceptible [MSSA] Staphylococcus aureus

Methods: In total, 209 S. aureus isolates were collected. The antibiotic susceptibility test was conducted using nine antibiotics according to the guidelines of Clinical and Laboratory Standards Institute. Phenotypic biofilm formation was performed with microtiter plate assay. The polymerase chain reaction was employed to detect icaA, icaD, icaB, icaC, clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, bbp, mecA, and SCCmec types as well as agr group genes with specific primers

Results: Sixty-four [30.62%] isolates were resistant to methicillin, and 54 [83%] MRSA harbored SCCmec III. Furthermore, 122 [58.3%] isolates belonged to agr group I. Twenty-six [36.1%] MRSA and 42 [28.9%] MSSA isolates were strong biofilm producers [no significant difference]. The prevalence of icaA, icaD, icaB, and icaC genes in MSSA isolates was 71, 41, 76, and 72%, respectively. The frequency of clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, and bbp in MSSA was 100, 100, 56, 46, 74, 54, 78%, 11, and 1%, respectively. However, in MRSA isolates, the frequency was 97, 97, 64, 51, 76, 56, 79, and 12% with no track of bbp, respectively

Conclusion: Statistical difference between MSSA and MRSA regarding biofilm formation and the frequency of all biofilm-encoding genes was not significant. The majority of the S. aureus isolates harbored clfA, clfB, eno, fib, icaA, and icaD genes

molecular survey of campylobacter jejuni and campylobacter coli virulence and diversity

The aim of this study was to determine the prevalence of virulence-associated genes and enterobacterial repetitive intergenic consensus PCR [ERIC-PCR] analysis of Campylobacter spp. isolated from children with diarrhea in Iran. A total of 200 stool specimens were obtained from children under 5 years during July 2012 to July 2013. Detection of C. jejuni and C. coli was performed by standard biochemical and molecular methods. The presence of virulence-associated genes and genetic diversity of isolates was examined using PCR and ERIC-PCR analyses. A total of 12 [6%] Campylobacter spp. were isolated from patients including 10 [4.5%] C. jejuni and 2 [1.5%] C.coli. The flaA, cadF and ciaB genes were present in 100% of isolates, while no plasmid of virB11 gene was present in their genome. The prevalence of invasion-associated marker was 100% among C. coli and was not detected in C. jejuni isolates. The distribution of both pldA and the genes associated with cytolethal distending toxin [CDT] was 58.3% in C. jejuni isolates. Seven distinct ERIC-PCR profiles were distinguished in three clusters using ERIC-PCR analysis. Genotyping analysis showed a relative correlation with geographic location of patients and virulence gene content of isolates. To our knowledge, this is the first molecular survey of Campylobacter spp. in Iran concerning genotyping and virulence gene content of both C. jejuni and C. coli. ERIC-PCR revealed appropriate discriminatory power for clustering C. jejuni isolates with identical virulence gene content. However, more studies are needed to clearly understand the pathogenesis properties of specific genotypes

Protective properties of nontoxic recombinant exotoxin A [domain I-II] against pseudomonas aeruginosa infection

Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. In this study, recombinant exotoxin A [domains I and II], [ExoA I-II] protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A [Domains I, II]. Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 [7.5 x 10[7] CFU] of clinical strain of mucoid P. aeruginosa. Sequencing of the cloned gene showed that the sequence of ExoA one-two gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA one-two increased survival times against intra-peritoneal challenge with an approximate 7.5 x 10[7] CFU [2 x LD50] of clinical strain of P. aeruginosa. Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa

Biological activity of recombinant accessory cholerae enterotoxin [Ace] on rabbit ileal loops and antibacterial assay

Vibrio cholerae [V. cholerae] causes a potentially lethal disease named cholera. The cholera enterotoxin [CT] is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin [Zot] and accessory cholera enterotoxin [Ace]. The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli [E. coli] and determination of some characteristics of the recombinant Ace protein. In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli [DH5 alpha] host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-beta-D-galctoside [IPTG] at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus [S. aureus], and Pseudomonas aeruginosa [P. aeruginosa]. The recombinant Ace protein with a molecular weight of 18 kDa [dimeric form] was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of >/= 200 micro g/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. This study described an E. coli cloning and expression system [E. coli BL21- pET-28a-ace] for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein

Cloning and expression of S1 subunit of pertussis toxin in Escherichia coli

Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human [whooping cough]. Pertussis toxin [PT] is the main component of current acellular pertussis vaccine and the S1 [subunit1] is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14, pET-22b[+] and pAED4. The possibility and level of expression of these constructs were investigated in BL21 [DE3] strain of Escherichia coli [E.coli] as expression host. The highest expression was in pET-22b[+]-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking [250 rpm]. Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway

[Cloning, expression, purification and antigenicity of recombinant UreB332-HpaA fusion protein from Helicobacter pylori]

Helicobacter pylori is a widely distributed Gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in developing a H. pylori vaccine is growing up rapidly. The aim of this study was to construct a recombinant vector containing fusion genes encoding a fragment of B subunit from Helicobacter pylori [H. pylori] urease [UreB332] and Helicobacter pylori adhesion A [HpaA] and expressed it in E. coli BL21, as well as determining its antigenicity as a vaccine candidate of H. pylori. The target genes encoding UreB332 and HpaA amplified from standard H. pylori chromosome by PCR, digested by restricted endonuclease enzyme and inserted into the prokaryotic expression vector pET28a [+] which was digested by corresponding restricted endonuclease enzyme. The target fusion protein was expressed in the BL21 [DE3] E. coli. Furthermore, UreB332-HpaA antigenicity was studied by western blotting after Ni-NTA agarose resin purification. Enzyme digestion analysis, PCR and sequencing showed that the target genes were inserted correctly into the recombinant vector. The fusion protein UreB332-HpaA was recognized by the rabbit anti H. pylori polyclonal antibody and the human sera infected with H. pylori. Our results in addition to favorable properties of HpaA and UreB antigens, support the application of rUreB332-HpaA fusion protein, as a good candidate for the development of H. pylori vaccine

Cloning, expression, purification and characterization of recorabinant UreB[229-561] from Helicobacter pylori

Helicobacter pylori is a widely distributed gram negative bacterium that infects the human stomach and duodenum. Some antibiotic regimens are subjected to cure the infection but the cost of drugs, poor patient compliance and emerging of antibiotic-resistant strains are limiting the usefulness of these antibiotic therapies. Therefore, interest in a H. pylori vaccine is growing up rapidly. We selected a fragment of B subunit of H. pylori urease enzyme consist of four important epitopes, involving in elevating host immune responses. This 1070bp fragment was amplified by PCR from genomic DNA isolated from H. pylori 22596 and then cloned into the pET28a expression vector. UreB229-561 was expressed and then affinity-purified by Ni2+-Sepharose resin. The recombinant UreB229-561 was reacted with the serum of/. pylori-infected human and rabbit anti-H. pylori polyclonal antibody in western-blot analysis. Having transformed competent E.coli DH5 alpha with ligation product of digested ureB fragment and pET28a, plasmid extraction from single colonies appeared in LB-agar plate after 18-24 h incubation at 37°C, using plasmid extraction kit [Bioneer, Korea]. Applying both infected human serum and rabbit anti-H. pylori polyclonal antibody, brown strip corresponding to the location of the recombinant protein appeared on PVDF membrane after adding DAB solution, hence confirming the antigenicity of the protein. This recombinant fragment showed urease activity. Our findings confirmed that a prokaryotic expression system of rUreB[229-561] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pET28 alpha- ureB[229-561]-BL21DE3 efficiently produces target recombinant protein in the form of dissoluble inclusion body. Therefore we can suggest that these epitopes can effectively be a vaccine candidate

Identification of methicillin-resistant Staphylococcus aureus by disk diffusion method, determination of MIC and PCR for mecA gene

Staphylococcus aureus is an important cause of serious infection in both hospital and the community. Methicillin-resistant S. aureus [MRSA] is associated with high morbidity and mortality rates with rapid development of resistance. There is a need for early and reliable detection of MRSA infection to direct antibiotic therapy, and more effectively control cross-infection. In this study, resistance to methicillin was detected by a disk diffusion method, the determination of MIC, and the PCR for mecA gene. A total of 174 S.aureus strains were isolated from different clinical specimens from three teaching Hospitals. Antibiotic susceptibility was determined by disk diffusion method, MIC for oxacillin was made by the agar dilution, and mecA gene was identified by specific primers. The prevalence of MRSA by three methods ranged from 47% to 50%, and mecA positive isolates were more resistant to all of the antibiotic tested than mecA negative isolates. All S. aureus isolates were resistant to penicillin, and susceptible to vancomycin. The results of agar dilution test indicated a low-level resistance to methicillin [MIC<64mg/l]. The distribution of MRSA isolates were uniform between three hospitals, and there were not significant differences in the presence of MRSA between isolates from different clinical specimens. The PCR method was the best test for routine detection of MRSA in the present study. An additional benefit of the mecA PCR is the potential to generate a susceptibility report, 24h earlier than the time of generation of results of conventional susceptibility testing methods