Jundishapur Journal of Microbiology
Year:2017 | Volume:10 | Issue:11|Papers Count:8

Background: Candida albicans genotypes derived from PCR-based techniques targeting 25S rDNA have been shown to correlate with antifungal susceptibility and virulence factors. However, this method has not been used in isolates recovered from recurrent vulvovaginal candidiasis. Comparing intron-based genotypes with the drug susceptibility of isolates recovered from recurrent vulvovaginitis may aid in epidemiological studies and better patient management. Objectives: The purpose of this study was to investigate the genetic diversity of isolates involved in vulvovaginal candidiasis and their susceptibility to seven antifungal agents. Methods: Candida genotyping based on the presence or absence of a group I intron by PCR targeting 25S rDNA and drug susceptibility assays using a modified Alamar Blue broth microdilution method were carried out. Fisher’ s exact test and the Mann-Whitney U test were performed, and P < 0. 05 was considered statistically significant. Results: Candida albicans was the predominant species among 384 Candida isolates followed by C. glabrata in acute and C. tropicalis in recurrent vulvovaginitis. Candida albicans from acute infections were more susceptible to fluconazole than isolates from recurrent cases but more resistant to miconazole and amphotericin B. The dominant genotype A was resistant to itraconazole and demonstrated higher minimal inhibitory concentrations (MICs) to 5-fluorocytosine and itraconazole. All C. albicans and nonalbicans species. with the group I intron were susceptible to 5-fluorocytosine. Conclusions: Candida albicans is the predominant cause of acute and recurrent vulvovaginal candidiasis, and drug susceptibility differed significantly among type of infection. Strain differences based on the group I intron may not be clinically significant in disease recurrence. However, the presence or absence of the group I intron has significant impact on antifungal MICs and resistance. Moreover, the group I intron in C. albicans and non-albicans may be the most important factor related to 5-fluorocytosine susceptibility.

Background: Lactobacillus plantarum is a widespread probiotic bacterium. Many plasmids from L. plantarum have been identified to encode some important phenotypic traits including carbohydrate metabolism, bacteriocin synthesis, and exopolysaccharide production. Objectives: The aim of this study was to identify and characterize the native plasmids from L. plantarum BM4. Methods: Lactobacillus plantarum BM4 was isolated from fermented meat in Guangxi Province, China, and characterized by 16S rRNA sequence. Four plasmids were isolated from L. plantarum BM4, sequenced, and characterized by the bioinformatics method. Moreover, the relative copy numbers of these plasmids were estimated using the droplet digital PCR method. Results: Four plasmids, designated as pBM1, pBM2, pBM3, andpBM4, wereisolatedfromL. plantarumBM4. By nucleotide sequencing, pBM1, pBM2, pBM3, and pBM4 were characterized as having sizes of 6069 bp, 7042 bp, 8131 bp, and 8892 bp, and G+C contents of 37. 5%, 36. 7%, 36. 4%, and 34. 5%, respectively. Nucleotide sequence analysis revealed 8, 10, 10, and 10 putative open reading frames (ORFs) for pBM1, pBM2, pBM3, and pBM4 plasmids, respectively. Based on sequence alignment, only pBM2 contained replication protein RepB and rep3, which contained a putative repeat origin of replication segment, indicating that the pBM2 belongs to the pUCL287 subfamily of theta-type replicons. Finally, the relative copy numbers of pBM1-4 were estimated to be 82, 24, 34, and 16, copies, respectively. Conclusions: Four novel plasmids were isolated from L. plantarum BM4 and characterized. These backbones can potentially be developed for use as a cloning or expressing vectors in biotechnology applications.

Background: Escherichia coli can cause human diseases and cow mastitis. Use of antibiotics in the treatment of E. coli infections causes drug residues in dairy products and develops antibiotic resistance. Thus, it is of prime importance to find a new solution for the treatment of E. coli infection. Objectives: The present study aimed at constructing a novel cell epitope-based polypeptide vaccine (OmpC-EP) against E. coli infection. Methods: Based on the outer membrane protein C (OmpC) of E. coli, we used ABCpred and BepiPred method to obtain its B cell epitopes, whereas nHLAPred and ProPred methods were used to obtain the CTL and Th cell epitopes, respectively. The predicted cell epitopes were recomposed using DNASTAR software to obtain a cell epitope-based polypeptide (OmpC-EP) of high antigenicity, which was purified by Ni-NTA flow resin. Purified OmpC-EP was mice immunized to prepare a polyclonal antibody; Western blotting analysis was used to detect antibody specificity. Pull down and ELISA detected the interaction between OmpC-EP antibodies and E. coli. Active immunity mice and challenge of E. coli were used to detect the immune protection of CM-TEP. Results: Anovel epitope peptide (OmpC-EP), with aMWof 12. 5kDawasdesigned, andNi-NTApurifiedOmpC-EPwasmiceimmunized to prepare a polyclonal antibody. OmpC-EP antiserum had a good specificity and directly interacted with E. coli. Specific immunity was activated in mice, and OmpC-EP displayed a significant immune protective effect (62. 5%), which was slightly higher than that of the OmpC protein (56. 3%) following infection with E. coli. Conclusions: The results of the present study revealed that OmpC-EP has a good immunogenicity and possesses a significant immune protective function. OmpC-EP is expected to be an approach to construct a vaccine against E. coli.

Background: Despite the considerable clinical role of Helicobacter pylori, its certain routes of transmission and origin have not been reported. Based on the argumentative hypothesis, foods play an imperative role in the spread of H. pylori to humans. Objectives: The current research was done to investigate the prevalence rate and distribution of Vacuolating Cytotoxin A and Cytotoxin Associated Gene A genotypes in the H. pylori strains isolated from meat, milk, and vegetables. Methods: A total of 340 food samples were collected and directly moved to the laboratory. Samples were cultured and H. pylori colonies were approved using the gram staining, urease test, and 16s rRNA-based polymerase chain reaction (PCR) amplification. Positive strains were tested for distribution of vacA and cagA genotypes using the multiplex-PCR. Results: Outof 340samples, 40(11. 76%) harbored H. pylori. Prevalence of H. pylori in meat, milk, andvegetable sampleswere7. 33%, 16%, and 12. 50%, respectively. Ovine milk (26%) was the most commonly contaminated sample. The most commonly detected genotypes were vacA s1a (87. 50%), vacA m1a (87. 50%), vacA s2 (82. 50%), cagA (80%), and vacAm2(62. 50%). Genotypes of S1am1a (62. 50%), s2m1a (55%), s1am2 (50%), s2m2 (45%), and m1am2 (42. 50%) were the most commonly detected combined genotypes. Conclusions: Milk, vegetables, and meat, are latent sources of H. pylori. Similarity in the genotyping pattern of H. pylori strains of various samples represents their similar sources of infection. Further studies are required for finding the exact sources of H. pylori strains.

Introduction: An increase in the number of infections with Klebsiella pneumoniae producing Extended-Spectrum -Lactamase (ESBL+) is a clinical issue, because there are no guidelines regarding the correct choice of an antibiotic and duration of treatment in kidney transplant recipients. The bacterial capacity to degrade almost all -lactam antibiotics, except carbapenems, causes resistance to standard treatment and makes long-term intravenous antibiotic therapy necessary. Case Presentation: This report describes the case of a 44-year-old patient after kidney transplantation, who developed recurrent urinary tract infections (UTIs) caused by ESBL-producing K. pneumoniae despite prolonged antibiotic targeted treatments with imipenem/cilastatin, meropenem, imipenem/cilastatin with amikacin and oral phosphomycin for UTI prophylaxis. Ineffectiveness of previous treatments caused the necessity to use a non-standard therapy of the consecutive UTI episode with high doses of amoxicillin combined with standard doses of clavulanic acid to break the bacteria’ s resistance. There was no recurrence of UTI and control urine cultures were sterile over the entire course of treatment, lasting 165 days, and throughout the follow-up period of more than 1 year. Conclusions: It has been shown in this case that UTI with the aetiology of K. pneumoniae could be treated on an outpatient basis with high doses of amoxicillin in combination with standard doses of clavulanic acid followed by prolonged antibiotic prophylaxis.

Background: The hepatitis E virus (HEV) accounts for the hepatitis E infection with a high mortality rate in pregnant women. Therefore, the design of the novel and effective vaccines seems essential and the DNA vaccine approach could be useful to achieve this anticipated goal. Objectives: The aim of this study was the cloning of a secretory form of truncated open reading frame 2 (ORF2) of HEV containing amino acids (aa) 112-607 into the eukaryotic expression pVAX1 Vector and evaluating of the expression of this recombinant protein in eukaryotic cells. Methods: The truncated ORF2 gene (aa 112-607) was cloned in the pVAX1 plasmid by restriction enzyme digest and confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into eukaryotic cells to express the recombinant protein. The expressed protein in the cell lysate and supernatant was evaluated by immunofluorescence assay (IFA) and western blot assay. Results: Colony polymerase chain reactions (PCR), restriction enzyme digestion, along with DNA sequencing of the recombinant plasmid were performed for confirmation of cloning tPA-PADRE-truncated ORF2 gene (aa 112-607) into pVAX1 eukaryotic expression vector. The appearance of the truncated ORF2 protein (56 kDa) in the eukaryotic cells was accepted by the western blot assay, reverse transcription polymerase chain reaction (RT-PCR) method, as well as IFA. Conclusions: All outcomes of the present research showed that pVAX1-tPA-PADRE-truncated ORF2 (aa 112-607, 56 kDa) recombinant plasmid was able to express truncated ORF2 from HEV as a potential candidate vaccine.

Background: Staphylococcus aureus is an important cause of hospital-acquired infections. The most important issue with S. aureus is that the isolates are getting increasingly methicillin-resistant. Rapid differentiation between methicillin-resistant and methicillinsusceptible S. aureus species is necessary to optimize treatment and minimize costs. Objectives: The current study aimed at evaluating the StaResMet® kit for rapid detection of methicillin resistance in S. aureus isolates. Methods: A total of 217 methicillin-resistant S. aureus (MRSA) and 252 methicillin-susceptible S. aureus (MSSA) isolates were tested using the StaResMet® kit. The test was performed according to the manufacturer’ s instructions. Results: The kit identified the MRSA isolates with 100% accuracy, and found that the minimum inhibitory concentrations (MICs) was > 32  g/mL cefoxitin for 133 of the isolates, 32  g/mL cefoxitin for 49, 16  g/mL cefoxitin for 8, and 8  g/mL cefoxitin for 11 of the isolates. Likewise, all 177 MSSA isolates were correctly identified using the kit, and the MICs against them were determined within the range of 1 to 4  g/mL cefoxitin. The MIC of cefoxitin can be determined in 6 hours using the StaResMet® kit. Conclusions: The obtained results indicated that the StaResMet® kit allowed the detection of MRSA isolates rapidly and reliably, and could be a valuable tool for microbiology laboratories with limited facilities. Authors believed that the routine use of this timesaving and easy-to-use test contributes to rapid clinical diagnoses and treatments.

Background: Acinetobacter baumannii is a leading cause of worldwide nosocomial infections. As many infections are caused by multidrug-resistant strains, the antibiotic-therapy of them has become greatly difficult. Objectives: The present study was aimed to evaluate the antimicrobial susceptibility of A. baumannii as well as cloning and expression of A. baumannii-smpA gene isolated from infectious patients. Methods: Sixty-seven samples were collected from different clinical infections wards especially intensive-care units (ICU). The A. baumannii was identified according to bacteriological standard method. The antibiotic resistance patterns of the isolates were analyzed by the disk diffusion method. The smpA was amplified by Polymerase chain reaction (PCR) following cloning and sub-cloning in T-vector and expression vector, respectively. Colony-PCR, double-digestion and DNA sequencing confirmed that smpA was cloned into the vectors. The expression of rSmpA in IPTG-induced E. coli-DE3 was examined by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Thirty out of 67 isolates (44. 78%) were positive for A. baumannii among which, more than 83% were resistance to all generation of Cephalosporins and the least resistance (10%) was observed for Colistin. The smpA-amplicon was 417 bp. Colony-PCR, double-digestion and sequencing showed that the target gene was inserted into the pTZ57RT and pET32a (+), successfully. Obtaining of a ~ 15 KDa band in SDS-PAGE showed the recombinant pET32a/smpA was highly expressed. Conclusions: These results indicated that high resistance to antibiotics among isolates and high conserved of smpA gene at sequence level among A. baumannii strains, therefore it can be used as an antigenic target for generation vaccine against multi drug resistant (MDR) A. baumannii infections.