Iranian Journal of Microbiology
Volume:11 Issue: 1, Feb 2019

Gut microbiota is the complex community of microorganisms that live in the digestive tracts of humans and other animals, including insects. The relationship between gut microbiota and human health is mutualistic and altered bacterial compositions in fecal and mucosal specimens of colon in patients with cancer compared to healthy subjects were observed. Thereby, studying the gut microbiota, their interactions with the host and their alterations in colorectal cancer (CRC) patients could be helpful to diagnose and treat the disease in earlier stages. In CRC research, the most common samples are feces and tumor tissues. Interestingly, scientists have quite different views regarding gut microbiota composition of feces and tissues. Some believe bacterial populations in feces and mucosa are completely distinct and differ in composition and diversity while some others declare similar variations. Actually, both types of specimens have some advantages and disadvantages in survey of gut microbiota. Fecal samples serve as a noninvasive approach for screening tests while mucosal associated samples are more powerful for identification of bacteria with adenoma and CRC initiation and growth. Here we have discussed the advantages and disadvantages of two type of specimens in CRC investigations and also discussed the similarities and differences of microbial composition between stool and tissue specimens.

Due to the widespread distribution of Listeria monocytogenes in environmental and animal sources and serious clinical complications in human, this study was aimed to isolate L. monocytogenes from water and clinical specimens by culture and PCR methods and to investigate the presence of hlyA and inlA virulence genes. Water and clinical samples of vaginal and fecal were screened for the presence of L. monocytogenes by phenotypic and standard biochemical tests. PCR amplification was performed on extracted DNA using primers based on the hlyA and inlA genes. A 733-bp fragment of inlA gene was used for investigation of polymorphism using RFLP analysis. In total, 45 phenotypically and molecularly confirmed L. monocytogenes strains were isolated from different sources including 30 (16.7%) from water, 9 (11.3%) from vaginal swabs and 6 (7.5%) from fecal samples. RFLP analysis of PCR products using AluI and Tsp509I restriction enzymes, generated two profiles with 8 to 10 bands ranging in size from 15 to 210 bp. The majority of water and clinical isolates were classified in profile 2. We demonstrated 45 L. monocytogenes isolates from tested water and clinical samples by phenotypic and molecular tests. The majority of the isolates were classified in the same RFLP profile, showing the water as a potential source of clinical complications in patients in the region of study.

Streptococcus pyogenes is recognized as an important pathogen of respiratory tract infections. The rapidly, emerging problem of antibiotic resistant Streptococcus pyogenes is a major issue nowadays. The present study aimed to evaluate the antibiotic susceptibility of Streptococcus pyogenes isolated from upper respiratory tract infections in tertiary care hospital of south Karnataka. A retrospective study was conducted over a period of two years. The specimens were processed by Gram staining and aerobic culture. The bacteria were isolated as per standard protocols. The minimum inhibitory values and extent of antibiotic resistance of commonly used antimicrobials were analysed for the isolated strains. A total of 2123 specimens were received from patients with respiratory tract infections, among which, 50 Streptococcus pyogenes isolates were obtained. Out of these, 8% were not sensitive to penicillin. Using VITEK 2 system, the prevalence of resistances to cefotaxime, erythromycin, tetracycline, levofloxacin, clindamycin and ceftriaxone were 4.2%, 83%, 51%, 8.9%, 40% and 5.3% respectively. It is important to know about the prevalence of resistance and rising MIC values of commonly used antibiotics regarding Streptococcus pyogenes to avoid therapeutic failures.

Francisella tularensis has a wide distribution in northern hemisphere of the world. Up to now, there was little information about the Francisella spp. situation in the environmental samples in Iran. In this study we aimed to determine the prevalence of Francisella spp. in the environmental samples in northwest of Iran. A total of 237 natural water samples from ponds, rivers, lakes, springs and other surface waters from north western provinces of Iran (Kurdistan and Western Azerbaijan) were collected from September to November 2015. All samples were cultured for Francisella and other bacterial species and Real Time TaqMan PCR was performed on the concentrated and DNA extracted samples. For detection of the presence of bacterial DNA in the samples, two different targets in the genome of Francisella, ISFtu2 and fopA were used. Among the tested surface water samples, 40 (17.09%; 95% CI: 12.67-22.33%) and 12 (5.13%; 95%CI: 2.81-8.56%) samples were positive for ISFtu2 and fopA respectively. None of them was positive in culture. The prevalence of Francisella spp. in the environmental samples in the west of Iran is high and it is comparable with Turkey, Iran’s neighboring country. Use of higher copy number genes or IS like ISFtu2 could improve the detection of this organism in the environmental samples.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen. The presence of several virulence factors such as exotoxin and exoenzyme genes and biofilm may contribute to its pathogenicity. The purpose of this study was to investigate the presence of toxA, exoU and exoS, the determination of biofilm production and antimicrobial susceptibility patterns among clinical isolates of P. aeruginosa. In this study, 75 isolates of P. aeruginosa were recovered from various clinical specimens. Antimicrobial susceptibility pattern of isolates were identified. Virulence genes toxA, exoU and exoS were determined using PCR. The ability of biofilm production was assessed. Antimicrobial susceptibility test showed that 12 strains were resistant to more than 8 antibiotics (17.14%). The most effective antibiotic was colistin as 98.6% of isolates were sensitive. The frequencies of exoU and exoS genes were detected as 36.6% and 55.7%, respectively. In addition, 98.6% of the isolates were biofilm producers. Exotoxin A was detected in sixty-eight isolates (95.7%). The findings of this study showed that, the presence of P. aeruginosa exotoxin and exoenzyme genes, particularly, the exoU gene is the most common virulence factors in the bacterial isolates from urine samples. Biofilm is a serious challenge in the treatment of P. aeruginosa infection.

Non-typhoidal Salmonellosis, a zoonotic infection associated with acute gastroenteritis is caused by non-typhoidal salmonellae (NTS). The study was carried out to determine the prevalence of NTS serovars and their antimicrobial resistance along with the presence of the virulence gene (invA gene) in poultry samples. This is a prospective cross-sectional study carried out at the Enteric Diseases Division, Kasturba Medical College, Manipal, South India from January 2016– December 2017. Poultry samples were collected randomly from two local poultry farms in Udupi district and processed following CDC standard protocol. From the 396 poultry meat samples, intestinal contents and faecal samples collected, 58 NTS serovars were isolated showing a prevalence of 14.64%. Salmonella Infantis, 43.1%, 25/58 was the commonest serovar. Resistance to ciprofloxacin 72.41%, ampicillin 32.8%, gentamicin 17.24%, cotrimoxazole 29.31% and amoxicillin-clavulanic acid 6.9% was observed. The invA gene was detected in 43 NTS isolates (74.13%). Poultry sources are recognized as a significant cause for non-typhoidal salmonellosis. Therefore, hygienic measures should be initiated to reduce the contamination of meat and poultry products with virulent strains of Salmonella that are of public health significance.

Leptospirosis is a zooanthroponosis caused by the genus of Leptospira. It is an emerging public health problem due to its increasing incidence. The achievement to a vaccine that prevent from entrance of Leptospira interrogans to the deeper tissues of the host is needed. This study aimed to investigate the immunogenicity of LcpA (rLcpA) and LenA (rLenA) recombinant proteins in combination with LTB (rLTB) recombinant protein as an adjuvant against leptospiral infection in hamsters. The genes encoding these proteins were cloned into pGH cloning vector and then lenA, lcpA and ltb genes subcloned into pET-15b and pET-28a expression vectors, respectively. The hamsters were immunized with the purified recombinant proteins and challenged with Leptospira interrogans for evaluation of their survival. The antibody responses to the recombinant proteins were determined by ELISA. Then, data entered into SPSS software. Statistical Kruskal–Wallis test was used to compare the significant differences among different groups. The groups with significant differences were further analyzed by post hoc tests. The p value < 0.05 statistically was considered significant. Immunized hamsters with rLenA-plus-rLTB, rLcpA-plus-rLTB and rLenA-plus-rLcpA-plus-rLTB proteins showed 60%, 74%, and 80% survival rates, respectively. A significant amount of interleukin-17 (IL-17), interleukin-4 (IL-4) and gamma interferon (IFNγ) cytokines were produced in immunized hamsters. Based on our findings, rLcpA and rLenA proteins in combination with rLTB can protect the hamsters against L. interrogans and effectively induce a protective antibody response. Thus, these proteins can be used as an additional prophylactic tool against leptospira.

Interrogation of the genomic relations between Iranian Mycoplasma agalactiae vaccine strains of Taliqan, Lorestan and Shiraz. Two MLVA (covering VNTR loci of 5, 9, 17 and 19) and MLST (comprising dnaA, gltX, gyrB, C, tufA genes) genotyping systems plus nucleotide structure analysis of P80 gene, was conducted. The shared MLVA pattern represented by the three strains differed to that of the Mag PG2 laboratory strain, only at locus VNTR19 where the PG2 genome hold a 3 bp longer stretch. In MLST analysis, at dnaA, gltX, gyrB, metS and tufA loci, the three strains displayed alleles 1, 21, 2, 2 and 1, respectively. At, gltX locus a new allele (21) was detected where a new sequence type (ST33) was identified. Besides, the trio strains hold an identical nucleotide structure in their ma-mp81 gene. In explanation, lack of efficient disease control measures, has possibly contributed in evolution of a clone or a few clones that gradually overwhelmed the population over the time. Besides, the similarity between the Iranian and the PG2 strains, might be due to homoplasy or farming exercises such as animal importation. Inclusion of further local isolates in next studies will help to assess these assumptions.

Expressions of lasA and lasB genes of Pseudomonas aeruginosa are associated with bacterium pathogenicity. The present study was aimed to assess the effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in P. aeruginosa. Pseudomonas aeruginosa isolates were cultured in Mueller Hinton broth containing sub-inhibitory concentrations of SKEO and total RNA extracted using Trizol method. cDNA was synthesized using random Hexamer primer and finally the expression of lasA and lasB genes carried out by real-time PCR. The MICs of SKEO extract for PA9, PA10, PA11, PA13, PA41 and PA42 isolates were 8, 8, 8, 9, 7 and 12 µg/ml, respectively. Statistical analysis for 6 isolates revealed that the reduction in expression of lasA and lasB genes under SKEO treatment was significant (P<0.05). The insignificantly increasing of lasB gene expression may lead to low virulent strains, for probably reason that the strain’s exotoxin A are destroyed in the high amount of protease. In conclusion, using of SKEO in burned patients infected with P. aeruginosa may be effective; however, it is better to assess the spectrum activity of SKEO, pharmacokinetics, potency and its toxicity in human cells.

Bioremediation is a process to reduce toxic heavy-metals, such as arsenic, in the environment using microorganisms. This study aimed to isolate arsenic remediating microbial strains from garbage leachates and to evaluate the effects of several factors on bioremediation by isolated strains. After isolating arsenic-resistant bacteria from garbage leachates and determining their MIC values, Taguchi design of experiments was used to evaluate the effect of arsenic concentration, pH solution, temperature, and contact time on arsenic bioremediation by isolated bacteria. The results revealed that 3 arsenic-resistant strains of genus Bacillus characterized as KL1, KL4, and KL6 had arsenic bioremediation activity. Based on the results, the highest bioremediation of arsenic by Bacillus sp. KL1 was obtained as 77% after 24 hours at 40°C, pH 5, and 150 ppm concentration. However, the maximum bioremediation of arsenic by KL4 (91.66%) and KL6 (88%) was achieved after 24 hours at 40°C, pH 5, and 60 ppm concentration and at 35°C, 90 ppm concentration, pH 5 after 36 hours, respectively. The results presented here may facilitate improvements in the eliminating arsenic from contaminated sites and reducing environmental pollutions.

rpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the rpoS gene. The aim of this study was to investigate if rpoS mutagenesis has impact on bacterial morphology in addition to cell division. A 0.603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy. After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that mutation in rpoS gene leads to decomposition of outer membrane of F. chinesis. A suitable vector to make suicide mutation in rpoS was constructed for F. chinesi.

Epstein-Barr virus (EBV) has infected more than 90% of adults worldwide. EBV infection is asymptomatic in healthy individuals and is controlled by a robust immune response while in individuals with weakened immunesystems including Hemodialysis (HD) patients and transplant recipients leads to serious illnesses. This study was aimed to investigate the frequency of EBV among the HD patients. The cross-sectional study was carried out on 84 HD patients. These sera were checked for anti-EBV (VCA) IgG Ab assessment using enzyme-linked immunosorbent assay (ELISA). The DNA was extracted from the sera samples and tested for EBV DNA using nested PCR. 52/84 (61.9%) of HD were males and 32/84 (38.1%) were females. The average age of participants was varying from 18 to 85 years while the mean age was 52 ± 1.57 SD years. 81 of 84 (96.42%); including 49/52 (94.23%) male and 32/32 (100%) female, were positive for anti-EBV (VCA) IgG antibody while 3 (3.58%) were negative. No significant differences were observed between the subjects regarding gender (P=0.28). EBV DNA was detected in 7 (8.33%) individuals, including 6 (11.53%) and 1 (3.12%) in male and female, respectively (P=0.24). Our study results showed that high prevalence of anti-EBV (VCA) IgG antibody (96.42%) were observed among the HD patients. Although the status of EBV latency was not performed, but it seems many of these patients are at risk of EBV-reactivation during the organ transplantation. As a result, it is recommended that the detection of EBNA-1 gene as a marker of EBV latency should be implemented for all HD patients to prevent EBV reactivation during organ transplantation.