Iranian Journal of Microbiology
Volume:12 Issue: 4, Aug 2020

In recent years, the prevalence of diseases caused by Vibrio spp. is increasing in the world, and among them species, Vibrio cholerae is the most important Vibrio associated with pandemic and epidemic cholera outbreaks. Therefore, the development of a reliable method for early and accurate detection of V. cholerae for management of diseases is a real need. Aptamers with the ability to detect targets with high specificity and accuracy can be one of the candidates used for the whole cell and thereby V. cholerae detection.

In this research high-affinity DNA aptamers against with two major serotypes of Inaba (ATCC 39315) and Ogawa (clinical sample) were selected from DNA aptamer library through 12 rounds of Systematic Evolution of Ligands by Exponential (SELEX) enrichment procedure using live cells as a target which monitored with flow cytometry.

The binding efficiency and dissociation constant of the isolated aptamers V.ch47 and V.ch27 were 56.4%, 53.3% and 15.404 ± 4.776 pM, 20.186 ± 3.655 pM, respectively. A sandwich Enzyme-linked aptamer sorbent assay (ELASA) was developed with the biotinylated V.ch47 aptamer and our previously developed nanobody anti-Lipopolysaccharides (LPS). We optimized this system with V. cholerae O1 and analyzed their cross reactivity with close physiological bacteria. The threshold of detection was obtained 104 CFU/ml in the sandwich ELASA process.

Our results showed that the sandwich ELASA is sensitive enough for the rapid detection of V. cholerae from other bacteria.

Cholixin (cholix toxin) is a novel exotoxin in Vibrio cholerae identified as an elongation factor II specific ADP-ribosyltransferase which inhibits protein synthesis in the eukaryotic cell. Previous researches have suggested that cholixin probably is an important virulence factor in non-O1/non-O139 V. cholerae (NAG) serotypes that could be related to extra-intestinal rather than intestinal infections. This study was aimed to investigate the frequency and genetic diversity of colixin gene (chxA) in clinical V. cholerae NAG isolates.

The presence of chxA gene in 44 clinical V. cholerae NAG isolates were screened using PCR through specific primers designed for the receptor-binding domain (RBD) of chxA gene. The five PCR products of chxA gene were sequenced.

This study showed that chxA gene presented in 19 V. cholerae NAG isolates. The sequences analysis of 5 out of 19 the partial chxA genes amplicon showed that 4 of them belonged to chxA I and the other one belonged to chxA II subtypes. Two distinct clusters were revealed for these isolates by phylogenic analysis, too.

The chxA gene contained high frequency among V. cholerae NAG isolates in Bushehr, Iran. The polymorphism study on RBD of cholixin gene is suggested as an appropriate method for phylogenic characterization of the various chxA gene subtypes.

Escherichia coli is responsible for various enteric and extraintestinal infections in animals and humans. Iron as an essential nutrient, has a proven role in pathogenicity of E. coli. Pathogenic E. coli benefits of having complicated systems for iron acquisition but our current knowledge is limited because of complexity of these systems. In the present study, three multiplex-PCR assays were developed to screen nine different virulence genes related to diverse iron acquisition systems in E. coli.

The multiplex-PCR systems were designed and optimized in three panels. Each panel includes a triplex-PCR cocktail. The panels are as follow: panel 1: iroN, iutA and fecA; panel 2: fyuA, sitA and irp2; and panel 3: iucD, chuA and tonB. A total of 39 pathogenic E. coli was screened according to the designed multiplex-PCR.

In total, the top three frequent genes were tonB (100%), fecA (66.6%) and sitA (58.9%). With the exception of fecA and tonB, comparing the prevalence of genes among different origin of isolates (human, cattle, poultry and pigeon) showed significant associations (P < 0.05). Moreover, the iroN, sitA and iucD genes were significantly prevalent (P < 0.05) among members of extraintestinal pathogenic E. coli in comparison with the group of diarrheagenic E. coli.

The current multiplex-PCR assays could be a valuable, rapid and economic tool to investigate diverse iron acquisition systems in E. coli for more precise virulence typing of pathogenic or commensal strains.

Enterohemorrhagic Escherichia coli (EHEC) causes bloody and non-bloody diarrhea, intestinal infection and extraintestinal complications in humans. This study aimed to isolate and evaluate the prevalence of E. coli O157: H7 and other Shiga toxin-producing E. coli (STEC) and identify the virulence genes (stx1, stx2, hly and eaeA) from patients with diarrhea. Also, the antibiotic resistance profile of the isolated strains was evaluated.

A total of 100 stool samples were collected from patients with acute diarrhea referring to the hospital and clinics in Isfahan County, Iran. Phenotypic tests and PCR assay were used for detection of E. coli O157: H7 and other Shiga toxin-producing E. coli. The presence of virulence genes (stx1, stx2, hly and eaeA) were identified by PCR. The antibiotic resistance profile of the isolates was determined using the agar disk diffusion method. The results were analyzed descriptively by Sigma stat version 4 software.

Seventy - eight out of 100 samples (78%) were contaminated with E. coli. E. coli O157 was isolated from five samples (6.4%), of which only two strains (2.56%) were identified as E. coli O157: H7. According to the results, out of two E. coli O157: H7 isolates, one (50%) isolate contained eaeA and two isolates (100%) contained Stx1, Stx2, hlyA genes. Out of three (3.84%) E. coli O157: HN, one of the isolate (33.3%) contained stx1 and, two isolates (66.7%) were positive for hlyA genes. Also, the results revealed that six strains (7.69%) were non-O157: H7 STEC, of which two isolates (33.3%) contained stx1 and four isolates (66.7%) were positive for stx2 and hlyA genes. The results of antibiogram tests revealed that all of the STEC isolates (100%) were sensitive to imipenem followed by kanamycin, gentamicin and nitrofurantoin (91%). High resistance (54.5%) to ampicillin and ciprofloxacin was observed among the STEC isolates.

The results of the current study showed that although the prevalence of E. coli O157: H7 was low among patients with diarrhea, the other STEC strains with relative resistance to antibiotics are more prevalent.

Clostridium difficile infection (CDI) has become a significant healthcare-associated infection throughout the world and is particularly important in developing countries. This study aimed to investigate clinical characterization and risk factors related to toxigenic C. difficile infection in adult and pediatric patients, antimicrobial susceptibility pattern. Also, to evaluate different diagnostic methods for rapid detection of C. difficile associated diarrhea (CDAD) in Egypt.

Stool samples were collected from 95 pediatric patients and 37 adult patients suffering from antibiotic associated diarrhea and were subjected to direct toxin immunoassay and culture on cycloserine/cefoxitin/fructose agar. The presence of tcdA and tcdB genes was tested by PCR.

Toxigenic C. difficile was isolated from pediatric and adult patients at a rate of 17.89% (17/95) and 27% (10/37) respectively. The sensitivity and specificity of direct PCR from stool are (100%, 100% and 82.4%, 100%) in adult and pediatric samples respectively. The susceptibility of C. difficile to vancomycin and metronidazole were found to be 66.7% and 48.2% respectively.

Diabetes mellitus, prior antibiotic treatment, hematological malignancy on chemotherapy, malnutrition, neutropenia and Ryle feeding are risk factors for development of CDAD. Tight restriction of unnecessary antibiotic uses is necessary in our locality. Direct detection of toxin genes in stool by PCR is sensitive and specific method for early detection of C. difficile.

The study was sought to detect the effect of Listeria monocytogenes on pregnant Iraqi women at Al-Diwaniya hospitals and determination of virulence genes and antimicrobial susceptibility of isolates.

360 specimens including blood, urine, vaginal and endocervical were collected from 90 patients with spontaneous abortions. Blood samples were displayed to immunological study and remaining specimens were subjected to bacteriological diagnosis. PCR was used to determine the virulence factors and antimicrobial resistance genes.

Fifteen positive samples (16.6%) of patients and thirteen isolates (14.5%) from patients were recognized based on ELISA and PCR assay respectively. The general isolation of L. monocytogenes strains in cases of abortive women was 13/270 (4.8%). L. monocytogenes strains were highly virulent because of presence of virulence factors associated genes, namely actA, hlyA, plcA and prfA in all strains. Multiple drug resistance (MAR) index values of 15.4% of isolates were >0.2.

It is necessary for conducting susceptibility testing and to select the suitable antibiotics and avoid the effects of these bacteria in pregnant women.

Stenotrophomonas maltophilia is a multidrug resistant opportunistic pathogen, which is normally present in hospital settings and has very high mortality rates.

A prospective study was conducted over a period of two years. The specimens were processed by Gram staining and aerobic culture. The bacteria were isolated using standard protocols. The extent of antibiotic resistance of commonly used antimicrobials and biofilm production were studied in the isolates.

A total of 80 strains of Stenotrophomonas maltophilia were isolated. The maximum sensitivity (93.8%) of these isolates was noticed for cotrimoxazole. 63.7% of strains were strong biofilm producers. The group given pathogen specific antibiotic showed better prognosis (P value ≤ 0.05).

Early diagnosis and proper management of cases infected with Stenotrophomonas maltophilia is important to avoid therapeutic failures.

Nowadays, high-level aminoglycosides and ampicillin resistant Enterococcus species are among the most common causes of nosocomial infections. The present study was conducted to determine the prevalence of high-level resistance to aminoglycosides and ampicillin among clinical isolates of Enterococcus species in Ardabil, Iran.

In this cross–sectional study, a total of 111 Enterococcus species were collected from different clinical specimens between 2013 and 2015. Enterococcus species were identified using standard phenotypic and genotypic methods. BHI agar screen and agar dilution methods were used for detection of high-level gentamicin and streptomycin resistance (HLGR and HLSR) and minimal inhibitory concentration (MIC) of ampicillin, respectively.

Of 111 clinical isolates, 59 (53.2%) and 25 (22.5%) isolates were E. faecalis and E. faecium, respectively, based on the PCR results. Totally, 60.3% and 56.7% of isolates were HLGR and HLSR, respectively, as well as 51.35% were HLGR plus HLSR. Among HLGR isolates, 36 (61.01%), 18 (72%) and 13 (48.14%) were E. faecium, E. faecalis and non-faecalis non-faecium species, respectively. Among HLSR isolates, 33 (55.93%), 16 (64%) and 14 (51.85%) were E. faecalis, E. faecium and non-faecalis non-faecium species, respectively. All HLGR isolates contained aac(6´)Ie-aph(2″)Ia gene. Overall, the prevalence of high-level ampicillin resistance among Enterococcus species was 17.1%. For E. faecalis, E. faecium and non-faecalis non-faecium species, ampicillin resistance rates were as follows: 11 (40.74%), 7 (28%) and 1 (1.69%), respectively. For aminoglycoside antibiotics, the resistance rate was significantly higher in E. faecium isolates and for ampicillin it was higher in E. faecalis isolates.

The frequency of high-level aminoglycoside resistant enterococcal isolates in our hospital was high and significant ampicillin resistance was noticed. This would require routine testing of enterococcal isolates for HLAR and ampicillin susceptibility.

Preterm delivery is an important subject in gynecology, obstetrics and pediatrics. It is defined as regular uterine contractions every five to eight minutes or less, lasting for 30 seconds. It is associated with progressive changes in the cervix, resulting in delivery after 22 weeks and before 37 weeks of gestation. This study aimed to evaluate the role of Chlamydia trachomatis infection in women with preterm delivery.

This case-control study was performed on 75 women with preterm delivery (case group) and 75 women with term delivery (control group). The research tools included a questionnaire, polymerase chain reaction (PCR) assay of cervical swab samples and ELISA assay of umbilical cord blood samples. Fisher’s exact test and t test were also performed to compare qualitative variables between the two groups.

In this study, the mean age of subjects was 26.55 ± 0.53 years in the control group and 26.76 ± 0.56 years in the case group. The prevalence of C. trachomatis in the cervical swab samples was 7 (9.33%) in the control group and 2 (2.67%) in the case group. There was no C. trachomatis IgM antibody in either of the groups, while there was 1 (1.33%) C. trachomatis IgG antibody in both groups.

The results of the present study showed that there was no significant relationship between C. trachomatis infection and preterm delivery.

Chlamydia psittaci, an obligate intracellular, Gram-negative zoonotic pathogen, has eight serovars and nine genotypes isolated from avian species with higher frequency in parrots and pigeons. The aim of this study was to characterize Chlamydia spp. using nested PCR and sequencing.

A total of 270 pharyngeal swab samples collected randomly from asymptomatic pigeons of 30 pigeon aviaries in Tehran province. DNA was extracted with specific kit and amplified by specific primers in the first PCR and outer membrane protein A (ompA) gene in the second PCR. Positive samples were sequenced and phylogenetic tree analyzed based on the ompA gene.

Records showed that 16 of 30 (53%) pigeon aviaries were positive for Chlamydia spp. Phylogenetic tree analysis revealed that 15 of 16 (93.7%) positive samples, belonged to C. psittaci genotype B whereas the other sample belonged to C. avium. C. psittaci detected in 50% of pigeon aviaries that is high rate in Tehran province.

As C. psittaci is a zoonosis and life threaten pathogen for human being, these results indicate the significance of it detection in asymptomatic pigeons. Also, this is the first report of Chlamydia avium presence in Iranian pigeons which its zoonotic potential is still unknown.

Streptococcus pneumoniae causes many lethal infections. Due to its reduced sensitivity to commonly used antibiotics, development of new strategies against pneumococcal infections seems to be necessary. We aimed to investigate immunodominant antigens in S. pneumoniae culture supernatant in order to develop novel targets for pneumococcal vaccines.

In this study S. pneumoniae ATCC49619 was sub-cultured into BHI broth from overnight culture at 37°C for 4 h. The supernatant proteins were precipitated using acetone precipitation method. A rabbit was intramuscularly immunized with alum adjuvant and 100 μg pneumococcal supernatant proteins, 6 times at 14 days' intervals to produce hyperimmune serum. ELISA assay was performed to determine the antibody level response to pneumococcal secretory proteins. Then dot blot applied for rapid evaluation of hyperimmune serum reactivity to pneumococcus supernatant proteins. The western blot was also used to determine the interaction of supernatant proteins with immunogenic rabbit's hyperimmune-serum.

According to the western blot analysis, the immunodominant protein had 140KDa molecular weight and designated as pneumococcal secretory protein140 (Psp140).

The Psp140 protein in the supernatant of S. pneumoniae culture is an immunodominant protein and it is likely related to pneumococcal secretory protein or surface exposed protein which released into culture supernatant during bacterial growth.

Streptomyces tendae is one of the most prolific actinobacteria with a wide range of biotechnological applications. Genomic data can help in better understanding and exploration of important microorganisms, however, there is a few genomic information available for this species.

Molecular identification, pH and salt tolerance of an actinobacterium, designated Streptomyces tendae UTMC 3329, isolated from a tea field soil were done. Also, genomic DNA was extracted and sequenced using Illumina platform with MPS (massively parallel sequencing) Illumina technology. Gene annotation and bioinformatic analysis were done using appropriate software and servers.

The draft genome is ~8.7 megabase pairs, containing 7557 predicted coding sequences. The strain was able to grow at pH 5-12 and 0-10% NaCl. The maximum growth rate of the bacterium was obtained at pH 7. The gene clusters involved in central carbon metabolism, phosphate regulation, transport system, stress responses were revealed. It was shown the presence of gene clusters of polyketides, ribosomally and non-ribosomally synthesized peptides. Various genes were found in xenobiotic degradation pathways and heavy metal resistance.

The current genomic information which reveals biological features, as well as the biotechnological potential of this acid and alkaline tolerant actinobacterium, can be implemented for further research on the species.

The respiratory tract is the most common site for developing fungal infections. People who have a weakened immune system are more susceptible to respiratory system involvement with fungi. Fungal infections of the respiratory tract are largely unrecognized and their true burden is elusive. Therefore, the aim of the current study was to evaluate the clinical spectrum, demographic characteristics, risk factors, and etiology of fungal respiratory infections in 384 patients hospitalized in pulmonary units of Razi hospital, Guilan province, Iran.

A total of 384 lung specimens (192 Bronchoalveolar lavages (BAL) and 192 sputa) were obtained from patients who met the inclusion criteria. All samples were analyzed by direct microscopy and culture. Fungal identification was accomplished by internal transcribed spacer (ITS) and beta-tubulin sequencing. Also, in patients suspected to invasive pulmonary aspergillosis BAL specimens were tested for galactomannan (GM) antigen. According to the host factors (clinical symptoms, radiology findings and predisposing factors which were defined as inclusion criteria), and the positive results in direct examination, culture and serology (GM for aspergillosis) the infection was confirmed.

Fungal respiratory infection was confirmed in 137 cases (35.67%) including 86 (62.77%) males and 51 (37.23%) females and the highest prevalence of infection was found in the age group of 46-72 years (n=75, 54.74%). Cough (n=129, 94.16%), dyspnea (n=111, 81.02%), purulent sputum (n=85, 62.04%) and weight loss (n=77, 56.2%) were the predominant symptoms. Tuberculosis (n=34, 24.81%), taking chemotherapy regimen (n=30, 21.89%) and diabetes mellitus (n=27, 19.70%) were the predominant underlying conditions. Candida albicans (37.22%) and Candida tropicalis (21.89%) represent the two most commonly isolated species in the current study. Furthermore, according to revised EORTC/MSG (2008) definitions for invasive fungal infections, from 5 cases of pulmonary aspergillosis, 2 (40%) cases of probable invasive pulmonary aspergillosis (IPA) and 3 (60%) cases of possible IPA were diagnosed.

According to the results of this study, infected infants with congenital CMV infection could identify at early stage by testing Guthrie cards (within 21 days of life). Furthermore, since there is a lack of CMV knowledge in our population, educating and effective counseling by obstetricians/ gynecologists to the pregnant women are recommended.

Infections caused by Chromobacterium violaceum are extremely rare but can be relatively fatal in septicemia. We report a case of a 76 year old female who presented with pustules in the skin and later developed into septicemia. She succumbed to the illness despite escalating the antibiotic therapy to meropenem. To the best of our knowledge this is the 16th case report from India.

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