First Record of Microbiomes of Sponges Collected From the Persian Gulf, Using Tag Pyrosequencing.

The Persian Gulf is a special habitat of marine sponges whose bacterial communities are under-investigated. Recently, next-generation sequencing technology has comprehensively improved the knowledge of marine sponge-associated bacteria. For the first time, this study aimed to evaluate the diversity of the Persian Gulf sponge-associated bacteria using tag pyrosequencing in Iran. In this study, 10 sponge samples from 6 different taxonomic orders were collected from the Persian Gulf using SCUBA diving. The diversity of the bacteria associated with the marine sponges was investigated using the 16S rRNA gene PCR-tagged pyrosequencing method. A total of 68,628 high-quality sequences were obtained and clustered at a 97% similarity into 724 unique operational taxonomic units (OTUs), representing 17 bacterial phyla. Cyanobacteria was the most abundant phylum in the sponges, followed by Proteobacteria, Chloroflexi, Acidobacteria, and Actinobacteria. Other phyla were detected as minor groups of bacteria. Bacterial community richness, Shannon, and Simpson indices revealed the highest diversity in sponge S11 (Dictyoceratida sp.) compared to other sponges. This study showed a diverse structure of bacterial communities associated with the Persian Gulf sponges. The dominance of Cyanobacteria may suggest an ecological importance of this phylum in the Persian Gulf sponges.

Distribution of pathogenicity island markers and virulence factors in new phylogenetic groups of uropathogenic Escherichia coli isolates.

The present study was aimed at investigating the relationship between the new Clermont's phylogenetic groups, virulence factors, and pathogenicity island markers (PAIs) among uropathogenic Escherichia coli (UPEC) in Iran. This cross-sectional study was carried out on 140 UPEC isolates collected from patients with urinary tract infections in Bushehr, Iran. All isolates were subjected to phylogenetic typing using a new quadruplex-PCR method. The presence of PAI markers and virulence factors in UPEC strains was evaluated by multiplex PCR. The most predominant virulence gene was fimH (85%), followed by iucC (61.4%), papC (38.6%), hlyA (22.1%), cnf-1 (18.6%), afa (10.7%), papG and neuC (each 9.3%), ibeA (3.6%), and sfa/foc (0.7%). The most common phylogenetic group was related to B2 (39.3%), and the least common to A (0.7%). The most prevalent PAI marker was PAI IV536 (77.14%), while markers for PAI III536 (13.57%), PAI IIJ96 (12.86%), and PAI II536 (12.14%) were the least frequent among the UPEC strains. Meanwhile, the PAI IJ96 marker was not detected. There was a significant association between the phylogenetic group B2 and all the studied virulence genes and PAI markers. To our knowledge, this is the first study to compare the relationship between new phylogenetic groups, virulence genes and PAI markers in UPEC strains in Iran. The phylogenetic group B2 was predominantly represented among the studied virulence genes and PAI markers, indicating the preference of particular strains to carry virulence genes.

First Molecular Identification of Symbiotic Archaea in a Sponge Collected from the Persian Gulf, Iran.

BACKGROUND: Marine sponges are associated with numerically vast and phylogenetically diverse microbial communities at different geographical locations. However, little is known about the archaeal diversity of sponges in the Persian Gulf. The present study was aimed to identify the symbiotic archaea with a sponge species gathered from the Persian Gulf, Iran. METHODS: Sponge sample was collected from a depth of 3 m offshore Bushehr, Persian Gulf, Iran. Metagenomic DNA was extracted using a hexadecyl trimethyl ammonium bromide (CTAB) method. The COI mtDNA marker was used for molecular taxonomy identification of sponge sample. Also, symbiotic archaea were identified using the culture-independent analysis of the 16S rRNA gene and PCR- cloning. RESULTS: In this study, analysis of multilocus DNA marker and morphological characteristics revealed that the sponge species belonged to Chondrilla australiensis isolate PG_BU4. PCR cloning and sequencing showed that all of the sequences of archaeal 16S rRNA gene libraries clustered into the uncultured archaeal group. CONCLUSION: The present study is the first report of the presence of the genus of Chondrilla in the Persian Gulf. Traditional taxonomy methods, when used along with molecular techniques, could play a significant role in the accurate taxonomy of sponges. Also, the uncultured archaea may promise a potential source for bioactive compounds. Further functional studies are needed to explore the role of the sponge-associated uncultured archaea as a part of the marine symbiosis.

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.

Phylogenetic groups of Escherichia coli strains from patients with urinary tract infection in Iran based on the new Clermont phylotyping method.

OBJECTIVES: In 2013, Clermont classified E. coli strains into eight phylogenetic groups using a new quadruplex PCR method. The aims of this study were to identify the phylogenetic groups of E. coli based on this method and to assess their antibiotic resistance patterns in Bushehr, Iran. METHODS: In this cross-sectional study, 140 E. coli isolates were subjected to phylogenetic typing by a quadruplex PCR method. Antimicrobial susceptibility testing was performed by disk diffusion method. RESULTS: Phylogenetic group B2 was most predominant (39.3%), followed by unknown (27.1%), E (9.3%), C and clade I (each 6.4%), B1 (5%), F and D (each 2.9%), and A (0.7%). The most common antibiotic resistance was related to amoxicillin (82.1%) and the least to meropenem (0.7%). 82.14% of isolates were multiple drug resistant (MDR). Antibiotic resistance was mainly detected in group B2 (50%). CONCLUSIONS: Our findings showed the high prevalence of MDR E. coli isolates with dominance of group B2. About 25% of E. coli isolates belong to the newly described phylogroups C, E, F, and clade I. Such studies need to be done also in other regions to provide greater understanding of the antibiotic resistance pattern and the prevalences of different phylogenetic groups.

Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran.

CagA is a major virulence factor of Helicobacter pylori involved in host cell modulation. The C-terminal part of CagA containing the EPIYA motifs is highly variable and is important for the biological activity of the protein. The aim of this study was consideration of the changes in cagA tyrosine phosphorylation motifs (TPMs) of H. pylori. A set of 302 H. pylori DNA samples from the Iranian population from 2006 to 2011 was selected for the proposed study. The cagA gene and its TPMs were assessed by using polymerase chain reaction (PCR) and specific primers. The prevalence of the cagA gene in our study ranged from 91.43% to 97.06% (with an average of 95.03%). Out of the cagA-positive samples, the prevalence of TPMs A and B increased from 12.5% and 23.44% to 71.2% and 63.63%, respectively. Also, the prevalence of samples infected with Western and East Asian types of H. pylori ranged from 64.06% to 5.73% for the Western type and 17.19% to 51.59% for the East Asian type. Overall, our results showed a high prevalence of the cagA gene. Also, it seems that cagA TPMs of H. pylori is undergoing a change from the Western type to the East Asian type in Iran.

The Association of Virulence Determinants of Uropathogenic Escherichia coli With Antibiotic Resistance.

BACKGROUND: The emergence of antimicrobial resistant strains of Escherichia coli has raised considerable interest in understanding the diversity and epidemiology of E. coli infections in humans. Virulence factors of E. coli determine the specific infections caused by this microorganism. OBJECTIVES: This study aimed to determine the prevalence of eight E. coli virulence factors and their association with antimicrobial resistance in bacteria isolated from patients with urinary tract infections (UTI). PATIENTS AND METHODS: One thousand patients with UTI were enrolled in this cross-sectional study. Antimicrobial susceptibility was examined by disc diffusion method according to CLSI guidelines. After DNA extraction, the materials were probed by PCR for eight virulence factors genes, namely fimH, hly, iucC, ibeA, sfa/foc, neuC, papC, and afa genes. RESULTS: The frequency of virulence factors papC, afa, sfa/foc, fimH, hly, neuC, ibeA, and iucC were 53.3%, 51.7%, 53.3%, 56.7%, 23.3%, 31.7%, 20%, and 73.3%, respectively. In addition, there was a high degree resistance to cotrimoxazole and nalidixic acid while a high degree of susceptibility to nitrofurantoin was detected. There was a statistically significant association between fimH gene and resistance to ciprofloxacin (P = 0.006), nalidixic acid (P = 0.025), and cotrimoxazole (P = 0.02). Such associations were found between ibeA gene and amikacin (P = 0.02) and cotrimoxazole (P = 0.02) as well as between afa gene and gentamycin (P = 0.05). CONCLUSIONS: The results showed that E. coli isolated from patients with UTI had eight virulence factors with high frequencies. Moreover, these results alleged a direct connection between virulence factors and antimicrobial resistance in E. coli.

Molecular detection of ESBLs production and antibiotic resistance patterns in Gram negative bacilli isolated from urinary tract infections.

BACKGROUND: ß-lactam resistance is more prevalent in Gram negative bacterial isolates worldwide, particularly in developing countries. In order to provide data relating to antibiotic therapy and resistance control, routine monitoring of corresponding antibiotic resistance genes is necessary. AIMS: The aim of this study was the characterization of ß-lactam resistance genes and its plasmid profile in bacteria isolated from urinary tract infection samples. MATERIALS AND METHODS: In this study, 298 Gram negative bacteria isolated from 6739 urine specimens were identified by biochemical standard tests. Antimicrobial susceptibility testing was performed by the disk diffusion method. Extended-spectrum ß-lactamase (ESBL)-producing strains were also detected by the double-disk synergy test. The presence of blaTEM and blaSHV genes in the strains studied was ascertained by polymerase chain reaction. RESULTS: Of all Gram negative bacteria, Escherichia coli (69.1%) was the most common strain, followed by Klebsiella sp. (12.1%), Enterobacter sp. (8.4%), Proteus sp. (4.4%), Citrobacter (4%) and Pseudomonas sp. (2%). The most antibiotic resistance was shown to tetracycline (95.16%), nalidixic acid (89.78%) and gentamycin (73.20%) antibiotics. Among all the strains tested, 35 isolates (11.75%) expressed ESBL activity. The prevalence of TEM and SHV positivity among these isolates was 34.29%, followed by TEM (31.43%), TEM and SHV negativity (20.0%) and SHV (14.29%), respectively. CONCLUSIONS: Regular monitoring of antimicrobial drug resistance seems necessary to improve our guidelines in the use of the empirical antibiotic therapy.

First molecular detection of group A rotavirus in urban and hospital sewage systems by nested-RT PCR in Shiraz, Iran.

BACKGROUND: Group A rotaviruses are the most significant cause of acute gastroenteritis in children worldwide. Rotaviruses are shed in high numbers and dispersed widely throughout bodies of water in the environment. This represents a significant health hazard for humans, mainly due to the stability of the viruses during wastewater treatment processes. This study was conducted to evaluate the prevalence of rotaviruses, to determine G genotypes of circulating rotaviruses and to assess the efficiency of rotavirus removal in urban and hospital sewage treatment plants in Shiraz, Iran. MATERIALS AND METHODS: During the period from October 2010 to June 2011, a total of sixty sewage samples from urban and hospital sewage disposal systems were collected by Grab Sampling in Shiraz, Iran. All the samples were concentrated in pellet form and two-phase methods and then group A rotaviruses were investigated with enzyme immunoassays (EIA). Rotavirus-positive specimens were genotyped by the nested RT-PCR and by using different types of specific primers. RESULTS: In total, rotaviruses were identified in 25% (15 cases) of sewage samples, representing 73.33% (11 cases) of influent and 26.67% (4 cases) of effluent systems. The frequency of rotavirus detection in autumn, winter and spring was 46.67%, 33.33% and 20%, respectively (P= 0.004). The most common circulating genotype was G1 (73.33%), followed by G1G4 (20%) and non-typeable (6.67%), respectively. CONCLUSIONS: The high prevalence of rotaviruses in urban and hospital sewage systems highlights the importance of environmental surveillance as a tool to detect new genotypes and to investigate the epidemiology of rotaviruses circulating in the community.

Importance of viral pathogens in children with acute gastroenteritis in the south of Iran.

BACKGROUND AND OBJECTIVES: Different types of viruses are the leading cause of acute diarrhea among infants and young children worldwide. Epidemiological surveillance of viral agents is critical for the develop.ment of effective preventive measures, including vaccines. This study aimed to determine the prevalence of the four major enteropathogenic viruses-rotavirus, norovirus, adenovirus and astrovirus-in children over 7 years of age. DESIGN AND SETTING: A cross-sectional descriptive study conducted on stool specimens of children with acute gastroenteritis admitted to the Pediatrics Unit of 17 Shahrivar Hospital in Borazjan, Iran from October 2008 to September 2010. PATIENTS AND METHODS: Acute gastroenteritis was defined as >=3 loose watery stools per 24 hours. A total of 375 stool samples were collected from hospitalized children aged < 7 years old with acute gastroenteritis. All samples were investigated by using enzyme-linked immunosorbent assay for the presence of viral antigens. RESULTS: Rotavirus was detected in 91 (24.3%) of the patients whereas the prevalence of norovirus, adenovirus and astrovirus was 12.5%, 5.1% and 2.4%, respectively. On average, 75.9% of children with viral diarrhea were younger than 2 years old (P=.023). All the strains of viral gastroenteritis studied peaked in the autumn, except for adenovirus which peaked in spring (P=.015). The most common clinical symptoms included diarrhea (92.2%), vomiting (68.7%), abdominal cramp (60.8%) and moderate dehydration (57.2%). CONCLUSION: Since nearly half of gastroenteritis cases (44.3%) were due to viral agents, testing for the viral antigens may guide the clinical approach to those patients with acute diarrhea particularly in the case of children less than 2 years old, and during cold seasons.

Molecular Epidemiology of Rotavirus Strains Circulating among Children with Gastroenteritis in Iran.

OBJECTIVE: This study was conducted to evaluate the prevalence of rotavirus disease and to investigate the genotypes of rotavirus strains causing acute gastroenteritis among children aged <5 years old in Marvdasht, Iran. METHODS: One hundred and forty-one children, aged 1 month to 5 years, afflicted with severe diarrhea were enrolled during January 2007 to December 2008. Their stool samples were studied with enzyme immunoassays (EIA) for group A rotaviruses. Rotavirus-positive specimens were genotyped by the Nested RT-PCR using different types of specific primers. FINDINGS: Out of total collected samples rotavirus infection was detected in 40 (28.37%). Of the rotavirus episodes, 72.91% occurred during the first 2 years of life (P=0.038). The highest prevalence of infection was identified in summer (52.50%) and the lowest in winter (7.50%). The most common clinical features included diarrhea (96.25%), vomiting (82.50%) and fever (45.0%). Mixed genotypes were the predominant G type (60.0%), followed by non-typeable (12.50%), G2 (12.50%), G4 (10.0%) and G1 (5.0%) genotypes. G3/G8 mixed infection is the first of these rotavirus genotypes to be reported in Iran. CONCLUSION: Regarding high frequency of rotavirus infection, continuous surveillance is needed to inform diarrhea prevention programs as well as to provide information about the occurrence of new rotavirus strains. This will assist policy makers in decision making on rotavirus vaccine introduction.

Burden and typing of rotavirus group a in children with acute gastroenteritis in shiraz, southern iran.

BACKGROUND: Human Rotavirus is a significant cause of severe gastroenteritis in infants and young children worldwide. In recent years, Rotavirus genotyping by RT-PCR has provided valuable information about the diversity of Rotaviruses circulating worldwide. The purpose of the present study is to monitor the prevalence of the different G types of Rotaviruses circulating in Shiraz, Southern Iran and detect any uncommon or novel types. METHODS: During the period from December 2007 to November 2008, a total of 138 stool samples were collected from children less than 5 years old who were hospitalized for acute gastroenteritis. Rotavirus-associated diarrhea was investigated in fecal specimens with enzyme immunoassays (EIA). Rotavirus-positive specimens were typed by the Nested RT-PCR and by using different types of specific primers. RESULTS: Out of the 138 collected samples, 34.78% (48 cases) tested positive for Rotavirus. The frequency of G1, G2 and G4 types was 6.25%, 2.08% and 27.08%, respectively. Mixed and non-typeable infections were detected in 33.34% and 31.25% of hospitalized children with acute diarrhea, respectively. This is the first time mixed Rotavirus infections with G1/G3 have been reported in Iran. CONCLUSION: The high frequency of Rotavirus detection indicates the severity and the burden of Rotavirus disease may be able to reduce through the implementation of an effective vaccine and continual surveillance for the detection of Rotavirus genotypes circulating in other regions of Iran. Regarding to the noticeable frequency of non-typeable and mixed infections, it is suggested to use the other specific primers and further studies to detection of other novel and unusual types.