محمدرضا پورشفیع

Intestinal colonization of the newborn is essential for establishment, maturation and maintenance of the gut mucosal barrier. The greatest difference between the microbiota of breast milk and Infant formula infants feeding is the numbers and species composition of Bifidobacteria. In this study, we tried to identify the native Bifidobacterium isolates obtained from the human’s breast milk and the feces of their paired infants in rural areas of Markazi Province in 2015. In this descriptive study, 28 samples from mothers’ milk and 28 samples from paired infants feces were collected and cultured. Suspicious colonies were picked up and confirmed by phenotypic identification and finally specific primers were designed for genotypic detection by PCR assay. Finally, the results were analyzed using spss18 software. Findings: In this study, amongst Fifty-six samples, 31 (55%) different Bifidobacterium species including 15 (36%) B. bifidum, 14 (34%) B. longum, and, 12 (29%) B. 1 were isolated. Out of which, 12 (29%) isolates including B. longum (6), B. breve (4) and B. bifidum (2) were shared between six mother-infant pairs. The correlation coefficient of bifidobacteria isolated from breast milk against their feces was +0.821 (p-value <0.05). By observing the 29 percent contribution and the positive correlation coefficient between breast milk and fecal bifidobacterium in our study, it probably be acknowledged that the main source of intestinal bifidobacterium in the early stages of birth is breast milk.

Meticillin resistant Staphylococcus aureus (MRSA) now is well documented as a nosocomial pathogen causing a variety of infections in human. Aminoglycosides are a class of bactericidal antibiotics which play an important role in treatment of staphylococcal infections. The aim of this study was to illustrate the phenotypic and genotypic resistance to aminoglycosides among MRSA strains isolated from 2 hospitals in Tehran, during 2011 and 2012. During a year, a total of 575 strains of S. aureus were collected and analyzed further. Resistance of strains to ozacillin and cefoxitin was determined using disk diffusion method in accordance with guidelines of CLSI. MRSA strains were collected and susceptibility of strains to 15 different antibiotics was determined. Minimum inhibitory concentrations (MICs) of oxacillin, vancomycin and gentamicin were evaluated using broth microdilution assay, and presence of mecA and pvl gene was showed by PCR method. For SCCmecand ccr typing, multiplex-PCR assays were employed and the genes encoding aminoglycoside modifying enzymes were detected. In this study, 127 strains were resistant to oxacillin and cefoxitin and also harbored mecAgene. The highest resistance was to penicillin (100%), erythromycin (91%), ciprofloxacin (90%), kanamycin (86%), tobramycin (84%) and clindamycin (81%) and also, 60% of strains were gentamicin resistant. On the other hand, all isolates showed susceptibility to vancomycin, lynezolid and quinupristin–dalfopristin. Thirty six and 32% of MRSA strains were resistant to high level of oxacilin and gentamicin (MIC≥ 512 μ g/ml). SCCmec type III and type 3 ccr were the dominant types among MRSA strains. the frequency of aac(6’)-Ie+aph(2’), ant(6)-Ia, ant(4’)-Ia and aph(3’)-IIIa, were detected successfully in 77%, 55%, 50% and 26% of strains. Moreover, the presence of pvl gene was limited to community acquired MRSA strains. Our findings illustrated the presence of aminoglycoside resistant strains of MRSA in hospitals in Tehran. These strains also showed high level resistance to other antibiotics and harbored SCCmec type III, indicating their hospital origin. Moreover, emergence of MRSA strains with high susceptibility to all classes of antibiotics, except for penicillin, are able to acquire antibiotic resistance genes, is an urgent for public health.

Staphylococcus aureus is a common cause of infection among human which contain broad spectrum of lysogenic phages and various virulence factors. Bacteriophages are involved in production of virulence factors via positive and negative lysogenic phage conversion. The aim of this study was to analyze different prophage types of methicillin resistant Staphylococcus aureus strains isolated from a sewage treatment plant (STP) in Tehran during 2010. Totally 653 isolates of S. aureus was collected from a STP in Tehran. All isolates were identified at the species level using specific primers. Susceptibility of isolates selected to oxacillin was determined using disc diffusion and Etest. Presence of mecA gene was checked by PCR. Primers for identification of 6 classes of prophages were used in a Multiplex-PCR assay. All S. aureus strains were confirmed using specific species primers. Amongst all isolates, 100 strains showed resistance to oxacillin. Resistance in 51% and 6% of MRSA isolates was very high and very low (MIC≥256 μg/ml) and (MIC≥4 μg/ml), respectively. One hundred percent of the MRSA isolates contained mecA gene. Five different prophage types and also 4 different prophage patterns were identified among MRSA isolates. All isolates contained at least 1 prophage types and 2 subtypes. The results indicating presence and persistence of clonal MRSA isolates in a sewage treatment plant in Tehran. Moreover, high prevalence of different classes of prophages among MRSA isolates indicating the potential of these strains to produce various virulence factors.

Pneumonia is caused by a range of microorganisms. The incidence of bacterial pneumonia can be divided into two categories: hospital-acquired pneumonia and community-acquired pneumonia. Community acquired Pneumonia is a related leading cause in different populations. Staphylococcus aureus and Klebsiella pneumonia are the most common cause of hospital-acquired pneumonia, and Streptococcus pneumonia and mycobacterium tuberculosis are Cause of community-acquired pneumonia. The aim of this was to investigate the common causative factors of respiratory tract infections include Streptococcus pneumonia, Staphylococcus aureus, Klebsiella pneumonia and mycobacterium tuberculosis using multiplex PCR was the main aim of this study. This study was experimental development. Multiplex PCR is a rapid, accurate molecular method for the simultaneous detection of two or more specific genes in a reaction.In this study, multiplex PCR method for simultaneous detection in the genome-specific sequences leading causes of pneumonia (Streptococcus pneumonia, mycobacterium tuberculosis, Staphylococcus aurous and Klebsiella pneumonia) was done. Specific genes that we chose in this study were: ply (pneumolysin) for Streptococcus pneumonia, nuc (thermonuclease) for the staphylococcus aureus, gyrA (gyrase A) for Klebsiella pneumoniae and pncA gene for Mycobacterium tuberculosis, also the 16s ribosomal gene was used as a positive control. After set-up procedure and observing specific bands, sequencing was confirmed the accuracy of observed bands.Our results showed that the detection of Streptococcus pneumoniae, Staphylococcus aureus, Klebsiellapneumoniae and Mycobacterium tuberculosis in a Multiplex PCR reaction is possible. In fact, this method can be used for direct and simultaneous detection of these agents in the respiratory specimens.

Staphylococcus aureus is known as community-acquired and nosocomial pathogen. Most of the isolates contain lysogenic phages and various virulence factors. The aim of the study was to analyze the antibiotic resistance pattern and detect the enterotoxin A gene among MRSA isolated from two hospitals in Tehran. Totally 94 isolates of methicillin resistant S. aureus were identified at the species level using specific primers. Susceptibility to seventeen antibiotics was determined using disc diffusion method and Minimum inhibitory concentration (MIC) of oxacillin and vancomycin in MRSA isolates were also detected using Etest. mecA and sea genes were detected using specific primers. Primers for identification of 6 classes of prophages were used in a Multiplex-PCR assay. The highest antibiotic resistance was observed to penicilli and followed by ciprofloxacin, erythromycin, kanamycin, amikacin, tobramycin, clindamycin and tetracycline. Five different prophage types were found in MRSA isolates and all MRSA isolates contained at least one prophage type. One hundred percent of the MRSA isolates contained sea and mecA genes. High prevalence of different classes of prophages, production of enterotoxin A and high resistance of MRSA isolates to first and second lines of treatments is a potential treat for public health.

Staphylococcus aureus is a common cause of infection among human and animals and known as community - acquired and nosocomial pathogen. Most of the isolates contain lysogenic phages which are responsible for production of various vir ulence factors. Methicillin resistance in S. aureus is related to mecA gene. mecA gene, it’s regulatory genes, resistance genes to other antibacterial agents and recombinase enzyme gene locus (ccr) are located on staphylococcal cassette chromosome (SCCmec). The aim of this study was to analyze the antibiotic resistance pattern, and typing of mecA gene cluster and ccr gene locus of MRSA strains isolated from patients in Isfahan from 2012 - 2013. Totally 293 Staphylococcus aureus isolate s were collected from 1 hospital in Karaj. All isolates were identified at the species level using specific primers. Susceptibility to 15 antibiotics was determined using disc diffusion method according to guidelines of Clinical Laboratory and Standard ins titute (CLSI). Minimum inhibitory concentration (MIC) of oxacillin and vancomycin in MRSA isolates were also detected using broth micro dilution assay according to CLSI recommendation. Primers for identification of 6 classes of prophages were used in a Mul tiplex - PCR assay. mecA gene was detected using specific primers. Multiplex - PCR assays were used for ccr and SCCmec typing. Among S. aureus isolates, 101 strains (34.5%) were selected as MRSA. The highest antibiotic resistance was observed to eryt hromycin (88%) and followed by ciprofloxacin (85%), clindamycin (84%) and tobramycin (81%) respectively. None of the isolates were resistant to vancomycin, linezolid and synercid. High (MIC≥128 μg/ml) level resistance to oxacillin was observed in 70% of th e isolates. Two different prophage types and 2 sub - types were found in MRSA isolates. All isolates contained mecA gene and 100% of MRSA isolates harbored SCCmecc type III and also type 3 ccr. High prevalence of different classes of prophages e ncoding a variety of virulence factors and high oxacillin resistance provide an important role for phages in the evolutionary development of virulence factors and also diversity in methicillin resistance cassette in MRSA isolates. The presence of SCCmec ty pe III indicating the high prevalence of hospital acquired MRSA isolates. Prevalence of these highly virulent isolates with high resistance to first and second lines of treatments is a potential treat for public health.

Urinary tract infection is the most common nosocomial infection worldwide. Microorganisms causing urinary tract infections are resistant to most of the antibiotics. Pseudomonas aeruginosa (P. aeruginosa) is one of the major causes of nosocomial infections and its antibiotic resistance leads to medical implications associated with urinary tract infections. The aim of this study was to assess the prevalence of P. aeruginosa strains to identify their in-vitro susceptibility to antimicrobial agents and to determine genetic diversity of them.

Urine samples were obtained from catheterized patients in Shohada and Labafi Nejad hospitals, Tehran, Iran, and cultured by standard loop method. The cultures with more than 105 CFU/ml were assumed as positive. After identification of bacterial species by biochemical tests, susceptibility of each isolate was assessed by disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In order to analyze bacterial genotypic diversity, pulsed-field gel electrophoresis (PFGE) was performed.

116 out of 163 urine samples were positive for bacterial isolates. Pseudomonas species were placed in third step of the most frequent isolates. Antibiotic sensitivity was observed against most of antimicrobial agents. Some of strains were genetically similar.

This study revealed that isolated strains were sensitive to wide range of antibiotic agents. In addition, common type strains were responsible of causing inter- and intra-hospital urinary tract infections in catheterized patients.

Pertussis is an acute respiratory infection with chronic paroxissmal cough. After initiation of EPI program in Iran in 1984 with more than 95% coverage of target group, burden of pertussis decreased dramaticaly in the country. In this article we discussed in detail on epidemiological aspects of pertussis in the country. According to the national guidelines for surveillance of pertussis, nasopharyngial specimen has been taken from all suspected cases and sent on transport media for lab confirmation to the Pasture Institute of Iran. Overall, 2052 suspected cases reported to the district health centers in the period of the study. The most prevalent age groups were less than 2 month, 2-11 month and 1-4 y with 22%, 36% and 24% respectively and just 10% were in above 10. 71% lived in urban areas and proportion of male and female was approximately equal. The cases reported mainly in spring and summer.In totally, 1629 clinical specimen tested in laboratory with 18 positive culture and 141 positive by RT-PCR. Detection rate of suspect and confirmed pertussis cases is increasing in Iran. Althogth 90% of cases were in less than 5 y/o, however it could be due to low suspision rate of physicians to pertussis in approach of adults with chronic cough.

Staphylococcus aureus is a common cause of infections among human and animals and known as community-acquired and nosocomial pathogen. MRSA infection has recently become a serious problem in anti-microbial chemotherapy. The aim of the study was to detect and analyze the antibiotic resistance pattern among MRSA isolated from six hospitals in Tehran in 2008-2011.

Totally 396 isolates of MRSA from clinical samples were collected from six hospitals in Tehran. All isolates were identified at the species level using specific primers. Susceptibility to seventeen antibiotics was determined using disc diffusion method. MIC of oxacillin and vancomycin in MRSA isolates was also done using Etest according to CLSI recommendation. PCR was used to detect mecA gene.

Using PCR all isolates were confirmed as MRSA. The highest level of resistance was observed to oxacillin, penicillin, ciprofloxacin, kanamycin, erythromycin, tobramycin and amikacin respectivelyMIC results showed that 86% of isolates showed high level resistant to oxacillin (MIC≥256 μg/ml). None of the MRSA isolates were resistant to vancomycin. One hundred percent of the isolates contained mecA gene.

This study showed that the prevalence of MRSA isolates is lower than the other studies in Iran. MRSA isolates showed resistance to broad spectrum of antibiotics. Synercid, linezolid, vancomycin and chloramphenicol are the most effective antibiotics against MRSA infections. Detection of mecA gene is a rapid and reliable method for identification of MRSA isolates.

Enterococci are normal microflora of the humans. They are identified as opportunistic pathogens and they are among the three major factors of hospital infections. Enterococci are able to form biofilms on the biotic and abiotic surfaces and this ability is one of the important factors to pathogenesis. The purpose of the present study was to evaluate biofilm formation and evaluation of antibiotic resistance among Enterococcus faecalis (E. f) isolates from clinical and environmental isolates in Tehran hospitals.

In total, 90 isolates of Enterococcus faecalis including 58 clinical and 32 environmental isolates of (E.F) were collected from 2 hospitals in Tehran. All enterococcal species isolates were identified by species-specific PCR. Antibiotic resistance pattern of 90 isolates of (E. f) isolates was determined by disk diffusion method according to the CLSI guidelines. Biofilm formation of (E. f) isolates was evaluated by microtiter plates method.

The highest resistant percents of clinical (E. f) isolates against tetracycline, erythromycin, co-trimoxazole, ciprofloxacin and gentamicin were 86%, 62%, 69%, 69% and 64%, respectively. Among environmental isolates the resistant percents were 75%, 44%, 25%, 22% and 25%, respectively. According to biofilm formation assay, 72% and 84.5% of clinical and environmental (E. f) isolates were positive, respectively. Samples that were able to form biofilms showed more antibiotic resistance than those that are not able to form biofilms.

The results of this study indicate high prevalence of antibiotic resistance and the ability of biofilm formation among Enterococcus faecalis. Considering the role of biofilms in increased antibiotic resistance and virulence, it is needed to carry out necessary measure to eliminate suitable conditions for biofilm formation which act as a shield against various conditions, including the host immune system and antibiotics.

V. cholerae, a Gram-negative bacterium is the causative agent of Cholera. V. cholerae O1 produces cholera toxin (CT) which is responsible for diarrhea. The zonula occludens toxin (Zot) acts on intestinal tight junctions to increase intestinal permeability. The accessory cholera enterotoxin (Ace) increases the potential difference across intestinal epithelium and alters ion transport. V. cholerae athogenicity depends on a combination of factors known as ability to produce a cholera toxin (CT) and to adhere and colonize small intestine through colonization factor known as toxin-coregulatedpilus (TCP). Thirty nine El Tor variants were obtained from outbreaks during 2005 in Iran. After detection of isolates by biochemical methods, and serotyping, chromosomal DNA was extracted by standard phenol/chloroform method. The oligonucleotide primers for each of the selected virulence-associated factors were designed based on available GenBank sequences for V. cholerae O1 E1 Tor for all the genes. PCR assay was performed and the PCR product was run and visualized in 1.5% agarose gels stained with ethidium bromide. In the present study, PCR was used to detect the presence of ctxA, Zot, Ace, and tcpA genes on 39 V. cholerae strains isolated from the summer epidemic 2005 in Iran. With the specific primers, the V. cholerae isolates were analysed by PCR for the presence of tcpA, ctxA, Ace and Zot genes. PCR showed that the genes encoding cholera toxin (ctxA), toxin co-regulated pilus (tcpA), accessory cholera enterotoxin (Ace) and zonula occludens toxin (Zot) were present in 89.74%, 84.6%, 100% and 100% of the isolates, respectively. Discussion & The results of our study confirm that strains without ctxA gene, Ace and Zot genes are important and can be the causative agent of cholera.

Cholera is a diarrheal disease caused by different serotypes of Vibrio cholerae. More than 200 O-antigen serotypes have been identified. Interestingly, only the O1 and O139 serotypes are known to cause epidemic disease. Different cases of Ogawa and Inaba serogroups have been found during last years in Iran. In this study, seroepidemiology of cholera in Iran between 2004 and 2008 was studied. In this descriptive study, fecal specimens (stool or rectal swabs) were cultured in a selective plating media like TCBS. Overnight growth of V. cholerae colony on TCBS was cultured on BHI. Colonies grown on BHI, used for biochemical and biotyping and serotyping tests. Among 93 clinical strains isolated during 5 consecutive years (2004-2008), different serogroups of V. cholerae O1 (Ogawa, Inaba and Hikojima) biotype El Tor were identified. This study showed significantly decreasing number of Ogawa strains compared with Inaba strains in recent years in Iran. However, some rare cases of Hikojima strains have been detected in Iran in 2007 and 2008.

Enterococci are natural inhabitants of the gastrointestinal tract of humans and animals. Over the past decade, enterococci have become one of the leading causes of nosocomial infections,because they have acquired resistance to many antimicrobial agents such as vancomycin and gentamicin.Development of high-level gentamicin resistance among enterococci represents a serious therapeutic problem as it precludes synergy between aminoglycosides and cell-wall active agents. High-level gentamicin resistance (MIC ≥ 500 mg/ml) in enterococci is predominantly mediated by aac (6')-Ie-aph (2'')-Ia gene.. The aim of present study was determination of high level gentamicin resistance in Enterococcus species isolated fromhealthy human in Tehran. After enrichment of the fecal samples collected from healthy volunteers in BHI broth containing gentamicin, the isolates were subcultured on m-Enterococcus agar containing gentamicin. The isolates were identified to the genus and species levels and MIC was determined. Antibiotic susceptibility of the isolates was tested by agar diffusion method for 8 various antibiotics. The aac(6')-Ie-aph(2'')-Ia gene was identified by PCR technique. gentamicin resistant enterococci(GRE) were isolated from 76(15.2%) of 500 fecal samples. The isolated species were: E. faecalis 45(59.2%), E. faecium 29(38.1%) and E. gallinarum 2(2.63%). GRE isolates were highly resistant to gentamicin(MIC ≥ 500 μg/ml). Eleven different antimicrobial resistance patterns were observed in this study. All of isolates carried the aac(6')-Ie-aph(2'')-Ia resistance gene. These results represent a rather high prevalence of gentamicin resistance enterococci as a normal flora in the community. The aac(6')-Ie-aph(2'')-Ia gene was the most common gene among the gentamicinresistant isolates evaluated in this stud.

The development of high-level resistance to aminoglycosides, penicillin and glycopeptides singly and in combination by enterococci, has important clinical implications. Enterococci are normal flora of intestine and are released into the environment through faeces. One of the investigation methods on enterococcal population is the screening of sewage treatment plants for presence of enterococci. The aim of this study was to determine clonal diversity among HLGR enterococcal isolates, obtained from the sewage treatment plants (STPs) in Tehran. From November 2005 to May 2006 a total of 140 enterococcal isolates were collected from 3 sewage treatment plants (STP) located at different parts of Tehran. Confirmation of species and detection of gentamicin resistance genes were performed by PCR method. Antimicrobial susceptibility and MIC determination tests were performed on the basis of CLSI standard. Php typing was used for typing of enterococcal HLGR isolates. E. faecalis (26%) and E. faecium (74%) were the most common species in STPs samples. E. faecium had the highest rate of resistance to tetracycline. The MICs for all of enterococcal HLGR isolates were ≥1024 µg/ml and most enterococci contained aac (6')-Ie-aph (2'')-Ia gene. Only one E. faecium isolate showed aph (2")-Ic gene. Php typing of sewage HLGR E. faecalis and E. faecium isolates yielded 16 and 50 patterns with a diversity index of 0.910 and 0.945, respectively. The presence of aac (6')-Ie-aph (2'')-Ia gene in most of HLGR isolates, suggested widespread dissemination of this gene in the enterococcal isolates in sewage. Clonal diversity in HLGR and MDR entrococcal population showed wastewater could be a possible reservoir for circulation of these isolates in the community.

Enterococci are members of the normal gut flora of animals and humans and are released into the environment directly or via sewage outlets. The aim of the study was to detect and analyze the biochemical diversity and antibiotic resistance of the enterococci strains in Tehran sewage. A total of 316 isolates of enterococci were selected on mEnterococcus agar medium and identified at the species level by the common biochemical tests. Antibiotic susceptibility test of isolates with six antibiotics and the MIC was also done using broth macro dilution assay. The results showed that 161, 94, 37, 12, 7, 3, 2 and 1 isolates were E. faecium, E. hirae, E. faecalis, E. gallinarum, E. mundtii, E. raffinosus, E. casseliflavus and E. avium respectively. The antibiotic resistance to the isolates were as follow: 5, 5, 6, 17, 21 and 35% of the isolates were resistant to vancomycin, gentamicin, chloramphenicol, tetracycline, ciprofloxacin and erythromycin respectively. MIC test showed that 15 of the isolates were highly resistant to vancomycin. Although Enterococcal species was considered to have a high distribution in Tehran sewage, but the presence of VRE was limited to E. faecium. Resistance to vancomycin and gentamicin was less than other antibiotics, and resistance to erythromycin and ciprofloxacin was higher.