Molecular characterization of intestinal carriage of carbapenem-resistant Enterobacteriaceae among inpatients at two Iranian university hospitals: first report of co-production of bla NDM-7 and bla OXA-48.

Gastrointestinal colonization of carbapenem-resistant Enterobacteriaceae (CRE) could serve as a reservoir for the transmission of these pathogens in the clinical setting. The aim of this study was to investigate the intestinal carriage of CRE and to analyze risk factors for CRE carriage. Rectal swabs were collected from 95 patients at two Iranian university hospitals. CRE screening was performed using selective media (CHROMagar and MacConkey agar). Polymerase chain reaction (PCR) was used to detect carbapenemase-encoding genes. Clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE). The rate of carriage of CRE in hospitalized patients was 37.9%. Overall, 54 CRE isolates were identified, of which 47 were carbapenemase-producers. All of the 54 CRE were detected using CHROMagar compared with 52 CRE detected using MacConkey agar. Fifteen patients were colonized by multiple CRE isolates. Three significant risk factors for CRE carriage were detected: intensive care unit (ICU) hospitalization, antibiotic exposure, and mechanical ventilation. bla OXA-48 was the most frequent carbapenemase detected, followed by bla NDM-1 and bla NDM-7. Eleven carbapenemase-producing Enterobacteriaceae (CPE) isolates co-harbored bla NDM-1 and bla OXA-48. Also, six CPE isolates co-harbored bla NDM-7 and bla OXA-48. We did not detect bla KPC, bla GES, bla IMP, or bla VIM. PFGE analysis showed that Escherichia coli clones were diverse, while Klebsiella pneumoniae isolates were divided into four clusters. Cluster I was the major clone carrying bla OXA-48 and bla CTX-M-15 genes. In our study, the carriage rate of CRE was high and the emergence of CPE isolates among patients is alarming. The implementation of adequate preventive measures such as active surveillance is urgently needed to control the spread of CPE in the healthcare setting.

Comparison of Two Different Methods for the Extraction of Outer Membrane Vesicles from the Bordetella pertussis as a Vaccine Candidate.

Despite the availability of a vaccine, pertussis is still a worldwide health problem. Outer membrane vesicles (OMVs) in gram-negative bacteria can stimulate the immune system due to several outer membrane proteins and are very good candidates in vaccine development. OMVs obtained from Bordetella pertussis contain several antigens, which are considered immunogenic, and could make them a potential candidate for vaccine production. The current study aimed to compare the current OMV extraction method (with ultracentrifuge) and a modified extraction method (without ultracentrifuge) and to evaluate the physicochemical properties as well as the expression of their main virulence factors. Vaccinal strain BP134 grown on Bordet Gengo agar were inoculated in Modified Stainer-Scholte medium for mass cultivation. OMVs were prepared using two different methods. They were then stained and examined with a transmission electron microscope. Protein contents were measured by the Bradford method, and then the protein profile was evaluated by SDS-PAGE. The presence of immunogenic antigens was detected by Western blotting. The size and shape of the OMVs obtained from the modified method without the use of ultracentrifuge were similar to the current method and had a size between 40 and 200 nm. The total protein yields of the OMV isolated using the current and modified methods were 800 and 600 µg/ml, respectively. Evaluating the protein profile of extracted OMVs showed the presence of different proteins. Finally, the presence of PTX, PRN, and FHA was observed in OMVs extracted from both methods. Comparison of the two OMV extraction methods showed that the obtained vesicles have a suitable and similar shape and size as well as the expression of three important pathogenic factors as immunogens. Despite the relatively low reduction in protein yield as the modified method does not require ultracentrifuge, this extraction method can be used as a suitable alternative for extracting the outer membrane vesicles from B. pertussis, especially in developing countries. It should be noted that further experiments including immunogenicity determination of OMVs obtained as vaccine candidates in animal models are required.

Assessment of Mouse Ileal loop Protection against Clinically Isolated Vibrio cholerae Outer Membrane Vesicles as a Vaccine Candidate.

Cholera, a life-threatening disease caused by the Gram-negative bacterium Vibrio cholera, remains a concern in developing countries. The present study investigated the immunogenicity and protective immunity of outer membrane vesicles (OMVs) and combination of OMV and killed whole cells (WC) of a local strain isolated from the last outbreak in Iran in addition to reference and local strains of V. cholerae El Tor O1 in comparison to Dukoral vaccine in mice model. The protein content, morphology, and size of extracted OMVs were evaluated by electrophoresis and microscopic analyses, respectively. The serum titers of total immunoglobulin G (IgG), IgG1, IgG2a, and immunoglobulin A (IgA) in addition to secretory IgA and total IgG in different mice groups were determined by enzyme-linked immunosorbent assay (ELISA). In addition, fluid accumulation (FA) assay regarding the resistance to live strain of V. cholerae in ligated ileal loops was carried out to determine immunogenicity by OMV or combination of OMV and WC in comparison to that reported for Dukoral vaccine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified OMVs indicated protein profiles within the range of 34-52 kDa. Furthermore, transmission electron microscopy demonstrated the spherical shaped vesicles of 50-200 nm. The results of ELISA showed significant titers of systemic and mucosal immune anti-OMV IgGs in immunized BALB/c mice with different vaccine regimens. Additionally, a notable increase in the FA ratio was demonstrated in this study. The obtained results of the present study revealed that the WC-OMV combination of local strain can induce a high level of antibody response indicating more protection than OMV or WC separately. Moreover, it can be considered an effective immunogen against V. cholerae.

High-level aminoglycoside resistance and distribution of aminoglycoside resistance genes among Enterococcus spp. clinical isolates in Tehran, Iran.

OBJECTIVES: Enterococci have gained attention during the past decade as important nosocomial pathogens. Their increasing prevalence has been paralleled by the occurrence of multidrug-resistant and high-level aminoglycoside-resistant strains. This study isolated Enterococcus spp. from hospital samples and determined their antibiotic resistance profile, focusing on aminoglycosides, and associated resistance mechanisms. METHODS: A total of 195 enterococci from hospital samples in Tehran were studied. Isolates were identified by biochemical reactions. Antimicrobial resistance was determined by disk diffusion. The vancomycin MIC for vancomycin-resistant isolates was determined by agar dilution. Detection of aminoglycoside resistance genes and intI1 and intI2 gene was performed by PCR. RESULTS: The majority of isolates were Enterococcus faecalis (65.1%), followed by Enterococcus faecium (31.8%), Enterococcus gallinarum (2.6%) and Enterococcus solitarius (0.5%). According to antibiogram results, 42.1% of isolates were high-level gentamicin-resistant (HLGR) and 40.5% were high-level streptomycin-resistant (HLSR). There was a high prevalence of aac(6')-Ie-aph(2")-Ia (96.3%) among HLGR isolates. ant(6)-Ia and aadA were identified in 93.7% and 64.6% of HLSR isolates, respectively. aph(2'')-Ic was detected in 7 isolates (3.6%) and aph(2'')-Ib in only 4 isolates (2.1%); no isolates harboured aph(2'')-Id, intI1 or intI2. CONCLUSION: Multidrug resistance was higher among HLGR and HLSR isolates compared with non-HLGR and non-HLSR isolates, which may result in limited treatment options. More than 50% of isolates were susceptible to aminoglycosides, thus correct identification in clinical laboratories and administration of these antibiotics can result in decreased used of antibiotics such as vancomycin and linezolid and help to reduce the emergence of resistance to these drugs.

Serovar determination, drug resistance patterns and plasmid profiles of Pseudomonas aeruginosa isolated from burn patients at two hospitals of Tehran (IRAN).

The serovars and drug susceptibility patterns of 265 isolates of Pseudomonas aeruginosa cultured from burn patients during 2001-2002 at Motahari and Tohid Hospital were determined. Distribution of serovars was different at two hospitals. Most of the isolates at Tohid Hospital belonged to serovar 0:1, but 21 and 13% of them were untypeable or polyagglutinable, respectively. Serovar 0:11 was the most prevalent serovar at Motahari Hospital. All the strains were multi resistant to tetracyclin, carbenicillin, amikacin, ceftazidime, sulfamethoxazol, ciprofloxacin, tobramycin, kanamycin, cefotaxime and gentamicin. Further analysis of the strains by plasmid profiling demonstrated that 95% of the isolates carried two megaplasmids. However, there was not any correlation between the serotyping and presence of plasmids. Changes in the drug susceptibility patterns and beta-lactamase production of some cured derivatives were observed after the strains lost their plasmids. The emergence of multi-drug resistant strains of P. aeruginosa is a serious concern in burn patients who are hospitalized in Tehran.

Molecular identification and detection of virulence genes among Pseudomonas aeruginosa isolated from different infectious origins.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa possesses a variety of virulence factors that may contribute to its pathogenicity. The aim of this study was to evaluate oprI, oprL and toxA genes for PCR identification of clinical P. aeruginosa. In order to find out any relation between special virulence factors and special manifestation of P. aeruginosa infections, we detected virulence factors among these isolates by PCR. Ribotyping was used to evaluate the clonal relationship between strains with the same genetic patterns of the genes studied. MATERIALS AND METHODS: In this study, 268 isolates of P. aeruginosa were recovered from burn, wound and pulmonary tract infections. The prevalence of oprI, oprL, toxA, lasB, exoS and nan1 genes was determined by PCR. One hundred and four isolates were selected randomly to investigate clonal diversity of the isolates with ribotyping using SmaI. RESULTS AND CONCLUSIONS: All P. aeruginosa isolates in this study carried oprI, oprL and lasB genes. Difference between exoS prevalence in isolates from pulmonary tract and burn isolates was statistically significant. Prevalence of nan1 and toxA gene was significantly higher in pulmonary tract and burn isolates, respectively. Ribotyping showed that most of the isolates (87%) belonged to clone A and B. Detection of oprI, oprL and toxA genes by PCR is recommended for molecular identification of P. aeruginosa. Determination of different virulence genes of P. aeruginosa isolates suggests that they are associated with different levels of intrinsic virulence and pathogenicity. Ribotyping showed that strains with the same genetic patterns of the genes do not necessarily have similar ribotype patterns.

Isolation and genetic characterization of metallo-ß-lactamase and carbapenamase producing strains of Acinetobacter baumannii from patients at Tehran hospitals.

BACKGROUND AND OBJECTIVE: Carbapenems are therapeutic choice against infections caused by gram-negative bacilli including strains of Acinetobacter baumannii. Resistance to these antibiotics is mediated by efflux pumps, porins, PBPs and ß-lactamases. The aim of this study was to determine the possibility of existence of MBLs, OXAs and GES-1 betalactamase genes among clinical isolates of Acinetobacter collected from Tehran hospitals. MATERIAL AND METHODS: Two hundred and three Acinetobacter isolates were collected from patient at Tehran hospitals. The isolates were identified using biochemical tests. The susceptibility to different antibiotics was evaluated by disk diffusion method and MICs of imipenem were determined using Micro broth dilution method (CLSI). PCR was performed for detection of bla(VIM-2), bla(SPM-1), bla(IMP-2), bla(GES-1), bla(OXA-51), bla(OXA-23) betalactamase genes. Clonal relatedness was estimated by PFGE with the restriction enzyme SmaI. RESULTS AND CONCLUSION: Of 100 isolates of imipenem resistant Acinetobacter spp. collected from Tehran hospitals in 2009 and 2010, 6 isolates produced metallo-beta-lactamases and 94 isolates produced OXA-type carbapenemase. The bla(SPM-1), bla(GES-1), bla(OXA-51), bla(OXA-23) genes were detected by PCR among 6, 2, 94 and 84 isolates of A. baumannii, respectively. The MICs of isolates to imipenem were 8-128 µg/mL. PFGE analysis of 29 bla(OXA-51) and bla(OXA-23)-positive A. baumannii isolates gave 6 different patterns. This is the first report of SPM-1 and GES-1 beta-lactamase producing A. baumannii. Production of the OXA-23, OXA-51, GES-1 and SPM-1 enzyme presents an emerging threat of carbapenem resistance among A. baumannii in Iran.

Antimicrobial resistance profile and presence of class I integrongs among Salmonella enterica serovars isolated from human clinical specimens in Tehran, Iran.

BACKGROUND AND OBJECTIVES: Salmonella is one of the leading causes of food-borne diseases. Increasing occurrence of antimicrobial resistance, especially multidrug-resistance, in Salmonella serovars is a major public health problem worldwide. This study was carried out to detect class I integrons and antibiotic resistance profiles in clinical isolates of Salmonella serovars collected from seven hospitals in Tehran during November 2009 to June 2010. MATERIALS AND METHODS: Antibiotic susceptibility profile of 19 antibiotics against 58 Salmonella isolates commonly used in humans was determined using disk diffusion assay. Minimum inhibitory concentration against ceftriaxone and ciprofloxacin was studied. PCR assays were used to detect class I integrons. RESULTS: Among 58 Salmonella isolates, 72.4% were Salmonella enterica serovar Enteritidis, 8.7% were Salmonella enterica serovar Typhimurium and 18.9% were other serovars. Of the total 58 Salmonella serovars, 43 (74.1%) were multidrug-resistant and showed resistance to three or more antibiotic families. Class I integrons were identified in 38 (88.3%) MDR Salmonella isolates. Ciprofloxacin minimum inhibitory concentration ranged between 0.125-2 g/ml for four isolates and other four isolates exhibited resistance to ceftriaxone (MIC 64-256 g /ml). CONCLUSION: The high prevalence of class I integrons was seen in our MDR Salmonella isolates and class I integrons might play an important role in the dissemination of antimicrobial resistance determinants.

Detection of Neisseria gonorrhoeae from vaginal swabs of Ewin, Rajaii shahr, Karaj and Varamin female prisoners by PCR and culture methods.

Isolation of N. gonorrhoeae by culture method is currently the gold standard for the definitive diagnosis of gonorrhoea. However, PCR techniques are being used more frequently as sensitivity and specificity of the newer tests are improved. In this study, 500 vaginal swabs from Ewin, Rajaii shahr, Karaj and Varamin female prisoners were used for detection of N. gonorrhoae by culture and PCR techniques. Five hundred vaginal swabs from Ewin, Rajaii shahr, Karaj and Varamin female prisoners were cultured in modified Thayer Martin in 37 degrees C with 5% CO2 for 72 h. Oxidase, catalase tests, biochemical tests such as maltose and glucose oxidation and gram staining, were used to confirm the isolated species. Amplification by PCR using 2 targets which are specific for N. gonorrhoeae, Ngu1 and Ngu2, were used to detect the presence of gonococcal specific DNA. Despite of finding some questionable samples as N. gonorrhoeae by using biochemical tests, PCR method confirmed that none of them were positive for N. gonorrhoeae. This study deals with detection of N. gonorrhoeae among woman prisoners in three main prisons in Tehran, Iran. The high specificity and sensitivity coupled with low cost and rapidity of the method (PCR) provided a substantial advantages over the time consuming culture methods currently used in hospitals and laboratories.