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Background: Bordetella pertussis, a highly contagious respiratory. Notably, the resurgence of pertussis has recently been associated with the lacking production of vaccine virulence factors. This study aimed to screen pertactin (Prn) and filamentous hemagglutinin (Fha) production in Iran with 50 years' whole cell vaccine (WCV) immunization program. Methods: Overall, 130 B. pertussis isolates collected from Pertussis Reference Laboratory of Iran during 2005-2018. Real-time PCR was performed by targeting IS481, ptxP, IS1001 and IS1002 for species confirmation of B. pertussis. Western-blot was used to evaluate the expression of virulence factors (pertactin and filamentous hemagglutinin). Results: All tested B. pertussis isolates expressed Prn and all except two isolates expressed Fha. We have sequenced genomes of these strains and identified differences compared with genome reference B. pertussis Tohama I. Conclusion: Many countries reporting Prn and Fha-deficiency due to acellular vaccine (ACV) pressure. Our results demonstrate in a country with WCV history, Fha-deficient isolates may rise independently. However, Prn-deficient isolates are more under the ACV pressure in B. pertussis isolates. Continues surveillance will provide a better understanding of the effect of WCV on the evolution of the pathogen deficiency.

Background and Objectives: Escherichia coli (E. coli) sequence type 131 (ST131) is associated with extended-spectrum beta-lactamase (ESBL) production and fluoroquinolone resistance. This study aimed to investigate the prevalence of ST131, ESBL, and plasmid-mediated quinolone resistance (PMQR) genes in the ciprofloxacin-resistant (CIPR ) and ESBL producers from women with UTI. Materials and Methods: The CIP-resistant ESBL producing (CIPR /ESBL+ ) E. coli isolates were screened for ST131-by specific PCR of mdh and gyrB. The ESBL and PMQR genes were screened by single PCR. The ST131 and non-ST131 isolates were selected to determine the mutations of gyrA and parC using PCR and sequencing, and also their genetic background by the Pasteur-MLST scheme. Results: Overall, 55% (33/60) CIPR /ESBL+ isolates were identified as ST131 (94% O25b-ST131). Resistance rate to ampicillin-sulbactam (70%), aztreonam (97%) and gentamicin (61%), the prevalence of aac(6′)-Ib-cr (66%), blaCTX-M-15 (82%), the profile of qnrS+aac(6′)-Ib-cr (30%), and the double mutation in the parC was significantly higher in ST131 than nonST131 isolates. The coexistence of PMQR and ESBL genes was found in more than 50% of ST131 and non-ST131 isolates. ST131 isolates differentiated into PST43 and PST506. Conclusion: Management of women with UTI caused by the CIPR /ESBL+ isolates (ST131) co-harbored PMQR, ESBL, and chromosomal mutations, is important for their effective therapy.

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a pathogen of global concern. In this study, both phenotypic and genotypic tests were used to detect hvKp. Antimicrobial resistance profiles and clonal relatedness of clinical isolates were also determined. We found that 62.6% of the isolates were tellurite resistant and among them iucA or iutA or peg344 as hvKp molecular markers, were positive. The blaSHV (81.4%), followed by blaCTX−M15 (75.5%) and blaTEM (67.6%), blaOXA−48 (33.7%), blaNDM−1 (32.3%) were detected, while blaKPC−1 was not present in any hvKp isolates. It was found that the majority of hvKp isolates belonged to capsular serotype K20 and ompK36 group C, which is related to CG23 (e.g. ST23). A high percentage of multidrug-resistant hvKp (MDR-hvKp) and high resistance to imipenem (66%) indicated that there is an urgent problem that should be addressed in the clinical settings.

Extended-Spectrum Beta–lactamase producing Enterobacteriales (ESBL-PE) in fecal carriage have become a global health concern. Detection of putative virulent ESBL-producing E.coli (ESBL-EC) isolates among asymptomatic carriers is a threatening issue in public health. The aim of this study was to investigate the intestinal carriage of ESBL-EC, phylo-groups and clonal relatedness among putative virulent groups of ESBL-EC isolated from fecal carriages. A total of 120 rectal swabs; 50.8% (61/120) from inpatients of intensive care unit (ICU) and 49.2% (59/120) from outpatients were collected. The ESBL-EC screening was performed by using MacConkey agar supplemented with cefotaxime. PCR assays were applied for determination of phylo-groups, detection of ESBL and carbapenemase genes. Conjugation experiment, plasmid replicon typing and Multilocus Sequence Typing (MLST) were performed for putative virulent phylo-groups. Totally, of 120 studied individuals, 60.0% (72/120) were carrier for ESBL-EC. The rate of blaCTX-M-15, blaTEM, blaSHV was 90.2% (65/72), 50.0% (36/72) and 5.5% (4/72), respectively. The frequency of phylo-groups A, B1, B2, C, D, and F were 20.8% (15/72), 6.9% (5/72), 20.8% (15/72), 2.7% (2/72), 13.8 (10/72) and 12.5% (9/72), respectively. In conjugation experiments, of 6 tested isolates, 5 had conjugative plasmids. The most prevalent plasmid types belonged to IncF incompatibility groups. The MLST analysis showed that the main sequence types among ESBL-EC isolates were ST769 and ST472. The current study provides novel information about the presence of the ESBL-EC isolates, particularly putative virulent phylo-groups among fecal carriages in Iran. Our data revealed that there was almost high ST heterogeneity among putative ESBL-EC isolates. In order to implementation of effective infection control program, detection of fecal carriage in appropriate time typically at the beginning of admission to the hospital is recommended.

Carmoisine is an azo dye widely used in many industries, and therefore frequently occurs in the effluent of many factories. To our knowledge, biological degradation of carmoisine has received little attention. The present study investigates the capability of Saccharomyces cerevisiae ATCC 9763 for degradation of carmoisine. Spectrophotometry data indicates that carmoisine (50 mg/l) was eliminated from the aqueous medium after approximately 7 h of incubation with Saccharomyces under anaerobic shaking conditions. Thin layer chromatography (TLC) revealed the removal of carmoisine as well as the appearance of aromatic amines in samples collected from the decolourized medium by S. cerevisiae and this was subsequently confirmed by Fourier transform infrared (FTIR) spectroscopy. Liquid chromatography mass spectrometry (LC/MS) was carried out on fractions from consecutive column chromatography and two-dimensional (2D) chromatography. LC/MS indicated degradation of carmoisine into its constituent aromatic amines. In addition, investigating the effect of environmental conditions on the decolourization process indicated that yeast extract could positively affect decolourization rates; shaking significantly accelerated decolourization and shortened the time required for complete biodecolourization from ≃ 8 days to ≃ 7 h; and Saccharomyces was able to consume sucrose as a carbon source and remove the carmoisine despite the presence of sunset yellow, which remained unaffected.

Pertussis caused by Bordetella pertussis, remains a public health problem worldwide, despite high vaccine coverage in infants and children in many countries. Iran has been using whole cell vaccine ...

Introduction: Pathogen adaptation is considered as one of the important reasons for the emergence of pertussis (whooping cough). Antigenic divergence between vaccine strains and clinical isolates of Bordetella pertussis has been occurred over the years. It is suggested that the predominant genomic profile of B. pertussis has an enough capacity to spread among the population. The aim of this study was to characterize a predominant B. pertussis strain isolated from Iranian patients during 2008-2015 period. Methods: Based on the epidemiologic results of B. pertussis circulating strains in Iran, a strain named BPIP91 with predominant genomic and virulence pattern was selected from Biobank of Pasteur Institute of Iran. The antibiotic susceptibility testing was done and the growth rate of this strain was analyzed. The lethal (LD50) and safety dose of infection of BPIP91 was also determined via mice intranasal infection. Results: Our results showed that BPIP91 was susceptible to erythromycin, azithromycin, clarithromycin, chloramphenicol, trimethoprim-sulfamethoxazole and rifampin antibiotics. The growth rate of BPIP91 was almost two-fold lower than the vaccine strain. In addition, the LD50 and infectious dose of BPIP91 strain were about 2 × 1010 and 4 × 106 colony forming units, respectively. Conclusion: In this study we obtained the growth curve, LD50 and intranasal infectious dose of a circulating strain with predominant genomic pattern in Iran. However, further examinations including determination of immunogenicity of this native strain in animal model is needed in order to evaluate its use as a vaccine strain candidate.

Objectives Carbapenem-resistant Klebsiella pneumoniae infections are associated with increased rates of treatment failure and death. Several studies have reported isolates with a combined hypervirulent and antimicrobial-resistant phenotype. Methods In this study, the molecular characteristics of hypervirulent K. pneumoniae (hvKP) isolated from mechanically-ventilated patients admitted to a toxicological intensive care unit (ICU) in Tehran, Iran, were examined. String test, antimicrobial susceptibility testing, virulence factors analysis and plasmid replicon typing were performed. The clonal relatedness of the isolates was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results hvKP accounted for 9.4% (5/53) of K. pneumoniae isolated from ventilator-associated pneumonia among patients admitted to the ICU with acute drug poisoning. The mortality rate was 7.5% (4/53) among K. pneumoniae-infected patients. All fatal K. pneumoniae were hvKP isolates, were resistant to imipenem and harboured an aacA7, blaVIM-2 and dhfrI cassette arrangement in a class 1 integron. The isolates were shown to be ST23 (Pasteur scheme) and exhibited similar PFGE patterns. Plasmid analysis revealed a class 1 integron harbouring blaVIM-2 located on an ca. 45-kb IncN plasmid. Conclusion Here we describe the emergence of VIM-2-producing hvKP serotype K1/ST23 in an outbreak with high mortality in a hospital toxicological ICU. It appears that we must alert and prepare the hospital’s surveillance system for the appearance, expansion and clinical importance of new K. pneumoniae clones associated with high antimicrobial resistance and robust virulence capabilities.

Introduction: Serum bactericidal assay is the gold standard index of protection against Vibrio cholerae. The outer membrane vesicles (OMVs) which are released during the bacterial growth have intrinsic immune stimulatory properties, based on their nature and composition. In this study, the induction of serum bactericidal activities in immunized BALB/c mice was determined using different vaccine regimens, using V. cholerae O1 (El Tor biotype) OMVs. Methods: A single clone of V. cholerae O1 (El Tor biotype) isolated during the 2005 outbreak in Iran, was used. A detergent-centrifugation procedure was used for OMVs production. Various vaccination regimens were inoculated into female mice via an oral route. The vaccine formulas included V. cholerae OMVs, killed whole cells of V. cholerae (WC), combination of WC-OMV and licensed cholera vaccine (Dukoral).The serum vibriocidal activity of mice sera was determined by measuring the complement-mediated lysis. Results: Electron microscopy of the purified OMVs from the isolated V. cholerae revealed the spherical-shaped vesicles of the size range 20-300 nm. In vitro reactivity of mice sera bactericidal capability against regimens of vaccination showed a significant immune response of antibody titers in comparison with negative control groups. Also, there was a significant increase in serum bactericidal titer of WC-OMV obtained from wild-type V. cholerae which had a satisfactory reactivity as Dukoral cholera vaccine. Conclusion: The results indicated that the combination of WC-OMV from the local strain is able to induce a high level of bactericidal antibody responses and it can be useful in optimization of the vaccine formula.

Background: Outer membrane vesicles (OMVs) release from Gram-negative bacteria and are interesting alternatives that can replace those vaccines that contain naturally incorporated bacterial surface antigens, lipopolysaccharides (LPS) and outer membrane proteins (OMPs). Nanoparticles can be used to encapsulate vesicles for slow release and prevent macromolecular degradation. Objective: Therefore, encapsulation of OMVs of B. pertussis into sodium alginate nanoparticles was the main aim of the current study. Methods: The OMVs of B. pertussis extracted and characterized by particle sizer, electron microscopy, SDSPAGE and Western blot assays. The extracted OMVs were encapsulated in sodium alginate nanoparticles (OMV-NP) using unique gelation process and injected into BALB/c mice. Immunogenicity indices such as different classes of antibodies and interleukins related to different T cell lines were evaluated in immunized mice by ELISA. The respiratory challenge was evaluated in the groups of mice. The existence of pertussis toxin (PTX), filamentous haemagglutinin (FHA) and PRN (pertactin) in B. pertussis OMVs was verified using SDSPAGE and Western blot analysis. Results: TEM electron microscopy showed the size of these OMVs to be around 20-80 nm. The OMVs with appropriate quality were encapsulated into sodium alginate nanoparticles (OMV-NP), and the final size was about 500 nm after encapsulation. Immunity indices were significantly higher in the OMV-NP receiving group. In challenge tests, the OMV-NP vaccine was able to show the highest rate of lung clearance compared to the control groups (OMV and wPV) at the lowest injection dose. Conclusion: The results indicate the potential of OMV-NP as a novel vaccine delivery system.

Pertussis also known as whooping cough is a respiratory infection in humans particularly with severe symptoms in infants and usually caused by Bordetella pertussis. However, Bordetella parapertussis can also cause a similar clinical syndrome. During 2012 to 2015, from nasal swabs sent from different provinces to the pertussis reference laboratory of Pasture Institute of Iran for pertussis confirmation, seven B. parapertussis isolates were identified by bacterial culture, biochemical tests, and the presence of IS1001 insertion in the genome. The expression of pertactin (Prn) as one the major virulence factor for bacterial adhesion was investigated using western blot. Moreover, the genomic characteristic of one recently collected isolate, IRBP134, from a seven-month infant was investigated using Illumina NextSeq sequencing protocol. The results revealed the genome with G+C content 65% and genome size 4.7 Mbp. A total of 81 single nucleotide polymorphisms and 13 short insertions and deletions were found in the genome compared to the B. parapertussis 12822 as a reference genome showing ongoing evolutionary changes. A phylogeny relationship of IRBP134 was also investigated using global B. parapertussis available genomes.

Here we investigated nationwide clinical Bordetella pertussis isolated during 2005–2017 from different provinces of Iran, a country with more than 50 years whole cell vaccine immunisation history. Our results revealed the ongoing increase in the population of ptxP3/fim3–2 B. pertussis isolates in different provinces which were differentiated into nine clades. The largest clade (clade 8) which was previously found to be prevalent in Tehran was also prevalent across the country and clade 5 with ptxP3/prn9 genotype has also increased in frequency (14% of all ptxP3 isolates) in recent years. Furthermore, we detected the first ptxP3 B. pertussis isolates that does not express filamentous hemagglutinin (FhaB) as one of the major antigens of the pathogen and a key component of the acellular pertussis vaccine.

ObjectivesPertussis remains endemic despite high vaccine coverage in infants and toddlers. Pertussis vaccines confer protection but immunity wanes overtime and boosters are needed in a lifetime. Iran, eligible for the Expanded Program on Immunization that includes the primary immunization, implemented two additional booster doses using a whole-cell vaccine (wPV) at 18 months-old and about 6 years-old. Duration of protection induced by the wPVs currently in use and their impact as pre-school booster are not well documented. This study aimed at assessing vaccination compliance and at estimating the duration of protection conferred by vaccination with wPV in children aged < 15 years in Tehran, Iran.MethodsDetailed information on vaccination history and capillary blood samples were obtained from 1047 children aged 3-15 years who completed the 3 doses-primary pertussis immunization, in Tehran. Anti-pertussis toxin IgG levels were quantified by ELISA.ResultsCompliance was very high with 93.3% of children who received the three primary and 1st booster doses in a timely manner. Timeliness of the 2nd booster was lower (63.3%). Rate of seropositive samples continuously and significantly increased from 1-2 to 5-6 years after 1st booster attaining 30.4% of children exhibiting serological sign of recent contact with B. pertussis. Second booster dating back 1 or 2 years was associated with high antibody titers, which significantly decreased within 3 years from injection. Among children who received 2nd booster injection more than 2 years before serum analysis, seroprevalence of pertussis infection was 8.4% and seropositivity rate was higher from the 10 years-old group.ConclusionSeropositivity in children aged 6-7 years with no 2nd booster supports the need for a vaccination at that age. Adolescent booster may also be considered.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has been increasingly reported and is now recognized as a public health concern. The aim of this study was to investigate the molecular epidemiology of CR-hvKp strains that were isolated from an Iranian hospital. A total of 74 non-duplicated carbapenem-resistant K. pneumoniae (CR-Kp) were collected from patients' clinical or surveillance cultures. Resistance/virulence genes were identified by PCR and sequencing. String test, capsular genotyping, conjugation assays, PCR-based replicon typing, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and were performed. All 74 CR-Kp isolates were carbapenemase producers, which co-carried multiple resistance genes such as bla

Background: New Delhi metallo-beta-lactamase-1 (NDM-1) is one of the most important emerging antibiotic resistance. Co-harboring three or four carbapenemases is rare and only a few reports exist in the literature. We described the characteristics of the large epidemic outbreaks and reports co-producing blaNDM-1 with the other carbapenemase genes in P. aeruginosa isolates. Methods: This present cross-sectional research was conducted on 369 P. aeruginosa isolates obtained from burn and general hospitals within years 2013 to 2016. Beta-lactamase classes A, B and D genes were identified by PCR method. Modified hodge test (MHT), double-disk potentiation tests (DDPT) and double disk synergy test (DDST) were performed for detection carbapenemase and metallo beta-lactamase (MBL) production of blaNDM-1 positive P. aeruginos isolates. Results: From 236 carbapenem-resistant P. aeruginosa (CRPA), 116 isolates have had MBL genes and twentynine isolates were found positive for blaNDM-1. In CRPA isolates, blaIMP-1, blaVIM-2 and blaOXA-10 were identified in 27.5%, 21.1% and 32.2% of isolates respectively, while co-producing blaNDM-1, blaIMP-1, blaOXA-10, co-producing blaNDM-1, blaVIM-2, blaOXA-10 and co-producing blaIMP-1, blaVIM-2 were determined in 11 (4.6%), 8 (3.4%) and 27 (11.4%) of isolates respectively. Conclusion: The finding of this co-existence of multiple carbapenemase resistance genes is threating for public health. Dipicolinic acid is a superior MBL inhibitor in DDPT antique than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P. aeruginosa

Background and Objectives: Despite widespread vaccination programs against pertussis, there has been a worldwide re- surgence of the disease in recent years. We aimed to investigate protein composition of outer membrane vesicles (OMV) of Bordetella pertussis (Bp) and to evaluate the immunogenicity of OMV antigens both in the vaccine and the dominant wild type strains in Iran. Materials and Methods: The OMV were purified from both vaccine and wild type strains. The immunoreactivity of the OMVs was investigated by exposing sera taken from the patients and the vaccinated infants. The protein profiles of OMVs were compared using two-dimensional electrophoresis. The LC-MS/MS was used to analyse and identify differentially ex- pressed protein spots. Results: The two type strains showed differences in their 2D gel protein profile. Further analysis of selected proteins from the dominant Iranian strains using LC-MS/MS demonstrated that the identified proteins fell into different functional catego- ries including (i) metabolism, (ii) membrane transport and secretion system, (iii) biosynthesis and degradation, (iv) adaption, adhesion, pathogenicity, conserved hypothetical and protection responses. Moreover, a number of immunogenic proteins were identified including Bp 2434 (serine protease) and Bp 1616 (putative DNA binding protein) from the vaccine and the wild type strains, respectively which could be considered as potential antigens for an OMV vaccine. Conclusion: OMV Bp could be considered as an alternative vaccine against pertussis, containing the bacterium’s protein antigens that can confer equal efficacy compared to a whole bacterial cell vaccine with advantages such as less side effects and lower costs than acellular pertussis vaccines.

The emergence of extensively drug-resistant (XDR) Acinetobacter baumannii strains and the limited number of efficacious antibiotics demonstrate an urgent need to develop novel agents to treat infections caused by this dangerous pathogen. To find antimicrobial peptides against A. baumannii growing either in planktonic or in biofilm mode, biopanning was carried out with a peptide library on five XDR A. baumannii strains grown in the medium containing human blood (blood biopanning) and biofilms formed by these strains (biofilm biopanning). Two groups of peptides were identified, among which two peptides N10 (from blood biopanning) and NB2 (from biofilm biopanning) were selected and synthesized for more assessments. The selected peptides showed significant binding to A. baumannii rather than to the human cell line Caco-2. Both peptides were effective against A. baumannii and showed antibacterial activities (minimum inhibitory concentration (MIC) 500 μg/ml). In the biofilm inhibition assay, NB2 reduced biofilm more efficiently (75%) than N10 (50%). The combination of the two peptides could function better than each peptide alone to prevent biofilm formation by A. baumannii. Supplementation of conventional therapy with a mixture of peptides targeting A. baumannii or using peptides to deliver antibiotics specifically to the site of infection may be promising to control A. baumannii-related diseases.

Objectives Despite the fact that the blaOXA-48 and blaNDM-1 genes have successfully disseminated among Klebsiella pneumoniae isolates worldwide, outbreaks remain unidentified in Iran. Here we examined the molecular epidemiology of 96 carbapenem-resistant K. pneumoniae recovered from an Iranian hospital. Methods A total of 96 non-replicate carbapenem-resistant K. pneumoniae were recovered from clinical specimens in a university hospital. Detection of ESBLs and carbapenemases produced by studied strains was performed using PCR and DNA sequencing. The bacterial isolates were assigned to clonal lineages by PFGE and MLST. In addition, plasmids were analysed by PCR-based replicon typing and conjugation assays. Results All isolates harboured blaOXA-48 and blaNDM-1 genes together or alone. Almost all strains also carried ESBL genes. Eighty-seven isolates of K. pneumoniae were categorized into seven pulsotypes. The predominant strain clusters/pulsotypes associated with the outbreak corresponded to ST11 (48/96) and ST893 (31/96). Plasmids carrying blaOXA-48 and blaNDM-1 were successfully transferred to Escherichia coli K12 as the recipient strain. blaOXA-48 was located on IncL/M plasmids of ∼39 kb, while blaNDM-1 was carried by either an IncFII plasmid of ∼50 kb or an untypeable plasmid of ∼4 or 10 kb. Conclusions We describe two separate outbreaks of blaOXA-48- and blaNDM-1-carrying K. pneumoniae strains associated with dissemination of the ST11 and ST893 clones, with the ICU acting as the epicentre. The spread of plasmids carrying carbapenemase genes resulting in fulminant antimicrobial resistance is a severe concern.

Background Colistin is one of the last-resort therapeutic antibiotics used to control carbapenem-resistant Klebsiella pneumoniae. However, the emergence of colistin-resistant strains with high mortality and morbidity has become a new challenge. Therefore, it is crucial to characterize the genotypic features of carbapenem- and colistin-resistant K. pneumoniae (CCKP) isolates to implement public health intervention strategies. Methods In this study, we analyzed the whole genome of an OXA-48-producing CCKP strain for molecular characterization of antimicrobial resistance (AMR) as well as comparative genome and plasmid analysis. Results The whole-genome analysis showed that IRAN-43 genome possessed 140 contigs with 5,585,691 bp length, GC content of 56.9% and N50 score of 200,728. Multi-locus sequence typing (MLST) analysis showed that this K. pneumoniae strain belonged to sequence type (ST) 11. Single amino acid substitutions associated with colistin resistance were found in PhoP (D150G), PmrB (R256G) and RamA (I25T). In addition, two plasmids harboring qnrS1, tetR, tetB (plasmid-1) and blaOXA-48 (plasmid-2) were discovered. No plasmid-mediated colistin resistance (mcr) genes were detected. Integrative conjugative element of K. pneumoniae (ICEKp)4 variant was detected as well as cps locus length of 22,156 bp encoding 17 open reading frames (ORFs) which belonged to K. pneumoniae capsular locus (KL)15-1. Conclusion CCKP has been increasingly reporting from Iran. High frequency of antibiotic resistance genes among ST11 suggests that stricter control measures are needed to prevent the spread of this clone in hospital settings.

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was blaOXA-48 (82.8%), followed by blaNDM-1 (31%), blaNDM-7 (6.9%) and blaOXA-181 (3.4%). Seven of the blaNDM positive isolates co-harbored blaOXA-48 carbapenemases. The blaNDM and blaOXA-48 were found in IncA/C and IncL/M conjugative plasmids, respectively. The blaCTX-M-15, qnrA and intI1 genes were also present in most isolates. The PFGE revealed genetic diversity among the 28 E. coli isolates, which belonged to six minor PFGE clusters and 14 isolates were singletons. The 26 isolates were distributed into 18 STs, of which two were dominant (ST648 and ST167). We identified one blaNDM-1-positive ST131 E. coli isolates that harbor the blaCTX-M-15 and blaTEM genes. Horizontal transfer of IncA/C and IncL/M plasmids has likely facilitated the spread of the blaOXA-48 and blaNDM genes among E. coli. Their clonal diversity and the presence of faecal carriers in isolates suggest an endemic spread of OXA-48 and NDM. Therefore, it emphasizes the critical importance of monitoring and controlling the spread of carbapenem resistant E. coli.

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.

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

Background and Objectives: Cholera disease remains an important global health problem affecting 3-5 million subjects worldwide. Outer membrane vesicles (OMVs) have been found in a variety of Gram-negative bacteria and act as protective transport vesicles. The aim of this study was to evaluate Immune responses against Vibrio cholerae O1 El Tor clinical strain OMV and compare it with killed whole cell (KWC), complex of (KWC-OMV) as well as the internationally licensed oral cholera vaccine, Dukoral, in serum and intestinal secretions of mice. Materials and Methods: OMVs were prepared by using modified detergent-centrifugation procedure from V. cholerae O1 El Tor clinical strain from 2005 outbreak. The ultrastructure and content of OMVs were investigated via the Scanning Elec- tron Microscopy (SEM) and SDS-PAGE analysis. Three doses of oral immunization were adjusted and total IgG and IgA in serum and intestinal secretion were measured by enzyme-linked immunosorbent assay (ELISA). Results: Extracted OMVs from the V. cholerae were spherical vesicles with a size ranging from 10 to 300 nm. OMV-im- munized mice showed an increased level of total IgG and IgA both in serum and intestinal secretion when compared to the negative controls. Also, there existed a higher level of secretory IgA than the total IgG, suggesting the most of protection against V. cholerae colonization provided by sIgA. Conclusion: Our findings revealed that oral immunization with V. cholerae OMVs might induce a long-term immunity, es- pecially when administered in combination with KWC. This study tested the adjuvant activity of OMVs and may be useful in future nano vaccine research.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has been increasingly reported and is now recognized as a public health concern. The aim of this study was to investigate the molecular epidemiology of CR-hvKp strains that were isolated from an Iranian hospital. A total of 74 non-duplicated carbapenem-resistant K. pneumoniae (CR-Kp) were collected from patients' clinical or surveillance cultures. Resistance/virulence genes were identified by PCR and sequencing. String test, capsular genotyping, conjugation assays, PCR-based replicon typing, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and were performed. All 74 CR-Kp isolates were carbapenemase producers, which co-carried multiple resistance genes such as blaCTX-M-15, blaTEM-1, blaSHV-type, qnrB1, and qnrS1. The most common carbapenemase gene was blaOXA-48 (67/74 90.5%), followed by blaNDM-1 (18/74 24.3%), and blaNDM-7 (3/74 4%). The blaOXA-48 and blaNDM-1 were found on IncL/M and IncFII conjugative plasmids, respectively. Of 74 CR-Kp isolates, 49 were positive for string test. Capsular genotyping revealed that 34 and 10 CR-Kp strains belonged to the K1 and K2 serotypes, respectively. rmpA was the most prevalent virulence gene detected in 64.8% of the isolates. Fifty two strains were identified as CR-hvKp. PFGE typing showed 5 different clusters with two major clusters B (39 isolates, 52.7%) associated with sequence type 11 (ST11), and A (21 isolates, 28.4%) associated with ST893. Furthermore, ST147, ST392, and ST15 carbapenemase producers have also been sporadically identified. One isolate belonging to ST11 was resistant to colistin and were negative for mcr-1-2-3 genes. Insertional inactivation of mgrB due to IS elements was observed in the colistin-resistant isolate. Our findings suggest that ST11 CR-hvKP strain has a clonal distribution in our hospital. Therefore, immediate implementation of infection-control measures may be the best way to prevent the spread of these clones.

Infections and outbreaks caused by multidrug-resistant Acinetobacter baumannii (MDR-AB) are prevalent and have been reported worldwide over the past 20 or more years. Class 1 integron in MDR-AB plays an important role in the spread of antibiotic resistance in clinical settings. This study has been conducted to evaluate the detection of metallo-β-lactamase, characterization of class 1 integron and determination of clonal relatedness among A. baumannii hospital isolates. Sixty-five clinical isolates of MDR-AB were recovered from two Iranian hospital's intensive care units from February to August 2013. Integrase (intI1) and blaIMP genes were detected in 70.8 % (n=46/65) and 9.23 % (n=6/65) of the isolates using PCR assay, respectively. No other metallo-β-lactamase genes (blaVIM, blaSIM and blaNDM) were detected. PCR sequencing of integron gene cassette revealed the following arrays: blaOXA10-aacA4-blaIMP-55-cmlA5 (as a novel array was designated In1243), aacC1 and aadA1. Analysis of blaIMP gene revealed a new allele designated as blaIMP-55. Gene transfer experiment by conjugation showed the 36 kb conjugative plasmid harbouring In1243. The clonal assessment by repetitive extragenic palindromic PCR demonstrated a high-degree relatedness among the strains, but strains harbouring In1243 displayed a different repetitive extragenic palindromic PCR profile. In this study, we found that a novel class 1 integron (In1243) that encoded a new blaIMP allele resided on a transferable plasmid in non-clonal strains of MDR-AB.

Background and study aim Colitis is a common complication after treatment with antibiotics such as β-lactams, quinolones, and aminoglycosides. Recently, Klebsiella oxytoca has been implicated in this type of diarrhoea. The prevalence and characterisations of K. oxytoca isolated from patients with antibiotic-associated diarrhoea were investigated. The K. oxytoca isolates were also tested for cytotoxin production. Patients and methods This study was conducted from May 2011 to Dec 2013. Faecal samples were collected from hospitalised patients receiving antibiotic treatment. Initial cultivation was performed on specific media. The clinical isolates were confirmed by polymerase chain reaction (PCR) using the specific K. oxytoca polygalacturonase (pehX) gene. The double-disc diffusion test was used to detect extended-spectrum beta-lactamase (ESBL)-producing strains. Tracking of ESBL-encoding genes was performed via PCR. The organism was cultured on Hep-2 cell lines for cytotoxin production. Results Out of 331 samples collected from patients, 40 were confirmed molecularly to be clinical isolates of K. oxytoca . Fourteen (35%) ESBL-producing strains were isolated using the double-disc diffusion method. Among the molecularly confirmed K. oxytoca isolates, seven (17.5%) tested positive for the bla SHV gene, 12 (30%) for bla TEM, 10 (25%) for bla CTX-M, three (7.5%) for bla OXA, nine (22.5%) for bla CTX-M-15, and seven (17.5%) for bla TEM-1. Five (12%) isolates showed cytotoxin activity below 30%, 12 (30%) strains showed moderate cytotoxin activity between 30% and 60%, and 23 (58%) strains showed cytotoxin activity ⩾60%. Conclusions The cytotoxin-producing K. oxytoca is found to be one of the causes of antibiotic-induced colitis. Discontinuing treatment and allowing normal intestinal flora to be established or prescribing appropriate medication after antibiogram can help patients with antibiotic-induced haemorrhagic colitis in a timely manner.

Infections due to Haemophilus influenzae result in tremendous global morbidity. The conjugated vaccines against H. influenzae type b (Hib) have dramatically reduced the incidence of invasive Hib disease in the routine immunization of infants. The several proteins used as vaccine candidates for this pathogen, but they don't produce efficient immune in animal models against all strains of H. influenzae. This study aimed to determine the diversity of hpd gene nucleotide sequences of Iranian native clinical isolates of H. influenzae as a native vaccine candidate compared to standard strains. Twenty isolates of H. influenzae recovered from different clinical specimens of patients admitted to Milad and Imam Khomeini hospitals, Tehran, Iran. Then, isolates detected and identified as H. influenzae using biochemical tests, and further confirmation through omp6 gene PCR. The hpd gene was amplified by PCR using gene-specific primers, and the amplicons digested with EcoR1. For four isolates, the Amplicon of hpd gene sequenced, and the sequences aligned with sequences harbored in GenBank. Subsequently, sequences were submitted to the EMBL site ( http://www.ebi.ac.uk/embl/ ). EcoR1 restriction enzyme pattern was the same among the 19 clinical isolates, and only one isolate was different. That different one with 3 out of 19 isolates were sequenced. The results showed that the nucleotide sequences and the deduced amino acid sequences for protein D in clinical isolates were highly conserved with similarities >95%. In conclusion, regarding high similarity up to 99% in clinical isolates, protein D can be a novel vaccine candidate against all types of H. influenza from Iran. This finding should be proved with more isolates, and also, evaluate the immunological features of protein D in animal models.

Increasing rate of silent intestinal carriers with extended-spectrum betalactamase (ESBL)-producing

Despite global efforts and widespread vaccination to control whooping cough (pertussis) caused by B. pertussis, the re-emergence of pertussis still is being reported all over the world. Antigenic divergence in B. pertussis virulence factors is one of the reasons of pertussis resurgence, resulting in dissimilarity of local and vaccine strains. In this study, clonal spread and variation of B. pertussis virulence factor in isolated strains from Iranian patients have been analyzed. A total of 100 B. pertussis isolates were obtained from Pertussis Reference Laboratory of Pasteur Institute of Iran. Real-time PCR were performed to confirm the B. pertussis strains. The genomic patterns of B. pertussis strains were analyzed by pulsed-field gel electrophoresis (PFGE). Predominant alleles of local strains were ptxP3, ptxA1, prn2, fim 2-1, fim3-2, and cya2. PFGE results showed 25 patterns clustered into 18 PFGE groups. A few similarities between the circulating isolates, vaccine, and standard strains were obtained. Significantly, 48% of the isolates showed dominant pattern with different allelic profiles from vaccine strains. According to the genomic profiles, the clonal spread was observed among the circulating strains. Predominant virulence factor profile was also comparable with other countries. It may be suggested that strain variation between vaccine and local strains may have an effect on pertussis resurgence in Iran like other parts of the world.

Introduction: Acinetobacter baumannii is associated with hospital-acquired infections. Outer membrane protein A of A.baumannii (AbOmpA) is a well-characterized virulence factor which has important roles in pathogenesis of this bacterium. Methods: Based on our PCR-sequencing of ompA gene in the clinical isolates, AbOmpA protein can be categorized into two types, named here type-1 and type-2. We preformed in silico analyses on beta-barrel domain of AbOmpA such as sequence alignments, prediction of 3D modeling and immunoinformatics. Results: High prevalence of AbOmpA type-1 were detected both in 21 multidrug-resistant (MDR) A. baumannii isolates collected from clinical settings (64% of total sequences) and in silico sequences extracted from Uniprot database (49.7% of total sequences). Multiple sequence alignments and phylogenic tree revealed AbOmpA sequences in comparison with OmpA of Enterobacteriaceae family were more heterogenic, especially at extracellular loops amino acid positions and were evolutionary far from this family. Characterization of in silico 3D molecular model of beta-barrel domain of AbOmpA type-1 showed this domain had four large extracellular loops which resemble to beta-barrel domain of Klebsiella pneumonia OmpA (KpOmpA). Immunoinformatics analyses showed the four extracellular loops had high scores as B and T cell antigenic epitopes especially in loop-1 and 3. Geometry analysis revealed extracellular loops effectively protrude to outer space of the cell. Conclusion: Beta-barrel domain of AbOmpA type-1 has all the characteristics of a promising vaccine and could be considered as an excellent target for a vaccine against MDR A. baumannii infection.

Introduction: Despite high level of vaccination against pertussis‚ whooping cough has re-emerged as a health threat, especially in infants. This could be related to expansion of Bordetella pertussis with novel alleles for virulence factors including the pertussis toxin promoter, ptxP3. Compared to ptxp1 strains‚ ptxp3 strains produce more pertussis toxin which results in immune suppression and virulence. The main aim of this study was the determination of dominant alleles of the promoter region of the gene coding for pertussis toxin in B. pertussis isolates in Iran. Methods: In this project, we studied the allelic variations in ptxP3 .This factor was sequenced and analyzed in 35 B. pertussis isolates from Iranian patients in 2011. Biochemical tests were performed to confirm the B. pertussis isolates. Ultimately, polymerase chain reactions and sequencing were done. Results: Our results showed that the predominant allele among the strains was ptxP3. One isolated strain (i.e. ptxP1) showed different results from the other strains. Also, B. pertussis 134 and 509 as common vaccine strains used in Iran were analyzed and they were identified to be ptxP1. Conclusion: Based on our results in recent years‚ the vaccine strains and the circulating strains do not share the same alleles which could be one of the causes of pertussis resurgence in the world. ptxP is a well-known toxin of B. pertussis which is responsible for binding to the host cell and the disruption of the cellular function. In particular‚ allelic variation in ptxP may play a role in adaption of B. pertussis.

The lipopolysaccharide (LPS) of Acinetobacter baumannii is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into A. baumannii ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1.

Acinetobacter baumannii is an important nosocomial pathogen which causes a wide range of infections. In this study, we addressed the role of class 1 integron, ISAba1 and ISAba125 associated with antimicrobial resistance in 72 clinical isolates of A. baumannii collected from clinical settings in Tehran, Iran. Moreover, to study the clonal relatedness of strains, repetitive extragenic palindromic-PCR (rep-PCR) assay was carried out. PCR revealed that blaOXA-51-like, blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, blaNDM, integrase gene (intI1), ISAba1, and ISAba125 were present in 86.11% (62/72), 84.72% (61/72), 30.55% (22/72), 0% (0/72), 0% (0/72), 58.33% (42/72), 97.22% (70/72), and 65.27% (47/72) of the strains, respectively. Sequencing of 39 internal variable regions of class 1 integrons showed seven gene cassette arrays, including aadA4-catB8-aadA1 (2.77%), aadA1-aadA4 (1.38%), aacC4-aadA1 (2.77%), aacC4 (22.22%), aadA1 (13.88%), aadA4 (5.55%), and catB8 (5.55%). We detected ISAba1 in the upstream of blaOXA-23-like, blaOXA-51-like, and blaADC in 54.16% (39/72), 9.72% (7/72), and 56.94% (41/72) of the strains, respectively. Whereas, there was a low frequency of disruptions in carO and dacD genes: 5.55% (4/72) and 4.16% (3/72). Rep-PCR analysis revealed that the isolates were genetically diverse. However, Cl-12 and Cl-15 were the largest clusters and they were recovered from various hospitals. Our analysis showed the high rates of class 1 integrons as a repertoire of aminoglycoside-modifying enzymes. It seems that linkages of ISAba1-blaOXA-23-like and ISAba1-blaOXA-69, and disruptions in carO or dacD can develop resistance to carbapenems among clinical isolates of A. baumannii.

Effectiveness of the whole-cell pertussis vaccine is apparent, but improvement in the quality of the vaccine is necessary to achieve strong immunogenicity with a low bacterial number content. Method Inactivated Bordetella pertussis (B. pertussis) cells entrapped microspheres were prepared via an emulsification method and analyzed for morphology, size, size distribution, loading efficiency, loading capacity, release kinetic, in vivo cytokines and antigen specific antibody subclasses. Results Bordetella pertussis encapsulated microspheres exhibited a smooth surface and spherical shape, mean particle size 151.1 µm, size distribution index 0.43, loading efficiency 89.6%, loading capacity 36.3% and release kinetic similar to the Korsmeyer-Peppas model. Splenocytes of animals immunized with new microsphere-based whole-cell vaccine produced greater quantities of IFN-γ and higher amounts of IL-4 and IL-5 cytokines compared to conventional adjuvant-adsorbed vaccines. Conventional adjuvant-adsorbed vaccines produced smaller quantities of IL-4 and IL-5. Bordetella pertussis entrapped microspheres induced both cell-mediated and humoral antibody in mice, evidenced by high levels of IgG2a and IgG1. IgG2a levels in mice were enhanced using the common aluminum phosphate-adsorbed B. pertussis whole-cell vaccine, and IgG1 levels did not increase significantly. Bordetella pertussis entrapped microspheres and common B. pertussis whole-cell vaccine samples enhanced total IgG levels in mice; however, B. pertussis-entrapped microspheres produced significantly higher levels of total IgG than other test samples. Conclusion Encapsulation of inactive B. pertussis cells in microspheres appears to be a suitable approach for improving the wP vaccine quality, in particular by decreasing its toxicity to obtain good cell-mediated and humoral immunogenicity with a low bacterial number content.

There is no doubt about the whole cell pertussis vaccine efficacy, but it is necessary to improve the vaccine quality specially to decrease its toxicity by obtaining good immunogenicity with low bacterial content. In this work, under optimum condition inactivated B. pertussis bacteria cells entrapped with alginate microparticles were fabricated and in vivo immunogenicity and ptency of new microparticle based vaccine were evaluated in mice. Microspheres loaded with inactive B. pertussis bacterium cells were prepared via an emulsification method and analyzed for morphology, size, polydispersity index, loading efficiency, loading capacity, release profile and in vivo potency. The inactivated bacterial suspension mixture prepared in this work was nontoxic and showed potent ED50 (1:333 of human dose) and preserved agglutinins 1, 2, 3. The optimum conditions for the preparation of microparticles were achieved at alginate concentration 3.8% (w/v), CaCl2 8% (w/v), PLL 0.1% (w/v), lipophilic surfactant 0.22 (%w/v), hydrophilic surfactant 3.6 (%w/v), cross linking time 3min, homogenization rate 600 rpm, and alginate to CaCl2 solution ratio 4. Both empty and B. pertussis loaded microparticles exhibited smooth surface texture and relatively spherical shape. The B. pertussis encapsulated microspheres fabricated under optimized conditions showed mean particle size 151.1 μm, polydispersity index 0.43, loading efficiency 89.6%, loading capacity 36.3%, and relatively constant release rate lasted to 15 days. In vivo immunogenicity and protection study against wild type challenge showed strongly higher potency (approximately 2.5 fold) of encapsulated B. pertussis organisms than non-encapsulated conventional aluminum hydroxide adsorbed vaccine. It can be concluded that microencapsulation of inactive B. pertussis cells appears to be a suitable approach for improving the wP vaccine quality, specially by obtaining good immunogenicity with low bacterial content.

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was bla

Purpose. Enterobacteriaceae is a large family of Gram-negative bacteria that are considered as normal gut flora. They are the most common human pathogens. The main objective of this study was to investigate the carbapenemase genes in clinical isolates of Enterobacteriaceae resistant to carbapenem antibiotics and determine their clonal relationship using pulsed-field gel electrophoresis (PFGE). Methodology. In the present study, bacteria were isolated and identified via conventional biochemical tests and API 20NE. Antibiotic susceptibility was evaluated by using the disc diffusion method and MIC was carried out using the E-test. Phenotypic determination of carbapenemases was performed by employing a modified Hodge test (MHT). Carbapenemase genes including IMP, VIM, KPC, NDM and OXA-48 were amplified by PCR. The relationships between their clonal types with l restriction enzyme were examined using PFGE. Results. Out of 40 isolates that were resistant or moderately susceptible to carbapenem antibiotics, 29 (72.5 %) strains were positive for carbapenem enzymes phenotypically. Moreover, six isolates contained carbapenemase genes including IMP, VIM, NDM and OXA-48, but the KPC gene was not found in any of the isolates. PFGE results showed that E. coli strains in our area were clustered into eight pulsotypes (A–H), Klebsiella spp. isolates five pulsotypes (A–E) and Proteus spp. had two pulsotypes (A, B). The high resistance to antimicrobial agents in the A, B and F pulsotypes was attributed to E. coli clinical isolates. Conclusions. Our results could reflect some hospital multidrug-resistant strains in nosocomial infections. The widespread emergence of carbapenem-resistant isolates has caused increasing concern in recent years. Therefore, specific strategies should be designed and evaluated for the control of resistant strains.

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of blaOXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of blaOXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and blaOXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and blaOXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded blaOXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE.

Sixty-five MDRAB isolates from clinical samples were collected. Molecular typing was carried out by random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR). Biofilm formation was assayed by the microtiter method.The sequence ofThe results suggest that

BACKGROUND Bordetella pertussis causes serious contagious infections, primarily in childhood. A whole-cell vaccine, diphtheria-tetanus-whole cell pertussis (DTwP), has been used to protect against pertussis in children in Iran, but the pertussis cases have been increasing during recent years. We determined the allelic variation level of housekeeping genes in isolates recovered from pertussis patients and vaccine strains used in national vaccination program. METHODS Five clinical isolates, 2 vaccine strains and a Tohama I strain were studied through multilocus sequence typing (MLST) of housekeeping genes. The relatedness between STs, the founder, single- and double-locus variants (SLVs, DLVs) was determined using eBURST algorithm. The concordance between the type assignments by MLST and PFGE was determined. RESULTS In the 5 clinical isolates, 2 STs were identified, ST2 and ST79. The vaccine strains displayed two distinct allelic profiles assigned to ST1 and ST2. ST2 was predicted as founder and the remaining STs were SLVs of ST2. MLST and PFGE type assignments were 86.6% concordant. CONCLUSIONS The clinical isolates of B. pertussis were different from vaccine strains used in the national vaccination program. This study confirms the low level of variation in housekeeping genes of B. pertussis. MLST of virulent antigenic genes needs to be applied as a complementary method for the characterization of new ST-harboring isolates that may predominate periodically. The combination of these data allows rapid and efficient surveillance of currently circulating isolates. These data might elucidate the future trends and considerations for vaccine formulation and design.

Multi-resistant Klebsiella pneumoniae has been considered a serious global threat. This study was initiated to investigate carbapenem resistance among K. pneumoniae isolates in Iran and to detect carbapenemases in resistant strains. From 2009 to 2012, 180 K. pneumoniae strains were collected from Tehran hospitals. Of the isolates, 42 isolates (23.3%) were resistant to meropenem, 29 isolates (16.1%) were resistant to ertapenem, and 14 isolates (7.7%) were resistant to imipenem. All of carbapenem-resistant isolates were also resistant to the third generation of cephalosporins. modified Hodge test was positive in 25 (59.5%) of carbapenem-resistant isolates showing carbapenemase production. bla(NDM) and bla(VIM) genes were identified in three and five carbapenem-resistant isolates, respectively. One isolate showed presence of bla(KPC) gene. Class 1 integrons were detected in 14 carbapenem-resistant isolates. The most important finding about class 1 integrons was identification of an integron containing metallo-β-lactamase gene VIM-1 that also harbored dfrA27 and arr3 genes. It is important to note that K. pneumoniae carbapenemase and New Delhi metallo-beta-lactamase-positive isolates identified in this study showed resistance to the majority of routine antimicrobial agents, including all β-lactams and other classes of antibiotics. To our knowledge, this is the first identification of bla(KPC) and bla(VIM-1) genes among isolates of K. pneumoniae in Iran.

Brevundimonase diminuta as an opportunistic environmental bacterium, due to the increase in isolation rate from clinical specimens and its antibiotic resistance is considered as a new threat to human health care. After performing conventional phenotypic methods and molecular approaches, antibiotics resistance pattern on the identified Brevundimonase isolates was performed as minimum bactericidal concentration by disk diffusion method. Out of 1084 nasopharyngeal specimens, 2/1084 (%0.18) Brevundimonase diminuta, and 1/1084 (%0.09) Brevundimonase subvibrioides were isolated. Evaluation of the resistance pattern from isolated Brevundimonase strains indicated that: levofloxacin, ciprofloxacin, ampicillin, amikacin (Excluding B. subvibrioides), chloroamphenicol and ceftazidime had poor susceptibility results. Also azythromycin, gentamycin, sulfamethoxazole/trimethoprim (Excluding B. subvibrioides), ciprofloxacin, kanamycin showed good susceptibility results against Brevundimonase isolated strains. In conclusion, Although, Brevundimonase diminuta rarely has been isolated from other clinical specimens, but, due to reports of resistance to some of antimicrobial agents in this genus all laboratories should be equipped for the identification and evaluation of susceptibility patterns of this species. It is first the report of isolated Brevundimonase diminuta from nasopharyngeal specimen from suspected patients to pertussis in Iran. Key words: Brevundimonase diminuta, nasopharyngeal specimens, 16srRNA primer.

Metallo-β-lactamases (MBLs) such as IMPs are broad-spectrum β-lactamases that inactivate virtually all β-lactam antibiotics including carbapenems. In this study, we investigated the hydrolytic activity, phylogenetic relationship, three dimensional (3D) structure including zinc binding motif of a new IMP variant (IMP-55) identified in a clinical strain of Acinetobacter baumannii (AB). AB strain 56 was isolated from an adult ICU of a teaching hospital in Kerman, Iran. It exhibited MIC 32μg/ml to imipenem and showed MBL activity. Hydrolytic property of the MBL enzyme was measured phenotypically. Presence of blaIMP gene encoded by class 1 integrons was detected by PCR-sequencing. Phylogenetic tree of IMP protein was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and 3D model including zinc binding motif was predicted by bioinformatics softwares. Analysis of IMP sequence led to the identification of a novel IMP-type designated as IMP-55 (GenBank: KU299753.1; UniprotKB: A0A0S2MTX2). Impact in term of hydrolytic activity compared to the closest variants suggested efficient imipenem hydrolysis by this enzyme. Evolutionary distance matrix assessment indicated that IMP-55 protein is not closely related to other A. baumannii IMPs, however, shared 98% homology with Escherichia coli IMP-30 (UniprotKB: A0A0C5PJR0) and Pseudomonas aeruginosa IMP-1 (UniprotKB: Q19KT1). It consisted of five α-helices, ten β-sheets and six loops. A monovalent zinc ion attached to core of enzyme via His95, His97, His157 and Cys176. Multiple amino acid sequence alignments and mutational trajectory with reported IMPs showed 4 amino acid substitutions at positions 12(Phe→Ile), 31(Asp→Glu), 172(Leu→Phe) and 185(Asn→Lys). We suggest that the pleiotropic effect of mutations due to frequent administration of imipenem is responsible for emergence of new IMP variant in our hospitals.