The emergence of the hypervirulent Klebsiella pneumoniae (hvKp) strains among circulating clonal complex 147 (CC147) harbouring blaNDM/OXA-48 carbapenemases in a tertiary care center of Iran.

BACKGROUND: Klebsiella pneumoniae is a public health concern because of its ability to develop multidrug resistance and hypervirulent genotypes, of those capsular types K1 and K2 cause community and nosocomial life-threatening infections. This study aimed to determine the antibiotic susceptibility patterns and genotypic traits of a collection of Klebsiella spp. isolates. Furthermore, the clonal relatedness of blaNDM producing strains was investigated. METHODS: During a 19-months  surveillance study, 122 Klebsiella spp. isolates were cultured from extraintestinal specimens of patients admitted to the tertiary referral hospital in Semnan, Iran. Isolates were identified using biochemical tests and subjected to determination of phylogroups, capsular types and virulence/resistance genes content. Hypervirulent K. pneumoniae (hvKp) strains were detected genotypically, and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting was used to determine the clonality of blaNDM producing strains. RESULTS: Multidrug resistant phenotype was detected in 75 (61.5%) isolates and amikacin was found as the most potent antibiotic with the susceptibility rate of 85.2%. The carbapenemase genes were detected in 45 (36.8%) strains, including 21 (17.2%) blaOXA-48, 7 (5.6%) blaNDM-1, 14 (11.4%) blaNDM-1/OXA-48 and 3 (2.4%) blaIMP- carrying strains, while 55 (45.08%) isolates showed carbapenem resistant phenotype. The first blaNDM-1 carrying strain was cultured from a sputum specimen on March 2015, while the last positive one was recovered from blood culture on September 2016. Most of the isolates (80.3%) belonged to phylogroup I, and blaNDM-1 was identified among all three phylogroups. The ERIC-PCR clustered the 101 blaNDM negative and 21 blaNDM-1 positive isolates into 25 and five clusters, respectively, and the latter group belonged to clonal complex 147 (CC147). One K1 and 15 K2 blaNDM-1 negative isolates were detected, of those three strains were identified as hvKp. Five K2 positive strains, including four blaOXA-48 producer and one hvKp sequence type 86 (ST86) were carbapenem resistant. Among carbapenem resistant isolates, CC147 strains harboured higher rates of siderophores iutA and ybtS. CONCLUSION: The present findings showed a hospital circulation of CC147 blaNDM-1 or blaNDM-1/OXA-48 producing strains, disseminated in different wards. The hvKp/ST86 strain expressing K2 capsular type and carbapenem resistant phenotype wasn't reported from Iran so far. So, it seems that we must be aware of the emergence and spread of new K. pneumoniae clones associated with resistant and hypermucoviscous phenotypes.

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.

Molecular characterization of the surface glycoprotein genes of highly pathogenic H5N1 avian influenza viruses detected in Iran in 2011.

Highly pathogenic avian influenza (HPAI) H5N1 virus is causing the death of a large number of wild birds and poultry. HPAI H5N1 was reported in the north of Iran in 2011. In this study, two A/Chicken/Iran/271/2011 and A/Duck/Iran/178/2011 viruses were genetically characterized by sequence analysis of Hemagglutinin (HA) and Neuraminidase (NA) genes. Phylogenetic analysis revealed that these viruses were different from previous Iranian isolates (Clade 2.2) and belonged to the subclade 2.3.2.1. The results showed that the detected viruses are almost identical to each other and closely related to HPAI H5N1 strains isolated in Mongolia in 2010. Based on the amino acid sequence analysis, these viruses at their HA cleavage sites contained the multibasic amino acid motif PQRERRRK-R/GLF lacking a lysine residue compared with the previous reports of the same motif. There is also a 20-amino acid deletion (resides 49-69) in the NA stalk similar to other viruses isolated after 2000. It seems that introduction of HPAI H5N1 to Iran might have happened by wild birds from Mongolian origin virus.

MIRU-VNTR analysis of the Mycobacterium tuberculosis isolates from three provinces of Iran.

BACKGROUND: Iran borders 2 high-burden tuberculosis (TB) countries to the east, and has the highest rates of TB in one of its eastern provinces. Limited information is available on the genetic diversity and transmission dynamics of Mycobacterium tuberculosis (MTB) in Iran. To examine the genetic diversity and transmission dynamics of MTB strains we genotyped a collection of isolates from different parts of Iran. METHODS: Standard 15-locus variable number tandem repeat (VNTR) typing was applied to genotype 121 MTB clinical isolates collected from 3 provinces of Iran, including Tehran (the capital of Iran), Sistan-Baluchestan (southeast province of Iran, with the highest rate of TB), and Kermanshah (western part of Iran with high TB/human immunodeficiency virus cases). Antibiotic susceptibility for all isolates was determined using the proportion method. RESULTS: Sixty-six distinct mycobacterial interspersed repetitive unit (MIRU)-VNTR patterns were detected among 121 isolates. Seventy-five strains grouped into 20 clusters, and 46 isolates were unique. The genetic diversity of strains from Sistan-Baluchestan was higher than that in the other provinces. All isolates from Tehran or Kermanshah that grouped into clusters shared identical patterns with Sistan-Baluchestan. The Hunter-Gaston discriminatory index (HGDI) was 0.972, indicating a high power of discrimination for MIRU-VNTR typing. The MIRU 16 and ETRA loci were designated as highly discriminative. The rates of monoresistance and multidrug resistance were 9.9% and 2.4%, respectively. CONCLUSIONS: MIRU-VNTR typing revealed high genetic diversity and suggests the possibility of transmission from Sistan-Baluchestan to other provinces of Iran. This method has potential for genetic analysis and for studying the transmission routes of TB.

Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens.

BACKGROUND: As a rapid diagnostic technique, the real-time polymerase chain reaction (PCR) can detect Mycobacterium tuberculosis with a high sensitivity and specificity. However, further studies are needed to confirm it as a standard method. In this study, we evaluated the cyp141 gene for the detection and quantification of M. tuberculosis in respiratory specimens and compared the results with direct microscopy and culture. METHODS: Sputum samples (n = 247) were collected from patients of the different provinces of Iran. DNA was extracted from clinical specimens and H37Rv strain. After measuring the standard strain DNA concentration by NanoDrop and using the Avogadro number, the DNA was diluted 6 times in order to obtain 1 × 10(6) to 10 template copies. A Taqman probe was designed for detection of the target in a real-time PCR using the specific primers. RESULTS: Of 247 samples, 135 (55%) were culture-negative. Of 112 (45%) culture-positive samples, 88 were positive by both smear and culture and 24 were smear-negative but culture-positive. The real-time PCR enumerated 1.5E + 02 to 4.3E+ 03, 8.5E + 03 to 5.5E + 04, 7.2E + 04 to 1.1E + 06, and 1.2E + 06 to 8.1E + 07 M. tuberculosis cells in the specimens with smear-negative, 1-plus, 2-plus, and 3-plus codes, respectively. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the real-time PCR were 90.2% (101/112), 97.8% (132/135), 97.1%, and 92.3%, respectively. CONCLUSIONS: The overall sensitivity and specificity, the results in comparison with those of the Xpert MTB/RIF kit, and the good correlation with molecular and phenotypic methods, show that cyp141 could be a good target for the quantification of M. tuberculosis in sputum and possibly other clinical specimens.

Isolation of High Level Macrolide Resistant Bordetella pertussis Without Transition Mutation at Domain V in Iran.

BACKGROUND: Bordetella pertussis, as a causative agent of whooping cough, due to the annual rise y of infection cases, failure of prophylaxis and treatment by macrolides, is considered as the new concern in the health care system. OBJECTIVES: The main objective of this study was the determination of single nucleotide polymorphisms (SNPs) at domain V, as the main binding site for macrolides, following the identification of high level macrolides resistant B. pertussis. MATERIALS AND METHODS: Following the identification of 11 recovered B. pertussis isolates, from a total of 1084 nasopharyngeal swabs, by using the biochemical and molecular methods, the activities of erythromycin, azithromycin and clarithromycin antibiotics against the recovered isolates were examined. Subsequently, A-G transition mutations in domain V were analyzed by molecular techniques, such as Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequencing. RESULTS: After susceptibility testing, one strain was detected as a high level macrolide resistant B. pertussis (Erythromycin = 128 µg/mL, Clarithromycin > 256 µg/mL). After sequencing and PCR-RFLP methods, transition mutations in positions 2047 and 2058 of the mentioned domain were not observed. CONCLUSIONS: Although previous studies have shown that A-G transition mutations in 23 SrRNA gene (domain V) are the main reason for the occurrence of high level macrolides resistance in B. pertussis, however, the mentioned single nucleotide polymorphisms (SNPs) have not been detected in our resistant strain. This is the first report of high level macrolide resistant B. pertussis, without SNPs in domain V, in Iran.

Detection of both vanA & vanB genes in vanA phenotypes of Enterococci by Taq Man RT-PCR.

Twenty seven isolates of vancomycin resistant Enterococci based on the disk diffusion and E- test have been screened; being found eight (0.3%) clinical isolates of vanA & vanB through Taq Man Real Time PCR assay. This study shows the presence of both vanA & vanB genotypes in vanA phenotypes clinical isolates in the three hospitals in Iran.

Metallo-beta-Lactamase VIM-1, SPM-1, and IMP-1 Genes Among Clinical Pseudomonas aeruginosa Species Isolated in Zahedan, Iran.

BACKGROUND: One of the major clinical problems regarding Pseudomonas aeruginosa is attributed to metallo-beta-lactamases (MBL). This group of enzymes is a subset of beta lactamases which belong to group B of Ambler classification and cause hydrolysis of carbapenems. Based on epidemiological studies conducted worldwide, it is proved that prevalence of genes coding MBLs in P. aeruginosa species are different in various geographic zones and even in various hospitals. Therefore, according to the clinical importance of organisms generating MBLs, it is necessary to identify and control these bacteria in hospitals for therapeutic purposes. OBJECTIVES: The current study aimed to investigate the Metallo-beta-Lactamase VIM-1, SPM-1, and IMP-1 genes among clinical P. aeruginosa species isolated in Zahedan, Iran. MATERIALS AND METHODS: The current study investigated the presence of MBL through phenotypic and genotypic methods and also the pattern of antibiotic resistance in P. aeruginosa species isolated in hospitals. The Minimum Inhibitory Concentration (MIC) against imipeneme was measured for 191 P. aeruginosa species isolated from Zahedan hospitals after identification through biochemical methods and determination of the antibiotic resistance pattern. Strains with MIC > 4 µg/mL were studied by phenotypic and genotypic methods. RESULTS: The rate of resistance against imipeneme was 5.7% and after carrying out the phenotypic experiments, nine species were identified as of MBL producer. Seven species were confirmed by Polymerase Chain Reaction (PCR) method. Gene VIM-1 was the predominant gene among the positive (antibiotic resistant) species. CONCLUSIONS: The study results showed that MBL genes were present in some of the species isolated from Zahedan hospitals. Regarding the importance of MBL producer bacteria in hospitals, quick identification and evaluation of these clinical species can be considered as an important and basic step for treatment and control of pseudomonad infections.

Duffy blood group genotypes among malaria Plasmodium vivax patients of Baoulch population in Southeastern Iran.

OBJECTIVE: To determine the distribution of Duffy blood group genotypes in Balouch population as a major ethnic group that living in a sub-tropical area in south East of Iran. METHODS: In this study, the Duffy blood group FY phenotypes were determined using indirect anti-globulin technique and also genotype by PCR-RFLP in 160 vivax malaria patients and 160 control individuals. RESULTS: The results showed that the most common Duffy genotype was FYA/FYB (46.6%) followed by FYA/FYA (15.3%), FYA/FYO (14.4%), FYB/FYO (11.9%), FYB/FYB (10%) and FYO/FYO (1.9%). In case individuals, frequency of FYA, FYB and FYO alleles were 0.471, 0.431 and 0.097, respectively compaired to 0.444, 0.353 and 0.203, respectively in control (non-infected) group. CONCLUSIONS: This data provide evidence that individuals with the FYA/FYB genotype have higher susceptibility to malaria and there are significant associations between Duffy blood group variants and susceptibility or resistance to vivax malaria.

Detection of Salivary Human Papilloma Viruses 16 and 18 (HPV) in Smoker Men in an Iranian Population by PCR: A Pilot Study.

BACKGROUND: Human papilloma virus (HPV) infection of the oropharynx is acquired through a variety of sexual and social forms of transmission. Recent epidemiologic evidence has suggested that HPV may be an independent risk factor for oropharyngeal cancers, but risk factors for persistent HPV infection in the oropharynx are unknown. More evidence is needed regarding the prevalence of oral high-risk HPV among healthy smoker and nonsmoker adults. OBJECTIVES: The aim of this study was to compare salivary infection with human papilloma virus types 16 and 18 between smokers and nonsmokers. PATIENTS AND METHODS: A hundred healthy adult subjects were selected from Zahedan dental school for this pilot study. DNA was isolated from saliva samples and screened for high-risk HPV strains of HPV 16 and 18. Then, further processed using Real Time PCR for quantification and confirmation of sensitivity and specificity of the test. Data was analyzed by t-test. RESULTS: There were no high-risk types of virus in patients and no significant differences between the groups (P = 1). CONCLUSIONS: It seems that smoking cannot increase the prevalence of high risk HPV 16, 18 in saliva samples.

Cytochrome CYP141: a new target for direct detection of Mycobacterium tuberculosis from clinical specimens.

Cytochrome P450 CYP141 is an intermediary metabolic and respiratory protein that interferes with oxidation reduction in Mycobacterium tuberculosis. This conserved protein has also been debated as a hypothetical target for therapeutics. We used the sequences of CYP141 gene to develop a PCR for rapid detection of Mycobacterium tuberculosis from respiratory specimens. The sensitivity of this PCR for culture positive-smear positive and culture positive-smear negative samples were 92% and 62.5%, respectively. The overall sensitivity and specificity of this PCR was 85.7% and 97.8%. As compared with other studies, it appears that the CYP141 gene is a good target for direct detection of M. tuberculosis from respiratory specimens.

Detection of both vanA & vanB genes in vanA phenotypes of Enterococci by Taq Man RT-PCR

Twenty seven isolates of vancomycin resistant Enterococci based on the disk diffusion and E- test have been screened; being found eight (0.3%) clinical isolates of vanA & vanB through Taq Man Real Time PCR assay. This study shows the presence of both vanA & vanB genotypes in vanA phenotypes clinical isolates in the three hospitals in Iran.