Carbapenemase genes distribution in clonal lineages of Acinetobacter baumannii: a comprehensive study on plasmids and chromosomes.

Background: The global spread of plasmids carrying carbapenemase genes within carbapenem resistant Acinetobacter baumannii (CRAB) strains poses a worldwide public health issue. In this study, we conducted a comprehensive genetic analysis of plasmids and chromosomes harboring the major carbapenemase genes (bla NDM, bla KPC, bla VIM, bla IMP, bla GES, bla OXA-58-like, bla OXA-24/40-like, bla OXA-143-like, and bla OXA-23-like) in CRAB strains using bioinformatic tools. Methods: We retrieved plasmids and chromosomes carrying the major carbapenemase genes from GenBank. The size, replicon type, and conjugal apparatus of the plasmids were also determined. Furthermore, allele types, co-existence of other antimicrobial resistance genes alongside carbapenemases in plasmids or chromosomes, co-occurrence of carbapenemase genes, gene repetition, and sequence types (ST) of whole genomes were characterized. Results: The database contained 113 plasmids and 38 chromosomes harboring carbapenemase genes. This investigation revealed that bla NDM and bla OXA-58-like were the predominant allele types in both the plasmids and chromosomes. Nine (7.96%) plasmids with bla NDM-1 were potentially conjugative. The most common replicon types of the plasmids were R3-T1, R3-T8, R3-T2, R3-T23, and RP-T1. The analysis revealed that bla NDM-1 and bla OXA-58-like genes possessed the highest variety of co-existence with other antibiotic resistance genes. The co-occurrence of dual carbapenemases was identified in 12 plasmids and 19 chromosomes. Carbapenemase gene repetitions were identified in 10 plasmids and one chromosome. Circular alignment revealed that the plasmids carrying the co-occurrence of bla NDM-1 and bla OXA-58 were more homogeneous. However, there was heterogeneity in certain regions of these plasmids. According to the minimum spanning tree (MST) results, the majority of the plasmids belonged to the genomes of ST2Pas, ST1Pas, ST422Pas, ST622Pas, and ST85Pas. Conclusion: A. baumannii appears to have a strong ability for genome plasticity to incorporate carbapenemase genes on its plasmids and chromosomes to develop resistance against carbapenems. Mobilizable plasmids harboring carbapenemases significantly contribute to the dissemination of these genes. The genetic structure of the plasmids revealed a strong associations of class I integrons, ISAba-like structures, Tn4401 elements, and aac (6')-Ib with carbapenemases. Furthermore, gene repetition may also be associated with carbapenem heteroresistance.

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection. METHODS: One hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA-51-like and ampC genes, and RAPD-PCR method. RESULTS: All isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA-51-like was detected in all isolates, and blaOXA-23-like was identified in 92.5% of isolates. However, blaOXA-143-like and blaOXA-58-like genes were not detected among isolates. Four distinct blaOXA-51-like alleles were determined as follows: blaOXA-317 (67.0%), blaOXA-90 (9.4%), blaOXA-69 (17.0%), and blaOXA-64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC-88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons. CONCLUSION: The present study demonstrated that there was a high prevalence of blaOXA-23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA-23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131.

BACKGROUND: Escherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children's infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran. METHODS: The E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing. RESULTS: Among 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern. CONCLUSIONS: Our results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

Recombinant GPEHT Fusion Protein Derived from HTLV-1 Proteins with Alum Adjuvant Induces a High Immune Response in Mice.

The human T-cell leukemia virus type 1 (HTLV-1) is a positive single-stranded RNA virus that belongs to the delta retrovirus family. As a result, a vaccine candidate that can be recognized by B cells and T cells is a good candidate for generating a durable immune response. Further, the GPEHT protein is a multi-epitope protein designed based on the Gag, Pol, Env, Hbz, and Tax proteins of HTLV-1. In developing a suitable and effective vaccine against HTLV-1, the selection of a designed protein (GPEHT) with the formulation of an alum adjuvant was conducted. In this study, we assessed the potential of a multi-epitope vaccine candidate for stimulating the immune response against HTLV-1. In assessing the type of stimulated immune reaction, total IgG, IgG1, and IgG2a isotypes, as well as the cytokines associated with Th1 (IFN-γ), Th2 (IL-4), and Th17 (IL-17), were analyzed. The outcomes showed that the particular antisera (total IgG) were more elevated in mice that received the GPEHT protein with the alum adjuvant than those in the PBS+Alum control. A subcutaneous vaccination with our chimera protein promoted high levels of IgG1 and IgG2a isotypes. Additionally, IFN-γ, IL-4, and IL-17 levels were significantly increased after spleen cell stimulation in mice that received the GPEHT protein. The immunogenic analyses revealed that the GPEHT vaccine candidate could generate humoral and cell-mediated immune reactions. Ultimately, this study suggests that GPEHT proteins developed with an alum adjuvant can soon be considered as a prospective vaccine to more accurately evaluate their protective efficacy against HTLV-1.

High frequency of carbapenem-resistant Enterobacteriaceae fecal carriage among ICU hospitalized patients from Southern Iran.

Objectives: The worldwide emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a major therapeutic concern to medical institutions. To date, no study has determined the frequency and risk factors of inpatients with CRE fecal carriage in Southern Iran. We studied the features of carbapenemase-producing Enterobacteriaceae (CPE) collected from the central ICU of a university hospital. Materials and Methods: Totally, 173 samples, including 124 stool samples from 46 ICU inpatients on admission and different follow-ups, 9 ICU staff, and 40 environmental samples were included. CRE was identified using microbiological methods. Antimicrobial susceptibility was investigated by using the disk diffusion method and E-test. Carbapenemase producers were detected using the mCIM method. Seven carbapenemase genes were characterized. The genetic relationship among 20 CPE was elucidated by PFGE. Results: The overall fecal carriage rate was 28.2%, while CRE acquisition was 6.1%. CRE were classified as Klebsiella pneumoniae (71.4%), Escherichia coli (23.8%), and Enterobacter aerogenes (4.8%). From 21 CRE, 20 (95.2%) produced carbapenemases, of which 10, 15, 10, 25, 5, and 65% were blaKPC, blaSME, blaIMP, blaVIM, blaNDM and blaOXA-48-positive, respectively. Out of 20 CPE, 14 different PFGE patterns were observed, categorized into six clusters, suggestive of non-clonal spread. No difference between the examined risk factors with CRE carriage was shown. Conclusion: The data indicate a high CRE fecal carriage rate among inpatients. Our findings implicate the widespread of OXA-48 carbapenemase together with heterogeneity among CRE with great concern for dissemination and therapeutic threat. Early diagnosis and monitoring of CRE among inpatients are urgent.

High heterogeneity of fecal carriage extended-spectrum beta-lactamase-producing E. coli isolated from iranian community and clinical settings.

BACKGROUND: Extended-spectrum beta-lactamase-producing enterobacteria (ESBL-PE) in carriers have become a global health problem. Using molecular typing techniques, including PFGE, could be useful to determine the source of bacterial dissemination. The current study aimed to investigate the intestinal carriage of ESBL-producing E. coli (ESBL-EC) and clonal relatedness among ESBL-EC isolated from hospitalized and outpatient fecal carriers in Iran. METHODS: A total of 120 rectal swabs were collected; 50.8% (61/120) from intensive care unit (ICU) inpatients and 49.2% (59/120) from outpatients. MacConkey agar enriched with cefotaxime was used to screen the ESBL-EC. PCR assays were performed to detect ESBL and carbapenemase genes. Pulse-fields gel electrophoresis (PFGE) was performed to assess clonal relatedness. RESULTS: Totally, 60.0% (72/120) were carrier for ESBL-EC. The rates of resistance against ceftazidime and cefepime were 90.2% (65/72) and 93.0% (67/72), respectively. The rates of blaCTX-M-15, blaTEM, blaSHV, blaNDM-1, blaOXA-48 and blaIMP was 90.2% (65/72), 50.0% (36/72), 5.5% (4/72), 4.1% (3/72), 4.1% (3/72) and 1.3% (1/72), respectively. Based on a cut-off 80%, 69 ESBL-EC isolates could be categorized in 10 mini-cluster and 47 isolates were considered as singletons. DISCUSSION: High heterogeneity among isolates from ESBL-EC suggests that this bacterium probably has a different source of dissemination. Screening of carriers in hospitals and communities could help the infection control program in public health.

Fha Deficient Bordetella pertussis Isolates in Iran with 50 Years Whole Cell Pertussis Vaccination.

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.

Evaluation of the immune response to a multi-epitope vaccine candidate in comparison with HlaH35L, MntC, and SACOL0723 proteins against MRSA infection.

Staphylococcus aureus is an important human opportunistic pathogen that can have a major influence on public health. Here, we aimed to evaluate different aspects of the immune response to a novel multi-epitope fusion protein (HMS) based on HlaH35L, MntC, and SACOL0723 proteins in comparison to the individual antigens. For this purpose, specific total IgG, IgG1, and IgG2a isotypes and the cytokines related to Th1, Th2, and Th17 were assessed. The Bio-efficiency of the fusion protein was evaluated by opsonic killing activity. The HMS fusion protein elicited a high specific IgG level and also induced a higher level of Th1, Th2, and Th17-related cytokines which were more polarized towards the Th1 and Th17 compared to individual antigens. The HMS-specific antisera also significantly promoted phagocytosis of S. aureus COL strain by mouse macrophages. In conclusion, the fusion protein might be an effective vaccine for potential protective immunity against a lethal infection of S. aureus in mice.

Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016.

Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored bla NDM-1 and bla OXA-48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying bla NDM-1 and bla OXA-48 were successfully transferred to an E. coli K12-recipient strain. The bla OXA-48 gene was located on an IncL/M conjugative plasmid, while the bla NDM-1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of bla NDM-1 and bla OXA-48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran.

Engineering of an Iranian Bordetella pertussis strain producing inactive pertussis toxin.

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough.Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX).Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering.Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91.Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.

Genomic epidemiology of Iranian Bordetella pertussis: 50 years after the implementation of whole cell vaccine.

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 for the last 50 years with more than 95% vaccination rate since 1988 and has experienced pertussis resurgence in recent years. Here, we sequenced 55 B. pertussis isolates mostly collected from three provinces with the highest number of pertussis cases in Iran, including Tehran, Mazandaran, and Eastern-Azarbayjan from the period of 2008-2016. Most isolates carried ptxP3/prn2 alleles (42/55, 76%), the same genotype as isolates circulating in acellular vaccine-administrating countries. The second most frequent genotype was ptxP3/prn9 (8/55, 14%). Only three isolates (5%) were ptxP1. Phylogenetic analysis showed that Iranian ptxP3 isolates can be divided into eight clades (Clades 1-8) with no temporal association. Most of the isolates from Tehran grouped together as one distinctive clade (Clade 8) with six unique single nucleotide polymorphisms (SNPs). In addition, the prn9 isolates were grouped together as Clade 5 with 12 clade-supporting SNPs. No pertactin deficient isolates were found among the 55 Iranian isolates. Our findings suggest that there is an ongoing adaptation and evolution of B. pertussis regardless of the types of vaccine used.

Strain variation and antigenic divergence among Bordetella pertussis circulating strains isolated from patients in Iran.

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.

First report of New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae in Iran.

New Delhi metallo-beta-lactamase (NDM-1) is a novel metallo-beta-lactamase (MBL). Sporadic cases of NDM-1 positive strains have been reported from different countries, suggesting a widespread dissemination. The aim of this study was the detection of MBLs in Enterobacteriaceae isolated from patients in Tehran hospitals. After identification tests, the susceptibility to the antibiotics was done by Kirby-Bauer method and broth microdilution. Carbapenem-resistant isolates were tested for carbapenemase production using the modified Hodge test (MHT). Carbapenem-resistant strains screened for bla(KPC) gene and genes encoding MBLs. Twenty-three isolates (6.3%) were resistant to meropenem, eleven isolates (3%) were resistant to ertapenem, and four isolates (1.1%) were resistant to imipenem. MHT was positive in 11 (47.8%) of the carbapenem-resistant isolates. In March 2011, we detected a multiple drug-resistant Klebsiella pneumoniae isolate that was resistant to all tested antibiotics except colistin. PCR confirmed that this isolate contained bla(NDM-1), bla(TEM), bla(SHV), and bla(CTX-M). This is the first report on the detection of MBL NDM-1 in Iran. The rapid spread of NDM-1-positive bacteria proved to be a major challenge for the treatment and control of infectious diseases.

PCR/RFLP-based allelic variants of streptokinase and their plasminogen activation potencies.

PCR-restriction fragment length polymorphism (PCR/RFLP)-based analysis of ß-domain variable region of streptokinase genes (sk) has previously identified 14 sk alleles (sk1-sk14) in group A (GAS), C (GCS) and G (GGS) streptococci isolates from a few geographically distinct regions. However, the relation of sk allelic variants to their plasminogen activation potencies remained as a matter of debate. Herein, employing the same PCR/RFLP assay, we analysed sk allelic variants of GAS and GCS/GGS isolates from Iranian patients. In total, 21 sk allelic variants including 14 new alleles (sk14-sk28) were identified. Results implied the horizontal gene transfer of sk fragments between GAS and GCS/GGS strains and did not prove the specificity of particular sk alleles to GCS/GGS or GAS groups. Measurement of streptokinase (SK) activity in streptococcal culture supernatants by colorimetric assay (S2251 substrate) ranged from 9 to 182 IU mL(-1). Although some strains with the highest SK activity were detected in definite variants, no significant correlation between sk alleles and plasminogen activation was detected (P value > 0.05). Analysis of DNA sequences and restriction site mapping of selective sk variants with similar SK activity pointed to the inadequacy of the currently available PCR/RFLP method for differentiation of critical/silent nucleotides to precisely categorize sk alleles for their functional properties.

Ribotyping of EPEC isolates from diarrheal and asymptomatic patients in Iran.

AIM: We intended to find out the diversity of EPEC isolates among asymptomatic or diarrheal children in Iran using ribotyping. BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is responsible for gastroenteritis especially in young children. PATIENTS AND METHODS: A total of 39 EPEC collected strains were serotyped and the presence of virulence genes as well as EAF plasmid among the strains was studied. Adherence assay was also performed. Clonal diversity of the isolates was investigated using ribotyping. RESULTS: Of 39 studied strains of E. coli, 6 serogroups of EPEC were represented. The presence of the stx gene was ascertained in 7 isolates and the eaeA, eaeB and bfpA genes were harbored by 5, 3 and 1 strains, respectively. Ribotyping yielded 9 different clusters. CONCLUSION: According to our results there was not a significant correlation between the results of serotyping and those of ribotyping. However, different serotypes of E. coli may belong to the same ribotype clusters and vice versa.