Identification and characterization of the type II toxin-antitoxin systems in the carbapenem-resistant Acinetobacterbaumannii.

Carbapenem -resistant A. baumannii (CRAB) is a major cause of both community-associated and nosocomial infections that are difficult to control and treat worldwide. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The functional diversity and ubiquitous distribution in bacterial genomes are causing significant attention toward TA systems in bacteria. However, there is no enough information on the prevalence and identity of TA systems in CRAB clinical isolates. This study aimed to identify type II toxin-antitoxin systems in carbapenem-resistant A. baumannii (CRAB) isolates. A total of 80 A. baumannii isolates were collected from different clinical samples. Antibiotic resistance patterns of A. baumannii isolates were evaluated phenotypically and genetically. The frequency of type II TA genes was evaluated in CRAB isolates using PCR. Moreover, the expression level of the most prevalent TA encoding genes in some clinical isolates were evaluated by RT-qPCR. To determine whether the SplT and SplA are functional, the growth of E. coli BL21 cells (DE3/pLysS) harboring pET28a, pET28a-splTA, and pET28a-splT were analyzed by kill-rescue assay. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin and levofloxacin, whereas, 72%, 81% and 87% were resistant to amikacin, carbapenems and tetracycline, respectively. The cheTA in 47 isolates (72.5%) and splTA in 39 isolates (60%) of 65 isolates were the most common genes encoding type II TA among CRAB isolates. RT-qPCR demonstrated that cheTA and splTA transcripts are produced in the clinical isolates. There was a significant correlation between the presence of splTA genes and blaOXA-24 in CRAB isolates. Over-expression of the splT gene in E. coli results in inhibition of bacterial growth, whereas co-expression of splTA effectively restores the growth. This study presents the first identification of the type II TA systems among the carbapenem -resistant A. baumannii isolates, in Iran.

Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran-Iran.

BACKGROUND: To investigate antibiotic resistance, the occurrence and distribution of class 1 and 2 integrons in multidrug- resistant Staphylococcus aureus isolates from hospitals in Tehran, Iran. The isolates were examined for susceptibility to antimicrobial agents. The mecA gene, class 1 and 2 integrons were detected by PCR. Integrase positive strains were further analysed for the presence of resistance gene cassettes using specific primers and were sequenced. RESULTS: Among 139 S.aureus isolates, 109 (78.4 %) and 112 (80.5 %) strains were considered as multidrug resistant and mecA positive, respectively. Class 1 integrons and internal variable regions were found in 72.6 % (101/139) and 97 % (98/101) and class 2 integrons and variable regions also in 35.2 % (49/139) and 65.3 % (32/49) of S.aureus clinical isolates, respectively. Twelve distinct cassette arrays were found, containing genes encoding resistance to ß-lactams, aminoglycosides, streptothricin, trimethoprim, chloramphenicol,a putative glucose dehydrogenase precursor and a protein with unknown function. Gene cassette arrays aadB, aadA2 and dhfrA1-sat2-aadA1 were common in S.aureus isolates. We detected a completely new gene cassettes which contained aadB, oxa2, aacA4, orfD-aacA4-catB8, aadB-catB3, orfD-aacA4 and aadB-aadA1-cmlA6 of class 1 and dhfrA1-sat2-aadA1, dhfrA11, dhfrA1-sat2 of class 2 integrons. CONCLUSIONS: This is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes among in S.aureus isolates from Iran.

Novel cassette array in a class 1 integron in clinical isolates of Acinetobacter baumannii from central Iran.

Antibiotic resistance in Acinetobacter baumannii is a major problem in the hospital and outbreaks caused by this organism have been reported frequently. The present study aimed at determining the antibiotic susceptibility patterns, the prevalence of different classes of integrons and the characterization of integron class 1 gene cassettes in Iranian A. baumannii isolates. A total of 63 non-duplicate A. baumannii isolates were collected from clinical and environmental specimens in the Vali-Asr hospital in the central province of Iran (March to September, 2011). The antimicrobial susceptibility for 15 antibiotics which are used conventionally was determined by disk diffusion. The presence of different integron classes was investigated by PCR and the size of gene cassettes in class 1 integrons was then determined by PCR as well. Moreover, integron cassette arrays of isolates were delineated by RFLP and sequencing amplicons with different lengths. Of 63 isolates 62 (98.4%) carried a class 1 integron. The prevalence of IntI2 was 15.9% and the length of the amplicons ranged from 500 bp to 3 kb. Sequencing of integrons of class 1 revealed the presence of many resistance genes (aadA, aacA, aacC, dfrA, bla(GES) and bla(IMP)). We identified a completely new gene cassette which contained aacA7-qacF-aadA5-bla(IMP), this cassette has not been reported previously in A. baumannii.

Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation.

BACKGROUND AND OBJECTIVES: This study aimed to investigate the accessible regions of the fimH mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense anti-virulence therapeutics against Uropathogenic Escherichia coli. MATERIALS AND METHODS: We predicted the secondary structure of the E. coli fimH mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of fimH were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the fimH Digoxigenin (DIG) labeled mRNA was assessed with dot-blot hybridization. RESULTS: When searching the fimH gene in the GenBank database, two lengths for this gene was discovered in different strains of E. coli. The difference was related to the nine bases in the first part of the gene utilizing either of two translation initiation sites. Based on the bioinformatics analyses, five regions lacking obvious stable secondary structures were selected in mRNA of fimH. The result of dot-blot hybridization exhibited strongest hybridization signal between the antisense oligonucleotide number one and fimH labeled mRNA, whereas hybridization signals were not seen for the negative control. CONCLUSION: The results obtained here demonstrate that the region contains start codon of fimH mRNA could act as the potential mRNA target site for anti-fimH antisense therapeutics. It is recommended in the future both of utilizing translation initiation sites be targeted with antisense oligomers compounds.

A Field Study of Plague and Tularemia in Rodents, Western Iran.

INTRODUCTION: Kurdistan Province in Iran is a historical focus for plague and tularemia. This study aimed at assessing the current status of these two foci by studying their rodent reservoirs. MATERIALS AND METHODS: Rodents were trapped and their ectoparasites were collected. The genus and species of both rodents and ectoparasites were determined. Serological analyses of rodent blood samples were done by enzyme-linked immunosorbent assay for plague and by standard tube agglutination assay for tularemia. Rodent spleen samples were subjected to bacterial culture, microscopic examination, and real-time PCR to search for active plague or tularemia infection. RESULTS: During this study, 245 rodents were trapped, of which the most abundant genera were Apodemus (40%), Mus (24.49%), and Meriones (12.65%). One hundred fifty-three fleas, 37 mites, and 54 ticks were collected on these rodents. The results of all direct and indirect tests were negative for plague. Serological tests were positive for tularemia in 4.8% of trapped rodents. DISCUSSION: This study is the first report on the presence of tularemia infection in rodents in Western Iran. Since Meriones persicus is a known reservoir for plague and tularemia, and this rodent carried plague and tularemia vectors in Marivan and Sanandaj districts, there is a real potential for the occurrence of these two diseases in this region.

High Prevalence of Class 1 to 3 Integrons Among Multidrug-Resistant Diarrheagenic Escherichia coli in Southwest of Iran.

OBJECTIVES: Horizontal transfer of integrons is one of the important factors that can contribute to the occurrence of multidrug-resistant (MDR) bacteria. This study aimed to determine the prevalence of integrons among MDR Escherichia coli strains isolated from stool specimens and investigate the associations between the existence of integrons and MDR properties in the southwest of Iran. METHODS: There were 164 E. coli strains isolated from January 2012 to June 2012. Fecal specimens identified as E. coli by the conventional methods. Subsequently the antibiotic resistance was assessed using Clinical and Laboratory Standard Institute criteria. The presence of class 1-3 integrons and embedded gene cassettes was verified using specific primers by multiplex polymerase chain reaction assay. RESULTS: Among a total of 164 studied samples, 69 (42.07%) isolates were multidrug resistant. Class 1 and class 2 integrons were present in 78.26% and 76.81% MDR isolates, respectively. For the first time in Iran, class 3 integron was observed in 26.09% MDR isolates. Significant correlations were identified between: class 1 integron and resistance to amikacin, gentamicin, chloramphenicol, ampicillin, tetracycline, nalidixic acid, and co-trimoxazole; class 2 integron and resistance to aminoglycosides, co-trimoxazole, cefalexin, ampicillin, and chloramphenicol; and class 3 integron and resistance to gentamicin, kanamycin, and streptomycin. CONCLUSION: Our results indicate that integrons are common among MDR isolates and they can be used as a marker for the identification of MDR isolates. Therefore, due to the possibility of a widespread outbreak of MDR isolates, molecular surveillance and sequencing of the integrons in other parts of the country is recommended.

Characterization of Plasmid-Mediated AmpC and Carbapenemases among Iranain Nosocomial Isolates of Klebsiella pneumoniae Using Phenotyping and Genotyping Methods.

OBJECTIVES: Plasmid-mediated AmpC ß-lactamases (PMABLs) and carbapenemases are emerging groups of antimicrobial-resistance determinants. The aims of the study were to evaluate the occurrence of PMABLs and carbapenemases in clinical isolates of Klebsiella pneumoniae and compare the test performance of various phenotypic methods for detection of these enzymes in Iran. METHODS: A total of 100 K. pneumoniae isolates were collected from clinical specimens obtained in Valiasr Hospital. AmpC production in all isolates was determined using the AmpC disk test, the cephamycin Hodge test, the AmpC Etest, and the boronic acid combined-disk test. In addition, carbapenemase production was determined using the modified Hodge test, the EDTA disk synergy test, and the boronic acid combined-disk test. The performances of various phenotypic methods were evaluated by the comparison of their results with polymerase chain reaction (PCR) method as the gold standard. RESULTS: Of the 100 isolates, 19 (19%) were demonstrated to harbor the PMABL-resistance gene by the multiplex PCR method. The PCR result indicated the presence of carbapenemase genes in 12 isolates. The performance of various phenotypic tests carried out for detection of carbapenemase-producing isolates varied widely, ranging in sensitivity from 30% to 100% and in specificity from 90.8% to 100%. CONCLUSION: This is the first report of MOX-type AmpC ß-lactamase and bla GES in K. pneumoniae in Iran. A comparison of the phenotypic methods showed that a combination of cefoxitin plus boronic acid is optimal for detecting plasmid-mediated AmpC enzymes in K. pneumoniae, whereas the implementation of molecular methods is often complex, requires specially trained personnel, and is associated with higher costs.

Molecular characterization of the first community-acquired methicillin-resistant Staphylococcus aureus strains from Central Iran.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has spread throughout the world with varying regional incidences and different staphylococcal cassette chromosome mec (SCCmec) elements in different genetic backgrounds. No information is available on CA-MRSA in Iran. A cross-sectional study was carried out among healthy students to investigate: (1) the prevalence of CA-MRSA in Central Iran, (2) the molecular epidemiology of such CA-MRSA strains, (3) the antimicrobial resistance patterns of the strains, and (4) the distribution of virulence genes in these CA-MRSA strains. METHODS: A total of 700 nasal swabs were collected and subjected to S. aureus and MRSA-specific isolation procedures. Antimicrobial resistance patterns were determined using the disk diffusion method, and molecular typing was carried out by multi-locus sequence typing (MLST), SCCmec typing, and Staphylococcus protein A (spa) typing for all CA-MRSA isolates. PCR was used to detect various virulence genes. RESULTS: One hundred fifty-four S. aureus strains were isolated from the anterior nares of 700 healthy students. According to the US Centers for Disease Control and Prevention definitions for CA-MRSA, seven (4.5%) isolates were confirmed as CA-MRSA. CA-MRSA isolates belonged to SCCmec types IV (n = 6) and V (n = 1). The predominant spa-type among the CA-MRSA isolates was t790 (n = 3), with single t660, t084, and t325 isolates; one isolate was not typeable. The predominant sequence type was ST22, t790, SCCmec IV in three isolates, and the four other sequence types were ST25, ST859, ST14, and ST15. CONCLUSIONS: Iranian CA-MRSA strains are genetically diverse with an elevated prevalence of t790/ST22 SCCmec IV isolates. These findings support the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of CA-MRSA in Iran.