The homogeneity of vanB gene cluster among enterococcal isolates in Iran

Aims:  This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran.
Methods and Results:  The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium , one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster.
Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters .
Conclusions:  Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species.
Significance and Impact of the study:  In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.     

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators. pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS 257 was not involved in the recombination events which gave rise to pRJ29.     

Prevalence of resistance genes to biocides in antibiotic-resistant Pseudomonas aeruginosa clinical isolates

Molecular Biology Reports - Biocides are frequently used as preservative, disinfectant and sterilizer against many microorganisms in hospitals, industry and home. However, the reduced...     

Prevalence and detection of metallo-β-lactamase (MBL)-producingPseudomonas aeruginosa strains from clinical isolates in Iran

Of the 126 isolates obtained from clinical specimens, seventy strains were selected because of resistance or reduced susceptibilities to imipenem and/or ceftazidime. Screening for detection of MBL-producing strains was performed in latter isolates by the Etest MBL strips. Isolates that were positive to Etest MBL strips were analysed by PCR. PCR was performed with specific DNA probes for detection of genes coding IMP or VIM enzymes and positive controls (MBL-producing strains). Finally, eight isolates ofPseudomonas aeruginosa were detected to carry a bla_VIM gene.     

Detection and characterization of mutations in genes related to isoniazid resistance in Mycobacterium tuberculosis clinical isolates from Iran

Molecular Biology Reports - The global rise in drug-resistant Mycobacterium tuberculosis (M.tb), and especially the significant prevalence of isoniazid (INH)-resistance constitute a significant...     

Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals

The production of antimicrobial activities as well as the presence of bacteriocin structural genes (entA, entB, entP, entQ, cylL, entAS-48, bac31, and entL50A/B) were studied in 140 non-selected faecal enterococcal isolates recovered from wild animals. Eight different indicator strains (including Listeria monocytogenes, Pediococcus pentosaceus, and different enterococcal species) were used for antimicrobial activity detection. Twenty-five of the 140 enterococci (18%) showed antimicrobial activity against L. monocytogenes and 33 additional isolates (24%) showed antimicrobial activity against other indicator strains, but Listeria. At least one bacteriocin structural gene was detected in 17 of the 25 enterococci with antimicrobial activity against L. monocytogenes and different combinations of entA, entB, entP, entQ, entL50A/B, and cylL genes were detected; entA and entB were the most prevalent detected genes, and they were generally associated. Bacteriocin structural genes were detected in 10 of 33 isolates with antimicrobial activity against indicator strains other than Listeria, and the cylL gene was the most prevalent one, especially in E. faecalis isolates.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.     

Production of bacteriocins by different enterococcal isolates

Bacteriocins produced by ruminal as well as environmental isolates were found to be heat-stable antimicrobial substances with a broad inhibitory spectrum (including Gramnegative species and sanitary-important species). The exponential phase of growth and pH 4–7 were optimal for their production. After purification, some of them were sequenced and specified.     

Susceptibility to biocides and the prevalence of biocides resistance genes in clinical multidrug-resistant Pseudomonas aeruginosa isolates from Hamadan,Iran

Molecular Biology Reports - This study aimed to investigate the association between biocides' reduced susceptibility and the presence of efflux pump genes including cepA, qacEΔ1 and qacE...     

Prevalence of OXA-type carbapenemase genes and genetic heterogeneity in clinical isolates of Acinetobacter spp. from Mangalore, India

The prevalence of OXA-type carbapenemase genes, ISAba1 insertion sequence, carbapenem resistance, biofilm forming ability and genetic heterogeneity in clinical isolates of Acinetobacter spp. from hospitals in Mangalore, South India was studied. Based on the presence of the bla(OXA-51) -like gene, the 62 isolates of Acinetobacter spp. were identified as 48 A. baumannii and 14 other Acinetobacter spp. The prevalence of bla(OXA-23) -like, bla(OXA-24) -like and bla(OXA-58) -like genes in A. baumannii was 47.9%, 22.9% and 4.2%, while in other Acinetobacter spp. it was 28.5%, 64.3% and 35.7% respectively. Several A. baumannii isolates (16/48) harbored the insertion sequence ISAba1 in the upstream region of the bla(OXA-23) -like gene. Resistance to meropenem was seen in 39.6% and 14.2% of A. baumannii and other Acinetobacter spp. isolates, respectively. The ability to form biofilm was observed to be higher among A. baumannii in comparison to other Acinetobacter spp. The present study shows that bla(OXA-23) -like genes are more common in A. baumannii,whereas bla(OXA-24) -like genes are common to other Acinetobacter spp. The study revealed genetic heterogeneity among the isolates, indicating multiple sources in the hospitals.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

Occurrence of virulence factors among human intestinal enterococcal isolates

AIMS: The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. METHODS AND RESULTS: The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. CONCLUSIONS: Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.     

Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid.

Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4-6 were identified which specify inhibitory activity on L. lactis indicator strains: one that could be confined to a 1.8-kb ScaI-ClaI fragment with low antagonistic activity and a 15-kb XbaI-SalI fragment specifying high antagonistic activity. The inhibitory substances produced by these two clones were sensitive to proteolysis. A 4-kb HindIII fragment derived from the 15-kb fragment strongly hybridized with the 1.8-kb fragment. The antagonistic activity specified by the 4-kb fragment was somewhat reduced as compared with that of the 15-kb fragment. A 1.3-kb ScaI-HindIII subfragment of the 4-kb fragment contained both the immunity and bacteriocin genes. Inhibition studies showed that the two bacteriocins had different specificities.     

Investigation of virulence genes in clinical isolates of Yersinia enterocolitica

In this study, we aimed to investigate the distribution of virulence genes in clinical isolates of pathogenic Yersinia enterocolitica. Two thousand six hundred stool samples were collected from 2600 patients with diarrhea, and were tested using the culture method and real-time PCR. Then, all isolates of pathogenic Y. enterocolitica cultured from the culture method were examined for virulence genes (inv, ail, ystA, ystB, ystC, yadA, virF) by PCR and for the presence of plasmid by four phenotypic tests. As a result, 160 pathogenic strains were successfully detected by the culture method, including bio/serotype 1A/unknown (4), 1B/unknown (8), 2/O:9 (39), 2/unknown (7), 3/O:3 (22), 3/unknown (6), 4/O:3 (55), 4/unknown (10) and 5/unknown (9). The positive rate of virulence genes tested in 160 isolates was inv (100%), ail (94%), ystA (93%), ystB (7.5%), ystC (5%), yadA (89%) and virF (82%) while the phenotypic test included autoagglutination (87%), binding of crystal violet (89%), calcium-dependent growth (74%) and Congo red absorption (78%), respectively. Finally, we found that not all pathogenic Y. enterocolitica necessarily carry all traditional virulence genes in both chromosomes and plasmids to cause illness. Perhaps, some of them, lacking some traditional virulence genes, contain other unknown virulence markers that interact with each other and play an important role in the diverse pathogenesis of pathogenic Y. enterocolitica.     

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.     

Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While P. aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P. aeruginosa strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in P. aeruginosa is discussed.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates

T. WATANABE, H. KUMATA, M. SASAMOTO AND M. SHIMIZU-KADOTA. 1992. Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.