Plasmid Profile Analysis Research Articles

Molecular characterization of Salmonella enterica serovar Typhimurium strains isolated from humans in Turkey

Multidrug resistant (MDR) Salmonella infections, especially infections due to Salmonella Typhimurium DT104 phage type strains are an important public health issue in many parts of the world. S.Typhimurium is the most common serotype isolated from clinical samples in Turkey but we have limited data about the phage types of these isolates. The aims of this study were to find out whether these MDR S.Typhimurium isolates are DT104 phage type isolates and have class 1 integrons and to investigate the relationships of these characteristics between plasmid and pulsed field gel electrophoresis (PFGE) profiles. A total of 66 S.Typhimurium stock strains selected from Enterobacteria Laboratory culture collections of Ankara University School of Medicine, Department of Medical Microbiology were investigated by plasmid profile analysis (PPA) and PFGE with the use of XbaI and SpeI enzymes. The presence of class 1 integrons and the phage type 104 were investigated by polymerase chain reaction (PCR). The strains used in the study were sporadically isolated cases from seven provinces after year 2000 with ACSSuT (63), ACGSSuTT/S (1), ACSSuTT/S (1) and ASSuTT/S (1) resistance types [ampicillin (A), chloramphenicol (C), gentamicin (G), streptomycin (S), sulphonamide (Su), tetracycline (T), trimethoprim/sulfamethoxazole (T/S)]. Of the isolates 65 were found as DT104 phage type. Forty-three S.Typhimurium DT104 isolates that carry class 1 integrons had five different bands between 350-1600 base pairs (bp); all of the isolates harbored 1-4 plasmids with sizes ranging from 1.0-180 kbp and 62 isolates had 90 kbp plasmid which was serotype specific and virulence related. S.Typhimurium DT104 isolates were grouped into five (X1-X5) and seven (S1-S7) profiles with XbaI and SpeI enzymes, respectively. When the profiles of the two enzymes were evaluated, 58 of the 65 (89.2%) isolates showed similar (X1.S1) profile. The molecular characteristics of the most S.Typhimurium isolates were clustered in similar groups when class 1 integron, plasmid and PFGE types were analyzed together. In this study we showed that nearly all S.Typhimurium isolates with five drug resistance pattern (ACSSuT) were DT104 isolates. PFGE profiles of these sporadic isolates suggested that they were epidemiologically related.

Incidence of antibiotic resistance and virulence determinants in Enterococcus faecium and Enterococcus faecalis strains, isolated from traditional cheeses in Turkey

Enterococcus faecalis (n = 15) and Enterococcus faecium (n = 33) strains isolated from traditional Turkish cheeses were tested for susceptibility to 11 different antimicrobial agents and for the presence of selected genes encoding resistance and 13 genes encoding virulence factors using PCR. Furthermore, the plasmid profile of enterococci was examined. All E. faecium and E. faecalis isolates were resistant to nalidixic acid and kanamycin. The percentages of other resistant E. faecium and E. faecalis isolates, respectively, were 97 and 100% to streptomycin, 15.1 and 20% to ampicillin, 9.1 and 26.7% to gentamycin, 12.1 and 46.6% to chloramphenicol, 12.1 and 60% to tetracycline, 75.7 and 93.3% to rifampicin, 84.8 and 80% to vancomycin, 97 and 100% to erytromycin and 72.7 and 60% to ciprofloxacin. efaA fm (100%) and ccf (90.1%) genes were the most common virulence genes identified among E. faecium isolates while efaA fs (100%), cpd (100%), ccf (93.3%) and cob (86.7%) genes among E. faecalis isolates. Cytolysin determinants (cylM, cylB, cylA) were not detected among tested strains. Plasmid profile analysis of Enterococcus spp. revealed plasmid DNA bands ranging in size from 2.4 to 35.8 kb.

Antibiotic resistance versus antimicrobial substances production by gram-negative foodborne pathogens isolated from minas frescal cheese: heads or tails?

In this study, 15 Gram-negative isolates from Minas Frescal cheese sold in commercial establishments in Rio de Janeiro, Brazil, were able to produce antimicrobial substances (AMSs). Seven, four, two, one, and one isolates identified as Yersinia, Acinetobacter, Enterobacter, Escherichia, and Hafnia genera, respectively, were considered potentially pathogenic. All 15 AMS(+) isolates were resistant to at least 1 antibiotic; however, 7 strains presented resistance to at least 3 antibiotics from different classes, exhibiting multiresistance profiles. The strains were also subjected to plasmid profile analysis. All isolates presented different plasmid forms with most ranging in size from 1 to 10 kb. Activity against various pathogens associated with food was tested and all 15 AMS(+) showed the same activity spectrum, inhibiting all Escherichia coli and Salmonella strains that were tested. Although restricted, the action spectrum of AMS-producing strains is extremely relevant to the food industry because Gram-negative bacteria such as E. coli and Salmonella spp. are most often associated with foodborne illnesses. The findings of this study reveal that even AMS produced by pathogens can have potential applications against other foodborne pathogens.

Plasmid Profile Analysis and Antibiotic Resistance Patterns of Shigella Isolates from Kwale County, Kenya

Shigellosis caused by multidrug-resistant (MDR) Shigella strains is an important cause of morbidity and mortality in developing countries. Shigella strains usually harbour heterogeneous population of plasmids which can confer resistance to different antibiotics. A hospital based survey was conducted to determine the pattern of antimi- crobial resistance and plasmid profiles in Shigella species from diarrheal patients in Kwale county. Twenty nine (8.1%) Shigella spp were obtained from 360 stool samples collected and each strain screened for antimicrobial resistance to common antimicrobial agents and presence of plasmids. High antibiotic resistance was found to Sulfamethoxazole-tri- methoprim (79%), Ampicillin (75.9%) and Streptomycin (75.9%). All isolates were sensitive to ciprofloxacin, ceph- alosporins and Aztreonam antibiotics. Ninety seven percent of the isolates screened contained one or more plasmids ranging from 1-6. Majority were small plasmids. Species specific plasmids of 3.2kb, 9.0kb, and 3.8kb were found in Shigella flexneri, Shigella dysenteriae and Shigella sonnei respectively, while 34.5% and 79.3% MDR strains harbored large (>100kb) and middle range self-transmissible plasmids (10-100kb). In all species, no association was found be- tween specific plasmid profile and antibiotic resistance patterns. Significant association was found between carriage of 80kb plasmid in S. dysenteriae (P<0.001), combined 48kb and 80kb plasmids in S. flexneri (P=0.002) and S. sonnei (P=0.041) and MDR phenotype. The middle range plasmids specified resistance to Sulfamethoxazole-trimethoprim, Ampicillin, Streptomycin and Tetracycline. This study reports the usefulness of plasmid profiling in detection and discrimination of Shigella strains in Kenya. The study also shows that middle range self-transferable R-plasmids are widespread among the circulating MDR Shigella strains indicating the important role of these genetic elements in the development and dissemination of multidrug resistance.

Prevalence of Toxins and Antimicrobial Resistance among &amp;lt;i&amp;gt;E. coli&amp;lt;/i&amp;gt; Isolated from Meat

Aim: The present study aims to evaluate the occurrence and characterize E. coli in meat and meat products marketed in Egypt based on their antimicrobial-resistance pattern and production of enterotoxins. Methods: A total of 250 meat samples, categorized as 80 fresh beef, 85 ground beef and 85 beef burger purchased from supermarkets and butchers’ shops were used for isolation of E. coli. All isolates were screened for antimicrobial susceptibility. Plasmid profile analyses were done. Polymerase chain reactions were performed for detection of enterotoxin-encoding genes (astA, eaeA, stx1 and stx2). Results: Twenty-five samples were isolated and identified as E. coli. 14 isolates were multidrug resistant. Plasmids isolation from all isolates revealed that 76% of these isolates harbored plasmids. astA gene was amplified in 7 isolates (28%). Eight (32%) isolates harbored eaeA gene. However, none of the isolates harbored stx1 or stx2 genes. Analysis of multiple drug resistant isolates revealed a significant relation between multiple drug resistance and both astA and eaeA. Conclusion: The study confirmed the prevalence of enterotoxin genes (astA and eaeA) in E. coli isolated from meat product and the association between the presence of these genes and multiple drug resistant phenomena.

A molecular epidemiological investigation of multistate outbreaks of Salmonella Enteritidis from clinical and environmental samples in Turkey, 2000-2010.

To analyze interrelated Salmonella Enteritidis strains isolated in Turkey. Fifty-five S. Enteritidis surveillance strains were isolated from human feces and environmental samples from different regions in Turkey between 2000 and 2010. Clinical isolates were selected from different outbreaks in the Turkish National Reference Laboratory. All isolates were submitted to antimicrobial susceptibility test, plasmid profile analysis, and XbaI-digested pulsed-field gel electrophoresis. The strains were scanned against 20 antibiotics and for 3 of them (amikacin, ciprofloxacin, gentamicin), all strains were found to be sensitive. Five isolates had no plasmid. Most of test strains carried the 57-kb plasmid in common and 15 genotypes were identified among the 55 isolates. Six genotypes were related closely, 3 genotypes were undistinguished, and 6 genotypes were unrelated. To our knowledge, this is the first report on the phenotypic and molecular characterization of S. Enteritidis isolates from both environmental samples and clinical isolates in Turkey.

Possible transfer of plasmid mediated third generation cephalosporin resistance between Escherichia coli and Shigella sonnei in the human gut

Choice of antibiotic for treatment of serious bacterial infection is rapidly diminishing by plasmid mediated transfer of antibiotic resistance. Here, we report a possible horizontal transfer of plasmid carrying third-generation-cephalosporin (TGC) resistance between Escherichia coli and Shigella sonnei. Two different types of colonies were identified in MacConkey agar plate from a faecal specimen collected from a patient with shigellosis. The colonies were identified as E. coli and S. sonnei. Both of the isolates were resistant to ampicillin, chloramphenicol, co-trimoxazole, erythromycin, azithromycin, nalidixic acid, ceftriaxone, cefixime, ceftazidime, cefotaxime and susceptible to co-amoxiclave, amikacin, imipenam, astreonam, levofloxacin, moxifloxacin, mecillinam. These two strains were positive for extended spectrum β-lactamase. We were able to transfer ESBL producing property from both ceftriaxone-resistant isolates to the ceftriaxone susceptible recipient E. coli K12 and S. sonnei. Plasmid profile analysis revealed that the first-generation E. coli K12 and S. sonnei transconjugants harbored a 50MDa R plasmid, as two-parent ESBL-producing S. sonnei and E. coli strains. Similar patterns of ESBL producing plasmid and transferable antimicrobial phenotype suggests that the ESBL producing plasmid might transferred between E. coli and S. sonnei through conjugation in the human gut.

Antimicrobial Susceptibility, Heavy Metals Tolerance and Plasmid Curing of &lt;i&gt;Shigella&lt;/i&gt; Species Isolated from El- Dakahlia, Egypt

The aim of this study was carried out determine the antibiotic susceptibility, heavy metals tolerance and plasmid curing of <i>Shigella</i> species isolating from diarrheal stool samples were collected from different hospitals in El- Dakahlia and Nile River waters, Egypt, from January 2012 to August 2014. After characterization and identification the results obtained show that 172 isolates isolated from stool samples belong to four <i>Shigella</i> species (<i>Shigella</i> <i>sonnei</i> 48.25 %, <i>Shigella flexneri</i> 29.65 %, <i>Shigella dysenteriae</i> 13.95 % and <i>Shigella boydii </i>8.13 %) while 5 isolates isolated from Nile River water were found belong to (<i>Shigella sonnei </i>40%,<i> Shigella dysenteriae </i>40 %<i> </i>and <i>Shigella flexneri</i> 20 %).The antibiotics susceptibility of <i>Shigella</i> sp. to11 antibiotics revealed that the most potent antibiotics were found co-amoxyclav, ciprofloxacin and ceftriaxone respectively while penicillin, ampicillin, co-trimoxazole and chloramphenicol respectively give low activity. The tolerance of<i> Shigella</i> sp. to heavy metals, (cadmium, nickel cobalt and zinc) revealed that all isolates sensitive to 1 and 0.1M concentration. Plasmid profile analysis of ten isolates <i>Shigella</i> <i>sonnei </i>shown that this isolates having numerous plasmid ranged from 8.5 to 4.3 kb. Treat isolates with SDS 2% for 24 hours to plasmid curing after recovery subject to antibiotic sensitivity and heavy metals tolerance. In conclusion, <i>Shigella</i>-associated diarrhea remains relatively common in Egypt and can be used ciprofloxacin and ceftriaxone for treat <i>Shigella</i> sp. infection. The heavy metal tolerance of <i>Shigella</i> sp .associated with resistance to antibiotics ampicillin, tetracycline and chloramphenicol. Present <i>Shigella</i> sp. in Nile River waters indicates polluted with sewage waters and becomes sources of some epidemic diseases.

Identification of the multi-resistance gene cfr in Escherichia coli isolates of animal origin.

Previous study indicated that the multi-resistance gene cfr was mainly found in gram-positive bacteria, such as Staphylococcus and Enterococcus, and was sporadically detected in Escherichia coli. Little is known about the prevalence and transmission mechanism of cfr in E. coli. In this study, the presence of cfr in E. coli isolates collected during 2010–2012 from food-producing animals in Guangdong Province of China was investigated, and the cfr-positive E. coli isolates were characterized by PFGE, plasmid profiling, and genetic environment analysis. Of the 839 E. coli isolates, 10 isolates from pig were cfr positive. All the cfr-positive isolates presented a multi-resistance phenotype and were genetically divergent as determined by PFGE. In 8 out of the 10 strains, the cfr gene was located on plasmids of ∼30 kb. Restriction digestion of the plasmids with EcoRI and sequence hybridization with a cfr-specific probe revealed that the cfr-harboring fragments ranged from 6 to 23 kb and a ∼18 kb cfr-carrying fragment was common for the plasmids that were ∼30 kb. Four different genetic environments of cfr were detected, in which cfr is flanked by two identical copies of IS26, which may loop out the intervening sequence through homologous recombination. Among the 8 plasmids of ∼30 kb, 7 plasmids shared the same genetic environment. These results demonstrate plasmid-carried cfr in E. coli and suggest that transposition and homologous recombination mediated by IS26 might have played a rule in the transfer of the cfr gene in E. coli.

KPC-3 Carbapenemase Harbored in FIIk Plasmid fromKlebsiella pneumoniaeST512 andEscherichia coliST43 in the Same Patient

Five carbapenem-resistant strains (three Klebsiella pneumoniae, one Escherichia coli, and one Enterobacter aerogenes) were isolated between 2009 and 2012 at the Verona University Hospital, Italy, during an epidemiological analysis of antibiotic resistance determinants and plasmid profiles in Enterobacteriaceae. Two out of the five strains, K. pneumoniae E530 and E. coli E558, were cultured from bile and abdominal drainage, respectively, of a single patient. The strains were resistant to beta-lactams and fluoroquinolones, and susceptible to tigecycline and colistin. All the strains harboured bla(KPC-3), bla(TEM-1), and bla(OXA-9), and the three K. pneumoniae additionally carried blaSHV-11 and aac(6')Ib. The bla(KPC-3) was inserted in transposon Tn4401a. All the strains hosted an FIIk-type plasmid, and the three K. pneumoniae coharboured an colE-type plasmid. Transconjugants, besides bla(KPC-3), harboured bla(TEM-1) and bla(OXA-9) genes on FIIk-type plasmid. K. pneumoniae E301 was ST258, while strain E530 and C525 belonged to the ST512, and E. coli E558 was ST43. To our best knowledge, this is the first report that strongly supports the transmission of bla(KPC-3) from ST512 K. pneumoniae to E. coli ST43 in a single patient, a phenomenon of both clinical and microbiological importance.

Virulence Properties of Multidrug Resistant Ocular Isolates of Acinetobacter baumannii

Purpose: Acinetobacter (A.) baumannii is an opportunistic pathogen and has been reported as a causative agent of ocular infections. The aim of this study is to identify virulence properties (biofilm formation, adhesion, invasion and cytotoxicity) and antibiotic resistance among A. baumannii isolates recovered from the eye.Materials and Methods: The Microscan Walk-Away®, an automated bacterial identification and susceptibility testing system was used to determine antibiotic resistance. Clonal relatedness was assessed by Pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. Conjugation experiments were carried out to determine the transfer of antibiotic resistance genes and PCR was used to confirm gene transfer. Virulence properties of the isolates were determined by biofilm formation using crystal violet and immunofluorescence staining, adherence and internalization using cultured corneal epithelial cells, and cytotoxicity by TUNEL-staining and LDH release assays.Results: All ocular isolates (n = 12) exhibited multidrug resistant (MDR) phenotype and one of the isolate (AB12) was resistant to 18 antibiotics (β-lactam, aminoglycosides, tetracycline, chloramphenicol and quinolones). The plasmid profile analysis showed the presence of multiple plasmids in each isolate and a total of 10 different profiles were observed. However, PFGE analysis was more discriminatory which revealed 12 distinct genotypes. Antibiotic resistance (tetracycline and quinolone) was transferable from the isolate AB12 to a recipient Escherichia coli J53. Ten isolates were strong biofilm producers and the remaining two (AB5 and AB7) were moderate producers. All isolates demonstrated adherence and invasive properties towards HCECs. A similar trend was observed in their ability to cause cell death and toxicity.Conclusions: Our results indicate that ocular isolates of A. baumannii are biofilm producers and adherent and invasive to corneal epithelium, a first step in the pathogenesis of ocular infection. In addition, they demonstrated plasmid-mediated transfer of MDR traits making them a reservoir of resistance genes at ocular surface.

Antibiotic Resistance Patterns and Plasmid Profiles of Methicillin Resistant Staphylococcus aureus Isolates from Human Samples

Antibiograms and plasmid profiles are commonly used to characterizemethicillin-resistant Staphylococcus aureus (MRSA) inepidemiologic studies. However, antibiograms are frequently inadequate to accomplish the differentiation. Plasmid profile being more informative has been reported to be useful in tracing the epidemiology of antibiotic resistance. Antibiotic resistance patterns and plasmid profiles of methicillin-resistant Staphylococcus aureus (MRSA) isolates from human specimens were investigated to determine the discriminatory power of plasmid profile analysis in conjunction with antibiotic susceptibility pattern. Specimens were analyzed using disc diffusion assay and restriction enzymes analysis of plasmid DNA procedure. The 51 methicillin-resistant Staphylococcus aureus (MRSA) isolates were grouped into 18 groups using their resistogram. Twenty four (47.1%) strains out the 51 methicillin-resistant Staphylococcus aureus (MRSA) isolates harbored plasmids. Single plasmid isolates were 14(27.5%), double plasmid isolates were 6(11.8%) while tripple plasmid isolates were 4(7.8%). The 24 isolates containing plasmids were categorized into 14 groups based on their resistogram. Plasmid profile showed Original Research Article British Microbiology Research Journal, 4(2): 185-194, 2014 186 greater similarity between isolates (10 profiles) than antibiotic resistance pattern which showed a higher disparity (14 patterns). However, resistance to various antimicrobial agents was not consistent with the presence of plasmids. No particular molecular size plasmid could be associated with any particular antimicrobial resistance patterns. Resistance was observed in isolates with various molecular size plasmids as well as in those that had no plasmids. Nonetheless, 2 pairs of isolates with the same plasmid profile also had similar (almost the same) resistance pattern. Plasmid profile analysis in conjunction with the antibiotic resistance typing is valuable in the epidemiological investigation of methicillin-resistant Staphylococcus aureus (MRSA).

Identification of Genomic Heterogeneity among Lactococcus lactis Strains by Plasmid Profiling, PFGE and 16S rDNA Sequence Analysis

Lactococcus lactis strains are used commonly as starters, which contribute to desirable flavour and texture properties known as strain-specific, in dairy industry. Genomic heterogeneity of 30 L. lactis strains originating from Turkey and characterized phenotypically were investigated in this study. Plasmid profiling, PFGE and 16S rDNA sequence analyses were performed to determine the genetic variability of strains. High degree of heterogeneity was detected among the L. lactis strains. Plasmid profiles of strains showed that compared to the plasmid free control strains, namely; L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1614, all tested strains carried one to ten plasmids with molecular size ranging from 1.5 to 41.5kb. The fingerprints of strains obtained by PFGE from digestion with ApaI, SmaI and I-CeuI restriction endonucleases of chromosomal DNA's were compared with each other. All strains out of four were grouped into a large cluster A with at least 44% similarity level. The other four strains formed a minor duster B, distinctively different from major cluster A. PFGE results were confirmed by 16S rDNA sequence analysis and strains included in cluster B were identified as members of different species. These results suggested that morphologic and biochemical methods should be verified by reliable molecular approaches for the purpose of strain typing. Also, PFGE was found suitable to determine genomic differentiations among inter- and intra species.

Bacteria Species Associated with Ugba (Pentaclethra macrophylla) Produced Traditionally and in the Laboratory and the Effect of Fermentation on Product of Oligosaccharide Hydrolysis

ABSTRACT Study on isolation of bacterial species associated with ugba fermentation was carried out. Bacterial species including Bacillus licheniformis, Bacillus sphaericus, Bacillus subtilis, Bacillus pumilus, Bacillus firmus, Escherichia coli, Klebsiella and Staphylococcus were isolated from traditionally fermented ugba (TPU) obtained from the market, whereas laboratory produced ugba (LPU) indicated the presence of Bacillus licheniformis, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis and Staphylococcus before boiling and fermentation. The absence of Bacillus pumilus and Staphylococcus was evident in LPU during post fermentation assay. Quantitative estimation of pentose and hexose sugars which are product of hydrolysis of galacto-oligosaccharide content of the test legume reduced from 1158 ppm – 346.4 ppm and 588 ppm – 516 ppm respectively. Greater loss was observed after boiling of LPU (564 and 486 mg/l) on comparison to TPU (94 and 10 mg/l) for pentose and hexose respectively. Plasmid profile analysis carried out showed that Bacillus species from fermented and unfermented ugba had bands indicating the presence in the organism of the genes coding for enzymes responsible for hydrolysis of oligosaccharides in the substrate. This study elucidates the need for good manufacturing practices and adequate boiling before consumption of ugba with the view to prove that there are other Bacillus species aside B. subtilis which can ferment ugba (Pentaclethra macrophylla) to give it its characteristic flavour and taste. It also quantified the effect of fermentation process on some dietary carbohydrate content of the ugba .

Prevalence and diversity of Escherichia coli isolated from a barley trial supplemented with bulky organic soil amendments: green compost and bovine slurry

A barley field trial supplemented with bulky organic soil amendments, municipal compost or bovine slurry was sampled for Escherichia coli to test the hypothesis that E.coli isolated from the soil or from barley plants were derived from bovine slurry. A qualitative analysis showed that a total of 12% of the bulk soil cores and 16% of harvested grain samples yielded E.coli. The strongest association for positive detection of E.coli from soil was with time of year and for slurry-treated plots, with irrigation. However, E.coli were detected in plots from all treatment types and not exclusively associated with bovine slurry. Phylogroup, plasmid profiling and population genetics analysis (multilocus sequence typing) revealed extensive genetic diversity. Identical sequence types for slurry and soil isolates were detected, indicative of direct transfer into the soil, although not frequently. Host interaction assays with selected isolates showed a variation in the ability to colonize barley roots, but not in interactions with bovine cells. The work has implications in appropriate use of E.coli as a faecal indicator as isolates were widespread and diverse, reinforcing the view that some are a natural part of the microflora in agricultural systems. Faecal deposition is considered to be the main process that introduces Escherichia coli into soil, giving rise to their use as a faecal indication species and the potential for cycling pathogens in agricultural systems. We found that bovine slurry was not the main source of E.coli in a barley trial and a high degree of diversity was present in the collection. The findings support the hypothesis that the population structure of E.coli in secondary habitats is shaped by the environment and highlight the drawbacks of its use as a faecal indicator species.

Prevalence of metallo-β-lactamase NDM-1-producing multi-drug resistant bacteria at two Pakistani hospitals and implications for public health

The rapid spread of metallo-β-lactamase producing clinical pathogens is a matter of great concern and with the addition of NDM-1 it poses more threat for public health as NDM-1 positive isolates show resistance to most of the antibiotics. The current study was carried out to determine the prevalence of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs), particularly NDM-1 in clinical multi-drug resistant isolates from two tertiary care hospitals in Pakistan. A total of 356 clinical isolates were included in the study where 301 isolates were collected from the Pakistan Institute of Medical Sciences (PIMS), Islamabad and 55 were collected from the Mayo Hospital Lahore. The isolates were screened for ESBLs and MBLs production by phenotypic method and PCR was performed to detect the presence of blaVIM, blaIMP and blaNDM-1 genes. Out of 356 clinical isolates, 160 showed carbapenem resistance. Of these 160 isolates, 131 displayed MBLs production as accessed by combined disk method. In MBLs producing organisms, PCR amplification confirmed 31 (23.6%) isolates harboring blaNDM-1 gene, 33 (25.1%) isolates having blaVIM gene and 2 (1.5%) isolates displaying blaIMP gene. Plasmid profile analysis of NDM-1 positive organisms showed variable number of plasmids which were stable during serial passages in antibiotic free media. The prevalence of ESBL producing organisms was recorded to be 87.5%. The results show a high level of NDM-1 positive organisms from variety of samples at both hospitals, implicating the spread of MBL genes in clinical isolates.

Effects of plasmid curing on Rhizobium spp.

Present study was carried out to determine the difference between the wild type rhizobial strains and plasmid cured strains for some important characteristics such as antibiotic resistance. The rhizobial strains were isolated from locally produced legumes of different varieties such as Pisum sativum, Sesbania aculeata, Vigna mungo, Phaseolus vulgaris, Lens culinaris and Arachis hypogea.They were purified and subjected to various morphological, cultural and biochemical analysis, and also to antibiogram. All the strains showed resistance against amoxicillin and bacitracin, and exhibited higher (50% or more) resistance against ampicillin, erythromycin, nalidixic acid and strptomycin. The isolates were then subjected to plasmid profile analysis. To determine appropriate dose for plasmid curing, Sesbania nodulating rhizobial strain was treated with different concentrations of acridine orange (plasmid curing agent). Rhizobial growth was found in yeast mannitol broth after curing with 30 ?g/ml of acridine orrange. The rhizobial strain was found to be sensitive against amoxicillin, streptomycin and ampicillin after plasmid curing and no exopolysaccharides were found. The present study reveals that the plasmid in rhizobial species might confer the antibiotic resistance and possibly involve in maintaining symbiotic relationships. DOI: http://dx.doi.org/10.3329/sjm.v2i1.15212 Stamford Journal of Microbiology, Vol.2(1) 2012: 34-37

Plasmid profile analysis and evaluation of antibiotic susceptibility of Staphylococcus aureus strains isolated from table chicken eggs

The aim of this study was to isolate and characterize Staphylococcus aureus bacteria present on the shell surfaces and in the contents of chicken eggs, taking into account their phenotypic properties, antibiotic susceptibility patterns, and the presence of plasmid DNA. The study included 90 table chicken eggs from laying farms situated in the vicinity of Lublin. A total of 105 bacterial strains identified as Staphylococcus were isolated from the material, of which 18 (17.14%) were of the species Staphylococcus aureus. All 18 S. aureus strains were found to be resistant to at least one of the antibiotics tested, while some (55.55%) showed resistance to five or more of the 17 therapeutic agents. The greatest number of strains showed resistance to erythromycin (66.66%), tetracycline (66.66%), oxytetracycline (61.11%), penicillin G (50%), and amoxicillin (44.44%). The plasmid profile analysis of the S. aureus strains made it possible to evaluate the dependence between antibiotic susceptibility and the presence of plasmids in particular isolates. The results showed that plasmids in various quantities and of varying molecular weights were isolated from 17 of the strains. Most often isolated were small plasmids, of 5.6 kb--from 11 of the S. aureus strains (61.11%), 2.5 kb--from 9 strains (50%), 4.1 kb--from 8 (44.44%), and 4.6 kb--from 7 (38.88%) of the strains.

Bronchopneumonia in wild boar (Sus scrofa) caused by Rhodococcus equi carrying the VapB type 8 plasmid

BackgroundRhodococcus equi is associated with pyogranulomatous infections, especially in foals, and this bacterium has also emerged as a pathogen for humans, particularly immunocompromised patients. R. equi infections in pigs, wild boar (Sus scrofa) and humans are mainly due to strains carrying the intermediate virulence (VapB) plasmid. In Brazil, R. equi carrying the VapB type 8 plasmid is the most common type recovered from humans co-infected with the human immunodeficiency virus (HIV). R. equi infection in pigs and wild boar is restricted predominantly to the lymphatic system, without any reports of pulmonary manifestations.FindingsThis report describes the microbiological and histopathological findings, and molecular characterization of R. equi in two bronchopneumonia cases in wild boar using PCR and plasmid profile analysis by digestion with restriction endonucleases. The histological findings were suggestive of pyogranulomatous infection, and the plasmid profile of both R. equi isolates enabled the characterization of the strains as VapB type 8.ConclusionsThis is the first report of bronchopneumonia in wild boar due to R. equi. The detection of the VapB type 8 plasmid in R. equi isolates emphasize that wild boar may be a potential source of pathogenic R. equi strains for humans.

Characterization of Shigella Strains Isolated in Ankara, Turkey by Antimicrobial Resistance Models, Plasmid Profile Analysis and Pulsed-Field Gel Electrophoresis

Shigella is one of the most important causative agents of diarrhea especially in childhood. Since man is the main reservoir of Shigella and human to human transmission is possible, Shigella can easily spread in public and cause outbreaks. In this study, a total of 60 Shigella strains isolated in Ankara, Turkey by years 2001, 2008 and 2009 were investigated by their antimicrobial susceptibility profiles, plasmid profile analysis (PPA) and pulsed-field gel electrophoresis (PFGE). For epidemiological investigation, the results obtained by antibiotic resistance typing (ART) which was the phenotyping method, was compared to the results of the genotyping methods which were PPA and PFGE. Of the isolates 49 (81.6%) were S.sonnei, 10 (16.6%) were S.flexneri and one was (1.6%) S.dysenteriae. Antimicrobial susceptibilities were evaluated by disc diffusion method and the highest resistance rates were found against trimethoprim/sulfamethoxazole (91.6%), followed by tetracycline (68.3%) and ampicillin (26.6%). Resistance against ampicillin, chloramphenicol and amoxycillin/clavulanic acid were found higher in S.flexneri isolates than S.sonnei (p< 0.001). All isolates were found to be susceptible to ciprofloxacin, gentamicin and ceftazidime. S.sonnei demonstrated 12 and S.flexneri demonstrated 4 antibiotic resistance models. All isolates were carrying plasmids with varying sizes and varying numbers between 1 to 7. S.sonnei isolates demonstrated 27 and S.flexneri isolates demonstrated 8 plasmid profiles. S.sonnei isolates were clustered in 4 patterns and S.flexneri were clustered in 5 patterns by PFGE. This method demonstrated obvious clonal similarity among S.sonnei strains isolated in Ankara and discriminative power (DP) was calculated as 0.26. PPA and ART demonstrated higher DP among S.sonnei strains (0.97 and 0.75, respectively). In this study gain or loss of instable genetic mobile elements were thought to be responsible for higher discriminative powers of PPA and ART methods. These typing methods were found to be appropriate for the epidemiological investigation of strains collected in a short time period. PFGE was found to be convenient for the evaluation of clonal relatedness of the strains, however, in such geographical areas where the same clone was in circulation, use of ART and/or PPA together with PFGE would be useful for precise discrimination of Shigella strains.