Repetitive Sequence-based PCR Research Articles

Molecular Characterization of Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae in the Countries of the Gulf Cooperation Council: Dominance of OXA-48 and NDM Producers

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Enterobacteriaceae (CRE) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic-resistant genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Sixty-two isolates which screened positive for potential carbapenemase production were assessed, and 45 were found to produce carbapenemase. The most common carbapenemases were of the OXA-48 (35 isolates) and NDM (16 isolates) types; 6 isolates were found to coproduce the OXA-48 and NDM types. No KPC-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with seven clusters of clonally related Klebsiella pneumoniae. Awareness of CRE in GCC countries has important implications for controlling the spread of CRE in the Middle East and in hospitals accommodating patients transferred from the region.

Epidemiology of Candida kefyr in Patients with Hematologic Malignancies

Candida kefyr is an emerging pathogen among patients with hematologic malignancies (HM). We performed a retrospective study at Johns Hopkins Hospital to evaluate the epidemiology of C. kefyr colonization and infection in HM patients between 2004 and 2010. Eighty-three patients were colonized and/or infected with C. kefyr, with 8 (9.6%) having invasive candidiasis (IC). The yearly incidence of C. kefyr colonization and candidemia increased over the study period (P < 0.01), particularly after 2009. In 2010, C. kefyr caused 16.7% of candidemia episodes. The monthly incidence of C. kefyr was higher during the summer throughout the study. In a cohort of patients with acute myelogenic leukemia receiving induction chemotherapy, risks for C. kefyr colonization included the summer season (odds ratio [OR], 3.1; P = 0.03); administration of an azole (OR, 0.06; P < 0.001) or amphotericin B (OR, 0.35; P = 0.05) was protective. Fingerprinting of 16 isolates by repetitive sequence-based PCR showed that all were different genotypes. The epidemiology of C. kefyr candidemia was evaluated in another hospital in Montreal, Canada; data confirmed higher rates of C. kefyr infection in the summer. C. kefyr appears to be increasing in HM patients, with prominent summer seasonality. These findings raise questions about the effect of antifungal agents and health care exposures (e.g., yogurt) on the epidemiology of this yeast.

B acteroides isolated from four mammalian hosts lack host‐specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns

One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade.

Pseudo-Outbreak ofKlebsiella oxytocaSpontaneous Bacterial Peritonitis Attributed to Contamination of Multidose Vials of Culture Medium Supplement

To determine the source of a cluster of Klebsiella oxytoca isolates cultured from peritoneal fluid of 3 patients with cirrhosis on a single day. Outbreak investigation and before-after study. A Veterans Affairs medical center. Epidemiologic investigation, analysis of antimicrobial susceptibility testing results and molecular typing of K. oxytoca isolates with repetitive sequence-based polymerase chain reaction (rep-PCR), review of microbiology laboratory procedures for processing peritoneal fluid cultures, and comparison of peritoneal fluid contamination rates 18 months before and after modification of laboratory procedures for culturing peritoneal fluid. Each of the peritoneal fluid samples that grew K. oxytoca was inoculated into blood culture bottles by different clinicians at different hospital locations. None of the patients had clinical findings suggestive of peritonitis or elevated polymorphonuclear cell counts in peritoneal fluid (range, 3-25 cells/μL). Molecular typing with rep-PCR demonstrated that the K. oxytoca isolates were genetically related (greater than 95% similarity). Laboratory procedures included the routine addition of a culture medium supplement of yeast extract and dextrose from a multidose vial into blood culture bottles with peritoneal fluid. After discontinuing use of the culture medium supplement, there was a marked reduction in the number of peritoneal fluid cultures deemed as contaminants (14.3% vs 0.9%; [Formula: see text]). A pseudo-outbreak of K. oxytoca peritonitis and high rates of contamination of peritoneal fluid were attributable to contamination of a multidose culture medium supplement. This article highlights the importance of discouraging the use of multidose vials in all clinical settings.

Molecular characteristics of vancomycin-resistant Enterococcus faecium from a tertiary care hospital in Chengdu, China

The aim of this study was to characterize vancomycin-resistant Enterococcus faecium (VREfm) isolates phenotypically and molecularly, and investigate associations between the virulence factors enterococcal surface protein (esp), hyaluronidase (hyl), and collagen adhesin (acm) and colonization/infection. A total of 126 E. faecium [66 VREfm and 60 vancomycin-susceptible (VSEfm)] were collected in West China Hospital. Nine E. faecium isolates (7 VREfm and 2 VSEfm) were selected at random for comparative study in a large region from China. Minimum inhibitory concentrations (MICs) were measured by Etest and agar dilution, vancomycin resistance genes (vanA, vanB, and vanC) and virulence genes (esp, acm, and hyl) were detected by polymerase chain reaction (PCR). Thirty-four VREfm underwent repetitive sequence-based PCR (rep-PCR) and multi-locus sequence typing (MLST). One linezolid-resistant isolate (MIC = 8 μg/ml) was found; none were tigecycline resistant. All 73 VREfm (28 infective strains and 45 intestinal colonizers) had the vanA gene and VanA phenotype. Positivity for esp, hyl, and acm in VREfm was 79.5, 46.6, and 86.3%, respectively, which was higher than in VSEfm (54.8, 27.4, and 56.5%, respectively). Among VSEfm, positivity for acm in isolates from pleural or cerebrospinal fluid (84.6%) was higher than that from blood (32.4%). There were 11 rep-PCR types (similarity >95%) and MLST revealed nine sequence types (STs) among the selected isolates. Most VREfm and all VSEfm belonged to clonal complex 17. A new ST was found, with allele sequence (15, 1, 38, 1, 1, 1, 1). In China, most VREfm seem to belong to the classical nosocomial CC17 clone, and many of them have acquired virulence genes, further strengthening a hospital-adapted type.

Heterogeneity of clinical and environmental isolates ofMycobacterium fortuitumusing repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients' isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ⩾2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

栃木県産ナス科青枯病菌Ralstonia solanacearumのナス属植物に対する病原性とPCR法による菌群判別の可能性の検討

Strains of Ralstonia solanacearum from eggplant and tomato fields in Tochigi Prefecture separated into four distinct groups (I, II, IV and one unknown) based on differences in pathogenicity to four species of Solanum plants. In repetitive sequence-based polymerase chain reaction (rep-PCR) DNA fingerprinting to discriminate the strains belonging to each pathogenicity group, two characteristic bands were identified. One was universally amplified from all tested strains, and the other was amplified only from strains in groups I, II and V. Based on the DNA sequences of the bands, we designed two more PCR primer sets (RsUp-F and RsUp-R, and RsDwn-F and RsDwn-R) that discriminated the strains in group I, II and V from those in III and IV.

Use of REP‐PCR and 16S rRNA gene sequencing for comparison of Mannheimia haemolytica isolates obtained from fatal cases of bovine respiratory disease in the USA and Australia

Assess the variability of Mannheimia haemolytica isolates obtained from fatal cases of bovine respiratory disease (BRD) in the USA and Australia using repetitive sequence-based PCR (REP-PCR) and sequencing of the 16S ribosomal RNA (rRNA) gene. We examined 22 isolates from the USA and 36 isolates from Australia using (GTG)5 and BOX-A1R REP-PCR primers, as well as sequencing a 700-base pair length of the 16S rRNA gene. The discriminatory ability of each typing method was assessed and correlation coefficients were calculated to assess concordance between the results of each approach. All methods appeared to discriminate among isolates, with BOX-A1R being the most sensitive and sequencing the least sensitive. Modest to moderate diversity was seen among the isolates, with as much variation within a continent as between the two. Using samples from diverse origins may permit extrapolation even to isolates with distant geographic and temporal relationships. Further, this information can serve as a baseline in assessing whether M. haemolytica is an opportunistic pathogen or if there are notable features that distinguish commensal isolates from those more likely to be associated with disease.

An Exploratory Study of Staphylococcus epidermidis Isolates from Environment and Patient Blood Cultures from an Intensive Care Unit

This exploratory study determined the epidemiological characteristics of Staphylococcus epidermidis from near touch sites and blood cultures in one Intensive Care Unit (ICU) between July and October 2009 in a large teaching hospital in Brazil. Five different touch sites around the environment of two different patients were sampled over four days and 24 blood cultures from 24 patients were obtained and analyzed. Isolates from near touch sites and blood cultures were compared by means of the repetitive extragenic palindromic sequence-based polymerase chain reaction (rep-PCR) and analyzed by the unweighted pair-group method with arithmetic mean (UPGMA) (BioNumerics, 6.0). Results found that ciprofloxacin- resistant Staphylococcus epidermidis on stethoscopes, bed rails ( P< . 004), and patient blood cultures indicated similarity (60-65%; IC: 85%). The similarities among near touch sites and patient isolates are unsurprising due to the commensal nature of Staphylococcus epidermidis, however it does emphasize the need for further research into environmental contamination and cleaning.

Epidemiological investigation of a nosocomial outbreak of multidrug-resistant Corynebacterium striatum at one Belgian university hospital

During an 8-month period, 24 Corynebacterium striatum isolates recovered from lower respiratory tract specimens of 10 hospitalized patients were characterized. The organisms were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and by 16S rRNA gene sequencing. The cluster of C. striatum exclusively affected patients who had been admitted to an intensive care unit and/or subsequently transferred to one medium-size respiratory care unit. Prolonged duration of hospitalization, advanced stage of chronic obstructive pulmonary disease, recent administration of antibiotics and exposure to an invasive diagnostic procedure were the most commonly found risk factors in these patients. Seven patients were colonized and three infected. All strains displayed a similar broad spectrum resistance to antimicrobial agents, remaining susceptible to vancomycin only. Typing analysis by MALDI-TOF MS and by semi-automated repetitive sequence-based PCR (DiversiLab typing) showed that all outbreak-associated C. striatum isolates clustered together in one single type while they differed markedly from epidemiologically unrelated C. striatum isolates. Pulsed-field gel electrophoresis (PFGE) profiles revealed three distinct PFGE types among the C. striatum isolates associated with the outbreak while all external strains except one belonged to a distinct type. We conclude that C. striatum is an opportunistic nosocomial pathogen in long-term hospitalized patients and can be at the origin of major outbreaks. The routine use of MALDI-TOF MS greatly facilitated the recognition/identification of this organism in clinical samples and this technique could also offer the potential to be used as an easy and rapid epidemiological typing tool for outbreak investigation.

Clonal Analysis of Staphylococcus hominis Strains Isolated from Hospitalized Patients

Staphylococcus hominis is a part of normal skin flora, but it is also a cause of nosocomial infections. The aim of this study was to investigate the genetic relatedness of 62 strains of S. hominis obtained from hospitalised patients during an 11-year period. For the discrimination of these clinical strains we used repetitive sequence-based PCR method (BOX-PCR) and multiple-locus variable-number tandem repeat analysis (MLVA). BOX-PCR analysis revealed a large genetic diversity among clinical strains and we did not find a predominant clone with the ability to persist in a hospital environment. MLVA is not as discriminatory as BOX fingerprinting and would not be a useful method for epidemiological studies.

Dissemination of blaVIM in Greece at the peak of the epidemic of 2005–2006: clonal expansion of Klebsiella pneumoniae clonal complex 147

VIM-producing Klebsiella pneumoniae (<n = 21) isolated from ten Greek hospitals during 2003–2007 were characterized with multilocus sequence typing (MLST), semi-automated repetitive sequence-based PCR (rep-PCR) (Diversilab), plasmid replicon typing, serotyping and screening for multiple resistance determinants. The isolates were selected to represent different strain clusters (defined by >80% similarity) according to pulsed-field gel electrophoresis. MLST identified three major clonal complexes (CCs); CC147 (n = 8), CC18 (n = 5) and CC14 (n = 3). Plasmid replicon typing showed that IncA/C and/or IncFIIK replicons were detected among isolates in each of the major CCs. Good concordance was observed between semi-automated-rep PCR genotyping and MLST.

Molecular epidemiology of Staphylococcus aureus colonization

Rodriguez et al performed a year-long prospective study of molecular epidemiology of Staphylococcus aureus colonization of 163 children with S aureus skin and soft tissue infections and their 562 household contacts. Samples of the infected site of index cases and swabs from potential colonizing sites of nares, axilla, and inguinal folds were obtained repeatedly from cases and once at case diagnosis from households. Strain relatedness was determined by repetitive sequence-based PCR testing. Results refuted a simplistic idea that a single strain affected a single patient or a single household, let alone multiple patients and multiple households. The study clarifies only the complexity of S aureus niches, potential modes of acquisition and transmission of colonizing organisms, and events that lead to infection. A better understanding of each will be required before postulating and studying strategies for control or eradication. Article page 105▶ Molecular Epidemiology of Staphylococcus aureus in Households of Children with Community-Associated S aureus Skin and Soft Tissue InfectionsThe Journal of PediatricsVol. 164Issue 1PreviewAlthough colonization traditionally is considered a risk factor for Staphylococcus aureus infection, the relationship between contemporary S aureus colonization and infection is not well characterized. We aimed to relate the presence of colonizing and disease-causing strains of S aureus within individuals and households. Full-Text PDF

Infections caused by fluoroquinolone-resistant Escherichia coli following transrectal ultrasound-guided biopsy of the prostate.

An increase in the number of infections with fluoroquinolone (FQ)-resistant Escherichia coli following transrectal ultrasound-guided biopsy of the prostate (TRUBP) was observed in Louis Stokes Cleveland Department of Veterans Affairs Medical Center. This study investigated whether these infections were caused by a single strain of E. coli possessing distinct resistance and virulence determinants. Of 15 patients with urinary tract infection, 5 were complicated with bacteraemia and 1 with prostate abscess. Thirteen FQ-resistant isolates demonstrated mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC but did not contain plasmid-mediated quinolone resistance determinants; blaCTX-M and blaCMY as well as genes coding for extended-spectrum β-lactamases were also absent. Genes encoding aminoglycoside-modifying enzymes were discovered in an isolate that was gentamicin-resistant. The most prevalent sequence type (ST) was ST43 (n = 7), corresponding to ST131 in Achtman's multilocus sequence typing (MLST) scheme. These isolates (i) were distinguished as >95% similar by repetitive sequence-based PCR (rep-PCR), (ii) belonged to the virulent phylogenetic group B2 and (iii) contained plasmid types FIB, FIA and Frep. Several other strain types were present (ST2, ST27, ST30, ST44, ST472, ST494, ST511 and ST627). Non-ST43 isolates infected patients with more co-morbidities but contained similar virulence factors (kpsMTII, iutA, papAH/papC and sfa/focDE). In our hospital, E. coli isolates causing TRUBP-related infection are quite heterogeneous (ST131 and other ST types) and are part of phylogenetic groups containing multiple virulence factors.

A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin Production

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

Blastoschizomyces capitatus’un Neden Olduğu Üç Fungemi Olgusu

Blastoschizomyces capitatus is a rare fungal pathogen that may lead to severe and fatal systemic infections especially in immunosuppressive individuals. B.capitatus strains have also been reported as the cause of hospital-acquired infections and outbreaks. In this report, three fungemia cases caused by B.capitatus with hematologic malignancies have been presented. The first case was a 20-year-old female with acute lymphoblastic leukemia, the second was a 26-year-old female with B-cell malignant lymphoma and the third was a 7-year-old male with B-cell acute lymphoblastic leukemia. All of the patients have been receiving chemotherapy, and treated with antibacterial and antifungal agents due to neutropenia. The blood cultures obtained from the second and third patients yielded B.capitatus although they were under empirical caspofungin therapy. Those patients have been treated with voriconazole and amphotericin B after the identification of B.capitatus, and clinical improvement were noted during their follow-up. However the first patient who was also under caspofungin therapy had died just before the isolation of B.capitatus from her blood culture. Conventional mycological methods [macroscopic and microscopic morphology, germ tube test, urea hydrolysis, carbohydrate assimilation tests (API 20C AUX; BioMerieux, France), growth temperature, cycloheximide sensitivity] were used for the identification of the isolates. The strains were identified as B.capitatus with the characteristics of annelloconidia formation, urease negativity, carbohydrate utilization, growth at 45°C and resistance to cycloheximide. Antifungal susceptibilities of isolates were determined by using microdilution method (for amphotericin B, fluconazole, itraconazole, voriconazole, ketoconazole) and E-test (for caspofungin). Minimum inhibitory concentration (MIC) values of the three B.capitatus strains were detected as 0.25, 0.125, 0.032 µg/ml for amphotericin B; 2, 2, 16 µg/ml for fluconazole; 0.064, 0.032, 0.032 µg/ml for itraconazole and 0.125, 0.064, 0.064 µg/ml for ketoconazole, respectively, while MIC values of all strains were 0.032 µg/ml for voriconazole and > 32 µg/ml for caspofungin. Since B.capitatus strains were isolated from the cases within about 15 days -sequentially-, the genotypes of the isolates were determined by repetitive sequence-based PCR (DiversiLab System; BioMerieux, France) to investigate the similarity rates. The results of analysis indicated 97% similarity between two (case 1 and 2) strains and 94.9% similarity in one strain (case 3) of B.capitatus, however the transmission route could not be clarified due to the absence of environmental sampling. In conclusion, B.capitatus should also be considered as a cause of systemic fungal infections in immunocompromised patients. Determination of the in vitro antifungal susceptibilities of clinical B.capitatus strains may contribute to the therapeutic approaches and epidemiological data.

Molecular Epidemiology of Staphylococcus aureus in Households of Children with Community-Associated S aureus Skin and Soft Tissue Infections

Although colonization traditionally is considered a risk factor for Staphylococcus aureus infection, the relationship between contemporary S aureus colonization and infection is not well characterized. We aimed to relate the presence of colonizing and disease-causing strains of S aureus within individuals and households. In a prospective study of 163 pediatric outpatients (cases) with community-associated S aureus skin and soft tissue infections in St Louis, infection isolates were obtained from cases and colonization cultures were obtained from cases and their household contacts (n = 562). Molecular typing by repetitive sequence-based polymerase chain reaction was used to compare infecting and colonizing isolates within each case. The infecting strain from each case was compared with S aureus strains colonizing household contacts. The colonization status of cases was followed for 12 months. A total of 27 distinct strain types were identified among the 1299 S aureus isolates evaluated. Between 1 and 6 distinct strain types were detected per household. A total of 110 cases (67%) were colonized at 1 or more body sites with the infecting strain. Of the 53 cases with an infecting strain that did not match a colonizing strain, 15 (28%) had 1 or more household contacts with a colonizing strain that matched the infecting strain. Intrafamilial strain-relatedness was observed in 105 families (64%). One-third of cases were colonized with a different strain type than the strain causing the skin and soft tissue infection. Fewer than one-third of cases with discordant infecting and colonizing isolates could be linked to the strain from another household contact, suggesting acquisition from sources outside the household.

A PCR-based assay for differentiating A- and A*-type strains of Xanthomonas citri subsp. citri, the causal agent of Asiatic citrus canker

By applying A- and A*-type strains of Xanthomonas citri subsp. citri (Xcc) in a repetitive sequence-based polymerase chain reaction (rep-PCR), two DNA amplicons, one unique to each strain, were evaluated as a probe against the DNA of Xcc strains. Two pairs of primers derived from these amplicons were tested in a PCR analysis. The results confirmed that primers Ms+/Ms− are useful for differentiating A-type from A*-type strains of Xcc. Also, a multiplex PCR with both set of primers can be used to distinguish three groups in Xcc populations: A-type strains and two subgroups of A* strains including Iranian and Thai A* strains.

Prevalence, characterization, and antimicrobial susceptibility of Salmonella Gallinarum isolated from eggs produced in conventional or organic farms in South Korea

To determine the prevalence of Salmonella serotype Enteritidis in eggs in South Korea, we conducted a microbiological survey of commercially available eggs produced in conventional or organic farms during the period from 2010 to 2012. The contents of 7,000 raw shell eggs (6,000 of conventional and 1,000 of organic origin) were examined to evaluate the extent and type of Salmonella Enteritidis contamination. A total of 26 salmonellae (7.4% of all pooled samples) were isolated from 350 homogenized pools, each containing the contents from 20 eggs. An unexpected and particularly surprising finding was that all the Salmonella isolates were serotyped as Salmonella Gallinarum. Salmonella Gallinarum was more common in eggs from organic farms: 10 of 50 egg pools (20.0%) from organic and 16 of 300 egg pools (5.3%) from conventional farms tested positive for Salmonella Gallinarum. However, organic and conventional isolates showed similar antimicrobial susceptibilities. All the isolates and a vaccine strain, SG 9R, which has been widely used in South Korea, were further characterized using the automated repetitive sequence-based PCR (rep-PCR) system, DiversiLab, to ascertain the molecular subtypes and to identify differences from the vaccine strain. The rep-PCR identified 2 distinct clusters among the 26 Salmonella Gallinarum isolates with a greater than 96% similarity index. These were clearly differentiated from the vaccine strain, SG 9R, with which there was a less than 86% similarity index. We found there was low genetic heterogeneity among isolates within each cluster and were able to distinguish wild type strains from the live vaccine strain (SG 9R) using the DiversiLab system.

Characterization of Metal Tolerant Serratia spp. Isolates from Sediments of Uranium Ore Deposit of Domiasiat in Northeast India

Three metal tolerant Serratia spp. isolates from sediments of pre-mined uranium ore deposit of Domiasiat, Meghalaya, India were characterized using morphological, biochemical and molecular methods. 16S rRNA gene analysis of the isolates identified one of the isolates (DB-10) as Serratia nematodiphila with 99.33 % pairwise similarity whereas remaining two isolates (DB-11 and DB-15) showed similarity with Serratia marcescens subsp. sakuensis with similarity of 98.78 and 99.54 % respectively. Randomly amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) studies of the isolates revealed genetic diversity among them. The isolates were found to tolerate all the five tested metals namely, uranium, cadmium, copper, zinc and lead and also could resist nine commonly used antibiotics namely, ampicillin, kanamycin, chloramphenicol, erythromycin, gentamicin, aztreonam, tetracycline, imipenem and ciprofloxin. All the three isolates were found to be superior in tolerance and resistance to the reference strain S. marcescens ATCC13880. The isolates showed high efficiency of uranium (VI) removal ranging from 90 to 92 % (21.4–21.9 mg/L) and from 65 to 70 % (309.4–333.2 mg/L) when challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions respectively within 1 h of incubation.