Geraldine Doran

Characterization of bacteriophages used in the Salmonella enterica serovar Enteritidis phage-typing scheme.

The 16 Salmonella enterica serovar Enteritidis (S. Enteritidis) typing phages (SETPs) used in the Laboratory of Enteric Pathogens (Health Protection Agency, London, UK) phage-typing scheme have not previously been characterized in detail. We have examined the adsorption properties of the phages with respect to a number of S. enterica serovars and defined phage morphology with electron microscopy. PFGE was used to estimate overall genome size and banding patterns generated by electrophoresis following restriction endonuclease digestion of the genome with HindIII were compared. PCR amplification and sequencing of selected genes was performed. The 16 phages comprise three morphotypes, Podoviridae (SETP1, 8, 10, 14, 15 and 16), Siphoviridae (SETP3, 5, 7, 11, 12 and 13) and Myoviridae (SETP2, 4, 6 and 9). All Podoviridae and Siphoviridae, but not Myoviridae, adsorbed to the O12 lipopolysaccharide antigen of Salmonella serogroups B (4,12) and D(1) (9,12). The genome sizes for the Podoviridae and Siphoviridae (PFGE-A) were approximately 42 kb. The genome size for Myoviridae SETP2, 4 and 9 was 36.5 kb, and for myovirus SETP6 was 27 kb. HindIII digestion of phage DNA produced 9 distinct patterns of 8 to 11 bands. Relationships between phages based on digest patterns were consistent with those defined by morphology. The Podoviridae had homologues of several P22 genes while the Siphoviridae had homologues of several genes present in the sequenced siphovirus SETP3 (EF177456). This study represents an initial step in characterizing the molecular basis that underlies the widely used S. Enteritidis typing scheme.

Salmonella enterica Serotype Bredeney: Antimicrobial Susceptibility and Molecular Diversity of Isolates from Ireland and Northern Ireland

ABSTRACT Salmonella enterica serotype Bredeney has emerged as the third most commonly identified serotype among human clinical isolates referred to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. A collection of 112 isolates of S. enterica serotype Bredeney collected during the period 1995 to 1999 from animal, food, and human sources from both Ireland and Northern Ireland were studied. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and DNA amplification fingerprinting (DAF) were performed on all isolates. Plasmid profiles were examined on a subset of 33 isolates. A high proportion (74%) of isolates were susceptible to all antimicrobial agents tested. Resistance to both sulfonamide and trimethoprim was observed in 21% of isolates, and resistance to multiple (five) antimicrobial agents was observed in a single isolate (0.9%). Eight different PFGE patterns were obtained, with 87% of isolates grouping as PFGE type A. PFGE type A was predominant in animals, food, and humans. There was good overall concordance between the groups identified by PFGE and DAF. Overall results indicate that most S. enterica serotype Bredeney isolates in Ireland and Northern Ireland from animal and human sources are clonally related.

First report of extended-spectrum-beta-lactamase-producing Salmonella enterica isolates in Ireland.

Production of extended-spectrum-s-lactamases (ESBLs) is predominantly associated with the Enterobacteriaceae , particularly Escherichia coli and Klebsiella pneumoniae . ESBL production in Salmonella spp. was first identified in 1988 ([11][1]) and is increasing in prevalence worldwide ([3][2], [18][

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium.

ABSTRACT Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.

Comparison of BACTEC MGIT 960 and BACTEC 460 for culture of Mycobacteria

We have compared the BACTEC 460 system with the BACTEC MGIT 960 system for culture of mycobacteria from 1800 routine clinical specimens. Rate of isolation of M. tuberculosis and time to detection of positive culture was comparable for both systems (BACTEC 460, 35 isolates, BACTEC MGIT 960, 34 isolates). Contamination of cultures was more common with the BACTEC MGIT 960 system. With intensification of the decontamination process an acceptable contamination rate was achieved in the BACTEC MGIT 960 system but time to detection of positive culture was increased by 1 to 2 days.

Role of subtyping in detecting Salmonella cross contamination in the laboratory

BackgroundWith the exception of M. tuberculosis, little has been published on the problems of cross-contamination in bacteriology laboratories. We performed a retrospective analysis of subtyping data from the National Salmonella Reference Laboratory (Ireland) from 2000–2007 to identify likely incidents of laboratory cross contamination.MethodsSerotyping and antimicrobial susceptibility testing was performed on all Salmonella isolates received in the NSRL. Phage typing was performed on all S. Typhimurium and S. Enteritidis isolates while multi-locus variance analysis (MLVA) was performed on selected S. Typhimurium isolates. Pulsed field gel electrophoresis (PFGE) using the PulseNet standard protocol was performed on selected isolates of various serovars.ResultsTwenty-three incidents involving fifty-six isolates were identified as likely to represent cross contamination. The probable sources of contamination identified were the laboratory positive control isolate (n = 13), other test isolates (n = 9) or proficiency test samples (n = 1).ConclusionThe scale of laboratory cross-contamination in bacteriology is most likely under recognized. Testing laboratories should be aware of the potential for cross-contamination, regularly review protocols to minimize its occurrence and consider it as a possibility when unexpected results are obtained.

Use of pulsed-field gel electrophoresis for comparison of similar but distinguishable isolates of Shigella sonnei collected in Ireland and Italy.

ABSTRACT Comparison of pulsed-field gel electrophoresis patterns (generated with XbaI and BlnI) of Shigella sonnei isolates from Ireland and Italy suggests that two possibly distantly related lineages are present in both countries. Smaller, more closely related groups, including isolates from Ireland and Italy, were also noted. These groups raise the possibility that the dissemination of clonal groups of S. sonnei may have occurred in recent years.

Failure To Detect Salmonella enterica Serovar Dublin on Aes Laboratoire Salmonella Agar Plate

Isolation and identification of Salmonella enterica from clinical specimens are an important component of the workload of clinical laboratories. Detection of S. enterica in specimens of feces is dependent on plating on selective and differential media both directly and following enrichment in selenite broth. Recently new differential media have become available (1, 4) including the Aes Laboratoire Salmonella Agar Plate (ASAP) medium (Aes Laboratoire). S. enterica produces distinctive pink to purple colonies on ASAP medium based on the enzymatic action of S. enterica C8 esterase on a chromogenic sub-strate, magenta-cap (5-bromo-6-chloro-3-indolylcaprylate).The medium also contains a second chromogen, X-β-d-glucopyranoside, which is hydrolyzed by β-d-glucosidase produced by Klebsiella spp. and Enterobacter spp. (blue to blue-green colonies). Serratia spp. produce both C8 esterase and beta-glucosidase, resulting in violet blue colonies. Most other species of bacteria result in white colonies. Following resuscitation from storage at −70°C on unselective medium, we subcultured a single colony of each of 320 isolates of S. enterica comprising S. enterica serovar Typhi (n = 6), S. enterica serovar Enteritidis (n = 99), S. enterica serovar Typhimurium (n = 83), and 34 other serovars (n = 132) on ASAP medium. All isolates produced colonies of the expected color, except for two isolates of S. enterica serovar Dublin, which produced white colonies. Thirty additional nonduplicate isolates of Salmonella serovar Dublin were subcultured onto ASAP medium. All 30 yielded white colonies. Isolates of Salmonella serovar Dublin were from diverse sources including humans and animals from Ireland and two isolates received as part of quality assurance panels from outside Ireland. Pulsed-field gel electrophoresis (PFGE) analysis (restriction enzyme XbaI, Pulse-Net protocol) on the 32 Salmonella serovar Dublin isolates showed six patterns differing by one or two bands. Twenty-six isolates were indistinguishable, and the remaining six isolates gave five distinguishable patterns. One isolate from outside Ireland was indistinguishable from most local isolates, but the other differed from the predominant pattern by one band. The failure of Salmonella serovar Dublin isolates to produce the expected pink to purple colonies suggests that these isolates do not produce C8 esterase sufficiently quickly or in sufficient quantity to result in color change after overnight incubation. After reincubation for a total of 72 h a faint pink color was noted for some isolates. Limited diversity of Salmonella serovar Dublin on PFGE analysis with XbaI has been reported previously (3). Likewise for S. enterica serovar Enteritidis (also a group D1 salmonella) isolates of different phage types and from different countries are often indistinguishable on PFGE with XbaI and additional enzymes (2). This collection included two isolates from outside Ireland that were very closely related to isolates from Ireland on PFGE. This collection of isolates is probably representative of the spectrum of the diversity that exists within Salmonella serovar Dublin. Failure to produce the expected color on ASAP medium is likely to be a frequent, though perhaps not universal, property of isolates of this serovar. These results indicate that Salmonella serovar Dublin may go undetected if laboratories use only ASAP medium as a differential medium for detection of S. enterica. Given the virulence of Salmonella serovar Dublin (5), its detection is particularly important.