Genetic Relationships among Reptilian and Mammalian Campylobacter fetus Strains Determined by Multilocus Sequence Typing

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared ∼90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.Campylobacter fetus is a human and animal pathogen which can be divided into two subspecies: subsp. fetus and subsp. venerealis (16). C. fetus subsp. fetus has a wide host range and causes abortions in sheep and cattle; C. fetus subsp. venerealis is host restricted, being isolated specifically from the bovine genital tract, and it causes fertility problems in cattle (5). C. fetus is an opportunistic pathogen in humans, particularly affecting severely immunocompromised patients. Initially, the bacterium can cause gastroenteritis; then, bacteremia can lead to septicemia and disseminated infections (1, 8). These two subspecies of mammalian C. fetus are referred to subsequently as “classical C. fetus.”A multilocus sequence typing (MLST) scheme has been developed for classical C. fetus (http://pubmlst.org/cfetus/) and used to genotype 140 isolates from humans and animals (14). The data showed that classical C. fetus is genetically homogeneous and clonal. C. fetus has also been isolated from reptiles (7), and DNA hybridization and nucleotide sequence data indicate that these reptile C. fetus isolates are genetically distinct from classical C. fetus (12). Reptile-like C. fetus strains have also been isolated from cases of human disease (11). In the present study, both the reptile and the human reptile-like strains are referred to collectively as “reptile C. fetus strains.”The MLST scheme for classical C. fetus was modified in this study to allow typing of reptile C. fetus (Table ​(Table1)1) and comparisons within and among Campylobacter species. The MLST method for classical C. fetus (15) was modified as follows. First, the annealing temperature of the PCR amplification was reduced to 47°C. Second, one of the oligonucleotide primers used to amplify the glyA locus (glyA2) was replaced with glyS4, 5′-AGGTGATTATCCGTTCCATCGC-3′, derived from the C. jejuni sequence. New allele and ST numbers were assigned, and the data were deposited at http://pubmlst.org/cfetus/. Data analysis was performed using the programs MEGA (http://www.megasoftware.net/) (9) and ClonalFrame (3). ClonalFrame is a model-based method for using multilocus sequence data to infer the clonal relationships of bacteria and the chromosomal position of homologous recombination events that disrupt a clonal pattern of inheritance.

TABLE 1.

MLST data for C. fetus reptile isolates recovered from humans and reptiles

Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson''s index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen causing considerable morbidity and mortality worldwide. In Scotland and the United Kingdom in general, the incidence of MRSA is high (∼35%) with two epidemic clones, EMRSA-15 (ST22) and EMRSA-16 (ST36/ST30), accounting for 70 and 20%, respectively, of isolates referred to the Scottish MRSA Reference Laboratory (SMRSARL). Molecular typing of clinical isolates is important to inform decisions regarding effective control measures. For over a decade, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA has been used in United Kingdom reference laboratories for outbreak investigations and epidemiological surveillance, owing to its high discriminatory power and validated interpretation scheme (18). However, it is laborious and time-consuming, with poor interlaboratory comparability, and has no common international nomenclature. Furthermore, ca. 50% of EMRSA-15 strains and 35% of EMRSA-16 strains are indistinguishable by PFGE (4). Accordingly, the method is unsuitable for the tracing of many epidemics caused by EMRSA-15 and EMRSA-16 strains.Various techniques have been developed to address some of the limitations of PFGE, including spa typing and the variable-number tandem-repeat (VNTR)-based methods, multilocus VNTR fingerprinting (MLVF) (15) and multilocus VNTR analysis (MLVA) (6, 13, 16). spa typing involves DNA sequencing of the polymorphic VNTR in the 3′ coding region of the S. aureus-specific staphylococcal protein A (spa) gene (7). The method is more rapid and less laborious than PFGE, and the output is a digital profile, which is easily comparable between laboratories (1). However, it is less discriminatory than PFGE (10, 17) and is unsuitable for investigating the transmission of MRSA in hospitals dominated by EMRSA-15 and EMRSA-16 (8).MLVF analyzes the variation in the number of tandem repeats in seven genes (clfA, clfB, sdrC, sdrD, sdrE, spa, and sspA) by multiplex PCR and has been reported to be highly discriminatory and reproducible (9, 10, 15, 19). Previously, Tenover et al. (19) and Moser et al. (11) demonstrated that MLVF can distinguish between strains with identical PFGE patterns.MLVA differs from MLVF in that the number of repeats at each locus is determined to produce a numerical profile that can be incorporated into electronic databases and easily shared between laboratories. Although several MLVA schemes have been described, which differ in the loci and PCR protocol used, the MLVA described by Schouls et al. (16) benefits from automated fragment sizing on a DNA sequencer.To date, the effectiveness of MLVF and MLVA for tracing hospital outbreaks has not been compared. The aim of the present study was to investigate the usefulness of MLVF and MLVA compared to PFGE for subtyping highly clonal EMRSA-15s (ST22) and EMRSA-16S (ST36/ST30) and for tracing hospital outbreaks of infection.     

Volunteering in Cambodia

In August 2004, three undergraduate nursing students from the University of Toronto's faculty of nursing travelled to Cambodia to volunteer as student nurses. Being immersed in another culture helped to develop the students' cultural competence, enhance their understanding of primary health care and strengthen clinical skills. Given the growing cultural diversity in Canada, developing s kills in these areas is particularly important. In this article, the author reflects on her experiences in Cambodia and explores the unique value that international placements offer.     

Intravascular ultrasonic analysis of atherosclerotic vessel remodeling and plaque distribution of stenotic left anterior descending coronary arterial bifurcation lesions upstream and downstream of the side branch

Bifurcation lesions remain a challenging lesion subset, even in the era of drug-eluting stents. The aim of this study was to investigate the longitudinal remodeling pattern and cross-sectional plaque location of bifurcation lesions. Seventy-four preintervention intravascular ultrasound studies of left anterior descending bifurcation lesions were analyzed, in which the lesion was located proximal (type A, n=32) or distal (type B, n=42) to the side branch. Vessel area and plaque area at the lesion (VAlesion and PAlesion) and at the reference site (VAreference and PAreference) were measured. The remodeling ratio was defined as VAlesion/VAreference, and the vessel compensation ratio was defined as (VAlesion-VAreference)/(PAlesion-PAreference). The geometric center of the lumen at the lesion site was identified, and the lesion site was divided into circumferential equal arcs to compare the cross-sectional distribution of percentage plaque area (100x[PAlesion/VAlesion]) between the 2 groups. The remodeling ratio (1.03+/-0.15 vs 0.94+/-0.14, p=0.01) and the vessel compensation ratio (0.0+/-0.36 vs -0.37+/-0.61, p<0.01) were significantly greater in type A than in type B lesions. The circumferential distribution pattern of percentage plaque area was significantly different between the groups (analysis of variance p<0.005), with greater percentage plaque area for the vessel wall opposite from the side branch in type B lesions (46.3+/-18.0% vs 54.6+/-15.4%, type A vs type B lesions, p<0.05). In conclusion, these results suggest that a major side branch may affect longitudinal lesion remodeling as well as the circumferential location of atherosclerotic plaque.     

Cysteinyl leukotriene levels in esophageal mucosal biopsies of children with eosinophilic inflammation: are they all the same?

OBJECTIVES: Allergic eosinophilic esophagitis (AEE) is characterized by intense eosinophilic inflammation of the esophageal mucosa. Cysteinyl leukotrienes (CysLT) are eosinophil chemoattractants. We studied CysLT levels in esophageal mucosa of children with AEE and controls. METHODS: CysLT levels (pg CysLT/microg protein) were quantified by Enzyme-linked Immunosorbent Assay (ELISA) on endoscopically obtained esophageal mucosal biopsies. RESULTS: Twelve children with AEE (eight boys, mean age 6.6 yr, range 1.0-14.5 yr) and 10 controls (six boys, mean age 9.56 yr, range 1.08-15.08 yr) were enrolled. None were on anti-LT or corticosteroid therapy. All controls had histologically normal mucosal biopsies of the esophagus, stomach, and duodenum. Patients with AEE had intense eosinophilic inflammation of the esophageal mucosa (mean 39 eosinophils/hpf, range 15-70 eosinophils/hpf) and a normal 24-h pH probe study. CysLT levels were similar between the two groups: mean levels were 12.44 (median 10.87, range 2.54-28.29) in AEE patients and 9.52 (median 9.26, range 1.71-21.64) in controls. CysLT levels did not correlate with the degree of esophageal eosinophilic inflammation. Incidentally, five patients with eosinophilic gastroduodenitis, in addition to esophagitis, were enrolled; their CysLT levels were statistically higher than those of controls. CONCLUSIONS: This is the first study to examine CysLT levels in esophageal mucosal biopsies of children with AEE and normal children. CysLT levels in AEE patients are similar to those in controls, and independent of the severity of inflammation. While this would argue against the use of CysLT antagonists in the treatment of AEE, further studies into the expression of the CysLT receptor itself are needed.     

Nursing and medical staff knowledge regarding the monitoring and management of accidental or exposure hypothermia in adult major trauma patients

Recording a patient's vital signs is a basic requirement that in part informs clinical decision-making. Practice suggests that recording a trauma patient's temperature is occasionally overlooked in the emergency department. A staff survey was undertaken to gain an appreciation of knowledge and understanding of the issues that surround accidental or exposure hypothermia in trauma patients. Results demonstrate that nurses and doctors are unsure of how to define hypothermia and are not conversant with simple ways to prevent heat loss or rewarm patients. Complications from hypothermia such as coagulopathy and metabolic acidosis were seldom identified. Issues that limit staff recording temperature include patient access and acuity, lack of knowledge and confidence and access to temperature-measuring devices. These results emphasize the need for regular education. Implications for clinical practice were considered; an algorithm to guide staff on ways to improve the monitoring and management of temperature in trauma patients was developed. Opportunities for ongoing and further research were identified.