Biomedical subjects
Publications and source records attributed to F R Cockerill.
Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of Staphylococcus lugdunensis among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as S. lugdunensis.
The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.
Staphylococcus lugdunensis, a coagulase-negative staphylococcus, is being increasingly recognized as the cause of serious infections. We report 2 cases of total knee arthroplasty infection caused by S lugdunensis. S lugdunensis frequently produces a clumping factor that can result in a positive slide (short) coagulase test result. If the microbiology laboratory does not use the tube coagulase (long) test to confirm the slide coagulase test result, the organism may be misidentified as Staphylococcus aureus. S lugdunensis is more virulent than other coagulase-negative staphylococci and in many clinical situations behaves like S aureus, further increasing the confusion. However, S lugdunensis differs from S aureus in that it is susceptible to most antibiotics. This fact may alert the microbiology laboratory or the clinician that the isolate is likely not S aureus and prompt further testing of a specific isolate. Accurate identification of S lugdunensis isolates facilitates studies to define the epidemiology and pathogenesis of prosthetic joint infection due to S lugdunensis and delineates optimal medical and surgical therapies.
OBJECTIVE: To evaluate the effects of 4 different drying methods to remove bacteria from washed hands. SUBJECTS AND METHODS: One hundred adult volunteers participated in this randomized prospective study. All bacterial counts were determined using a modified glove-juice sampling procedure. The difference was determined between the amounts of bacteria on hands artificially contaminated with the bacterium Micrococcus luteus before washing with a nonantibacterial soap and after drying by 4 different methods (cloth towels accessed by a rotary dispenser, paper towels from a stack on the hand-washing sink, warm forced air from a mechanical hand-activated dryer, and spontaneous room air evaporation). The results were analyzed using a nonparametric analysis (the Friedman test). By this method, changes in bacterial colony-forming unit values for each drying method were ranked for each subject. RESULTS: The results for 99 subjects were evaluable. No statistically significant differences were noted in the numbers of colony-forming units for each drying method (P = .72). CONCLUSION: These data demonstrate no statistically significant differences in the efficiency of 4 different hand-drying methods for removing bacteria from washed hands.
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Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and </=0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.
A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.
PURPOSE: Preretinal neovascularization has been previously observed in neonatal rats with spontaneously occurring diarrhea. This neovascularization appears analogous to retinopathy of prematurity (ROP), which occurs in human neonates. A new enterococcus species, designated Enterococcus rattus, has been isolated from the duodenum of these rats. In the present controlled study, the effect of the enteropathy induced by this organism on the retinal vasculature in the neonatal rat was further investigated. METHODS: One hundred fifty newborn Sprague-Dawley rats were randomly assigned to 6 expanded litters (n = 25). On the second day of life, animals were gavaged with either 100 microl of E. rattus suspension (1.0 X 10(7) colony forming units, inoculated group, n = 100 rats) or 100 microl saline (control group, n = 50 rats). All rats were raised in room air and were killed on day 13 of life. Duodenal and blood samples were cultured. The retinal vasculature was assessed using fluorescent microscopy and ADPase staining in a masked manner. Two additional inoculated litters and one control litter were studied for evaluation of arterial blood gases and validation of the grading method for preretinal neovascularization. RESULTS: One hundred percent of rats in the inoculated group developed severe diarrhea and had duodenal cultures positive for E. rattus compared with 0% in the control group. Preretinal neovascularization similar to ROP occurred in 55% of rats in the inoculated group compared with 2% in the control group (P = 0.001). Retinal vascular areas were reduced in the inoculated group (mean +/- SD, 89% +/- 5% versus 96% +/- 2%; P < 0.001). Rats in the inoculated group demonstrated severe growth retardation (final weight, 9.7 +/- 2.2 versus 16.7 +/- 2.7 g, P < 0.001). Inoculated animals also experienced acidosis (pH 7.31 +/- 0.06 versus 7.39 +/- 0.06 control, P = 0.04). CONCLUSIONS: A previously undescribed enterococcal enteropathy was associated with preretinal neovascularization similar to ROP in the neonatal rat. This supports an independent role for factors other than inspired oxygen in the development of ROP.
We report the activity of LY333328 against 35 clinical isolates of vancomycin-resistant enterococci (including organisms carrying the vanA, vanB, vanC-1, and vanC-2/3 genes, as determined by PCR), 33 clinical isolates of methicillin-resistant S. aureus, and 29 clinical isolates of high-level penicillin-resistant S. pneumoniae. All isolates of vancomycin-resistant enterococci were inhibited by 2 micrograms/mL LY333328, and 8 micrograms/mL LY333328 was bactericidal against all isolates tested. All isolates of methicillin-resistant S. aureus were inhibited by 1 microgram/mL LY333328, and 4 micrograms/mL LY333328 was bactericidal against all methicillin-resistant S. aureus isolates tested. All isolates of penicillin-resistant S. pneumoniae were inhibited by < 0.125 microgram/mL LY333328, and 0.25 microgram/mL LY333328 was bactericidal against all S. pneumoniae isolates tested. LY333328 is a promising new glycopeptide antimicrobial agent.
During surveillance for various tickborne pathogens in the upper Midwest during the summer and early fall of 1995, a Bartonella-like agent was detected in the blood of mice that were concurrently infected with Borrelia burgdorferi or Babesia microti (or both). The organism was isolated in pure culture after inoculation of blood from wild-caught mice into C.B-17 scid/scid mice. Phylogenetic analysis of the 16S rRNA and the citrate synthase genes showed that the novel Bartonella species and a Bartonella isolate from a mouse captured on Martha's Vineyard, Massachusetts, were closely related to each other and secondarily related to Bartonella grahamii and Bartonella vinsonii. Further analysis of Peromyscus leucopus blood and tissue samples demonstrated that the novel Bartonella species was exclusively found in conjunction with B. burgdorferi and B. microti. Patent coinfection with these agents may be relatively frequent in naturally infected mice.
We performed a comprehensive analysis of the molecular, serological, and clinical features of 16 consecutive cases of invasive streptococcal disease (ISD). The majority of cases were linked to two group A streptococcus (GAS) clones closely related by pulsed-field gel electrophoresis (PFGE) and designated as PFGE-1 and PFGE-1.1. These clones, serotyped as M-3, T-3/B3264, carried an allelic variant of the gene that encodes pyrogenic exotoxin A (speA3) and the gene that encodes streptococcal superantigen (SSA) but different emm alleles that encode M-protein. The characteristics and clinical features of patients were similar to those described in previous reports, regardless of the responsible GAS clone. However, worse clinical outcomes (shock and death) were more frequent when patients infected with PFGE1/1.1 clones were considered as a group and compared with all other patients as a group. One striking feature in some patients with deep tissue infection was a lack of inflammatory cells despite the presence of numerous streptococci. An evaluation of PFGE profiles of GAS isolated elsewhere demonstrated that the PFGE-1 clone has caused invasive disease in other locations in the United States and in Japan.
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The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.
We studied the DNA sequence variation of van genes of 34 isolates of Enterococcus spp. The isolates containing the vanB gene exhibited between 0 and 41 base pair changes per 801 bp studied when the vanB sequences were compared to that of the reference strain Enterococcus faecalis V583. The isolates carrying the vanC-2 gene exhibited between 0 and 23 base pair changes per 346 bp studied when the vanC-2 sequences were compared to that of the reference strain E. casseliflavus ATCC 25788. Little variation was noted in the vanA and vanC-1 genes.
The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.
Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples (n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (> or = 11,000 versus > or = 30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.
AIMS: Recent studies suggest that Helicobacter pylori is an invasive enteropathogen. However, the efficiency with which this pathogen invades mammalian cells remains unknown. Therefore, this study was designed to investigate the invasion frequencies of HEp-2 cells by clinical strains of H pylori. METHODS: An acridine orange assay and cultured HEp-2 cell monolayers were used to determine the HEp-2 cell penetration frequencies of 17 clinical isolates and one American Type Culture Collection (ATCC) strain of H pylori, and single clinical strains of Yersinia enterocolitica, Shigella flexneri, and a non-invasive ATCC Escherichia coli strain. RESULTS: The acridine orange assay demonstrated that invasion frequencies of HEp-2 cells by all H pylori isolates were significant and, in most instances, exceeded those for the S flexneri strain and equalled those for the Y enterocolitica strain. The assay also showed that internalised H pylori organisms remained viable for at least six hours, the maximum time that bacteria and HEp-2 cells were co-incubated. CONCLUSIONS: These results may have important implications for treatment and prevention strategies for this gastric pathogen. Furthermore, the acridine orange assay may be useful for assessing, in vitro, the ability of conventional and newer antibiotics, alone or in combination, to kill intracellular H pylori organisms.
Detection of antimicrobial resistance is important so that clinicians can make rational decisions about optimal antimicrobial therapy for their patients. During the past decade, new types of antimicrobial resistance have emerged, some of which present new challenges for the clinical microbiology laboratory. In most cases, conventional culture-based testing methods continue to be useful. In other situations in which the organism responsible for infection grows slowly (for example, Mycobacterium tuberculosis), culture methods are technically difficult (such as for human immunodeficiency virus), or genotypes are inconsistently expressed (for instance, methicillin resistance in staphylococci), genetic susceptibility testing methods may offer special advantages. Determining serum concentrations of antimicrobial agents may be useful both to ensure adequacy of treatment and to prevent toxicity. In this review, methods are described for conventional and genetic tests used to guide antimicrobial therapy.