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Biomedical subjects

J T Magee

Publications and source records attributed to J T Magee.

At least 19 recordsLinked to original sources

Resistotyping of campylobacters: fulfilling a need.

A 9-month trial of a simple typing scheme for ¿thermophilic' enteric campylobacter isolates at a large Public Health Laboratory is described. Resistotyping was performed with six agents in a method modified by Bolton and colleagues from an earlier scheme, and biotyping was performed by a modified Lior scheme involving three tests. Reproducibility was excellent in both schemes, with test variation < 2%. Five household clusters and one larger presumptive milk-borne outbreak were identified in this scheme, and confirmed in pyrolysis mass spectrometry. The 328 isolates from new patients, excluding duplication from these clusters, were divided into 35 resistotypes with the largest group comprising 22% of isolates. In combined bio- and resistotyping, 86 types were found, with the largest group comprising 9.5% of isolates. The results are contrasted with salmonella sero- and phage-typing, where, on the same basis, the 176 isolates in the same period were divided into 40 groups, with the largest comprising 45% of isolates. Resistotyping, with or without additional biotyping, proved to be a convenient, simple, rapid, highly discriminatory, reproducible and inexpensive method well suited to use in local laboratories. It is a strong candidate for first-line national and local surveillance of campylobacter infections, fulfilling a need for monitoring of this important cause of enteric disease.

Adult

Temperature-dependent aminoglycoside resistance in Stenotrophomonas (Xanthomonas) maltophilia; alterations in protein and lipopolysaccharide with growth temperature.

Clinical strains of Stenotrophomonas (Xanthomonas) maltophilia often show large, growth temperature-dependent, variations in their susceptibility (TDVS) to aminoglycoside antibiotics. Strains showing more than a fourfold increase in susceptibility between 30 degrees and 37 degrees C (TDVS+ strains; n = 23) were contrasted with those showing lesser variation (TDVS- strains; n = 15) in studies of growth temperature-dependent variation in protein and cell-wall lipopolysaccharide (LPS) electrophoresis patterns in an attempt to determine the mechanism of TDVS. Several proteins showed increased intensity with increasing growth temperature. These comprised bands at c. 65, 55, 42.5, 26 and 21.5 kDa in the whole cell proteins, an outer membrane protein band at c. 21.5 kDa, and cytoplasmic membrane protein bands at c. 42.5 and 27 kDa. Two whole cell protein bands at c. 30 and 24 kDa and three outer membrane protein bands at c. 45, 30 and 24 kDa decreased in intensity with increasing growth temperature. However, there was no correlation with the extent of variation in susceptibility, either in the extent of temperature dependent changes in protein banding patterns, or the presence or absence of specific protein bands. By contrast, temperature-dependent variation in LPS patterns correlated well with TDVS. TDVS+ strains yielded intense ladder patterns of more than 30 discrete bands, and the mean molecular weight of the ladder pattern was markedly higher at growth temperatures < or = 30 degrees C, than at > or = 37 degrees C. TDVS- strains gave a clearly distinct high mol. wt LPS banding pattern showing fewer, less intense bands and a smaller and less consistent shift in mean molecular weight with temperature. Strains which were clearly resistant at 30 degrees and 37 degrees C, had a high mol. wt. polysaccharide component but an absence of the typical LPS-ladder pattern. We conclude that the temperature-dependent variation in the aminoglycoside susceptibility of this species was not correlated with any detectable change in protein composition, but correlated well with changes in LPS structure.

Aminoglycosides

Analysis of Bacteroides species by pyrolysis mass spectrometry.

Two methods for the classification of isolates of the Bacteroides fragilis group were compared in a study of 133 clinical isolates and 10 reference strains. There was broad similarity between the pattern of clusters in pyrolysis mass spectrometry (PyMS), which reflects whole-cell composition, and that in conventional tests, which reflect nutritional interactions with the environment. A single large cluster was resolved by both approaches; this cluster comprised mainly strains identified as B. fragilis and was surrounded by a multiplicity of small clusters (42 in PyMS and 24 in conventional tests). Individual clusters corresponding to strains identified as Bacteroides thetaiotaomicron and Bacteroides vulgatus, respectively, were found by both techniques. However, for the remainder of strains, little correlation was evident between clustering in PyMS and that in conventional tests. Further comparison with data obtained by sodium dodecyl sulfate- polyacrylamide gel electrophoresis for this collection of strains showed equal levels of disagreement between classification based on whole-cell protein and each of the classifications described here. In previous studies of other genera, PyMS-based and conventional test-based classifications showed good agreement. It appears that the B. fragilis group presents intractable problems in phenotypic characterization.

Bacterial Typing Techniques

Growth temperature-dependent variation of cell envelope lipids and antibiotic susceptibility in Stenotrophomonas (Xanthomonas) maltophilia.

Clinical isolates of Stenotrophomonas (Xanthomonas) maltophilia showed growth temperature-dependent variation in susceptibility (TDVS) to aminoglycoside antibiotics between 30 degrees C and 37 degrees C, but little or no TDVS effect for polymixin B, colistin, ceftazidime, chloramphenicol and piperacillin. When phenylethanol was added at sub-inhibitory concentrations, the TDVS effect was eliminated. Gas liquid chromatography showed that 13-methyl tetradecanoate (i-15;0), was the predominant fatty acid, and was present in lower proportions in cells grown at 30 degrees C than 37 degrees C, by contrast to the unsaturated acids, which were found in increased proportions in cells grown at 30 degrees C. However, the extent of these shifts in composition did not correlate with the extent of the TDVS effect in individual strains. Membrane analysis by spin label-electron spin resonance spectroscopy showed that strains exhibiting TDVS had significantly decreased membrane fluidity compared with susceptible strains at 30 degrees C. Furthermore, analysis of the outer and cytoplasmic membranes from the strains with TDVS revealed that in organisms grown at 30 degrees C, the outer membrane remained in a more rigid conformation than the cytoplasmic membrane. We conclude that resistance of S. maltophilia to aminoglycoside antibiotics at 30 degrees C correlates with changes in the conformation of the outer membrane so that binding and/or uptake of the antibiotic is inhibited.

Anti-Bacterial Agents

A numerical taxonomic study of the gram-positive anaerobic cocci.

Clinical (101) and collection (26) strains of gram-positive anaerobic cocci were examined in conventional tests and pyrolysis mass spectrometry (PMS). Numerical classifications based upon conventional test reaction patterns (CTRPs) and PMS showed 27 and 22 clusters, respectively. Cross-tabulation of cluster membership in the two classifications showed excellent correlation, with the combined classifications showing clear groups corresponding to the currently recognised species Peptostreptococcus anaerobius, P. heliotrinreducens, P. hydrogenalis, P. indolicus, P. lactolyticus, P. magnus, P. micros and Peptococcus niger. Strains of P. prevotii and P. tetradius clustered together in a heterogeneous group of saccharolytic organisms. However, strains previously identified as P. asaccharolyticus were divided into three distinct groups in PMS, two of which differed only in indole-associated pyrolysis products. A further four groups and several single-member clusters were distinct from these species. PMS data supported the validity of identification by pre-formed enzyme profiles and confirmed that Hare group III is synonymous with P. hydrogenalis, Hare group IV with P. magnus, and the "ADH group" with P. vaginalis. There is clearly a need for a taxonomic revision of the genus Peptostreptococcus, which probably encompasses several generic groups.

Bacteria, Anaerobic

Pyrolysis typing of isolates from a recurrence of systemic cryptococcosis.

Cryptococcal meningitis was diagnosed in a 71-year-old male diabetic patient with underlying ischaemic heart disease, asthma and bilateral axillo-femoral vascular grafts. After treatment with fluconazole for 2 months, the patient appeared to be cured. Two years later he presented with an aneurysm of the right graft that was resected and replaced with a new graft segment. Cryptococcus neoformans var. neoformans was grown from post-operative blood cultures and samples of the excised graft. The patient was treated with fluconazole and discharged after 6 weeks. Multiple isolates from both episodes had been preserved, and these, together with isolates from other UK patients, were cultured in duplicate, blind coded and characterised by pyrolysis mass spectrometry (PMS). Duplicate culture and re-isolate sets formed tight clusters, with each patient set clearly distinct. Sets of isolates from the two episodes in this patient formed a single tight cluster and were indistinguishable by PMS. These results support the contention that C. neoformans infection can be reactivated after being dormant for a prolonged period.

Aged

An investigation of a nosocomial outbreak of Clostridium difficile by pyrolysis mass spectrometry.

Isolates from a presumptive nosocomial outbreak of Clostridium difficile infection at a large teaching hospital were typed by pyrolysis mass spectrometry (PMS) and antibiograms. One isolate, from the putative index case, was dissimilar from the outbreak strain, but 24 isolates from 16 patients were indistinguishable by both methods. The outbreak centred on two wards for the acute care of the elderly, with a few cases elsewhere. Transfer of patients appeared to be the route of transmission between wards. There was a significant fall in the incidence of cases following intervention by the Infection Control Unit. This included ward inspection, advice on antibiotic usage and advice on prevention of faecal-oral transfer, particularly by proper handwashing. Subsequent monitoring of C. difficile infection showed a background of sporadic, dissimilar isolates with occasional apparent cross-infection incidents limited to a few patients. In suspected outbreaks, patterns of antibiotic susceptibility may be useful in initial screening, before referral for more sophisticated typing. There was excellent correlation between PMS results, antibiograms and epidemiological information.

Aged

Classification of black-pigmented anaerobes by pyrolysis mass spectrometry (PMS) and conventional tests (CTs).

Clinical (66: dental 53; vaginal 4; wound 9) and reference (5*) strains of pigmented Gram-negative anaerobic bacilli were examined in pyrolysis mass spectrometry (PMS) and conventional tests (CTs). The strains were identified in CTs as: Prevotella intermedia (48*); Pr. melaninogenica (1); Pr. corporis (7); Porphyromonas asaccharolytica (12*); P. endodontalis (1*) and P. gingivalis (2*). Numerical classification based on CTs resolved five clusters comprising strains identified as (I) Pr. corporis, (II) Pr. melaninogenica, (III) Pr. intermedia, (IV) P. gingivalis and (V) P. asaccharolytica and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity in the order Pr. intermedia, Pr. corporis, P. asaccharolytica, in agreement with the ordering of homogeneity for these species in CTs. PMS clusters corresponding the Porphyromonas spp. were clearly distinct from those of Prevotella spp. PMS and CT classifications disagreed on cluster membership for only six of the strains. PMS identification from blind challenge sets agreed with conventional identification for 64 of 67 strains.

Bacterial Typing Techniques

A numerical taxonomic study of the "Streptococcus milleri" group based upon conventional phenotypic tests and pyrolysis mass spectrometry.

Clinical strains presumptively identified as Streptococcus milleri (60), and blind coded collection strains (21) were characterised in conventional tests and pyrolysis mass spectrometry. Comparison of the clusters found by these two approaches revealed five clearly distinct centres of variation. Three corresponded to the DNA homology groups suggested by Whiley and Hardie (1989) as representing the species S. anginosus, S. intermedius and S. constellatus; a fourth comprised three Lancefield group C beta-haemolytic strains; the fifth may represent a biotype of S. anginosus. The characteristics of the latter group are described.

Cluster Analysis

Classification of oral pigmented anaerobic bacilli by pyrolysis mass spectrometry and biochemical tests.

Clinical (66) and reference (5) strains of pigmented gram-negative anaerobic bacilli, identified as Prevotella intermedia (47), Pr. melaninogenica (1), Pr. corpora (8), Porphyromonas asaccharolyticus (12), P. endodontalis (1) and P. gingivalis (2), were examined by pyrolysis mass spectrometry (PMS) and in conventional tests. Numerical classification based on conventional test reaction patterns (CTRPs) resolved five clusters, four comprising strains identified as Pr. intermedia, Pr. corpora, Pr. melaninogenica, and P. gingivalis respectively, and one comprising strains identified as P. asaccharolyticus and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity of chemical composition in the order Pr. intermedia, Pr. corpora, P. asaccharolyticus, which agreed with the order of homogeneity in CTRPs. PMS clusters corresponding to the genus Porphyromonas were clearly distinct from those of the genus Prevotella. PMS and CTRP classification disagreed on cluster membership for six strains. PMS identification from blind challenge sets was in agreement with conventional identification for 64 of 67 strains.

Cluster Analysis

A pyrolysis mass spectrometry study of the non-pigmented Prevotella species.

Collection strains (21) and non-pigmented clinical isolates (96) provisionally identified as Prevotella spp. were classified numerically on the basis of pyrolysis mass spectrometry (PMS) data and reaction patterns in conventional tests (CTRPs) for volatile and non-volatile fatty acids, pre-formed enzymes and biochemical activity. PMS and CTRP classifications were compared with a previous classification based on visual analysis of SDS-PAGE patterns. Although the order of clusters differed, cross-tabulation of cluster membership revealed strong correlations between classifications. Cluster membership in the PMS classification correlated particularly well with SDS-PAGE results. CTRP clusters corresponded largely to the recognised species of Prevotella, but PMS and SDS-PAGE divided two species into sub-groups: two in P. buccae and five in P. veroralis. The latter subgroups could be discriminated by small but consistent differences in CTRPs. An undesignated, well differentiated cluster of strains appeared closest to the main group of P. buccae strains in PMS and CTRPs. B. (P.) capillus could not be distinguished from P. buccae; these species are regarded as synonymous. Strains of P. zoogleoformans and B. (P.) pentosaceus were well separated from other strains in PMS. A complex comprising clusters of P. disiens, P. oralis, P. veroralis, P. loescheii and a further undesignated group similar to P. melaninogenica was well differentiated from P. buccae and P. oris in PMS; clusters corresponding to P. bivia, P. corporis, P. intermedia and P. denticola formed another complex.

Bacterial Typing Techniques

Typing of Streptococcus pyogenes by pyrolysis mass spectrometry.

Strains of Streptococcus pyogenes from an outbreak in an oncology ward (13) and routine isolates from sporadic cases (6) were examined blind by pyrolysis mass spectrometry (Py-MS), extending previous work on epidemiological typing. This outbreak appeared more complex than one reported previously, but Py-MS and conventional typing results were in complete agreement. The results confirm the potential of Py-MS as a rapid method for identification at strain level in studies of cross infection.

Bacterial Typing Techniques

Micrococcus and Stomatococcus spp. from human infections.

Infections with Micrococcus spp. in six patients and Stomatococcus mucilaginosus in one patient are described. Two of the Micrococcus infections occurred in leukaemic patients with indwelling lines, six episodes occurred in three patients undergoing continuous ambulatory peritoneal dialysis and one occurred in a patient with a ventriculo-peritoneal shunt. Stomatococcus was isolated from fluid draining from a sub-dural haematoma. Colony morphology, oxidase reaction and resistance to nitrofuration were useful in differentiation of micrococci from staphylococci. Incidence and risk factors for micrococcal infections appear similar to those for infections with coagulase-negative staphylococci (CNS), but the literature is confused due to changes in the definitions of these genera.

Adolescent

A three year survey of clinical isolates in the United Kingdom and their antimicrobial susceptibility.

A United Kingdom national survey of clinical isolates and their antimicrobial susceptibility was performed, with 61 participating hospital laboratories, between 1986 and 1989. Each centre used Microbe Base, a commercial suite of micro-computer programs which can record and analyse antimicrobial susceptibility data Informative on 366,853 bacterial isolates and their antimicrobial susceptibility was received from hospital and domiciliary specimens; Candida spp. accounted for a further 9121 isolates. The sites of origin were urine 51%, skin and soft tissue 21%, lower respiratory tract 8%, genital tract 7%, ear, nose and throat 6%, eye 3%, blood 1.5% and faeces 1%. Gram-negative bacteria accounted for 242,307 isolates, the main species were Escherichia coli 48%, Proteus spp. 9%, Pseudomonas spp. 7%, Haemophilus influenzae 6% and Klebsiella spp. 4%. Gram-positive bacteria numbered 124,546 with a predominance of Staphylococcus aureus 42%, beta-haemolytic streptococci 20%, Enterococcus spp. 12%, coagulase negative staphylococci 10% and Streptococcus pneumoniae 5%. All pneumococci were sensitive to penicillin, and methicillin resistant in Staph. aureus was only 2%. Twelve per cent of H. influenzae strains were resistant to ampicillin. There were no significant levels of gentamicin resistance in Gram-negative bacilli.

Drug Resistance, Microbial

A pyrolysis-mass spectrometry study of Corynebacterium spp.

Clinical (66) and collection (38) strains of Corynebacterium spp., including C. jeikeium and CDC group D2, and of Listeria monocytogenes were examined. Conventional characters used in species identification were assessed by a microbiochemical method, and pyrolysis-mass spectrometry (Py-MS) was performed with a Horizon Instruments PYMS 200X. Classification based on Py-MS data yielded clusters that corresponded with species identification and classification groups from conventional data. One small group of clinical strains, homogeneous in conventional tests and Py-MS, comprised isolates from sputum samples from patients undergoing ventilation; they were similar to collection strains of C. renale and C. striatum; the latter species has been implicated in chest infection. Another group, similar to C. minutissimum in both systems, comprised clinical strains isolated from urogenital specimens. L. monocytogenes strains were clearly distinct from Corynebacterium spp. Groups comprising CDC D2 strains and C. jeikeium were resolved, and were similar to other Corynebacterium spp. Two collection strains of C. xerosis were distinct in conventional tests and Py-MS.

Bacterial Typing Techniques