Effects of duplicate and screening isolates on surveillance of community and hospital antibiotic resistance.

OBJECTIVES: To investigate common contentions that duplicate and screening isolates consistently show marked excess resistance, and that inclusion of such isolates significantly distorts regional resistance estimates. METHODS: Two Welsh surveys of antibiotic resistance for routine diagnostic isolates were analysed, comprising 309,129 isolates of six common community pathogens and 85,061 ward isolates of 11 common hospital pathogens. Duplicate isolates were defined as isolates from the same patient of the same pathogen with an indistinguishable susceptibility pattern, excluding the initial isolate. Significance was assessed from 95% confidence limits of the difference between resistance estimates. RESULTS: Duplicate isolates comprised approximately 20% of total isolates. For the 195 antibiotic-pathogen combinations investigated, differences in resistance between duplicate and non-duplicate isolates were statistically significant for 93. Only 54 combinations showed significantly increased resistance amongst duplicates, and only 30 of these showed a difference >5%. Comparisons of de-duplicated with un-de-duplicated regional resistance estimates showed significant differences for only 18 of 195 antibiotic-pathogen combinations; none were sufficient to alter judgement on clinical use. Screening isolates produced little disturbance of resistance estimates for Staphylococcus aureus, with the exception of flucloxacillin resistance, where inclusion of screening and duplicate isolates resulted in an increase of 4.4% for both community and hospital resistance estimates. CONCLUSIONS: The contentions were incorrect for these regional surveys. However, the proportion (and so effects) of screening and duplicate isolates may be greater in surveys of units with frequent repetitive sampling practice (burns, ITU, cystic fibrosis), or pathogens subjected to unusually intensive infection control sampling.

Pyrolysis mass spectrometry: data processing in classification studies.

A scheme for numerical processing of pyrolysis mass spectrometry (Py-MS) data is detailed, along with methods for combining the results of conventional phenotypic and Py-MS taxonomic surveys. The importance of this combined approach in polyphasic taxonomy is emphasised; it yields data on cell composition and the nutritional and physiological interactions of strains with their environment. Large collections of strains can be surveyed rapidly and economically, yielding presumptive classifications, which may then be confirmed with a few representative strains in more demanding, difficult and expensive approaches. An objective, non-arbitrary method of establishing suitable cut-off points to delineate group structures in dendrograms is also described.

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.

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.

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.

Identification of staphylococci by pyrolysis gas-liquid chromatography.

The identification of bacteria by pyrolysis gas-liquid chromatography (PGLC) was evaluated with the genus Staphylococcus. A matrix containing the characteristics of seven species of Staphylococcus was constructed from the results obtained with 42 named strains identified by biochemical methods. An identification program based upon this database was evaluated in tests with 100 isolates of catalase-positive oxidase-negative gram-positive cocci identified by biochemical and PGLC systems. Agreement between the two systems was 90%. Pure cultures of unknown isolates were identified within 30 min by PGLC using a microcomputer data acquisition and analysis program. A taxonomic analysis of the data is presented, and methods for quality control of PGLC, prolongation of column life, and data analysis are described and discussed.