The resistance ratchet: theoretical implications of cyclic selection pressure.

OBJECTIVES: To investigate the effects of cyclic antibiotic selection pressure on resistance in a simple mathematical model. METHODS: The model assumed that resistance in microbial ecologies changes slowly with changing selection pressure, at a rate proportional to the difference between the current resistance level and the resistance level that would be in equilibrium with current selection pressure. The maximum rate of increase in resistance during periods of increasing selection was assumed to be greater than the maximum rate of decrease during decreased selection. RESULTS: Under a simulated annual cyclic selection pressure variation of 40%, with maximum resistance rise and fall rates of 10 and 0.5%, respectively, resistance rose above the level expected from the mean selection pressure by small ratchet-like increments. Over 50 simulated years, resistance increased to 62%, rather than the 50% expected from the mean level of selection. Welsh community prescribing for a selection of antibiotics showed a seasonal cyclic variation of 13-45%. CONCLUSIONS: The intuitive assumption that cyclic selective pressure would produce resistance levels commensurate with the mean selection pressure was contradicted; rather resistance drifted towards a level commensurate with maximum selection pressure. If the ratchet effect exists in reality, it may produce unexpected excess resistance, particularly in the community for antibiotics used in respiratory infection, where cycling is pronounced, or in ITU antibiotic rotation. It should be most pronounced for resistance systems with strong asymmetry between rates of adaptation under rising and falling selection pressure. Non-linear dynamic systems in physics and ecology are notorious for producing counter-intuitive effects; resistance epidemiology may be similar.

Finding a strategy: the case for co-operative research on resistance epidemiology.

Progress on rational intervention to prevent increasing antibiotic resistance has been slow. We suggest that this is because the science of resistance epidemiology has received little attention, and that a systematic, co-operative investigation of this area might yield a relevant knowledge base, analogous to the basis for effective public health intervention in infectious disease given by infection epidemiology. The steps required to progress this approach in the UK are discussed, along with a summary of what is known and speculation on what might emerge.

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.

Laboratory testing policies and their effects on routine surveillance of community antimicrobial resistance.

OBJECTIVE: To investigate the effects of laboratory testing policies, particularly selective testing, rule-based reporting and isolate identification, on estimates of community antimicrobial resistance. MATERIALS AND METHODS: Antibiotic resistance estimates were analysed from an all-Wales dataset for approximately 300 000 community isolates of common pathogens. RESULTS: Selective testing policies were often associated with markedly increased resistance, particularly for second-line testing. Site-specific testing tended to yield variant resistance estimates for eye and ear isolates. Estimates from rule-based reporting deviated markedly from test-result-based reporting. Urinary isolates reported as Escherichia coli showed greater susceptibility than those reported as undifferentiated urinary 'coliforms'. The proportion of isolates tested for an antibiotic by a laboratory was a useful indicator of selective testing in this dataset. Selective testing policies had invariably been applied where the proportion of isolates of a species tested against an antibiotic was <90%. As this proportion fell with increasingly selective policies, divergence from pooled-all-Wales non-selective estimates tended to increase, with a bias to increased resistance. CONCLUSIONS: Selective testing, rule-based reporting and urinary coliform identification policies all had significant effects upon resistance estimates. Triage based upon the proportion of isolates tested seemed a useful tool in assigning analysis resources. Where <20% of isolates were tested, selective policies with inherent bias to increased resistance were common, the low number of isolates gave high potential sampling errors, and little confidence could be placed in the resistance estimate. Where 20-90% of isolates were tested, detailed analysis sometimes revealed resistance estimates that might be usefully retrieved. Where >/=90% of isolates were tested, there was no evidence of selective testing, and inter-laboratory variation in estimates appeared to be safely ascribable to other effects, e.g. methodology or real variation in resistance levels.

Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.

SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.

Clinical significance of the emergence of bacterial resistance in the hospital environment.

Antibiotic resistance is an increasing threat in hospitals and both morbidity and mortality from infections are greater when caused by drug-resistant organisms. Whilst hospitals are universally blamed for this increase, there is an insufficient appreciation of external sources of resistance, such as when patients are admitted to hospitals from long-term care facilities in the community. The use of antibiotics in family practice and animal husbandry has also been linked to drug resistance being encountered in the hospital setting. Justifiable hospital antibiotic use, which can be life saving, may lead to 'collateral damage' with the emergence of resistance in non-target bacteria in the bowel, for example, with subsequent spread by cross-infection. At a management level, antibiotic resistance can have a significant impact on the ability of hospitals to maintain services since cohorting of patients and ward closures from outbreaks add to continuing bed shortages and waiting lists. Hospital laboratories must review their standard operating procedures since some resistance mechanisms may be missed by current methods of antibiotic susceptibility testing. With increasing public concern from press reports of 'multiresistant Staphylococcus aureus killer virus' and other drug-resistant organisms, there will inevitably be a push by national authorities for more surveillance data on antibiotic resistance; however, the cost-effectiveness of different surveillance strategies should be considered. Clinical governance and risk management are dominant themes in the National Health Service and hospital hygiene and antibiotic resistance are likely to feature prominently in audits related to these themes in the near future.

Clinical significance of the emergence of bacterial resistance in the hospital environment.

Antibiotic resistance is an increasing threat in hospitals and both morbidity and mortality from infections are greater when caused by drug-resistant organisms. Whilst hospitals are universally blamed for this increase, there is an insufficient appreciation of external sources of resistance, such as when patients are admitted to hospitals from long-term care facilities in the community. The use of antibiotics in family practice and animal husbandry has also been linked to drug resistance being encountered in the hospital setting. Justifiable hospital antibiotic use, which can be life saving, may lead to 'collateral damage' with the emergence of resistance in non-target bacteria in the bowel, for example, with subsequent spread by cross-infection. At a management level, antibiotic resistance can have a significant impact on the ability of hospitals to maintain services since cohorting of patients and ward closures from outbreaks add to continuing bed shortages and waiting lists. Hospital laboratories must review their standard operating procedures since some resistance mechanisms may be missed by current methods of antibiotic susceptibility testing. With increasing public concern from press reports of 'multiresistant Staphylococcus aureus killer virus' and other drug-resistant organisms, there will inevitably be a push by national authorities for more surveillance data on antibiotic resistance; however, the cost-effectiveness of different surveillance strategies should be considered. Clinical governance and risk management are dominant themes in the National Health Service and hospital hygiene and antibiotic resistance are likely to feature prominently in audits related to these themes in the near future.

Factors associated with antibiotic resistance in coliform organisms from community urinary tract infection in Wales.

Routine susceptibility data for urinary coliform isolates from community practice were analysed in comparison with dispensed antibiotic prescriptions for all conditions and social deprivation data for Bro Taf and North Wales Health Authorities for financial years 1996--1998. Prescribing rates and resistance rates varied widely between practices. Among isolates from practices with high usage of an antibiotic, rates of resistance to that antibiotic tended to be high, and usage correlated significantly with resistance between practice population units. Cross-correlations were found between usage of one antibiotic and resistance to another, particularly for trimethoprim and ampicillin. Usage, particularly of trimethoprim, was associated with multi-resistance to up to four antibiotics. Resistance was more frequent in isolates from males, children and the elderly. Ampicillin resistance correlated with social deprivation. Analyses including or excluding potential repeat isolates yielded closely similar results. Indices reflecting sampling behaviour (laboratory coliform positivity rates, positivity per 1000 registered patients, specimens submitted per 1000 registered patients) varied widely between surgeries, suggesting lack of consensus on urine sampling policies. These indices showed only weak correlations with usage or resistance. Associations between resistance and usage were compared for isolates from two patient subsets that were likely to differ in their proportions of non-Escherichia coli isolates: female patients aged 16--55 years; and males, children and patients aged >55 years. The latter showed higher base levels of resistance, but the associations of resistance with usage were statistically indistinguishable for the two populations. The results suggest that usage of antibiotics in a practice population may affect the rate of urinary infection caused by resistant coliform organisms in that population.

The diagnosis of ventilator-associated pneumonia using non-bronchoscopic, non-directed lung lavages.

OBJECTIVES: (1)To assess the diagnostic utility of quantitative cultures of non-bronchoscopic lung lavage (NBL) in ventilator-associated pneumonia and evaluate the role of the Bacterial Index; (2) To assess the predictive value of NBL surveillance quantitative cultures in ventilated patients; (3) To evaluate the Clinical Pulmonary Infection Score (CPIS) system in ventilated patients. DESIGN: A prospective comparison of NBL with bronchoscopic bronchoalveolar lavage and protected specimen brush. SETTING: Three intensive care units in academic tertiary care centres. PATIENTS: 145 adults ventilated for 72 h, with and without clinical signs of pneumonia. INTERVENTIONS: Sampling of lower airway secretions by NBL, bronchoalveolar lavage and protected specimen brush. MAIN OUTCOME MEASURES: Diagnostic reliability of quantitative cultures, Bacterial Index and CPIS. RESULTS: 34 episodes of clinical ventilator-associated pneumonia were documented in 32 patients. 9 episodes were confirmed by concordant blood/pleural culture or post-mortem lung examination. Qualitative concordance of the predominant pathogen between sequential NBL: bronchoalveolar lavage and protected specimen brush was 83 %. Sensitivity and specificity of non-directed bronchial lavage at a threshold of 10(4) CFU/ml were 68 % and 70 % respectively (p = 0. 003) and were comparable with the bronchoscopic methods. However, the low positive predictive value of NBL indicates that quantitation in the absence of clinical signs is unlikely to be useful. Bacterial Index did not improve discriminatory power of quantitation compared with bacterial load of predominant organism. Mean CPIS for confirmed pneumonia cases was 8.4 +/- 1.01, significantly higher than for non-pneumonia observations (p = < 0.0001). CONCLUSION: NBL is a simple, safe, cheap, readily-available method of diagnosing ventilator-associated pneumonia with comparable diagnostic accuracy to bronchoscopic techniques. Quantitation of respiratory tract cultures can exclude pneumonia in patients with equivocal clinical signs. The diagnostic threshold should vary depending on the length of ventilation, likelihood of pneumonia and antibiotic administration. The Bacterial Index is a flawed mathematical device that has no contributory role in pneumonia diagnosis. The CPIS has some diagnostic role in selected cohorts of ventilated patients.

Development of amplified 16S ribosomal DNA restriction analysis for identification of Actinomyces species and comparison with pyrolysis-mass spectrometry and conventional biochemical tests.

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin.

Proposed new bacterial taxa and proposed changes of bacterial names published during 1997 and considered to be of interest to medical or veterinary bacteriology.

A list of names of bacteria published or validated in 1997 is presented. Comments are made of the tendency to base names of new taxa on a single bacterial strain and the consequences for reliable descriptions that this tendency implies.

PCR-ribotyping and pyrolysis mass spectrometry fingerprinting of environmental and hospital isolates of Clostridium difficile.

The relationships between environmental isolates of Clostridium difficile were examined by two typing methods, PCR ribotyping and pyrolysis mass spectrometry (PyMS). The 184 isolates were divided into 23 different PCR ribotypes, 13 of which were producers of toxins A and B; the remaining 10 types did not produce either toxin A or B. PyMS analysis resolved 31 groups with 60 (32.5%) isolates in one group (group 9). In both methods most of the isolates showed similar clustering. PCR ribotypes of the environmental isolates were compared with those of clinical isolates that had been typed previously. Seventeen PCR types (13 toxigenic PCR types and four non-toxigenic types) were found in both sets of isolates.

A comparative study of Fusobacterium necrophorum strains from human and animal sources by phenotypic reactions, pyrolysis mass spectrometry and SDS-PAGE.

Fusobacterium necrophorum strains from human infection (21) were compared with strains from animals (17 biotype A, 2 biotype AB, 4 biotype B, 1 biotype unknown), and the type strain NCTC 10575 in conventional tests reaction patterns (CTRPs), SDS-PAGE and pyrolysis mass spectrometry (PMS). Classifications from the three approaches showed one major consensus group comprising all human strains, and another comprising animal biotype A strains. Animal biotype B strains and one animal strain, designated with some doubt to biotype A, were outliers of the consensus 'human strain' group. Again, animal biotype AB strains were outliers of the consensus 'animal biotype A group', as was the type strain, which was clearly atypical in conventional tests and PMS. Colonial and microscopic characters showed good discrimination between the major consensus groups. However, only haemagglutination and the API-ZYM leucine arylamidase of the biochemical tests discriminated well between these groups. The 'animal biotype A group' clearly corresponds to F. necrophorum subsp. necrophorum, but synonymy of F. necrophorum subsp. funduliforme with the group of human strains was less certain. The latter subspecies was described solely on the basis of animal strains, all of biotype B, but each of four animal biotype B strains in this study was an outlier of the 'human strain group' in one or more of the characterisation approaches. Strains of F. necrophorum causing human infection were clearly distinct from the biotype A strains commonly found in animal infection. This has implications for the validity of animal models of human necrobacillosis. In view of these differences, it would be useful to have a validated designation for strains causing human infection. However, it would be premature to assume that the definition of F. necrophorum subsp. funduliforme encompasses the human strains in the absence of confirmatory DNA-homology and 16S rRNA-sequencing studies.

Characterization of smooth and rough morphotypes of Peptostreptococcus micros.

Isolation of the smooth (Sm) morphotype of Peptostreptococcus micros, a suspected oral pathogen, is sometimes accompanied by isolation of a rough (Rg) morphotype of P. micros. The Rg type readily changes to a Sm-like variant (RgSm) in broth culture. Sm and Rg isolates and RgSm variants were compared to determine whether these three types are the result of phase variation. The RgSm variants resembled the Sm morphotype in colony morphology; furthermore, the Sm type and the RgSm type did not have the fibrillar surface structures characteristic of the Rg type, and the Sm and RgSm types were more hydrophobic than the Rg type. However, when we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell proteins, serotyping data, pyrolysis mass spectrometry data, 16S ribosomal DNA sequences, and hemolytic activities, the RgSm variants and the Rg isolates were very similar and were clearly distinct from the Sm isolates. These results suggest that the Rg and RgSm types form a cluster distinct from the Sm type and thus provide evidence that P. micros can be differentiated into two groups, one consisting of the Sm type and the other consisting of the Rg and RgSm types.

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.

A numerical taxonomic study of Streptococcus sanguis, S. mitis and similar organisms using conventional tests and pyrolysis mass spectrometry.

Numerical taxonomic methods were applied to the results of pyrolysis mass spectrometry (Py-MS) and conventional test reaction patterns (CTRPs) obtained with 46 strains of oral streptococci identified as representative of six species. Of these: Streptococcus gordonii strains formed a single homogeneous well-differentiated cluster both in the Py-MS and CTRP analyses; S. oralis strains were more diverse, forming 2 CTRP and 3 Py-MS clusters; S. crista, S. parasanguis and S. sanguis formed clear, but diverse and poorly differentiated clusters on the basis of CTRPs, and were poorly differentiated in the Py-MS analysis; and S. mitis strains were clearly diverse in both analyses. Peritrichously fimbriate ('tufted') S. mitis strains formed a tight cluster, well differentiated from the type strain in both Py-MS and CTRP analyses, and may represent a previously unrecognised centre of variation.

Epidemiological typing of Klebsiella pneumoniae by pyrolysis mass spectrometry.

Thirteen isolates of ceftazidime-resistant Klebsiella pneumoniae from a suspected cross-infection outbreak involving patients on an intensive care unit and a haematology ward were examined in pyrolysis-mass spectrometry (Py-MS), along with eight concurrent non-outbreak-associated clinical isolates of klebsiellae as controls. Py-MS showed tight clustering of the suspected outbreak isolates, suggesting cross-infection with a single strain. Non-outbreak isolates were clearly distinct from one another and from the outbreak strain. The results confirm that Py-MS is a powerful tool for rapid strain comparison in investigations of cross-infection incidents.