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To harness the work already done by the community frailty team in establishing advanced care plans for frail care home residents. Identifying patients with these plans at the front door to ensure clinical teams can be best placed to deliver care which is tailored to patients’ own wishes. East Sussex Healthcare NHS Trust has a large elderly …

Background Aseptic loosening and periprosthetic osteolysis are frequent complications in total hip arthroplasty requiring revision surgery. Highly cross-linked polyethylene (HXLPE) implants have improved wear resistance, permitting larger femoral heads. However, such implants may experience surface cracking, mechanical failure, and oxidative damage. Vitamin E-infused HXLPE (VEPE) implants were therefore developed to reduce oxidation without compromising mechanical strength. We addressed the following questions: (1) Does femoral head size affect the midterm annual polyethylene wear rates of VEPE acetabular cups? (2) Does femoral head size affect the midterm migration rates of VEPE acetabular cups? (3) Are clinical outcomes affected by femoral head size? Hypothesis Annual wear rate, migration rate, and clinical outcomes of VEPE acetabular cups are independent of femoral head size. Patients and methods This was a prospective, multicentre, observational study of patients that underwent total hip arthroplasty. Hips were grouped according to the size of femoral head implanted (28 mm, 32 mm, and 36 mm). We determined annual wear rate and migration rate of VEPE acetabular cups using the Einzel-Bild-Rontgen-Analyse software. Clinically, we assessed the Harris Hip Score and visual analog score for pain and satisfaction. Results We followed 253 patients (267 hips) for a mean of 55.0 ± 20.6 months in the 28 mm, 46.2 ± 21.4 months in the 32 mm, and 43.8 ± 22.6 months in the 36 mm group. The annual wear rate was 0.025 mm per year from 1 year to the last follow-up and remained similar between the groups (p > 0.05). Also, mean two-dimensional migration rates did not exceed 0.05 mm from 2 years to the last follow-up and remained similar between the groups (p = 0.355). Finally, clinical outcomes also did not differ between the groups (p > 0.05). Two patients required revision surgery. Discussion Femoral head size did not influence midterm annual wear rate, migration rate, and clinical outcomes of VEPE acetabular cups. Furthermore, wear and cup migration rates were below the reported values leading to osteolysis and aseptic loosening. Nevertheless, studies with extended follow-up periods will be necessary to confirm these results in the long term. Level of evidence IV.

Introduction: In the developed world,Clostridium difficile infection (CDI) is the mostimportant cause of nosocomial infectiousdiarrhea. In addition to providingepidemiological data and helping to indicatethat a local outbreak may be occurring,laboratory tests are used to augment clinicaldecisions on individual patients. Very rarely dodiagnostic tests provide results at the point of decision making; in the intervening periodbetween requesting investigations on a patientwith suspected CDI and return of the laboratoryresult, decisions must be made regardingpatient isolation and treatment.Methods: A 22-month, real-world feasibilitystudy was conducted in patients with clinicallysignificant diarrhea, in a London Hospitalbetween March 2011 and January 2013, inthree older persons’ wards and two intensivecare units (ICUs) to determine acceptability,ease of use, change in turnaround time andclinical utility of a rapid, polymerase chainreaction (PCR)-based point-of-care test (POCT)(Cepheid GeneXpert, Sunnyvale, California,USA) for diagnosis of Clostridium difficile. Nursesin the older persons’ ward and laboratorytechnicians in the ICU were trained toperform the test. Residual samples were sent tothe centralized laboratory for parallel testingusing a two-step algorithm.Results: A total of 335 samples were tested usingthe POCT with a median turnaround time of1.85 h compared with 18 h for the centralizedlaboratory test. Overall agreement withcentralized laboratory testing was 98.1%.Discrepant samples were more frequent on elderly wards than ICU. Overall 20/335 (6%)processing errors were encountered and werehighest in the first few months of the study.Significantly more processing errors occurred onthe older persons’ wards 13/102 (12.7%) than onICU 7/271 (2.6%). Older persons’ patients whohad POCT were significantly less likely to have atest requested for bacterial stool culture (3.1% vs.10.9% p = 0.044). This difference was notobserved in the ICU patients. No otherdifferences in ancillary test requesting,mortality or length of stay were observed.Conclusions: The majority of users reportedthat the POCT was easy to perform and was anacceptable part of their job. POCT using thissystem is feasible and acceptable to nursing staffand technicians working within these twohospital-based settings.

We describe the identification and control of an outbreak of gentamicin resistant, meticillin susceptible Staphylococcus aureus (GR-MSSA) on a 36-bed neonatal unit (NNU) in London. Control measures included admission and weekly screening for GR-MSSA, cohorting affected babies, environmental and staff screening, hydrogen peroxide vapour (HPV) for terminal disinfection of cohort rooms, and reinforcement of hand hygiene. Seventeen babies were affected by the outbreak strain over ten months; seven were infected and ten were asymptomatic carriers. The outbreak strain was gentamicin resistant and all isolates were indistinguishable by pulsed-field gel electrophoresis. The outbreak strains spread rapidly and were associated with a high rate of bacteraemia (35% of 17 affected patients had bacteraemia vs. 10% of 284 patients with MSSA prior to the outbreak, p=0.007). None of 113 staff members tested were colonised with GR-MSSA. GR-MSSA was recovered from 11.5% of 87 environmental surfaces in cohort rooms, 7.1% of 28 communal surfaces and 4.1% of 74 surfaces after conventional terminal disinfection. None of 64 surfaces sampled after HPV decontamination yielded GR-MSSA. Recovery of GR-MSSA from two high level sites suggested that the organism could have been transmitted via air. Occasional breakdown in hand hygiene compliance and contaminated environmental surfaces probably contributed to transmission.

Summary Background Meticillin-resistant Staphylococcus aureus (MRSA) is frequently endemic in healthcare settings and may be transmitted by person-to-person spread. Asymptomatic MRSA carriers are potential, unsuspected sources for transmission and some of them may be identified by admission screening. Aim To assess whether rapid point-of-care screening (POCS) for MRSA at hospital admission may be associated with a reduction in MRSA acquisition rates when compared with slower laboratory-based methods. Methods A cluster-randomized cross-over trial was conducted in four admission wards of an acute London tertiary care hospital. Polymerase chain reaction-based POCS screening was compared with conventional culture screening. Patients were screened on ward admission and discharge, and the MRSA acquisition rate on the admission wards was calculated as the primary outcome measure. Results In all, 10,017 patients were included; 4978 in the control arm, 5039 in the POCS arm. The MRSA carriage rate on admission was 1.7%. POCS reduced the median reporting time from 40.4 to 3.7 h (P

Infection control for hospital pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) often takes the form of a package of interventions, including the use of patient isolation and decolonization treatment. Such interventions, though widely used, have generated controversy because of their significant resource implications and the lack of robust evidence with regard to their effectiveness at reducing transmission. The aim of this study was to estimate the effectiveness of isolation and decolonization measures in reducing MRSA transmission in hospital general wards. Prospectively collected MRSA surveillance data from 10 general wards at Guy's and St. Thomas' hospitals, London, United Kingdom, in 2006-2007 were used, comprising 14,035 patient episodes. Data were analyzed with a Markov chain Monte Carlo algorithm to model transmission dynamics. The combined effect of isolation and decolonization was estimated to reduce transmission by 64% (95% confidence interval: 37, 79). Undetected MRSA-positive patients were estimated to be the source of 75% (95% confidence interval: 67, 86) of total transmission events. Isolation measures combined with decolonization treatment were strongly associated with a reduction in MRSA transmission in hospital general wards. These findings provide support for active methods of MRSA control, but further research is needed to determine the relative importance of isolation and decolonization in preventing transmission.

Studies in the 1970s and 1980s suggested that environmental surface contamination played a negligible role in the endemic transmission of healthcare-associated infections. However, recent studies have demonstrated that several major nosocomial pathogens are shed by patients and contaminate hospital surfaces at concentrations sufficient for transmission, survive for extended periods, persist despite attempts to disinfect or remove them, and can be transferred to the hands of healthcare workers. Evidence is accumulating that contaminated surfaces make an important contribution to the epidemic and endemic transmission ofClostridium difficile,vancomycin-resistant enterococci, methicillin-resistantStaphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa,and norovirus and that improved environmental decontamination contributes to the control of outbreaks. Efforts to improve environmental hygiene should include enhancing the efficacy of cleaning and disinfection and reducing the shedding of pathogens. Further high-quality studies are needed to clarify the role played by surfaces in nosocomial transmission and to determine the effectiveness of different interventions in reducing associated infection rates.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) often produce Panton–Valentine leukocin (PVL), which is encoded by two co-transcribed genes located on lysogenized bacteriophages. Six PVL-encoding temperate phages have been described and single nucleotide polymorphisms (SNPs) in the PVL genes have been reported. In the present study, 22 PVL-positive CA-MRSA isolates were chosen to reflect the diversity of multilocus sequence type (MLST) clonal complexes (CC) identified in our hospital. Isolates were characterized by antimicrobial resistance profile, staphylococcal cassette chromosome mec (SCCmec) and spa type, pulsed-field gel electrophoresis profile and MLST. Primers were designed to sequence the lukSF-PV genes. PVL-encoding phages were characterized using a PCR-based assay. SNPs were identified at seven locations in the lukSF-PV genes, which varied with S. aureus MLST lineage. One SNP was nonsynonymous. All CC80 and some CC1 isolates carried FSa2mw; CC8, CC88 and CC154 isolates harboured PVL-encoding elongated head-type phages; and some CC59 isolates harboured a FSa2958-like phage. Novel or variant phages were present in CC5 and some CC1 and CC59 isolates. The PVL gene sequence and the PVL-encoding phage varied with lineage. Further work is required to determine whether PVL sequence and/or phage variations result in biological differences.

The clinical utility of real-time PCR screening assays for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay's positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C−) assays. P+C− results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C− results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C− patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.

Despite increasing antimicrobial resistance and multiple drug resistance in clinical isolates of both Gram-positive and Gram-negative bacteria, there are few novel antimicrobial agents in development. The few new agents that have been recently licensed have tended to have narrow spectra of activity, focused on Gram-positive pathogens, especially methicillin-resistant Staphylococcus aureus (MRSA). This situation is rightly causing concern among clinicians and public health authorities worldwide. This article reviews available data on three new antibacterials currently in development. The cephalosporin ceftobiprole is active against MRSA, Enterococcus faecalis and penicillin-resistant Streptococcus pneumoniae, but otherwise has a spectrum of activity similar to that of other recent cephalosporins. In a clinical trial, ceftobiprole was non-inferior to vancomycin for the treatment of MRSA-associated complicated skin and skin structure infections (cSSSIs). Doripenem, a new carbapenem, has some activity against MRSA, but otherwise has an anti-Gram-positive spectrum of activity similar to that of imipenem and an anti-Gram-negative spectrum similar to that of meropenem. In a clinical trial, it was non-inferior to meropenem for the treatment of complicated intra-abdominal infections. Iclaprim is a dihydrofolate reductase inhibitor with greatly enhanced activity, as compared with trimethoprim, against a range of Gram-positive and Gram-negative pathogens. The limited literature concerning this agent has concentrated on its potential role in the treatment of infections with Gram-positive bacteria. A clinical trial has demonstrated the non-inferiority of iclaprim, as compared with linezolid, in the treatment of cSSSIs, including those associated with MRSA.

Over the last decade, methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as serious pathogens. These strains are often multiresistant to several antibiotic classes and are a major cause of serious hospital- and now community-acquired infections and associated morbidity and mortality. As a result of increasing antimicrobial resistance, glycopeptides, such as vancomycin, are widely used as first-line therapy for serious MRSA infections. However, the emergence of glycopeptide tolerance and resistance has complicated treatment and there remains a clinical need for new antibiotics with suitable pharmacokinetic properties with activity against MRSA and other gram-positive pathogens. Infections caused by MRSA and other bacteria usually respond as well to bacteriostatic agents as to bactericidal ones. Nevertheless, there is evidence that rapid bacterial killing has potential clinical advantages over bacteriostatic therapy in certain infections. Daptomycin, the first of the cyclic lipopeptides, shows rapid bactericidal activity against S. aureus, including strains tolerant or resistant to other agents. This review outlines the methods by which bactericidal and bacteriostatic properties are defined and tested, discusses the potential importance of bactericidal therapy in MRSA and other infections and examines the potential role of daptomycin in treatment.

Background: Acromegalic patients have slow colonic transit, increased rates of deoxycholic acid formation, and an increased prevalence of cholesterol gall stones, especially during long term octreotide treatment. However, the effects of this prolonged large bowel transit time on the numbers of faecal anaerobes and the activities of the enzyme systems which biotransform conjugated cholic acid into unconjugated deoxycholic acid (cholylglycine hydrolase and 7α-dehydroxylase) are unknown. Methods: Therefore, in 10 non-acromegalic controls, 11 acromegalic patients not treated with octreotide, and 11 acromegalics on long term (8–48 months) octreotide (100–200 μg three times daily subcutaneously), we measured large bowel transit time and, in freshly voided faeces, the activities of the two bile acid metabolising enzymes, and related the results to the proportion of deoxycholic acid in fasting serum. Moreover, in patients with acromegaly, we measured quantitative bacteriology in faeces. Results: Mean large bowel transit time in acromegalics not treated with octreotide (35 (SEM 6.5) hours) was 66% longer than that in non-acromegalic controls (21 (3.1) hours; NS) and became further prolonged during octreotide treatment (48 (6.6) hours; p

Objectives: To investigate the changes in resistance frequencies of common Gram-negative bacteria in a London teaching hospital. Methods: Antimicrobial susceptibilities were analysed for the 6 years 1995-2000. Gentamicin-resistant isolates from 1995 and 2000 were typed by a repetitive element sequence-based PCR (Rep-PCR) method. Results: Resistance rates for all agents and all organisms were higher in isolates from inpatients than in those from outpatients or general practice. For most agents and most species there was a trend for a highly significant linear increase in resistance over the study period, and there was significant cross-resistance between different agents. Increases in resistance were especially marked in Klebsiella, Enterobacter and Acinetobacter spp., organisms that tend to cause outbreaks of hospital cross-infection. For example, the increases in gentamicin resistance in isolates from inpatients was from 2.9% to 23.5% for Klebsiella spp., from 0.3% to 20.8% for Enterobacter spp. and from 10.1% to 42.2% for Acinetobacter spp. There was much less increase in acquired resistance in Escherichia coli and Pseudomonas aeruginosa, organisms that tend to cause endogenous infections, with gentamicin resistance in isolates from inpatients increasing from 0.4% to 3.2% for E. coli and decreasing from 4.6% to 3.6% for P. aeruginosa. Rep-PCR typing showed consid- erable diversity amongst gentamicin-resistant isolates of E. coli and P. aeruginosa, but dominance by a limited number of presumably epidemic types of gentamicin-resistant isolates of the other species. Conclusions: Multiple antibiotic resistance has increased dramatically in some hospital isolates, and appears to be associated with hospital cross-infection.

Clinical trials have shown that linezolid (600 mg twice daily in adults) is safe and generally well tolerated for up to 28 days. Drug-related adverse events, which are typically mild to moderate in intensity and of limited duration, include diarrhoea, nausea and headache in adults, and diar- rhoea, loose stools and vomiting in children. Clostridium difficile-related complications with linezolid are uncommon. Linezolid is a weak, reversible monoamine oxidase inhibitor: foods containing high concentrations of tyramine should be avoided, and linezolid should be used with caution in patients taking adrenergic or serotonergic agents or in those with uncontrolled hypertension. In the majority of patients, linezolid has minimal adverse effects on blood chem- istry or haematology. There have been case reports of reversible thrombocytopenia, anaemia and neutropenia associated with linezolid therapy. In Phase III studies, 2.4% of patients treated with linezolid and 1.5% of patients treated with comparator drugs developed reversible thrombo- cytopenia (P = 0.066), but there was no evidence of an increased risk of agranulocytosis, aplastic anaemia or other irreversible blood dyscrasias. Reduced platelet counts were associ- ated with linezolid treatment for ≥2 weeks; complete blood counts should be monitored weekly in patients receiving linezolid for more than 14 days and treatment should be discontinued if there is evidence of myelosuppression.

Since the mid-1980s the number of cases of TB notified within the U.K. has continued to rise although the contribution of HIV to this rise remains unclear. A 12-month prospective cohort study was conducted at chest and HIV clinics in four hospitals in Lambeth, Southwark and Lewisham (LSL), an area of South London, to determine the proportion of patients with culture-proven TB infected with HIV. Secondary aims were to determine the proportion of patients with TB and undiagnosed HIV at first presentation to chest clinics, to determine the proportion of patients presenting with TB as an AIDS defining illness (ADI) and to identify risk factors for co-infection with TB and HIV. In chest clinics, demographic data and left-over blood from patients aged 16 or over with culture-proven TB was collected, anonymised and HIV tested. In HIV clinics, demographic data on patients with TB already known to be HIV seropositive were also obtained. Twenty-one patients (13%, 95% CI—8–19%) of 159 with culture-proven TB were infected with HIV. Four (3%) of 133 patients at first presentation to chest clinics had undiagnosed HIV; two were subsequently diagnosed. Of the 21 patients with TB and HIV, nine (43%) presented with TB as an ADI. Patients with TB and HIV were significantly more likely to be aged between 35 and 55 years compared to HIV seronegative patients [12/21 (57%) vs. 38/138 (28%), P =0.006]. None of the patients from the Indian Subcontinent were HIV seropositive [0/21 vs. 25/138 (18%), P =0.047]. At the present time, universal HIV testing of patients with culture-proven TB in chest clinics within the U.K. is unlikely to significantly reduce the number of patients with undiagnosed HIV.

Objective: A recently proposed guideline from the NCCLS recommends that results only from the first isolate of a species per patient be used in calculation of percentage susceptibilities to antimicrobial agents. Because this is apparently based on the comparison of various calculation methods for results for oxacillin against a fairly small number of isolates of Staphylococcus aureus, we have applied these methods to a wider range of antibiotic/organism combinations. Methods: Antibiotic susceptibility results from our hospital laboratory database were analysed. Rates of antimicrobial susceptibility were calculated using the various criteria proposed by the NCCLS, including exclusion of results from duplicate isolates and surveillance specimens from the calculations. Results and conclusion: Analysis of results for methicillin against S. aureus, gentamicin against Klebsiella spp., vancomycin against enterococci (all in-patient specimens), and amoxicillin and cefuroxime against Escherichia coli (general practice specimens) confirm that, if duplicates and surveillance specimens are excluded, results obtained with the various patient- and episode- based methods for the calculation of percentage susceptibility are very similar. Because of its simplicity and unambiguity, we agree with the suggestion of the NCCLS group that results for the 'first isolate of a given species per patient per analysis period, irrespective of body site, antimicrobial susceptibility profile or other phenotypic characteristics' should be used in the calculation of susceptibility frequencies.

To investigate the effect of screening specimens and different criteria for exclusion of duplicate isolates when surveillance of antimicrobial resistances is performed.Trends in resistance were analysed for recent isolates of selected organisms from Guy's and St Thomas' Hospitals with the use of various criteria for the exclusion of duplicates, including time since the last isolate and antibiogram pattern, and the effect of excluding screening specimens.There was a significant difference of about 8% in the apparent frequency of methicillin resistance in Staphylococcus aureus in inpatients if the time limit for duplicates was set at 5 rather than 30 days; it was about 10% if a 5 day limit was compared with a 365 day limit. There was also a significant difference, of 6-10%, in apparent resistance frequencies if isolates from screening specimens were excluded. Apparent gentamicin resistance rates in Klebsiella spp. varied between 11% and 28%, and the number of apparent patient isolates of gentamicinresistant organisms varied by up to 35%, depending on the duplicate exclusion criteria chosen. Effects were smaller, though still significant, for vancomycin resistance in Enterococcus spp. There was little effect for amoxicillin or cefuroxime resistance in Escherichia coli isolates from general practitioners, where the proportion of duplicates was small.Improved surveillance of antibiotic resistance is needed. However, care needs to be taken in setting the criteria for classifying isolates as duplicates and in comparing results where these criteria may be different or unknown.

Conventional disinfection methods are limited by reliance on the operator to ensure appropriate selection, formulation, distribution and contact time of the agent. ‘No-touch’ automated room disinfection (NTD) systems remove or reduce reliance on operators and so they have the potential to improve the efficacy of terminal disinfection. The most commonly used systems are hydrogen peroxide vapour (H2O2 vapour), aerosolised hydrogen peroxide (aHP) and ultraviolet (UV) radiation. These systems have important differences in their active agent, delivery mechanism, efficacy, process time and ease of use. The choice of NTD system should be influenced by the intended application, the evidence base for effectiveness, practicalities of implementation and cost constraints.

BACKGROUND—Deoxycholic acid (DCA), implicated in the pathogenesis of gall stones and colorectal cancer, is mainly formed by bacterial deconjugation (cholylglycine hydrolase (CGH)) and 7α-dehydroxylation (7α-dehydroxylase (7α-DH)) of conjugated cholic acid (CA) in the caecum/proximal colon. Despite this, most previous studies of CGH and 7α-DH have been in faeces rather than in caecal contents. In bacteria, CA increases 7α-DH activity by substrate-enzyme induction but little is known about CA concentrations or CA/7α-DH induction in the human colon. AIMS AND METHODS—Therefore, in fresh "faeces", and in caecal aspirates obtained during colonoscopy from 20 patients, we: (i) compared the activities of CGH and 7α-DH, (ii) measured 7α-DH in patients with "low" and "high" percentages of DCA in fasting serum (less than and greater than the median), (iii) studied CA concentrations in the right and left halves of the colon, and examined the relationships between (iv) 7α-DH activity and CA concentration in caecal samples (evidence of substrate-enzyme induction), and (v) 7α-DH and per cent DCA in serum. RESULTS—Although mean CGH activity in the proximal colon (18.3 (SEM 4.40) ×10−2 U/mg protein) was comparable with that in "faeces" (16.0 (4.10) ×10− 2 U/mg protein) , mean 7α-DH in the caecum (8.54 (1.08) ×10-4 U/mg protein) was higher (p

The Osmetech Microbial Analyzer (OMA) is an automated headspace analyzer fitted with a novel detector system consisting of an array of polymer sensors, each of which responds to different volatile organic compounds. The system can be used for screening clinical urine specimens for significant bacteriuria by sampling urine headspace and subjecting the output of the multiple-detector response to principal component analysis. The OMA readily distinguished artificially infected urine samples from sterile controls. The OMA was then used to analyze 534 unselected clinical urine specimens, of which 21.5% had significant bacteriuria (containing >10 5 CFU of bacteria/ml). The sensitivity and specificity of the OMA compared with conventional culture were 83.5 and 87.6%, respectively. The OMA is a promising automated system for the rapid routine screening of urine specimens, and further clinical trials are in progress.

Near instantaneous detection of pathogens from clinical material, combined with simultaneous prediction of their antimicrobial resistance profiles, would revolutionize the impact of microbiology on the management of infection. Array-based assays allow a range of characteristics to be rapidly and simultaneously determined. At present these systems have found their primary role as research tools for the monitoring of mRNA expression in the form of DNA microarrays or 'chips'. As fabrication costs reduce and validated targeted arrays are developed, it is inevitable they will be used for more routine applications. Microfluidics offers the exciting possibility of combining purification, amplification and detection in a single disposable device; microarrays are particularly suitable for use within these systems. Arrays will become an important tool for clinical diagnostics.

Background & Aims: Many patients with cholesterol gallbladder stones (GBS) have a high percentage of deoxycholic acid (DCA) in gallbladder bile (all of which are in the conjugated form), probably as a result of prolonged large bowel transit times (LBTT). However, whether the prolonged LBTT increases DCA formation, solubilization, or absorption (or all 3) is not known. Methods: In 40 subjects (20 with GBS; age range, 24–74 years), we measured LBTT using radiopaque markers, and intestinal luminal pH by radiotelemetry. We also measured quantitative anaerobic bacteriology and the activities of 2 bile acid–metabolizing enzymes in fresh cecal aspirates obtained during clinically indicated unprepared colonoscopy, and related these results to the percentage of DCA in fasting serum measured by gas chromatography–mass spectrometry. Results: Compared with controls, GBS patients had longer LBTT (mean 23.1 ± SEM 2.8 h vs. 36.5 ± 3.3 h; P 9 vs. 5.9 ± 1.5 × 10 9 cfu/mL) and Gram-positive (9.5 ± 3.1 × 10 8 vs. 18.0 ± 4.1 × 10 8 cfu/mL; P −4 U/mg protein) in the cecal aspirates. They also had higher intracolonic pH values ( P P Conclusions: Slow colonic transit (more time), increased Gram-positive anaerobes (more bacteria), and greater 7α-DH activity (more enzyme) favor enhanced DCA formation; transit-induced increases in distal colonic luminal pH favor enhanced DCA solubilization/bioavailability; and increases in LBTT (more time) again favor DCA absorption. GASTROENTEROLOGY 2000;119:806-815

Tea tree oil has recently emerged as an effective topical antimicrobial agent active against a wide range of organisms. Tea tree oil may have a clinical application in both the hospital and community, especially for clearance of methicillin-resistant Staphylococcus aureus (MRSA) carriage or as a hand disinfectant to prevent cross-infection with Gram-positive and Gramnegative epidemic organisms. Our study, based on the time-kill approach, determined the kill rate of tea tree oil against several multidrug-resistant organisms, including MRSA, glycopeptide-resistant enterococci, aminoglycoside-resistant klebsiellae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, and also against sensitive microorganisms. The study was performed with two chemically different tea tree oils. One was a standard oil and the other was Clone 88 extracted from a specially bred tree, which has been selected and bred for increased activity and decreased skin irritation. Our results confirm that the cloned oil had increased antimicrobial activity when compared with the standard oil. Most results indicated that the susceptibility pattern and Gram reaction of the organism did not influence the kill rate. A rapid killing time (less than 60 min) was achieved with both tea tree oils with most isolates, but MRSA was killed more slowly than other organisms.

Aims: To investigate bacterial contamination on hand-touch surfaces in the public transport system and in public areas of a hospital in central London. Methods and Results: Dipslides were used to sample 118 hand-touch surfaces in buses, trains, stations, hotels and public areas of a hospital in central London. Total aerobic counts were determined, and Staphylococcus aureus isolates were identified and characterized. Bacteria were cultured from 112 (95%) of sites at a median concentration of 12 CFU cm−2. Methicillin-susceptible Staph. aureus (MSSA) was cultured from nine (8%) of sites; no sites grew methicillin-resistant Staph. aureus (MRSA). Conclusions: Hand-touch sites in London are frequently contaminated with bacteria and can harbour MSSA, but none of the sites tested were contaminated with MRSA. Significance and Impact of the Study: Hand-touch sites can become contaminated with staphylococci and may be fomites for the transmission of bacteria between humans. Such sites could provide a reservoir for community-associated MRSA (CA-MRSA) in high prevalence areas but were not present in London, a geographical area with a low incidence of CA-MRSA.

Representative isolates from 10 distinct extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae that caused hospital outbreaks in the United Kingdom from 1991 to 1994 were examined for relationships between their enzymes and plasmids. The β-lactamases were identified by a combination of isoelectric focusing and gene sequencing. SHV-2 β-lactamase was produced by isolates from four outbreaks, SHV-5 was involved in three, and SHV-4, TEM-15, and TEM-26 were involved in one outbreak each. All of the extended-spectrum β-lactamases were encoded by self-transmissible plasmids, with sizes ranging from about 70 to 160 kb. No similarities between the restriction digest patterns of the extended-spectrum β-lactamase-encoding plasmids were detected, except to some extent between those that produced TEM-15 and TEM-26. Thus, outbreaks of hospital infection with these organisms in the United Kingdom from 1991 to 1994 involved distinct organisms and resistance plasmids and appeared to be unrelated.

Some isolates of SHV-5 beta-lactamase-producing Klebsiella pneumoniae K2 from a single-strain outbreak of cross-infection produced approximately five-fold more beta-lactamase than others. We investigated three possible genetic mechanisms of this hyperproduction: the presence of a more powerful promoter, an increase in plasmid copy number or an amplification of the gene on a plasmid. No differences between low and high beta-lactamase producers were detected in the promoter region of the SHV-5 beta-lactamase gene, which closely resembled that of SHV-2. SHV-5 beta-lactamase production was encoded on a low copy number plasmid, but DNA-DNA hybridization with an SHV-specific probe detected a higher gene dose in hyperproducers. The beta-lactamase hyperproduction was unstable on repeated subculture, with a reduction of about 75% after 100 generations. Hyperproducing mutants of a low-producing Klebsiella and its Escherichia coli K-12 transconjugants could be selected in vitro at a frequency of 10(-5) to 10(-6) and these variants had an increased SHV-5 beta-lactamase gene copy number on low copy number plasmids. We conclude that hyperproduction of the extended-spectrum beta-lactamase was caused by gene amplification that could be easily lost or gained in vitro. Since the change to hyperproduction occurred at a high frequency and hyperproducers showed increased resistance to many beta-lactams and beta-lactam/beta-lactamase inhibitor combinations, we suspect that these variants may readily be selected in patients during antibiotic therapy.

Among Escherichia coli organisms isolated at St. Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant). Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates. MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder. The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2. Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid. Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2. Three isolates produced inhibitor-resistant TEM-group enzymes. In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34. Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40. The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41. The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.

Vancomycin is first-line therapy for healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSIs) in many countries. It is therefore of concern that some studies have reported increases in MRSA vancomycin minimum inhibitory concentrations (V-MICs) within the susceptible range (≤2 μg/mL) [1, 2], a phenomenon called “MIC creep.” However, this has not been evident in other studies [3, 4], and it remains unclear whether observed increases represent replacement by intrinsically more resistant clones [5] or an increase in the population V-MIC profile within clones [5]. Concerns about V-MIC creep are compounded by reports that bloodstream and other infections caused by MRSA isolates with high V-MICs (1.5–2 μg/mL) are associated with a worse outcome [6–8], which may be only in part explained by vancomycin treatment parameters and the V-MIC. For example, high V-MIC isolates have been associated with less shock [8], a lower inflammatory response [9], and lower pathogenicity in a Galleria mellonella infection model [10]; however, this would suggest lower mortality for high V-MIC isolates, which has indeed been observed in one study [11]. These reports might be explained by low V-MIC strains having an inflammation-inducing phenotype causing acute sepsis, and high V-MIC strains having cell wall changes leading to persistence and immune evasion [9, 12]. Such a proposal is consistent with data emerging from genotypic and phenotypic analysis of MRSA strains with reduced vancomycin susceptibility [13]. Another important consideration is bacterial genotype, which has been linked with differences in population V-MIC profile and clinically significant events such as bacteremia, persistent bacteremia, or the ability to cause hematogenous complications [14–16]. Thus, differences in V-MIC and genotype may both affect disease pathogenesis and clinical outcomes. The aims of this study were 2-fold: to determine whether vancomycin creep has occurred in a large genotyped collection of MRSA bloodstream isolates from a single center and to test the hypothesis that strains with a high V-MIC are associated with an increased rate of hematogenous complications.

Rifampin is the most potent drug used in the treatment of disease due to Mycobacterium kansasii . A 69-bp fragment of rpoB , the gene that encodes the β subunit of the bacterial RNA polymerase, was sequenced and found to be identical in five rifampin-susceptible clinical isolates of M. kansasii . This sequence showed 87% homology with the Mycobacterium tuberculosis gene, with an identical deduced amino acid sequence. In contrast, missense mutations were detected in the same fragment amplified from five rifampin-resistant isolates. A rifampin-resistant strain generated in vitro also harbored an rpoB gene missense mutation that was not present in the parent isolate. All mutations detected (in codons 513, 526, and 531) have previously been described in rifampin-resistant M. tuberculosis isolates. Rifampin MICs determined by E-test were 256 mg/liter for all rifampin-resistant ones. In addition, four of the five rifampin-resistant isolates were also resistant to rifabutin. We have thus shown a strong association between rpoB gene missense mutations and rifampin resistance in M. kansasii . Although our results are derived from a small number of isolates and confirmation with larger numbers would be useful, they strongly suggest that mutations within rpoB form the molecular basis of rifampin resistance in this species.

This meeting focused on infections in humans and animals due to methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing bacteria and Clostridium difficile, and their corresponding treatments. MRSA is predominantly a human pathogen, and molecular typing has revealed that certain clones have spread widely both between humans and from humans to animals. ESBL-producing bacteria, particularly those that express the CTX-M β-lactamases, have been disseminated worldwide. Whilst such strains are usually isolated from humans, some animal isolates also produce CTX-M enzymes. In humans, one clone of CTX-M-producing Escherichia coli, sequence type (ST)131, has been particularly successful. C. difficile, often ribotype 027, commonly colonizes the hospital environment and causes serious infections in humans. In animals, ribotype 078 is more often found, and is an important cause of diarrhoea in piglets. There is a concern that the numbers of MRSA or other antimicrobial-resistant bacteria might increase further when human isolates become established in animals, as this can amplify the numbers of such bacteria by dissemination within animal groups with subsequent spread back to humans. Certain antimicrobials have been implicated in the selection of MRSA, ESBL-producing bacteria and predisposition to infection by C. difficile. Guidelines for treatment and prevention of infections by MRSA, ESBL-producing bacteria and C. difficile were discussed and evidence-based policies were recommended for both humans and animals.

We investigated hypermutability in Klebsiella pneumoniae and its association with ciprofloxacin resistance and mutations in the quinolone resistance-determining region (QRDR). Sixty-four strains of K. pneumoniae isolated in London, UK, between 1995 and 2002 with widely differing ciprofloxacin minimum inhibitory concentrations (MICs) and known gyrA and parC sequences were tested for mutation frequencies by selection with rifampicin. Only three hypermutable (frequency ≥10 −6 ) strains were identified, with ciprofloxacin MICs of 0.25μg/mL, 8μg/mL and 64μg/mL. There was no relationship between hypermutation and the ciprofloxacin MIC or QRDR mutations. Screening selected strains with streptomycin did not reveal any hypermutators, and screening with ciprofloxacin identified only two of the three hypermutators identified by rifampicin. Hypermutation in K. pneumoniae is uncommon and does not contribute to accumulation of QRDR mutations or directly to ciprofloxacin resistance.

The public health burden caused by methicillin-resistant Staphylococcus aureus (MRSA) infections is now widely recognized, and is a cause of public alarm. Effective MRSA risk management in the healthcare system as well as in the community should rely on accurate detection of reservoirs and sources of transmission, as well as on close monitoring of the impact of interventions on disease incidence and bacterial dissemination. MRSA carrier screening and disease surveillance, coupled with molecular typing, are key information tools for integrated MRSA control and individual risk assessment. These tools should be tailored to the distinct needs of local interventions and national prevention programmes. Surveillance schemes should primarily inform local staff and serve as quality assurance about MRSA risk management. New technologies, including the use of selective culture media and real-time PCR assays, allow faster detection of MRSA carriers upon admission or during stay in healthcare institutions. More research is needed to ascertain their cost-effectiveness for MRSA control. Likewise, tremendous progress has been made concerning molecular typing methods, with optimization and standardization of sequence-based technologies offering broad applicability and high throughput. However, no single S. aureus typing method is yet providing fully reliable information within the range of discrimination needed for public health action. Further refinement of genotyping methods and international harmonization of surveillance and typing schemes must be achieved to facilitate global MRSA control.

Sir, I appreciated the recent and very comprehensive guidelines for managing hospital-acquired pneumonia by Masterton et al. Malnutrition can greatly increase pneumonia morbidity and mortality rates in both children and adults. Malnutrition is responsible for an estimated 52% of the 2 million impoverished children who die annually worldwide due to pneumonia. However, the relationship between malnutrition and pneumonia in adults in the developed world is often overlooked. Various studies have reported malnutrition for hospitalized elderly to range from 42% to 91% in the developed world. Several studies have reported that rates of both hospital-acquired pneumonia and nosocomial infections, in general, are significantly higher in malnourished versus well-nourished patients. A study of 578 nursing home elderly reported that rates of both pneumonia infection and antibiotic consumption were significantly higher in residents with low serum zinc levels. Many acutely ill patients require enteral tube feeding, which may present some aspiration risk. A study of 4049 mechanically ventilated patients reported that enteral feeding within 48 h of intubation was associated with a significant reduction in hospital mortality (27.8% versus 34.2%, P 1⁄4 0.0005), even though rates of aspiration pneumonia were increased (12.6% versus 9.5%, P 1⁄4 0.007) when compared with patients who received later enteral feeding. Use of ‘immunonutrition’ enteral formulas containing omega 3 fatty acids and extra levels of glutamine, zinc, copper, selenium and vitamins may reduce the risk of pneumonia among the acutely ill. Meta-analysis of 11 studies reported that the use of such ‘immunonutrition’ formulas was associated with a 46% reduced risk of hospital-acquired pneumonia when compared with standard formulas (P 1⁄4 0.007). A study of 165 mechanically ventilated patients with severe sepsis reported that use of an enteral formula with omega 3 fatty acids and extra vitamins was associated with significantly lower mortality and significantly fewer days on mechanical ventilation when compared with patients on standard formula. Good nutrition can play a major role in preventing and treating hospital-acquired pneumonia. Much more research on the relationship between nutrition and pneumonia should be published and the results incorporated into future pneumonia guidelines.

OBJECTIVE: To determine whether introducing a rapid test for meticillin resistant Staphylococcus aureus (MRSA) screening leads to a reduction in MRSA acquisition on hospital general wards. DESIGN: Cluster randomised crossover trial. SETTING: Medical, surgical, elderly care, and oncology wards of a London teaching hospital on two sites. MAIN OUTCOME MEASURE: MRSA acquisition rate (proportion of patients negative for MRSA who became MRSA positive). PARTICIPANTS: All patients admitted to the study wards who were MRSA negative on admission and screened for MRSA on discharge. INTERVENTION: Rapid polymerase chain reaction based screening test for MRSA compared with conventional culture. RESULTS: Of 9608 patients admitted to study wards, 8374 met entry criteria and 6888 had full data (82.3%); 3335 in the control arm and 3553 in the rapid test arm. The overall MRSA carriage rate on admission was 6.7%. Rapid tests led to a reduction in median reporting time from admission, from 46 to 22 hours (P

These guidelines have been developed by a Working Party convened on behalf of the British Society for Antimicrobial Chemotherapy. Their aim is to provide general practitioners and other community- and hospital-based healthcare professionals with pragmatic advice about when to suspect MRSA infection in the community, when and what cultures should be performed and what should be the management options, including the need for hospitalization.

Since the mid 1980s the number of notified cases of TB in the UK has continued to rise with the largest increases noted in London and inner city areas.1 King George Hospital in Goodmayes, Essex provides clinical services to a population of approximately 230 000; 17% are non-white subjects including immigrants from countries with high rates of M tuberculosis infection and drug resistance. From September 1996 to July 1997 47 adult cases of culture proven TB were identified including seven with drug resistant isolates. None was identified by contact tracing. A previous TB audit of African born patients revealed …

We evaluated toxigenic Clostridium difficile detection by a lateral flow assay for antigen and toxin, an enzyme immunoassay, and two commercial PCR methods. Compared to the cell cytotoxicity neutralization assay and toxigenic culture, both toxin detection methods lacked sensitivity. PCR following combined antigen and toxin detection provided the most useful diagnostic information.

We read with interest the paper “Evaluation of the C.Diff Quik Chek Complete Assay, a New Glutamate Dehydrogenase and A/B Toxin Combination Lateral Flow Assay for Use in Rapid, Simple Diagnosis of Clostridium difficile Disease” by Sharp and colleagues (10). Interest in the laboratory diagnosis of Clostridium difficile has been encouraged by research that has shown the commonly used toxin enzyme immunoassays (EIAs) to be inadequate when used alone (1, 9). We agree with Sharp et al. (10) that glutamate dehydrogenase (GDH) is a very sensitive (but poorly specific) assay and can accurately rule out the presence of C. difficile in stool samples. Sharp and colleagues (10) used a commercially available combined GDH and toxin A/B assay (C.Diff Quik Chek Complete) in the first step of their testing algorithm, as others have done (2, 8, 11). However, we note that there were no true-positive samples that were GDH negative but EIA toxin positive in the 284 specimens they tested. We made the same observation in our own study of 500 specimens (3), and others have reported similar results (6, 7, 11). Thus, it is very rare to have a GDH-negative, EIA toxin-positive result for a true-positive sample. Therefore, we believe the toxin component of the C.Diff Quik Chek Complete assay is redundant. We prefer to use the GDH-only assay (C.Diff Chek-60), which is less expensive and allows for automated processing using the Dynex DS2 platform. This saves hands-on time and avoids possible misinterpretation of ambiguous results, since it produces a numerical result. We note that Sharp and colleagues had one false-positive result with their testing algorithm, giving a sensitivity and specificity of 100% and 99.6%, respectively (10). Using those authors' figures, we calculated performance characteristics using our preferred algorithm of screening with the C.Diff Chek-60 GDH assay, followed by confirmation with GeneXpert PCR, and found a sensitivity and specificity of 100% and 100%, respectively. Furthermore, the GeneXpert system can simultaneously indicate the presence of the potentially “hypervirulent” ribotype 027 strain (4), giving important epidemiological information not provided by the C.Diff Quik Chek Complete test. Using the Sharp and colleagues' figures for material cost per test (10), we also calculated the cost of each algorithm for testing 284 specimens. Those authors suggested the algorithm would cost $4,200.64, and ours cost slightly less at $3,923.30 (Table ​(Table1).1). Turnaround times for these algorithms would not differ significantly (depending upon degree of batching), but significant reductions may be possible by using GeneXpert as a point-of-care test. Further study is warranted in this area. TABLE 1. Material cost comparison for two suggested diagnostic algorithms It is clear that the GDH test is sufficiently sensitive to work well as an initial screen, but we do not believe any benefit is derived by coupling it with a toxin EIA. Furthermore, a dedicated, automated GDH test is easier to interpret. In England, we are now seeing a decrease in the prevalence of Clostridium difficile infection (which is a mandatory reportable disease) (5), and this will inevitably have an impact upon the positive and negative predictive values of any diagnostic tests. It is critical, therefore, that we have robust, accurate methods of identifying patients with this organism.

Gemifloxacin is a new quinolone and, like moxifloxacin, trovafloxacin, grepafloxacin and clinafloxacin, is more potent in vitro than ciprofloxacin or ofloxacin against Gram-positive aerobes. Gemifloxacin was the most potent of the quinolones tested against streptococci and most ciprofloxacin-resistant pneumococci were susceptible to gemifloxacin. Gemifloxacin, like moxifloxacin, trovafloxacin, grepafloxacin and clinafloxacin, was more potent than ciprofloxacin or ofloxacin against all staphylococci and many ciprofloxacin-resistant isolates were susceptible to these quinolones. Against Gram-negative aerobes gemifloxacin was as potent as or slightly less potent than ciprofloxacin, and isolates resistant to ciprofloxacin were also resistant to gemifloxacin and to moxifloxacin, trovafloxacin and grepafloxacin. Gemifloxacin was also the most potent quinolone against Gram-positive anaerobes and fusobacteria but trovafloxacin was the most potent agent tested against other Gram-negative anaerobes.

The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.

Extended-spectrum TEM β-lactamases (ESBLs) do not usually confer resistance to β-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla TEM-1 gene to introduce into the TEM-1 β-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164→Ser), TEM-26 (Arg164→Ser plus Glu104→Lys), TEM-19 (Gly238→Ser), and TEM-15 (Gly238→Ser plus Glu104→Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244→Cys), TEM-33 (Met69→Leu), and TEM-35 (Met69→Leu and Asn276→Asp). Introduction of the Arg244→Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met69→Leu or Met69→Leu plus Asn276→Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 β-lactamases. With the exception of the TEM-12 β-lactamase, introduction of the Met69→Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 β-lactamase. However, introduction of the double substitution Met69→Leu plus Asn276→Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM β-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.

Three cefoxitin-resistant Escherichia coli isolates from stool specimens of a patient with leukemia were either resistant, intermediate, or sensitive to imipenem. Conjugation experiments showed that cefoxitin resistance, but not imipenem resistance, was transferable. All isolates were shown by isoelectric focusing to produce two β-lactamases with isoelectric points of 5.4 (TEM-1, confirmed by sequencing of a PCR product) and >8.5 (consistent with a class C β-lactamase). The gene coding for the unknown β-lactamase was cloned and sequenced and revealed an enzyme which had 99.9% sequence identity with the plasmid-determined class C β-lactamase CMY-2. The cloned β-lactamase gene differed from bla CMY-2 at one nucleotide position that resulted in an amino acid change, tryptophan to arginine at position 221. We propose that this enzyme be designated CMY-4. Both the imipenem-resistant and -intermediate isolates lacked a 38-kDa outer membrane protein (OMP) that was present in the imipenem-sensitive isolate. The lack of an OMP alone did not explain the difference in carbapenem susceptibilities observed. However, measurement of β-lactamase activities (including measurements under conditions where TEM-1 β-lactamase was inhibited) indicated that the imipenem-intermediate isolate expressed six- to eightfold less β-lactamase than did the other isolates. This study illustrates that carbapenem resistance in E. coli can arise from high-level expression of plasmid-mediated class C β-lactamase combined with an OMP deficiency. Furthermore, in the presence of an OMP deficiency, the level of expression of a plasmid-mediated class C β-lactamase is an important factor in determining whether E. coli isolates are fully resistant to carbapenems.

Objective To establish the extent of inter-hospital spread of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb and to determine the most suitable method for typing local strains. Methods We analyzed a collection of 33 MRSA isolates from three Zagreb hospitals together with five unrelated British MRSA isolates by antibiogram typing, bacteriophage typing, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) after digestion with Sma I restriction endonuclease. Bacteriophage typing was done with the international set of S. aureus typing phages. RAPD and PFGE profiles were analyzed visually and by using the ‘GelCompar' computer program. Results Antibiogram typing provided eight profiles. Thirty (91%) of the 33 Croatian strains of MRSA were non-typable by phage typing. Visual analysis of RAPD products identified six, and visual analysis of PFGE fragments nine, distinct profiles. Computer analysis of RAPD data separated British isolates from the Croatian ones, but did not cluster the visually determined RAPD types. PFGE computer analysis separated British isolates and clustered isolates in concordance with visual interpretation. Thirty-one of the 38 isolates (82%) were visually grouped in the same clusters by both molecular methods. The dominant strain was present in each of the three hospitals. Conclusions Bacteriophage typing was unhelpful for the analysis of Croatian MRSA, since most strains were untypable with the international set of bacteriophages. RAPD and PFGE were more successful in typing the organisms and showed evidence of inter-hospital spread of one predominant MRSA strain in all three Zagreb hospitals. Thus RAPD and PFGE proved to be a useful aid in elucidating the epidemiology of MRSA infection in Zagreb hospitals and should be established in Croatia for typing MRSA.

Treatment failures with vancomycin prompted us to investigate the phenomenon of tolerance to glycopeptides in recent clinical isolates of Staphylococcus aureus. We used both MBC/MIC determinations and time-kill measurements to study tolerance to vancomycin and teicoplanin in 35 blood or heart valve isolates of S. aureus from patients with endocarditis or bacteraemia. There was generally good agreement between vancomycin tolerance indicated by an MBC:MIC ratio of > or =32 and by < or =90% kill after 6 h incubation in the presence of 20 mg/L vancomycin. However, two isolates were tolerant according to their MBC:MIC ratios but non-tolerant as judged by time-kill measurements. Seven of 15 methicillin-resistant S. aureus (MRSA) isolates but only two of 20 methicillin-susceptible ones were tolerant as judged by time-kill experiments (chi2 = 4.27 with Yates' correction, P = 0.04). Seven of the 16 isolates from patients with endocarditis were tolerant, compared with only two of the 19 isolates from patients with other conditions (chi2 = 3.43 with Yates' correction, P = 0.06). Within the endocarditis and non-endocarditis subgroups, tolerance was associated more frequently with methicillin resistance than with susceptibility, but the numbers were too small for the differences to be statistically significant. Most of the vancomycin-tolerant isolates were also tolerant to teicoplanin. We conclude that glycopeptide tolerance is a real phenomenon in S. aureus, particularly amongst MRSA isolates, and can be reliably determined by our method of time-kill analysis. Tolerance may compromise glycopeptide therapy of serious S. aureus infection and should be taken into account when deciding treatment.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.

Left to their own devices, doctors don’t always do the right thing Obstetricians were outraged when, in 1846, Ignaz Semmelweis reduced mortality from puerperal fever in Viennese women from 16% to 3% by making doctors and medical students disinfect their hands between performing postmortems and delivering babies: they could not accept any criticism of their professional practice. Semmelweis lost his job and died in a lunatic asylum, while his dirty, deluded, and dangerous colleagues abandoned his policies, continued with their distinguished careers, and returned puerperal mortality to its previous appalling level. Of course they did not know then, as we do now, that puerperal fever is caused by group A streptococcus, or that normal human skin is colonised by high concentrations of bacteria that transfer to the hands of staff during routine patient care and then on to other patients.1 2 They would have been shocked to discover that we now have incontrovertible evidence that hand decontamination significantly reduces the transfer of pathogens and the incidence of hospital and healthcare associated infections,2 and that Semmelweis has been vindicated. Between the 1890s and the 1950s, the epidemiology of common bacterial pathogens was elucidated. This led to the universal introduction of standard hygiene measures, such as handwashing, no touch technique, gloving and gowning, instrument sterilisation, environmental …

We have recently found that one of the β-lactamases reported in the paper by Stapleton et al. on incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli (1) was misidentified. Redetermination of the DNA sequence of the TEM gene from strain 51864, in both the French and British laboratories, has revealed that the nucleotides corresponding to the amino acid at position 244 are AGC. Thus, the amino acid at this position is serine rather than threonine and the enzyme is TEM-30 and not TEM-41.