Prevalence and role of efflux pump activity in ciprofloxacin resistance in clinical isolates of Klebsiella pneumoniae.

We investigated the prevalence and role of efflux pump activity and possible drug influx resistance in ciprofloxacin susceptibility amongst 26 distinct clinical isolates of Klebsiella pneumoniae of varying ciprofloxacin susceptibilities and known quinolone resistance-determining region (QRDR) genotypes. Cellular [(14)C]ciprofloxacin accumulation patterns and the amount of cell-associated [(14)C]ciprofloxacin of mid-logarithmic phase cells were determined before and after challenging with the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Most isolates (24/26), and all with ciprofloxacin minimum inhibitory concentrations (MICs) >1 µg/ml, had efflux activity that could extrude up to 90% of cell-associated [(14)C]ciprofloxacin; none had significant influx resistance. In isolates with no QRDR mutations, efflux alone reduced ciprofloxacin susceptibility. In isolates with QRDR mutations, the efflux activity varied: in one isolate with no efflux activity, the most common fluoroquinolone resistance-causing QRDR mutation did not bring about clinically significant ciprofloxacin resistance; isolates with multiple mutations had high MICs and, usually, high levels of efflux activity. Fluoroquinolone efflux activity is much more common in clinical isolates of K. pneumoniae than previously reported and it can contribute to decreased ciprofloxacin susceptibility.

Utility of antimicrobial susceptibility-based algorithms for the presumptive identification of genotypically-defined community-associated methicillin-resistant Staphylococcus aureus at a London teaching hospital.

PURPOSE: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains are classically characterised by susceptibility to most non-ß-lactam antimicrobial agents. We sought to determine whether antimicrobial susceptibility (AMS)-based algorithms could be used to presumptively identify CA-MRSA in a hospital MRSA collection. METHODS: Over a three-month period, all MRSA were tested for AMS, staphylococcal cassette chromosome mec (SCCmec) type, presence of the Panton-Valentine leukocidin (PVL) genes and spa type. CA-MRSA isolates were defined genotypically using a combination of spa and SCCmec type. AMS based algorithms were developed and tested for their sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: Ciprofloxacin susceptibility (p < 0.001) and fusidic acid resistance (p = 0.044) were independent predictors of CA-MRSA in a multivariate model. Although 98.5% of HA-MRSA were ciprofloxacin resistant, so too were 36.6% of CA-MRSA. Algorithms based on ciprofloxacin-susceptibility and fusidic acid resistance performed best, with specificity and NPV >90% and sensitivity and PPV >70%. CONCLUSIONS: Our data indicate that while ciprofloxacin-susceptible isolates are likely to be CA-MRSA, the use of ciprofloxacin-susceptibility as a marker of CA-MRSA would miss approximately one third of CA-MRSA isolates. Therefore, AMS patterns have limited utility for the identification of genetically-defined CA-MRSA in our setting.

Comparison of community-associated methicillin-resistant Staphylococcus aureus from teaching hospitals in London and the USA, 2004-2006: where is USA300 in the UK?

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is increasingly common in the USA, but rare in the UK. We compared CA-MRSA from the UK and USA to examine differences in the molecular epidemiology. We investigated patients presenting with MRSA in the first 72 h of hospital admission or in out-patient settings in a UK and a US hospital from January 2004 to March 2006. Fluoroquinolone susceptibility was used as a screening marker to select presumptive CA-MRSA. One hundred and eighteen and 49 such strains were identified, representing a prevalence of 0.1 and 0.2 isolates per 1,000 patient days in the UK and US respectively. Panton-Valentine leukocidin (PVL)-positive ST8-IVa (USA300)-type strains predominated among 43 surviving US isolates, whereas PVL-negative ST1-IVa predominated among 71 surviving UK isolates. There are striking differences between the molecular epidemiology of CA-MRSA in UK and US hospitals, which may have implications for control.

Bacterial contamination on touch surfaces in the public transport system and in public areas of a hospital in London.

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.

Identification and control of an outbreak of ciprofloxacin-susceptible EMRSA-15 on a neonatal unit.

We report the identification and control of an outbreak of a ciprofloxacin-susceptible strain of UK epidemic meticillin-resistant Staphylococcus aureus (EMRSA)-15 on a neonatal unit (NNU). All babies were screened for MRSA on admission using ciprofloxacin-containing media which did not detect the outbreak strain. The first identified case was a premature baby who developed MRSA bacteraemia with associated tibial osteomyelitis and multiple subcutaneous abscesses. The outbreak strain was subsequently identified in the nasopharyngeal secretions of a second child who was not clinically infected. Screening of all patients on the NNU using non-ciprofloxacin-media identified two other colonised babies. All four patient isolates were EMRSA-15, spa type t022, SCCmec IV, Panton-Valentine leucocidin (PVL) negative, indistinguishable by pulsed-field gel electrophoresis and susceptible to all non-beta-lactam antimicrobials tested. The outbreak strain was cultured from four of 48 environmental sites in a communal milk-expressing room. Unsupervised movement of mothers to and from the milk-expressing room may have contributed to the outbreak. Control measures included cohort isolation of affected babies, improved environmental cleaning, increased emphasis on hand hygiene and education of mothers. Ciprofloxacin-containing media should be used with caution for MRSA screening in settings where ciprofloxacin-susceptible strains (including community-associated MRSA) are increasing in prevalence.

Point-of-care universal screening for meticillin-resistant Staphylococcus aureus: a cluster-randomized cross-over trial.

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 < 0.001). MRSA was acquired on the admission wards by 23 (0.46%) patients in the control arm and by 24 (0.48%) in the intervention arm, acquisition rates of 5.39 and 4.60 per 1000 days respectively. After taking account of predefined confounding factors, the adjusted incidence rate ratio (IRR) for change in trend for MRSA acquisition was 0.961 (95% confidence interval: 0.766-1.206). The adjusted IRR for step change for MRSA acquisition was 0.98 (0.304-3.162). CONCLUSION: POCS produces a significantly faster result but has no effect on MRSA acquisition on admission wards compared with culture screening. Where compliance with infection prevention and control is high and MRSA carriage is low, POCS has no additional impact on MRSA acquisition rates over the first one to four days of admission compared with conventional culture screening.

Enhanced surveillance of meticillin-resistant Staphylococcus aureus bacteraemia in a London teaching hospital.

In 2001, the UK Department of Health introduced mandatory surveillance of meticillin-resistant Staphylococcus aureus (MRSA) bacteraemias (blood-culture-positive episodes) in English hospitals. We performed enhanced surveillance in their hospital between April 2001 and March 2003 to determine the epidemiology of MRSA bacteraemia across different specialities. There were 267 MRSA-blood-culture-positive episodes, giving a rate of 0.37 per 1000 occupied bed-days (OBD). Thirty-three (12.4%) episodes were false positives due to contaminants and 15 (5.6%) originated in the community or at another institution. Thirty-one (11.6%) episodes were in outpatients or occurred after recent discharge and were designated 'hospital associated'. The remaining 188 cases were clinically significant hospital-acquired episodes in inpatients, with a rate of 0.26 per 1000 OBDs. The highest rates were in the intensive therapy unit (ITU; 2.74 per 1000 OBDs) and the high-dependency unit (HDU; 1.68 per 1000 OBDs). Fifty-five non-ITU, non-HDU episodes occurred in patients who had been discharged from ITU or HDU prior to the development of bacteraemia but during the same admission. The number of MRSA bacteraemias related to ITU/HDU suggests that these wards may be hubs of MRSA infection. Haematology, oncology and renal (HOR) patients had the greatest number of hospital-associated episodes. The most common source of MRSA bacteraemia was a vascular access device (VAD) (108 episodes, 57%, 64% of which were central lines). The high bacteraemia rates in ITU, HDU and HOR patients were associated with high usage of VADs. The majority of episodes occurred in patients who were newly colonized with MRSA after admission. Thus, in this hospital, VADs and stays in ITU or HDU are important risk factors for bacteraemia, and VAD care and prevention of cross-infection are priorities for intervention. We recommend that the mandatory national surveillance scheme should collect additional data on MRSA bacteraemia to provide information for a national strategy for MRSA control and to allow appropriate comparison between institutions.

Guidelines for the control of glycopeptide-resistant enterococci in hospitals.

The increase since the mid 1980s in glycopeptide resistant enterococci (GRE) raised concerns about the limited options for antimicrobial therapy, the implications for ever-increasing numbers of immunocompromised hospitalised patients, and fuelled fears, now realised, for the transfer of glycopeptide resistance to more pathogenic bacteria, such as Staphylococcus aureus. These issues underlined the need for guidelines for the emergence and control of GRE in the hospital setting. This Hospital Infection Society (HIS) and Infection Control Nurses Association (ICNA) working party report reviews the literature relating to GRE prevention and control. It provides guidance on microbiological investigation, treatment and management, including antimicrobial prescribing and infection control measures. Evidence identified to support recommendations has been categorized. A risk assessment approach is recommended and areas for research and development identified.

Clinical impact and relevance of antibiotic resistance.

Increasing antimicrobial resistance and multiple resistance have resulted in increasing difficulties in the treatment of bacterial infections. Resistance leads to inappropriate empirical therapy, delay in starting effective treatment, and the use of less effective, more toxic, and more expensive drugs. Although studies are not always consistent, antimicrobial resistance in the infecting organisms is associated with treatment failure, prolonged or additional hospitalization, increased costs of care, and increased mortality. Additional costs and lost bed days are incurred by the need to control the spread of antimicrobial-resistant organisms within hospitals. All this has significant direct impact on patients and their families and also secondary effects on the cost effectiveness of healthcare delivery. There is an urgent need to control antimicrobial resistance by improved antibiotic usage and reduction of hospital cross-infection.

Significant reduction of endemic MRSA acquisition and infection in cardiothoracic patients by means of an enhanced targeted infection control programme.

Due to increasing methicillin-resistant Staphylococcus aureus (MRSA) infection in cardiothoracic patients at St Thomas' Hospital, an enhanced infection control programme was introduced in September 2000. It was based on UK national guidelines on the control of MRSA and targeted additional identified risk factors for surgical site infection (SSI). It included recognition of the problem by senior staff and their taking responsibility for it; intensive support, education and advice from the infection control team; improved ward and theatre hygiene; pre-admission, admission and weekly MRSA screening; isolation and clearance treatment; nursing care pathways for MRSA colonized patients; and teicoplanin plus gentamicin surgical prophylaxis. The effectiveness of the programme was assessed by retrospective analysis of computerized patient data for the 16 months before and after the introduction of the programme. There was no significant change in the number of operations or the proportion of patients admitted with MRSA, although nine patients were cleared of carriage before admission. However, there were significant falls in the proportion of patients acquiring MRSA on the ward [38/1036 to 14/921, P=0.003, RR 2.4 (95%CI 1.32-4.42)] and in the rate of bloodstream MRSA infections [12/1075 to 2/956, P=0.014, RR 5.34 (95%CI 1.20-23.78)]. Sternal and leg wound infections both halved (from 28/1075 to 13/956 and 16/1075 to 7/956, respectively) but this did not reach statistical significance. These results demonstrate that an enhanced, targeted infection control programme based on the UK national guidelines, SSI prevention guidelines and local risk assessment can reduce the incidence of nosocomial MRSA acquisition and invasive infection in cardiothoracic patients in the face of continuing endemic risk.

Diagnosis of tuberculous pleural effusion by the detection of tuberculostearic acid in pleural aspirates.

Detection of TBSA was attempted in pleural aspirates of 74 patients with tuberculous and 44 patients with nontuberculous pleural effusion by gas chromatography/mass spectrometry with selected ion monitoring. The results were disappointing with a test sensitivity of 67.6 percent and a specificity of 52.3 percent. In contrast, histologic examination of pleural biopsies gave a diagnostic sensitivity of 71.0 percent. Pleural biopsy remains a better investigational procedure for the diagnosis of tuberculous pleural effusion.

DNA array technology and diagnostic microbiology.

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.

Comparison of methods for the diagnosis of typhoid fever.

Over five years the Bactec radiometric blood culture method yielded Salmonella typhi in 41 of 45 confirmed cases of typhoid fever, 90% of which were from the first culture set taken. Blood clot culture was positive in 18 (41%) of 44 confirmed cases and stool culture in 24 (59%) of 41. The yield from 2189 Widal clot cultures was only 0.03%. There were 68 positive results in 2258 unpaired Widal tests: 23 of them were falsely positive and 13 falsely negative, but in 11 out of 68 cases the Widal was the only positive laboratory test. It is concluded that routine clot culture is not cost effective if a sensitive blood culture method is used, and that the Widal test is useful only in selected patients.

Evaluation of the Microbact-24E bacterial identification system.

The Microbact 24E (MB24E) system is a commercial microsystem for the identification of common clinical isolates of Enterobacteriaceae and non-fermenting Gram negative bacilli, and consists of dehydrated substrates distributed in the wells of microtitre trays. This system was compared with the API20E for the identification of 386 bacterial isolates, which included 284 clinical and 102 environmental organisms. There was 97% and 91% agreement for the identification of clinical isolates of Enterobacteriaceae and other Gram negative bacilli, respectively. The identification of environmental isolates by both systems was less satisfactory. The API20E has a more extensive database than the MB24E and is thus more reliable for the identification of rare or unusual organisms, but the MB24E is cheaper, is easy and convenient to use, and is suitable for a routine microbiology laboratory.

Evaluation of three commercial detection systems for Mycobacterium tuberculosis where clinical diagnosis is difficult.

AIMS: To assess the performance of three commercially available Mycobacterium tuberculosis detection systems employing nucleic acid amplification, when applied directly to respiratory and non-respiratory specimens from patients where the diagnosis of tuberculosis is difficult using clinical and traditional bacteriological methods. METHODS: 42 respiratory and 21 non-respiratory specimens were concentrated, examined with auramine staining, and cultured on Lowenstein-Jensen slopes. These specimens were also assayed using the Amplicor Mycobacterium tuberculosis test (AM) (Roche Diagnostic Systems), the Amplified Mycobacterium tuberculosis direct test (AMD) (Gen-Probe), and the LCx Mycobacterium tuberculosis assay (LMA) (Abbott Laboratories). RESULTS: All three amplification systems used in this study gave specificities of 100% when used on respiratory specimens. When used on non-respiratory specimens, AM and LMA gave specificities of 100% and AMD 75%. With respiratory specimens the AM, AMD, and LMA systems gave sensitivities of 75%, 65.2%, and 79.2%, respectively. With non-respiratory specimens the sensitivities were 50%, 66.7%, and 60%, while with smear negative, culture positive specimens they were 33.3%, 66.7%, and 55.6%. Positive predictive values of 100% were seen with all specimens except non-respiratory specimens assayed using AMD where the value was 66.7%. CONCLUSIONS: The manufacturers of these systems recommend that they should only be used for the direct analysis of respiratory specimens, and the US Food and Drug Administration has approved them for use only with smear positive specimens. This study confirms that sensitivities are lower for non-respiratory and smear negative specimens, but positive predictive values are high. Provided they are interpreted with caution, positive results with these tests in respiratory and non-respiratory specimens are useful in tuberculous patients who are otherwise difficult to diagnose. Each amplification has advantages and disadvantages compared with the others.

Necrotising fasciitis caused by Vibrio vulnificus.

A case of necrotising fasciitis caused by Vibrio vulnificus is described. The need for early recognition and aggressive surgical treatment are highlighted, and the necrotising infections due to V vulnificus described in the published work are reviewed.

Evaluation of Cathra system for identifying gram negative aerobic bacteria.

The Cathra system is a commercial multipoint inoculation method for the identification of aerobic Gram negative bacteria. The system uses a replicator technique in which 21 different agar media can be inoculated simultaneously with 36 organisms. Identifications are made by use of a special computer database. The performance of this system was compared with that of the API 20E for the identification of 372 clinical isolates of Enterobacteriaceae and 133 miscellaneous Gram negative bacteria. For enterobacteria, the Cathra system was in 97% agreement with API 20E at species level and 98% at genus level. For miscellaneous Gram negative strains the two systems were in 59% agreement at species level and 77% at genus level. The Cathra system is suitable for use in diagnostic laboratories, especially those with a heavy workload and a wish to use break-point sensitivity testing. The identification database for miscellaneous Gram negative organisms, however, needs to be expanded.

Diagnosis of nasopharyngeal tuberculosis by detection of tuberculostearic acid in formalin fixed, paraffin wax embedded tissue biopsy specimens.

The use of gas chromatography and mass spectrometry with selected ion monitoring detected tuberculostearic acid (TBSA) in 10 of 12 formalin fixed, paraffin wax embedded nasopharyngeal and head and neck biopsy specimens from patients with confirmed tuberculosis and carcinoma, and in one of 50 control specimens (giving a sensitivity of 83% and a specificity of 98%). The two false negative cases had very small tissue fragments and the patient with a false positive result may have had pulmonary tuberculosis. Tuberculostearic acid (TBSA) was also detected in nine of 16 specimens from the head and neck region with non-caseating granulomas suspected, but not confirmed, to be tuberculosis. It is concluded that nasopharyngeal tuberculosis is relatively common in Hong Kong and should be considered when biopsy specimens show granulomas. The detection of TBSA in tissue biopsy specimens is a useful, rapid method for the diagnosis of tuberculosis and other mycobacterial infections, and can be conveniently performed within two days on formalin fixed and paraffin wax embedded material.