J. Flynn

Extended spectrum beta-lactamase production and fluorquinolone resistance in pathogens associated with community acquired urinary tract infection

We have evaluated the susceptibility of 199 pathogens isolated in pure culture from consecutive urine samples submitted from the community. Rates of susceptibility for all organisms were ampicillin, 48%; amoxycillin/clavulanic acid, 88%; cephalothin, 57%; cefuroxime axetil, 74%; nalidixic acid, 85%; ciprofloxacin, 99%; nitrofurantoin, 78%; and trimethoprim, 67%. Ciprofloxacin resistance and production of extended spectrum beta-lactamase enzymes were detected in Escherichia coli strains isolated from patients in the community.

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

AIMS: To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. METHODS: Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. RESULTS: M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. CONCLUSIONS: The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Multiplex PCR for identifying mycobacterial isolates

AIMS--To develop a multiplex polymerase chain reaction (PCR) method to facilitate identification of mycobacterial isolates. METHODS--Type strains of 14 species of mycobacteria and 56 clinical isolates were lysed by boiling in TE Triton. The lysate (5 microliters) was used directly in a PCR reaction incorporating three pairs of PCR primers expected to amplify fragments from the genome of (a) all mycobacteria, (b) Mycobacterium tuberculosis complex only and (c) M avium only. PCR products were visualised by electrophoresis on agarose gels. RESULTS--Multiplex PCR applied to 14 type strains yielded patterns on electrophoresis which permitted identification of the mycobacterial isolates as M tuberculosis complex, M avium or as mycobacteria other than the former. The identification of 56 clinical isolates by multiplex PCR was consistent with other methods and was accomplished in less than one working day. CONCLUSIONS--This method may facilitate rapid and convenient identification of most clinical isolates of mycobacteria by PCR and gel electrophoresis. Further evaluation is warranted.

Evaluation of a PCR assay for detection of Mycobacterium tuberculosis in clinical specimens.

A polymerase chain reaction (PCR) assay for detection of M. tuberculosis was optimized for application to clinical specimens, which were prepared for amplification by boiling in buffer. The buffer contained a synthetic DNA fragment to determine if DNA amplification from the individual prepared specimens was subject to inhibition because of substances present in the specimen, or by the process of specimen preparation or storage. The PCR test was less sensitive than direct microscopy (75% as against 87.5%) and had a specificity of 97%. Invalid results due to inhibition of amplification occurred in 12% of specimens. Incorporation of the internal standard into the specimen preparation buffer ensures that any step in the process which inhibits DNA amplification is detected in the failure of amplification of the internal standard. The use of internal standard in this way should be considered in developing diagnostic protocols.

Tuberculosis in the west of Ireland 1986-1990.

A review of mycobacteria isolated from clinical samples from the Western Health Board Area (WHBA) for the years 1986 to 1990 was performed to establish the pattern of mycobacterial infection. The incidence of microbiologically proven cases of tuberculosis (13.3/100,000) and of sputum smear positive cases (6.2/100,000) was determined and correlated with notification data. M. bovis accounts for 6.3% of cases of microbiologically confirmed tuberculosis in this area. Resistance to one or more of the first line anti-tuberculous drugs was noted in 5.3 % of isolates (excluding pyrazinamide resistance in M. bovis). Clinically significant isolates of mycobacteria other than tuberculosis (MOTT) were rare throughout the five year period. The overall incidence of microbiologically proven cases of tuberculosis is relatively high and M. bovis and drug resistant isolates of M. tuberculosis are relatively common.

A comparison of antimicrobial sensitivities of urinary pathogens for the years 1980 and 1990.

Urinary tract infection is a major cause of morbidity in both the hospital and community which often requires empirical therapy. We have retrospectively studied laboratory diagnosed urinary tract infections for the years 1980 and 1990 to document the common pathogens and antimicrobial susceptibility patterns. In 1990 a significantly lower proportion of specimens yieldedProteus sp. orKlebsiella sp. than was the case in 1980. This was true of specimens from both the hospital and the community. There was an increase in the proportion of specimens yieldingPseudomonas sp. and coagulase negative Staphylococci (CNS). Significant changes in the antimicrobial susceptibility of urinary pathogens are also noted. In particular a greater proportion of isolates from the community were sensitive to cephalothin in 1990, while fewer isolates were sensitive to nalidixic acid and gentamicin. A greater proportion of isolates from hospital practice were sensitive to ampicillin,to cephalothin and to trimethoprim in 1990 while fewer isolates were sensitive to gentamicin. In relation to nitrofurantoin no significant change was noted. In respect of isolates from both community and hospital practice the agents ofloxacin, co amoxiclav (not available in 1980) and gentamicin are those which are most consistently active.

PCR based fingerprinting of Enterobacter cloacae

An outbreak of lower respiratory tract infection with Enterobacter cloacae occurred in an intensive care unit in a university teaching hospital. Random amplification of polymorphic DNA (RAPD) was used to assist in the investigation of the outbreak. The technique was readily applied to this organism and permitted differentiation between strains which had identical biochemical profiles and antibiograms. The versatility of this technique makes it attractive for use in hospitals where fingerprinting of any one of the many Gram-negative rods associated with nosocomial infection may be required from time to time.

The Microbial Flora of In-Use Blood Pressure Cuffs

SummaryThe capacity of blood pressure cuffs to act as vehicles of hospital infection has been recognised. We describe the microbial flora of in-use DINAMAP blood pressure cuffs used in the operating theatres and one recovery room in a teaching hospital. Our results show significant microbial contamination of in-use blood pressure cuffs.

Evaluation of a PCR based method for identification of mycobacterial isolates

In recent years an increasing number of mycobacterial species have been described as causing disease in humans. Identification of isolates to the species level is essential for evaluation of the significance of an isolate. Conventional methods for identification based on selective inhibitors of growth and biochemical reactions are slow. We have evaluated a novel method, PCR restriction enzyme pattern analysis (PRA), for identification of mycobacterial isolates. Fifty three cultures including a mixed culture ofM. tuberculosis andM. avium and four mycobacterial cultures contaminated with rapidly growing organisms of other genera were studied. The method permits identification of most isolates within 1 to 2 days, including some contaminated cultures, and results were generally in accordance with those obtained by conventional methods.

In-vitro activity of piperacillin/tazobactam relative to other antibiotics against blood culture isolates

Resistance of bacteria to antibiotics is an increasing problem in many countries. Accurate locally relevant information is essential for detection and control of emerging resistance and to facilitate choice of empirical antibiotic therapy in the immediate management of seriously ill patients. We have determined the minimum inhibitory concentration of piperacillin/tazobactam for 97 strains of bacteria (55 Enterobacteriaceae, 13 non-fermentative Gram-negative bacilli, 22Staphylococcus aureus), 6Enterococcus faecalis and 1Bacillus cereus) isolated from blood cultures and compared its activity to that of amoxycillin, co-amoxiclav, cephalothin, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, piperacillin, cefotaxime. The strains were consecutive non-fastidious isolates with the following qualifications: coagulase negative staphylococci and diphtheroids were excluded and the number of Staphylococcus aureus isolates was limited to 12 methicillin-resistant and 10 methicillin-sensitive strains. Multiple isolates of the same species from individual patients were not included. The minimum inhibition concentrations of methicillin, penicillin, teichoplanin and vancomycin were also determined for specific groups of organisms. MICs were determined by the Etest method (AB Biodisk, Solna, Sweden) on Mueller Hinton agar. The MICs of appropriate American Type Culture Collection control strains were determined. Based on the interpretative criteria of the National Committee for Clinical Laboratory Standards (USA), 87 per cent of Gram-negative bacilli were susceptible to piperacillin/tazobactam compared with amoxycillin 26 per cent, cephalothin 35 per cent, co-amoxiclav 54 per cent, piperacillin 56 per cent, cefotaxime 69 per cent, ceftazidime 84 per cent, gentamicin 85 per cent and ciprofloxacin 91 per cent. Of all isolates 75 per cent were sensitive to piperacillin/tazobactam, compared with amoxycillin 22 per cent, cephalothin 35 per cent, piperacillin 41 per cent, co-amoxiclav 52 per cent, cefotaxime 59 per cent, ceftazidime 60 per cent, gentamicin 74 per cent and ciprofloxacin 77 per cent. Two isolates (1E. coli and 1Klebsiella pneumoniae) with antibiograms consistent with the relatively new resistance phenomenon of extended spectrum beta-Iactamase production were identified. The spectrum of activity of piperacillin-tazobactam for empirical antibiotic therapy is significantly greater than that of piperacillin alone and is similar to that of ciprofloxacin and gentamicin.