Prevalence of glycopeptide and aminoglycoside resistance in Enterococcus and Listeria spp. in low microbial load diets of neutropenic hospital patients.

Low microbial load diets for patients with haematological malignancy were examined for enterococci and listeria using pre-enrichment, enrichment and selective plating. Enterococci were highly prevalent and their ecology diverse; 100/211 samples yielded 132 isolates made up of 67 strains distinguishable by PFGE. Listeria monocytogenes was not found. Screening of enterococci for antibiotic resistance showed low level vancomycin resistance (6-12 microg/ml) in six isolates of E. gallinarum and high level streptomycin resistance (> or = 1000 microg/ml) in eight isolates from various foods. No strains showing high level glycopeptide or gentamicin resistance were found. The high prevalence of enterococci in food processed for safety indicates a possible route for the acquisition of antibiotic-resistant strains by vulnerable hospital patients.

Quality control of culture media--perspectives and problems.

The Working Party on Culture Media (WPCM) of the International Union of Microbiological Societies (IUMS) has promoted and facilitated the development of methods for quality control of culture media used for the detection and enumeration of food-borne microorganisms since the early 1980s. While progress has been made in establishing protocols to test for productivity and selectivity of media, problems associated with the influence of food constituents and background microflora, as well as the presence of sublethally injured cells in test foods, are yet to be fully addressed before optimum methods for assessing the quality of media can be defined. However, for various reasons, the development of standardised procedures which account for the influence of food constituents or state of debilitation of target microorganisms may not be practical or even desirable.

A formal system of approval for monographs in the pharmacopoeia of culture media--statement from the IUMS-ICFMH working party on culture media.

A categorization system for monographs on culture media is outlined which will lead to more rapid publication and a formal division into three classifications; draft, proposed and approved. This should assist the quality assurance and accreditation processes in food microbiology laboratories.

Subtyping of Listeria monocytogenes on the basis of plasmid profiles and arsenic and cadmium susceptibility.

The susceptibilities to arsenic and cadmium together with the detection of plasmid DNA were evaluated for use as epidemiological markers for the subtyping of Listeria monocytogenes. Plasmid DNA was detected in 34% of 322 apparently unrelated isolates of L. monocytogenes. The resistance to cadmium and arsenic differentiated 565 apparently unrelated cultures into four groups, the smallest being 5% of cultures resistant to both agents, and the largest (53%) being sensitive to cadmium and resistant to arsenic. The resistance patterns to these agents and the presence of plasmid DNA varied markedly between the serotypes of the cultures. The detection of plasmid DNA was strongly associated with cadmium resistance in serogroup 1/2 cultures, but not within those of serogroup 4. Arsenic resistance was not associated with plasmid DNA. All methods were sufficiently stable to be useful for epidemiology investigations. The techniques described here offer simple methods which can be easily utilized in laboratories without a specialized expertise for this bacterium.

Increased prevalence of Listeria monocytogenes in the faeces of patients receiving long-term H2-antagonists.

BACKGROUND: Human listeriosis is an uncommon infection caused by the Gram-positive organism Listeria monocytogenes. OBJECTIVE: To investigate the effects of therapeutic gastric acid suppression on faecal isolation of L. monocytogenes and the incidence of human listeriosis. METHODS: Five stool specimens from each of 20 patients on continuous H2-antagonist therapy and two faecal samples from each of 47 healthy controls were investigated for the presence of Listeria spp. RESULTS: A higher faecal isolation rate of L. monocytogenes was detected amongst the patients (20%) compared with the controls (2.1%) (P < 0.025). All subjects with stools positive for Listeria spp. were female, this sex difference being significant in the patient group (P < 0.0036) compared with controls. No patient, however, developed listeriosis. CONCLUSION: Patients on long-term gastric acid suppressive therapy may be at increased risk of faecal carriage of L. monocytogenes.

The WHO multicenter study on Listeria monocytogenes subtyping: random amplification of polymorphic DNA (RAPD).

As part of a WHO multicenter study on Listeria monocytogenes subtyping methods the random amplification of polymorphic DNA (RAPD)-technique was evaluated. Six participants were asked to use a standard protocol to analyse a set of 80 L. monocytogenes strains. This set contained 22 groups of epidemiologically linked isolates and 11 pairs of duplicate strains. Using three different 10-mer primers the median reproducibility of the RAPD-results obtained by the six participants was 86.5% (range 0-100%). Failure in reproducibility was mainly due to results obtained with one particular primer. The number of epidemiological groups found to be homogeneous varied from 1-22 (median 16). However, for some groups an inhomogeneity was found by the majority of participants. The overall correlation between the results from the different participants ranged from 32 to 85%.

Culture media and methods for the isolation of Listeria monocytogenes.

The recovery of low numbers of Listeria monocytogenes from foods and environmental samples requires the use of enrichment cultures followed by selective plating and, where injured organisms are likely to be present, a pre-enrichment step. The development of selective and enrichment media for L. monocytogenes is traced and currently used media are discussed. Comparisons of media and methods for the culture of L. monocytogenes are reported but no single method can be recommended for all situations. Guidance is given on the choice of media and methods which is governed by the type of sample, number and nature of competing flora and cost.

Media for 'total' Enterobacteriaceae, coliforms and Escherichia coli.

Selective and enrichment media for the isolation and enumeration of 'total' Enterobacteriaceae, coliforms, faecal coliforms and E. coli from foods are described. The effects of time and temperature of incubation are discussed. Reports of some comparative studies of these media and factors to be considered in the selection of media for this group of organisms are noted and the difficulties associated with the isolation and enumeration of pathogenic serotypes of E. coli considered.

Bacteriocin (monocin) interactions among Listeria monocytogenes strains.

Monocin interactions of 97 strains of Listeria monocytogenes were assessed using an improved production method and standardisation of the monocins against the type strain of L. ivanovii. Monocins were resistant to trypsin, sensitive to heating at 56 degrees C for 30 min and stable at 4 degrees C. Only serovar 4 strains acted as indicators. A typing system using 8 producer and 11 indicator strains showed poor discrimination.

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp.

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.

Comparison of latex and haemolysin tests for determination of anti-streptolysin O (ASO) antibodies.

A latex agglutination test was compared with the micro-titration haemolysin inhibition method for the detection of anti-streptolysin O (ASO) antibodies in 428 serum samples. After slight modification of the latex method to produce maximal agglutination good agreement was shown between the results obtained by the two methods. The latex test had a sensitivity of 83.6%, a specificity of 93.3%, a predictive positive value of 86.5% and a predictive negative value of 91.6%. It was convenient, required less labour than the haemolysin test, and permitted economic testing of small numbers of sera.

Comparison of culture media for detecting methicillin resistance in Staphylococcus aureus and coagulase negative staphylococci.

The detection of methicillin resistance was examined in 51 strains of Staphylococcus aureus and 135 strains of coagulase negative staphylococci using Isosensitest, Diagnostic Sensitivity Test (DST), Mueller-Hinton (MH), Columbia, and Sensitest agars. MH agar with 5% added sodium chloride incubated at 35 degrees C was the most effective in detecting resistance in S aureus, and Columbia agar with 5% added sodium chloride incubated at 35 degrees C was most effective for coagulase negative staphylococci. For clinical purposes, a provisional report of sensitivity for S aureus could be issued after 18 hours; with coagulase negative staphylococci, only resistant strains could be reported at this time. For definitive results cultures must be examined after 40 hours of incubation.

Automated bacteriuria screening using the Berthold LB 950 luminescence analyser.

The Berthold LB950 Automatic Luminescence Analyser was used to estimate bacterial adenosine triphosphate in urine. The system provided a rapid (15 min) and fully automated screening test for bacteriuria at the 10(5) CFU/ml level. Bioluminescence results for 1040 urines were compared with viable counts using two reference culture methods and frequency distributions of bacterial counts and adenosine triphosphate levels were calculated. With a specificity of 79% the automated method showed a sensitivity of 84% using a pour plate reference count and 91% using a standard loop reference count. When contaminated urines were excluded the sensitivity improved to 98%. The automated bioluminescence test, though expensive, was shown to work well with good quality specimens.

A note on the use of dextran in blood cultures monitored by conductance methods.

The incorporation of dextran into broth for the detection of bacteraemia by conductance monitoring is recommended to eliminate the effect of sedimenting blood cells which may mask early signals from bacteria. Broth containing ten times the recommended concentration of dextran was tested with a wide range of bacteria without demonstrating any reduction in growth or rate of change of conductance.

Detection of bacteriuria by bioluminescence: effect of pre-analysis centrifugation of specimens.

Three bioluminescence-based, rapid methods of detecting significant bacteriuria were applied in parallel to 514 urine specimens. The results were compared with those of a quantitative pour plate viable count method, defined as positive if greater than or equal to 10(5) c.f.u./ml of urine were observed. When adjusted to yield 21% falsely positive results the three rapid methods yielded 24%, 21% and 19% falsely negative results. If specimens with evidence of urethral or vaginal contamination were excluded (237 specimens remaining) the three methods yielded respectively 14%, 8% and 13% falsely negative results. A major source of disagreement between the bioluminescence-based methods and quantitative culture thus appeared to be contaminated urine specimens.

Synovial fluid lactate and the diagnosis of septic arthritis.

To assess the value of synovial fluid lactate estimation in the diagnosis of septic arthritis, 238 specimens received for routine culture and 75 reference samples were examined using a rapid enzyme technique. Samples were collected without special treatment and the effect of delay in transport to the laboratory investigated. Raised levels were found in all cases of untreated septic arthritis, in six out of ten partially treated patients and in 19 out of 219 non-septic fluids. Special treatment of the sample was unnecessary if it was examined within six hours of aspiration. The predictive value of a negative result was 98 per cent and the value of the test appeared to be in the rapid exclusion of sepsis in untreated patients.