Analysis of prevalence, risk factors and molecular epidemiology of Clostridium difficile infection in Kuwait over a 3-year period.

We conducted a prospective study to evaluate the prevalence and epidemiology of CDI in Kuwait government hospitals over a 3-year period, January 2003 to December 2005, to determine the ribotypes responsible for CDI and to estimate the prevalence of ribotype 027. We also conducted a case-control study to identify the risk factors in our patient population. A total of 697 stool samples from patients with suspected CDI were obtained and sent to Anaerobe Reference Laboratory, Faculty of Medicine, Kuwait University for Clostridium difficile toxin detection, culture and PCR ribotyping. During the period, 73 (10.5%) out of 697 patients met the case definition of CDI. Of these, 56 (76.7%) were hospital-acquired and 17 (23.3%) were from outpatient clinics. Thus, the prevalence of hospital-acquired CDI amongst patients with diarrhoea was 8% over the study period; the prevalence in 2003, 2004 and 2005 was 9.7%, 7.8% and 7.2%, respectively. Our data showed that 42.9% of the CDI patients were above 60 years, of which >79% were aged 71 years and above. Patients with CDI were more likely than the controls to have been exposed to immunosuppressive drugs and feeding via nasogastric tube. The most common ribotypes isolated during this study were 002, 001, 126 and 140 and they represent 55.1% of all isolates. PCR ribotype 027 was not isolated.

Distribution and antimicrobial susceptibility patterns of Clostridium difficile PCR ribotypes in English hospitals, 2007-08.

A surveillance study designed to provide a representative sample of the strains of Clostridium difficile causing infections in hospitals in England was in operation from April 2007 to the end of March 2008. Six hundred and seventy-seven isolates were obtained from 186 hospitals in the nine geographical regions of England as recognised by the Health Protection Agency's Regional Microbiology Network. Typing studies revealed that PCR ribotype 027 is now the most common strain isolated from symptomatic patients, accounting for over 41.3% of isolates in English hospitals. Type 106 was the second most common strain (20.2%) and Type 001, which was once the most common strain associated with hospital outbreaks, has now been reduced to only 7.8% of the total. A mixture of 44 other PCR ribotypes accounted for the remaining 28.9% of isolates. This represents a changing distribution of strains when compared to a previous study performed two years earlier which showed roughly equal proportions of types 106, 001 and 027. Antimicrobial susceptibility testing by the E test method revealed significantly lower susceptibility to metronidazole in the more common strains when compared to the less common ribotypes, although none were classified as clinically resistant. Similarly, no resistance to vancomycin was detected. However, common PCR ribotypes were more resistant to moxifloxacin and erythromycin than the less common strains, which may indicate a selective advantage for resistance to these agents, and combined resistance to these two agents was a good indicator of a common ribotype.

Comparison of laboratory methods in the diagnosis of urinary tract infection.

Three methods of semiquantitative culture and two techniques of microscopy were compared with a surface viable count for the detection of significant bacteriuria in one thousand midstream specimens of urine. The results obtained with the blotting-paper-strip method on MacConkey agar and with Uricult dip-slides correlated well with the results of the surface viable count, and both methods were suitable for routine use. The blotting-paper-strip method was preferred for laboratory use because of expense but dip-slides are useful for general practice and outpatient clinics. Semi-quantitative culture by Microstix dip-strips gave less accurate results, and the nitrite test area detected only a small proportion of infected specimens. The microscopic examination of a Gram-stained film of the centrifuged deposit of urine specimens yielded more useful information and was more reproducible than examination of a wet film of the untreated urine.

Laboratory assessment of physical and chemical methods of preserving urine specimens.

Three methods of preserving simulated specimens of urine were studied with six test strains of bacteria. Viable counts were measured by a surface viable count and by the filter-paper-strip method during a holding period of 72 hours. Refrigeration at approximately 4 degrees C was effective and reliable. Boric acid (1-8%) at room temperature was toxic for the strain of Escherichia coli at a density of 10(7) cfu/ml but this may not be significant at the higher concentration of bacterial cells often found in clinical specimens. NaCl-polyvinylpyrrolidone (PVP) solutions containing PVP of mol. wt 44 000 or 700 000 were not effective; they were toxic for the Gram-negative strains and did not retard the growth of Micrococcus subgroup 3. The two methods of measuring viable counts were compared for specimens held under different conditions; the specificity of the filter-paper-strip method was high but the sensitivity was low when many of the specimens contained approximately 10(5) cfu/ml.

Recovery of anaerobic bacteria from small inocula: a model for blood culture studies.

The recovery of anaerobic, facultative anaerobic, and aerobic pathogens from very small inocula was studied under conditions that could be related to routine blood culture procedures. Results with Brain Heart Infusion broth were unsatisfactory. Freshly prepared Brewer thioglycollate medium gave apparently good results, but there are disadvantages when this medium is used for blood culture. Results with Difco Thiol broth were disappointing. A modification of Robertson's cooked-meat broth supplemented with Brain Heart Infusion gave good recovery and sustained viability with a wide range of test organisms including exacting strains. The findings raise points of practical importance.

Bacteraemia due to a rifampicin-resistant strain of Bacteroides fragilis.

A strain of Bacteroides fragilis with high-level chromosomal resistance to rifampicin was isolated by blood culture from a patient with bacteraemia after gastrointestinal surgery. He had been receiving antituberculous therapy with rifampicin for nine months. This resistance led to some difficulty in the recognition and identification of the isolate by methods that depended upon antibiotic sensitivity patterns.

The isolation and identification of Bacteroides spp. from the normal human faecal flora.

Gram-negative anaerobic bacilli of the Bacteroides group were isolated on an enriched selective medium from specimens of faeces from 20 normal healthy adults and identified by conventional bacteriological methods. A heavy growth of Bacteroides spp. was obtained from all specimens and 10 representative colonies from each subject were identified. Most isolates (84%) belonged to the B. fragilis group. The commonest species identified in this group were B vulgatus and B. thetaiotaomicron (22% each), B. distasonis (18%) and the B. eggerthii/variabilis complex (14%), and only 9% were B. fragilis. B. vulgatus was isolated from 80% of subjects, B. thetaiotaomicron and B. distasonis from 70% each and the B. eggerthii/variabilis complex from 65%, but B. fragilis was detected in only 45% of specimens. Asaccharolytic species were isolated in smaller numbers from 14 (70%) subjects, but only five strains of the B. melaninogenicus/oralis group were isolated and fusobacteria were not detected.

The isolation and identification of Bacteroides ssp. from the normal human vaginal flora.

The occurrence of gram-negative anaerobic bacilli in the normal vaginal flora was studied in 20 normal healthy women attending a family-planning clinic. A swab was taken from the cervix and posterior fornix and Bacteroides spp. were isolated on a selective medium from 13 (65%) subjects. A heavy growth of Bacteroides was obtained from 11 specimens but only a few colonies were isolated from two specimens. Where possible, 10 representative colonies from each subject were studied and 113 isolates were identified by conventional bacteriological tests. Most isolates (78%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were the B. bivius/disiens complex (42%), B. melaninogenicus ss. intermedius (22%) and ss. melaninogenicus (16%). Asaccharolytic strains were isolated in smaller numbers from 54% of subjects, but only five strains of the B. fragilis group were isolated from two subjects; fusobacteria were not detected.

The isolation and identification of Bacteroides spp. from the normal human gingival flora.

Gram-negative anaerobic bacilli were isolated on an enriched selective medium from specimens of subgingival plaque from 20 normal, healthy adults without periodontal disease. a heavy growth of Bacteroides and Fusobacterium spp. was obtained from all specimens and 10 representative colonies from each subject were selected for identification by conventional bacteriological methods. Results were obtained in these tests with 188 isolates; some strains of the B. melaninogenicus/oralis group would not grow in pure culture on solid media and showed satellitism in mixed culture, and some strains of fusobacteria did not survive repeated subculture. Most isolates (68%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were B. oralis (41%), B. melaninogenicus ss. melaninogenicus (26%) and ss. intermedius (17%). B. oralis was isolated from all subjects and at least one strain of B. melaninogenicus was isolated from 18 out of 20 subjects. Only eight strains of the B. fragilis group were detected and they probably represented transient carriage. Fusobacterium spp. and L. buccalis were isolated from 15 out of 20 subjects and accounted for 21% of the isolates studied, but most could not be identified specifically by the set of tests used. Asaccharolytic species of Bacteroides were isolated in small numbers from seven subjects.

Pigment production by Bacteroides species with reference to sub-classification.

All six reference strains of Bacteroides species, 36 laboratory isolates conforming to this group, and individual strains of Escherichia coli, Proteus mirabilis, Salmonella typhimurium and Clostridum welchii produced a dense black pigment, identified as ferrus sulphide, when grown in cooked-meat media containing cystine and ferrous sulphate. This was an indicator effect resulting from the production of H2S by the bacteria in the presence of ferrous ions and was unrelated to the characteristic pigment produced by strains of B. melaninogenicus when grown on blood agar. A pigment was extracted by ultrasonic disintegration of washed cells of three reference strains of B. melanino-genicus grown for 1 week in horse-blood broth and on human-blood agar. It was intracellular or cell-associated, soluble in water and had the spectrophotometric characteristics of a derivative of haemoglobin. No such pigment was extracted from strains of B. fragilis or B. necrophorus by similar procedures. Pigment production is a stable characteristic of those strains of Bacteroides called B. melaninogenicus and it is a significant property in the classification of the Bacteroides group. However, the pigment-producing strains are not a homogenous species, and there were considerable differences between the results of biochemical tests and antibograms obtained with the three strains of B. melaninogenicus.

The development of the bacterial flora in normal neonates.

The development of the bacterial flora of neonates during the first week of life was studied in 23 babies. Specimens of meconium or faeces were collected and swabs taken from the umbilicus and mouth on days, 1, 2, 3 and 6. The bacteria present were isolated on a variety of plain and selective media. The predominant faecal organisms by the end of the first week were anaerobes. Bifidobacteria were isolated from all the neonates and bacteroides and clostridia were isolated from 78.3%. Bifidobacteria and bacteroides were present in large numbers; other species were isolated in smaller numbers. Enterococci were isolated from all neonates, enterobacteria from 82.6%, anaerobic cocci from 52.2%, and streptococci and staphylococci from 34.8% each. Staphylococcus aureus was the predominant species isolated from the umbilicus; it was isolated from 21.7% of neonates on the first day rising to 87.0% by the sixth day and represented 49% of isolates from this site. S. albus, streptococci, enterococci and Escherichia coli were each isolated from a few neonates. Viridans streptococci (31.4% of isolates) and Streptococcus salivarius (25.1%) were the commonest species recovered from the mouth. They were present from 8 h after birth; S. albus and Neisseria spp. were isolated later on the first day, and anaerobic species of Veillonella and Bifidobacterium appeared on the second day.

Characterisation of non-pigmented species of the genus Prevotella by polyacrylamide gel electrophoresis.

Gram-negative anaerobic bacilli previously known as the melaninogenicus-oralis group of Bacteroides have been assigned to a new genus, Prevotella. The non-pigmented members of this genus share several general characteristics and cannot be readily distinguished by routine tests. A polyacrylamide slab gel electrophoresis procedure, with visual analysis of protein patterns, was used to compare cellular protein patterns from clinical isolates with those from collection (reference) strains. Reference strains of P. oralis, P. veroralis, P. buccalis, P. oris, P. buccae, P. zoogleoformans, P. bivia, P. disiens, P. oulora, B. (P.) capillus and B. (P.) pentosaceus, and 91 non-pigmented isolates from patients with adult periodontal disease were examined by conventional biochemical tests, gas-liquid chromatography (GLC) and enzyme tests, and whole-cell protein profiles were obtained by SDS-PAGE. There was close correlation between patterns of results in biochemical and GLC tests and the SDS-PAGE profiles, and the species were readily distinguished in SDS-PAGE. The periodontal isolates were assigned to 10 groups by conventional test reaction patterns and nine groups by SDS-PAGE; the profiles of 79 isolates corresponded to those of seven species reference strains. By SDS-PAGE, clinical isolates of P. buccae (42 isolates) and P. oralis (eight isolates) showed good similarity with reference strains. However, for P. veroralis (15), P. oris (7), P. bivia (4), P. zoogleoformans (2) and P. buccalis (1), clinical isolates showed some minor variations from reference strains. Twelve isolates remained undesignated in SDS-PAGE analysis. Variant SDS-PAGE profiles divided clinical isolates of P. buccae into two subgroups and those of P. veroralis into five subgroups.

Transferable plasmid-mediated antibiotic resistance in Bacteroides.

Plasmid-mediated resistance to chloramphenicol (Chlr), erythromycin (Eryr), tetracycline (Tetr) and clindamycin (Clindr) was transferred from three clinical isolates of Bacteroides fragilis and one faecal isolate of B. thetaiotaomicron to strains of B. fragilis, B. distasonis and Escherichia coli, and subsequently to B. fragilis and E. coli second-and third-stage recipients in series. Successful transfer was achieved by membrane-filter and centrifugation techniques that provide stable cell-to-cell contact but not by simple mixed broth culture. Chlr Eryr Tetr and Clindr Eryr were transferred at high frequency (1.9 x 10(-3)-1.8 x 10(-4)) but Tetr was transferred at low frequencies (1-1.6 x 10(-6)). Segregation of resistance markers was observed with selection for Tetr when donors were Chlr Eryr Tetr and Chlr Tetr. All transcipients were identical with the parent recipient strains but had the resistance markers of the donor strains. Resistance to antibiotics other than tetracycline was cured by growth with subinhibitory concentrations of aminoacridines and ethidium bromide for 24 h; cure of solitary Tetr required longer incubation (21 days). Identical plasmid DNA bands were demonstrated by agarose-gel electrophoresis in all the donor and corresponding transcipient strains but plasmids were not found in the recipient strains or in strains cured of resistance. Plasmid-mediated transferable antiobiotic resistance in Bacteroides spp. may compromise the treatment of infections and may provide a reservoir of antibiotic resistance in the intestinal flora.

The assessment of faecal flora in patients with inflammatory bowel disease by a simplified bacteriological technique.

A semi-quantitative bacteriological method was used to study faecal flora in 42 patients with Crohn's disease, 37 with ulcerative colitis and 21 healthy controls. Faecal homogenates were plated on primary isolation plates by a technique that allowed the growth of various microbial isolates to be assessed on a visual 1(+)-5+ score. This method was first calibrated against a standard quantitative bacteriological technique, which confirmed the reliability and reproducibility of the results obtained by the simpler method. Patients with clinically active Crohn's disease (22) had significantly higher total aerobe scores than patients with quiescent disease (20) (p less than or equal to 0.006) or ulcerative colitis (p less than or equal to 0.04) or normal controls (p less than or equal to 0.02). The scores of Escherichia coli were parallel to those of total aerobes. Lactobacillus and bifidobacteria scores were significantly reduced in patients with Crohn's disease compared to those with ulcerative colitis and controls. The anaerobic flora in both Crohn's disease and ulcerative colitis was indistinguishable from that of controls. Bacteroides vulgatus and B. fragilis were the predominant bacteroides in all groups. Patients with ulcerative colitis, regardless of disease activity, harboured faecal flora that did not differ from that of normal controls. The abnormal faecal flora in Crohn's disease did not correlate with established clinical and laboratory indicators of disease activity.

Identification of Bacteroides species from adult periodontal disease.

Samples from deep (4-7 mm) periodontal pockets were collected from 17 patients with adult-type periodontal disease and one with the juvenile form of the disease. They were streaked immediately on selective and non-selective media and incubated anaerobically for 96 h. There was a heavy growth of Bacteroides spp. from most samples and 10 representative colonies from each sample were sub-cultured for identification. In a total of 149 isolates from patients with adult-type disease, the commonest species were B. oralis (40), B. asaccharolyticus (35), B. intermedius (31), B. fragilis (12) and B. ureolyticus (10); B. gingivalis was not detected. The distribution of species was not distorted by multiple identical isolates from individual patients. There was a heavy growth of a single species, B. ureolyticus, from the patient with juvenile-type disease.

Classification of oral pigmented anaerobic bacilli by pyrolysis mass spectrometry and biochemical tests.

Clinical (66) and reference (5) strains of pigmented gram-negative anaerobic bacilli, identified as Prevotella intermedia (47), Pr. melaninogenica (1), Pr. corpora (8), Porphyromonas asaccharolyticus (12), P. endodontalis (1) and P. gingivalis (2), were examined by pyrolysis mass spectrometry (PMS) and in conventional tests. Numerical classification based on conventional test reaction patterns (CTRPs) resolved five clusters, four comprising strains identified as Pr. intermedia, Pr. corpora, Pr. melaninogenica, and P. gingivalis respectively, and one comprising strains identified as P. asaccharolyticus and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity of chemical composition in the order Pr. intermedia, Pr. corpora, P. asaccharolyticus, which agreed with the order of homogeneity in CTRPs. PMS clusters corresponding to the genus Porphyromonas were clearly distinct from those of the genus Prevotella. PMS and CTRP classification disagreed on cluster membership for six strains. PMS identification from blind challenge sets was in agreement with conventional identification for 64 of 67 strains.

Tropical and exotic infections. Proceedings of the 5th Liverpool Tropical School Bayer Symposium on Microbial Diseases. 14 February 1998.

In summary, MDR strains of S. typhi are both epidemic and endemic in many countries in Asia and MDR S. paratyphi A has recently emerged in Pakistan. Multiple clones may be present in a given area at any time. Fluoroquinolones and third generation cephalosporins have been used widely over the past decade to treat MDR strains. The clinical superiority of fluoroquinolones is now threatened by the rapid emergence of chromosomally mediated resistance and cephalosporin resistance is also being reported. Whether these problems can be overcome by the use of newer fluoroquinolones or cephalosporins remains to be seen. Meanwhile, furazolidone and azithromycin deserve further trials, and clinical and molecular surveillance of resistance patterns remains essential.

Gas-lipuid chromatographic analysis of metabolic products in the identification of bacteroidaceae of clinical interest.

The acid end-products of 185 isolates from the family Bacteroidaceae were separated and analysed by gas-liquid chromatography on broth cultures. Different media were evaluated and definitive studies were performed in a fully supplemented complex medium. The limitations of this approach to the identification of a wide range of strains from various clinical sources were determined and the results were compared with those of a series of morphological, biochemical, tolerance and antibiotic-resistance tests. All test strains were identified to generic level by simple microscopic and colonial observations and GLC analysis; additional tests were required to allow species or subspecies identification of most strains. Population differences were detected between some species or subspecies isolated from different clinical sites by quantitative analyses of fatty acids, but individual strains could not always be separated because of overlapping ranges of distribution of acids that were common products of more than one species or subspecies. Small differences in minor products between different species or subspecies were variable and are not considered adequate for discrimination at these taxonomic levels without support from other observations. The potential application of the GLC technique to the rapid and accurate identification of these organisms in hospital laboratories is considered.

Fusobacterium necrophorum infections in England and Wales 1990-2000.

In response to a marked increase in both the number of Fusobacterium necrophorum bacteraemia reports to the PHLS Communicable Disease Surveillance Centre and the number of F. necrophorum isolates referred to the PHLS Anaerobe Reference Unit in 1999, the data from both sources on F. necrophorum infections were reviewed for the decade 1990-2000. There were 208 reports of F. necrophorum bacteraemia (average 19/year; range 14-34/year) with a peak in incidence in the late winter months; 68% were from male patients and the peak age range was 16-23 years. Of 205 referred isolates of F. necrophorum, 122 (59%) were from blood cultures and these represented 58% of the bacteraemia reports; the others were from brain and soft tissue abscesses, pleural and joint fluids, eyes, ears and lymphatic tissue. The average number of referrals was 19/year (range 9-37/year). The peak year for bacteraemia reports (34) and isolate referrals (37) was 1999; this increase was not sustained in 2000. All isolates were susceptible to metronidazole, but 2% were resistant to penicillin and 15% to erythromycin. F. necrophorum continues to be a regular but uncommon cause of bacteraemia and metastatic abscesses following an acute sore throat, especially in young, otherwise healthy adults.

The effects of pH on colonic bacteria grown in continuous culture.

A model of the proximal colon was used to investigate the effects of pH on fermentation by colonic bacteria in vitro. Twelve continuous anaerobic cultures of human faecal bacteria were maintained at constant pH in a medium simulating ileostomy effluent. Five cultures were maintained at pH 7, five at pH 6, and two at pH 5. The pH of each of three further cultures was altered after they had reached steady state, either from 7 to 6 and then to 5, or from 5 to 6 to 7. Both experimental designs showed that the pH exerted an important effect on bacterial metabolism without causing major changes in bacterial populations. Osmolality was lower in cultures run at a low pH. Total volatile fatty acid concentration was decreased at pH 5, and the production of propionic acid rather than acetic acid was favoured at pH 6. Changing the pH had no significant influence on the production of ammonia in these systems.