Oropharyngeal Gram-negative bacillary carriage: a survey of 120 healthy individuals.

BACKGROUND: The presence of aerobic Gram-negative bacilli (AGNB) in the oropharynx can be either temporary or persistent. Prolonged colonization (ie, carriage) is distinguished from transient presence (ie, acquisition), which often occurs in healthy individuals but less frequently in those with underlying disease. Prevalence rates of up to 61.1% quoted previously for healthy individuals were obtained by using single sample surveys, which fail to differentiate acquisition from carriage. STUDY OBJECTIVES: To illustrate the need to distinguish carriage from acquisition in a healthy population at risk of acquisition of AGNB, and to show that although differing groups of healthy individuals may acquire oropharyngeal AGNB at differing frequencies, carriage is rare in healthy individuals. PARTICIPANTS: Two oral rinses were obtained within a 2-day interval from 120 healthy individuals comprising 40 nurses, 40 students, and 40 laboratory-associated persons. DESIGN: Two hundred forty oral rinses were quantitatively (1:10 dilution series) cultured for AGNB by using broth enrichment. MEASUREMENTS AND RESULTS: The rate of AGNB carriage based on two consecutive samples positive for the same AGNB was 6.6%; the rate of AGNB acquisition based on one positive sample was 35.8%. The concentrations of all carried and acquired AGNB were < or = 103 cfu/mL. AGNB acquisition was significantly higher in students (52.5%) compared to nurses (32.5%) and laboratory-associated persons (22.5%; p < 0.05). CONCLUSION: Healthy individuals rarely carry oropharyngeal AGNB, suggesting effective oropharyngeal clearance in a healthy population predisposed to acquisition. Apparently, the oropharyngeal mucosa in healthy individuals is not receptive to adhesins of AGNB, resulting in rapid elimination of these bacteria.

The production of neuraminidase and fucosidase by Helicobacter pylori: their possible relationship to pathogenicity.

The pathogenicity of enterobacteria often correlates with their production of neuraminidase (sialidase). Forty-nine Helicobacter pylori isolates have therefore been examined for their production of neuraminidase and other glycosidases. All 49 isolates produced considerable neuraminidase (median 228 IU/microg protein, interquartile range 121-370), pH optimum 7.5. Nine of the 49 also produced fucosidase (median 23 IU/microg protein, interquartile range 12-39), pH optimum 7.0. Production of these enzymes did not correlate with bacterial Cag A expression or duodenal ulceration. Neutrophils exposed to neuraminidase show increased adherence to endothelium so the neuraminidase production by H. pylori could partly explain the predominant neutrophil inflammatory infiltrate seen in H. pylori-associated gastritis. Inhibition of this enzyme by use of neuraminidase-inhibitors could be a useful therapeutic approach.

Correlation between intracellular pharmacological activation of nucleoside analogues and HIV suppression in vitro.

Following intracellular activation of HIV nucleoside analogue reverse transcriptase inhibitors, their triphosphates (ddNTPs) compete with endogenous nucleoside triphosphates (dNTPs) for incorporation into proviral DNA. In this study we have examined the effect of combinations of two thymidine analogues, stavudine (d4T) and zidovudine (ZDV), and two cytidine analogues, lamivudine (3TC) and zalcitabine (ddC) on intracellular drug activation and on the relevant competing dNTP in uninfected and persistently HIV-infected cells. Endogenous triphosphates of deoxycytidine (dCTP) and deoxythymidine (dTTP) were measured using a template primer assay and the ratio of ddNTP:dNTP was calculated. Antiviral activity of two-drug combinations was also assayed by p24 ELISA. A significant reduction in d4T triphosphate (d4TTP) [0.11+/-0.09 pmol/10(6) cells to undetectable (<0.01); P=0.039] in the presence of equimolar concentrations of ZDV and d4T, resulted in a decrease in the d4TTP/dTTP ratio of 90%. ZDVTP/dTTP was not significantly altered in the presence of d4T. 3TC (10 microM) reduced total ddC phosphates by 57% and ddCTP/dCTP by 27%. 3TC phosphorylation was comparatively unaffected by ddC, up to a concentration of 10 microM ddC (>100 times the plasma concentration achieved following standard dosing). 3TC plus ddC resulted in greater p24 inhibition than 3TC or ddC alone (P<0.001). Combining one thymidine analogue (ZDV or d4T) with one cytidine analogue (3TC or ddC) resulted in greater inhibition of p24 inhibition than with any single agent. From a pharmacological viewpoint, the combination of ZDV plus d4T should be avoided, but in vitro the combination of 3TC plus ddC confers modest benefit over either drug alone. This in vitro study illustrates that decreases in ddNTP/dNTP are consistent with a reduction in antiviral effect.

Branhamella catarrhalis colonization in preschool asthmatics.

Branhamella catarrhalis has been associated with exacerbations of chronic bronchitis and asthma in adults. To investigate the possible role of B. catarrhalis in asthma of early childhood, we took posterior pharyngeal swabs from 24 normal children, 20 well asthmatics, and 20 acutely wheezy asthmatics, all between 1 and 4 years of age. On culture, 33% of the normal children were colonized with B. catarrhalis; colonization rates in the well asthmatics (70%) and in the wheezy asthmatics (75%) were significantly higher than in normals. The nature of this association requires further study.

Oropharyngeal gram-negative bacillary carriage in chronic obstructive pulmonary disease: relation to severity of disease.

The prolonged presence of aerobic Gram-negative bacilli (AGNB) in the oropharynx is termed 'carriage'. AGNB carriage rates are low in populations of healthy individuals. Previously, severity of underlying disease has been positively correlated with oropharyngeal AGNB carriage rate. Overgrowth of AGNB at the oropharynx poses a significant risk of endogenous infection in end-stage chronic obstructive pulmonary disease (COPD) patients. The aims of this study were to undertake an epidemiological survey of the oropharyngeal flora of COPD patients and to correlate oropharyngeal carriage of AGNB with severity of disease. Two oral rinses were obtained, within a 2-day interval, from 40 COPD patients comprising three disease severity groups: 1. mild, 2. moderate and 3. severe. Eighty oral rinses were quantitatively (1:10 dilution series) cultured for AGNB and yeasts using broth enrichment. The mean AGNB carriage rate was 15%. AGNB carriage rates of 0, 7.7 and 29.4% were observed within the mild, moderate and severe disease groups, respectively. The mean yeast carriage rate was 33.3%. Yeast carriage rates of 33.3, 15.4 and 64.7% were observed within the mild, moderate and severe disease groups, respectively. Carriage of Staphylococcus aureus was 5%. Rates of oropharyngeal carriage of AGNB (1/23 vs. 5/17) and yeasts (5/23 vs. 11/17) were significantly higher within the severe disease group than in non-severe disease groups. Oropharyngeal carriage of AGNB in end-stage COPD patients (forced expiratory volume in 1 sec, FEV1 < 50% predicted) presents a potential source of Gram-negative endogenous pneumonia. This outcome may be promoted by intubation and some flora-suppressing antibiotic therapies.

Does the fibrin coat around a central venous catheter influence catheter-related sepsis?

The purpose of this study was to determine whether the fibrin sheath that develops around an intravenous silicone catheter influences catheter-related sepsis. A rat model with a central intravenous silicone catheter was used. Staphylococcus aureus, dose 1 x 10(7) colony forming units (cfu), were injected via the tail vein, either immediately after catheter insertion (group 1, n = 23) or after the catheter had been in situ for at least 7 days (group 2, n = 22). Blood cultures were done on at 24 hours and 7 days. Animals were killed on day 7 and the catheter was removed for culture (Maki and broth) and scanning electronmicroscopy (SEM). The was no significant difference (P > .05) between the number of positive blood cultures in groups 1 and 2 at 24 hours (16 v 9) and 7 days (12 v 6). In group 1 there were significantly more positive catheter cultures by both methods (23 v 16 in group 2; P < .05) and more cfu/per centimeter catheter (group 1 mean, 520, range, 197 to 600; group 2 mean 195, range 9 to 600; P < .001). In group 1, 12 animals had catheter sepsis compared with 5 in group 2 (P = NS). On SEM a fibrinous sheath was identified on all catheters removed on day 7 but not on 5 catheters inserted and removed after 10 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Multicentre randomised double bind crossover trial on contamination of conventional ties and bow ties in routine obstetric and gynaecological practice.

OBJECTIVE: To assess level of contamination of neckwear worn by gynaecologists and obstetricians during routine working week. DESIGN: Multicentre randomised double blind crossover trial. Participants wore the same conventional ties for three days in one week and bow ties for the same period in second week. SETTING: Two teaching and three district general hospitals in the midlands, Wales, and north England. SUBJECTS: 15 registrars and senior registrars. INTERVENTIONS: A swab soaked in sterile saline was taken from specific area on ties at end of first and third working days and sent in transport medium for culture on chocolatised blood and MacConkey agar for 48 hours. MAIN OUTCOME MEASURES: Level of bacteriological growth assessed semiquantitatively (0 for no contamination; for heavy contamination) after swabs had been cultured. At end of study the participants completed a questionnaire to assess their attitude toward wearing different types of necktie. RESULTS: 12 doctors (80%) completed the study. Although bow ties were significantly less contaminated at end of first working day (z = -2.354, p = 0.019), this difference was not maintained; there was no difference in level of contamination on third day. Level of contamination did not increase between first and third day of wearing the same garment. One of the 10 doctors who returned the questionnaire found the bow tie very uncomfortable. All participants would consider wearing a bow tie if it proved to be less contaminated than a conventional tie. CONCLUSIONS: Although a significant difference in contamination was established between conventional and bow ties on first day of study, this difference was not confirmed on third day and there is unlikely to be any real association between tie type and bacterial contamination. Because of its negative image and difficulty to tie, the bow tie will probably remain a minority fashion.

Comparison of four methods for detection of rotavirus in faeces.

Faecal samples from 325 children with gastroenteritis and 23 children without gastroenteritis were examined for the presence of human rotavirus (HRV) using four different methods. Using the WHO-ELISA, HRV was found in the stools of 98 (30%) symptomatic and 2 (9%) asymptomatic children. A latex particle agglutination test had the highest sensitivity (92%) but the lowest specificity (96%). Both electron microscopy and polyacrylamide gel electrophoresis of HRV RNA (RNA-PAGE) were highly specific (100%) but of lower sensitivity (73% and 84% respectively). Of the four methods tested latex particle agglutination is the simplest and since it requires little extra equipment is ideally suited for bedside tests in tropical countries. It is, however, not cheap. An alternative is to use RNA-PAGE which will require some equipment and a power supply but which is relatively cheap and will also provide epidemiological data.

Ceftazidime treatment in cystic fibrosis: resistant organisms in sputum and faeces.

Ceftazidime was used as the only intravenous agent for treating lower respiratory tract infections in patients with cystic fibrosis. The risks of inducing beta lactamases and conferring antibiotic resistance are high when monotherapy is used; so the emergence of resistant bacteria was studied prospectively in the sputum of 120 patients. The mean age of patients was 9.0 (range 0.3-25) years and there were equal number of male and female patients. Pseudomonas aeruginosa was the only ceftazidime resistant bacterium to be isolated from the respiratory tract, and was identified only in chronically colonised patients. Ceftazidime resistance occurred in 103 (14%) of 750 P aeruginosa isolates, and in 16 of 36 chronically colonised patients. Ceftazidime resistant organisms were isolated from the faeces of 17 of 64 patients investigated. Eighty two per cent of the resistant faecal organisms were single isolates: the same resistant organism in faeces was isolated from successive samples in only two patients. In no case was the ceftazidime resistant enteric isolate the same as that from sputum. Patients chronically colonised by P aeruginosa did not harbour ceftazidime resistant enteric organisms any more than non-colonised patients. The use of ceftazidime as a single intravenous agent in treating chest exacerbations in cystic fibrosis does not induce a reservoir of ceftazidime resistant bacteria.

Long-term potentiation in the interpositus and vestibular nuclei in the rat.

Previous unpublished experiments from this laboratory had revealed only post-activation depression effects in the cerebellar cortex when its inputs were activated by high frequency trains. In the experiments reported in this paper, we found reliable long-term potentiation (LTP) effects in the deep nuclei (interpositus and vestibular) when stimulation trains were applied to the white matter at the point where inferior peduncle fibers enter the cerebellum. LTP effects were found in both acute and chronic preparations. In the chronic preparations, LTP lasted for at least 8 days in all but one animal.

A novel mucin sulphatase from human faeces: its identification, purification and characterization.

1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3-7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15,000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.

Bacterial factors in the formation of renal scars. An experimental study on the role of Escherichia coli P-fimbriation and hydrophobicity.

A model capable of comparing the effects of bacterial virulence factors on renal scarring in vivo has been developed using the female piglet. By creating, at open surgery, unilateral vesicoureteric reflux (VUR) and quantifying scarring both by uptake of an isotope bound to functioning renal parenchyma and by planimetry of the surface area scarred, the effects of 2 organisms, a P-fimbriate Escherichia coli and an E. coli K1 have been compared. The P-fimbriate E. coli was shown to express P-fimbriae in freshly voided urine, was more hydrophobic and produced smaller scars. This indicates that neither the hydrophobicity nor P-fimbriation of the organism causing urinary tract infection (UTI) is of prime importance for the development of renal scars and is evidence against the "big bang" theory for the development of renal scars. Studies on the association of UTI with VUR showed that infection with both E. coli under study led to VUR on the side contralateral to the side undergoing surgery. It seems likely that a non-specific effect of UTI, such as bladder oedema, is responsible for this acquired VUR. An effect of the 2 bacteria under study on the lower urinary tract was observed in that infection with the P-fimbriate E. coli allowed the retention of an intravesical wax plug, whereas infection with E. coli K1 did not. Epidemiological data have shown that the majority of upper urinary tract infections in children are associated with UTI by P-fimbriate organisms. Such an association may be explained in part by an effect of P-fimbriate bacteria on lower urinary tract function rather than an effect on the upper urinary tract.

Reconsideration of the roles of urinary infection and vesicoureteric reflux in the pathogenesis of renal scarring.

In an experimental study of female piglets with surgically created unilateral vesicoureteric reflux, Escherichia coli were inoculated into the bladder and, at later sacrifice, bacterial culture was undertaken of renal parenchyma from the refluxing and non-refluxing kidneys. Positive cultures of the same E. coli were obtained from 33% of refluxing kidneys with pyelonephritic renal scars, 23% of refluxing kidneys without scars and 21% of non-refluxing kidneys. Although other aspects of the experiments confirmed that a combination of urinary infection, vesicoureteric reflux and intra-renal reflux is a necessary precondition for renal scarring, these findings indicate that reflux plays a role in the pathogenesis of renal scarring over and above a means whereby pathogens gain access from the lower urinary tract to the renal substance. Possible mechanisms are discussed.

Meningococcal disease in Malawi: studies on the genetic relatedness of the bacteria.

Seventy-seven meningococci, isolated from patients and carriers during a large epidemic of meningococcal meningitis in Malawi, were characterized in terms of antimicrobial susceptibility, plasmid content and multi-locus enzyme electrophoresis (MLEE). All the isolates were sensitive to chloramphenicol but six had high enough minimum inhibitory concentrations of penicillin (> or = 2 mg/litre) to render them clinically resistant. Only one isolate was sensitive to sulphonamides but all the isolates were sensitive to rifampicin and ciprofloxacin, two drugs that would be suitable alternatives in prophylaxis. None of the isolates carried plasmids. MLEE indicated that 32 (80%) of the cerebrospinal fluid isolates and 22 (69%) of those from carriers were closely related genetically (in two electropherotypes that differed at only one allele). The Malawian group A meningococci differed from three Ethiopian isolates by two or three alleles, indicating that direct spread from the sub-Saharan meningitis belt to Malawi was unlikely.

Faecal mucinase activity assessed in inflammatory bowel disease using 14C threonine labelled mucin substrate.

Previous studies have shown the presence in faeces of sulphatases, sialidases, glycosidases, and proteases relevant to mucus degradation, but the relative role of these enzymes in the degradation of colonic mucus has been unclear. A total mucinase assay using 14C threonine biologically labelled human colonic mucin as substrate was therefore developed in this study. Faecal mucinase activity of a pooled normal faecal filtrate was capable of removing 80% of the 14C threonine label from mucin within eight hours incubation, but 20% remained intact despite prolonged incubation. The pH profile of mucinase activity is broad (pH 4.5-9.5) suggesting contribution from multiple enzymes. Mucinase activity was reduced by preincubation with 100 micrograms/ml chymostatin (82.8%), 0.5 mg/ml EDTA (91.6%), and 4 g/l bismuth subsalicylate (72.0%). All 55 faecal samples studied contained detectable mucinase activity, measured as dpm release/micrograms protein/hour, which was greater in samples from patients with ulcerative colitis (n = 17, median 52.7, interquartile range 32.9-66.9), than controls (n = 26, 34.4, 26.8-40.4, p < 0.02) or patients with Crohn's disease (n = 12, 35.5, 17.5-55.7, p < 0.05). There was, however, no significant difference in faecal mucinase activity between inactive and active ulcerative colitis. These results suggest that faecal mucinase activity is one factor contributing to the thin mucus layer in ulcerative colitis and represents a potential target for drug treatment.

Cloxacillin and sodium fusidate in management of shunt infections.

The effectiveness of two oral antibiotics, cloxacillin and sodium fusidate, has been evaluated in the treatment of shunt infections among 37 patients allocated at random to two treatment groups. Both proved to be safe bactericidal agents giving adequate serum M.I.C. when taken by mouth. Treatment should always be started on the basis of the clinical presentation without waiting for the bacteriologist's report. The commonest infecting organism is Staphylococcus aureus. Nine shunts were lost in this study, eight through Staphylococcus aureus infection. The nasal carrier state is of considerable importance in perpetuating these shunt infections.

Zidovudine phosphorylation and mitochondrial toxicity in vitro.

Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDVTP) by the host's intracellular enzymes. The initial phosphorylation step is conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for thymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondrial damage. It has been suggested that ZDV cytotoxicity correlates with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metabolism of ZDV in five lymphocyte/monocyte cell lines, U937, BSM, MOLT 4, JJAHN, and RAJI (4 x 10(6) cells), was investigated following 24 h incubation with [(3)H]ZDV (1.2 microCi; 0.1 microM) and cytotoxicity was determined by the MTT assay. Cytotoxicity was closely related to intracellular concentrations of the major metabolite (ZDVMP) but not with the active metabolite ZDVTP. ZDVMP was the only metabolite detected following incubation of viable mitochondria isolated from U937 cells with ZDV (1.2 microCi; 0.1 microM; 24 h) with mitochondrial levels of 0.27 +/- 0.11 pmol/microg protein (mean +/- SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells there was an increase in ZDV cytotoxicity compared to unstimulated cells. The results suggest that the mitochondrial isozyme of thymidine kinase (TK2) plays only a minor part in ZDVMP formation. Following PHA stimulation, activation of the cytosolic thymidine kinase isozyme (TK1) is associated with increased toxicity of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol.