Performance of converted pressure cookers and two conventional jars for anaerobic bacterial culture.

The simple conversion of commercial pressure cookers into inexpensive anaerobic jars is described. These containers were shown to be as good as the small conventional BBL polycarbonate GasPak and large vented 150 gas-replacement jars when assessed by means of three biological indicators: Pseudomonas aeruginosa, Bacteroides melaninogenicus, and Bacteroides fragilis. Ps. aeruginosa seeded on Simmond's citrate agar was shown to be the most sensitive indicator of the three for traces of oxygen.

Phenomenon of resistance to Augmentin associated with sensitivity to ampicillin: occurrence and explanation.

Ampicillin was found to be some tenfold more active than amoxycillin against Enterobacter cloacae. This finding explains the observation that some Ent cloacae strains are sensitive to ampicillin in the disc test but resistant to Augmentin. Ampicillin was also found to be more active than amoxycillin against Citrobacter freundii and Serratia marcescens. In view of these findings, the practice of using ampicillin discs to predict sensitivity to amoxycillin should be reconsidered. The use of both ampicillin and amoxycillin discs is appropriate if errors are to be avoided.

Pre-opsonisation of Escherichia coli induces resistance to neutrophil killing in serum and urine: relationship to growth phase.

Tests of phagocytosis and killing by polymorphonuclear neutrophil leucocytes (PMNL) are usually done with pre-opsonised organisms. Phagocytosis of 11 strains of Escherichia coli, pre-opsonised, and in the stationary phase, resulted in the killing of only one strain although all the organisms were phagocytosed. However, when the same strains were added unopsonised to a PMNL-serum mixture, eight were killed after phagocytosis. With two of these strains, the amount of killing was inversely proportional to the time of pre-oposonisation. E. coli incubated for 30 min in dilute peptone water in Hanks's Balanced Salts Solution before phagocytosis also became resistant to killing; bacterial division did not occur during this period. Experiments with bacteria in urine confirmed these findings and showed that E. coli exposed to serum or urine before phagocytosis became resistant to killing by PMNL. E. coli rapidly changes its sensitivity to phagolysosome killing during transition from stationary to lag phase in a nutrient medium. This resistance is retained through the exponential phase but is lost during the stationary phase. The killing of Pseudomonas, Enterobacter, and Acinetobacter by PMNL was unaffected by varying the method of opsonisation or the phase of growth. If this phenomenon occurs in vivo it may affect the outcome of infections caused by strains of E. coli that survive killing by PMNL.

Effect of bacterial products including endotoxin on neutrophil function in infected urine.

Experiments were performed to determine the effects of products of bacterial growth (including endotoxin) on phagocytosis and intracellular killing by polymorphonuclear leucocytes (PMNL) in urine. Bacteriologically filtered supernates of two strains of Escherichia coli grown in urine were added in varying amounts to mixtures of PMNL and E. coli, also in urine. Phagocytosis of the two strains was reduced from > 90% in controls to 66% and 48%, respectively, in the presence of undiluted culture filtrate (containing endotoxin 2-2.5 micrograms/ml). Intracellular killing was also decreased and was abolished by dilutions corresponding to endotoxin concentrations of 0.6 and 0.75 micrograms/ml. When PMNL exposed to these inhibitory dilutions were resuspended in fresh urine, their phagocytic ability was fully restored and 13-24% of their killing activity was regained. A minimum concentration of commercially purified E. coli endotoxin of 200 micrograms/ml was required to abolished PMNL killing, with phagocytosis uninhibited. The results strongly suggest that bacterial growth metabolites, not endotoxin, are responsible for the depression of phagocytosis and intracellular killing in infected urine. A moderate dilution of the bacterial products in urine permits good PMNL function. Extrapolating this to the clinical situation, diluting the urine by water loading (as recommended for patients with urinary infections) should ensure efficient activity of PMNL under in-vivo conditions providing urinary pH and osmolality are not adversely affected.

Rapid screening for urease inhibitors.

Two methods are described that are suitable for the rapid screening of compounds as urease inhibitors. The first utilizes an electrode sensitive to NH4+ ions; the second is dependent on pH rise. A feature of both is a direct readout of reaction rate.

Opsonophagocytosis in infected urine: relation to pH and osmolality.

Polymorphonuclear neutrophils (PMN) in freshly voided urines from 20 symptomatic bacteriuric patients were examined. Although the PMN were viable (median 85%), in only 2 cases could phagocytosis of the infecting organisms be demonstrated, even after the addition of serum opsonins. Polymorphonuclear neutrophils from urines of 12 patients were also unable to phagocytose added opsonized Staphylococcus aureus. These urines were found to be of pH < 6.0 and/or osmolality > 700, or < 180 mOsm. However, the phagocytic function of these PMN was restored when transferred to Hanks balanced salt solution (HBSS). By contrast, most PMN in urines of suitable pH (> or = 6.0) and osmolality (between 200 to 700 mOsm.) phagocytosed the opsonized S. aureus. When bacteria cultured from the infected urine were incubated in the same urine and then transferred to HBSS, in 17 of 19 cases opsonization occurred and the organisms were phagocytosed when PMN, isolated from blood, were added. IgG appeared to be the prime opsonin in the urines, and heat-stable opsonins for S. aureus were also present. It is concluded that lack of opsonization is not a major cause of the absence of phagocytosis by urinary PMN. Low pH and adverse osmolality are largely responsible, correction of which may restore PMN function in vivo.

Effect of alkalinisation and increased fluid intake on bacterial phagocytosis and killing in urine.

The effect of alkalinisation and increased fluid intake on bacterial phagocytosis and killing in urine was studied. Phagocytosis of Escherichia coli and Staphylococcus saprophyticus by polymorphonuclear neutrophils (PMN) took place in only one of three first voided early morning urine samples from volunteers, and no bacterial killing occurred. This was attributed to the high osmolality (690 to 720 mOsm) and low pH of the early morning urine; two samples were pH 5.8 and the third, in which phagocytosis occurred, was pH 6.4. Afternoon urine samples from the same volunteers had lower osmolality and a higher pH, with a high rate of phagocytosis (> or = 88%) and 55-69% killing. PMN remained viable (mean 94%) after exposure for 1 h to both early morning and afternoon urine. Volunteers taking 4 g sodium citrate showed a mean rise of urinary pH of 1 unit. Water loading in patients with urinary tract infections produced a bacteria to neutrophil ratio conducive to bacterial killing, bacterial counts being reduced by a mean of 2.5 logs after 1-3 h without a corresponding reduction in neutrophils. Thus, raising the pH and reducing the osmolality of urine increases the ability of neutrophils to eliminate infecting organisms.

Effect of pH and osmolality on in vitro phagocytosis and killing by neutrophils in urine.

Phagocytosis and intracellular killing of two strains of Escherichia coli and a Staphylococcus saprophyticus by polymorphonuclear neutrophils (PMN) in pooled sterile urine at three osmolalities (800, 485, and 200 mosM/kg of H2O) between pHs 5 and 8 was investigated. Urine at 800 mosM virtually abolished phagocytosis of both E. coli strains, regardless of pH, and reduced the phagocytosis of S. saprophyticus to 30%; no killing of any organisms took place at this osmolality. On the other hand, phagocytosis was a good in urine as in Hanks balanced salt solution at both 485 and 200 mosM between pHs 6 and 8. Phagocytosis of all three strains was virtually abolished at pH 5. Killing of the strains by PMN was optimal between pHs 6.5 and 7.5 in urine at 485 mosM (being at least 90% of the control values in Hanks balanced salt solution), whereas at 200 mosM killing was reduced to 50 to 70% of these values. Reduced killing of all three strains occurred at pH 8, whereas at pH 6 only S. saprophyticus was killed. Thus, the bactericidal activity of PMN in urine was more sensitive than phagocytic function to alterations in pH. The dominant modulating factor affecting PMN function in urine of 500 mosM or less was pH, but osmolality had a greater influence at 800 mosM. Thus, raising the pH of urine and reducing the osmolality may increase the ability of natural defense mechanisms to eliminate infecting organisms.

Antibody-coated bacteria in urine: criterion for a positive test and its value in defining a higher risk of treatment failure.

Midstream urines from 237 patients with significant bacteriuria were examined for antibody-coated bacteria (ACB): 113 urines gave a positive result. When more than 25 000 organisms from the ACB-positive urines were scanned either fewer than 100 or more than 250 fluorescent bacteria (ie, greater than 1%) were found: thus the distribution of the fluorescent bacteria was bimodal. To compare the effectiveness of therapy in patients with ACB-positive and ACB-negative bacteriuria, 120 randomly selected patients were given a 7-day course of appropriate therapy. The results were assessed after 6 weeks. In 66 ACB-negative patients the cure rate was 84.8%, which was not significantly different from that found in 26 patients (80.7%) whose urine contained ACB in small numbers (less than 1%). In contrast, in 28 patients whose urine contained greater than 1% ACB the cure rate was only 36%. There was no correlation between the immunoglobulin class coating the bacteria and the response to treatment. The presence of greater than 1% ACB in the midstream urine thus identifies patients who are at high risk of treatment failure.

Periurethral enterobacterial carriage preceding urinary infection.

The periurethral enterobacterial flora was identified before infective episodes in 56 patients with recurrent urinary infection. There were 91 episodes of infection, with colonisation by aerobic gram-negative bacilli in 60. In only 31 (34%) episodes were patients colonised with the infective strain. In 31 episodes there was no colonisation of the perineum and in 29 there was heterologous colonisation. In another group of 54 women investigated during an enterobacterial infection of the urine there was colonisation with the infecting organism in 55 (86%) of 64 episodes; in 2 there was no colonisation; and 7 (11%) were associated with a heterologous strain. Women who have recurrent urinary infections are susceptible to perineal and periurethral colonisation with gram-negative bacteria but the infection need not be with the colonising enterobacteria.

A clinical comparison between Macrodantin and trimethoprim for prophylaxis in women with recurrent urinary infections.

Seventy-two patients with a history of at least three attacks of urinary infection in the previous 12 months were assigned randomly to long-term prophylaxis with 100 mg at night of either Macrodantin (34 patients) or trimethoprim (38 patients). The mean interval between symptomatic attacks while on either treatment was increased three-fold compared with the pretreatment period. Macrodantin was significantly more effective (P less than 0.05) at preventing bacteriuria. Prophylaxis was equally effective in patients with and without a radiological abnormality. Side effects were significantly more common (P less than 0.05) in the group taking Macrodantin. In patients taking trimethoprim acquisition of resistance by faecal coliforms occurred at a rate of about 5%/month, and breakthrough infections were almost exclusively caused by trimethoprim-resistant coliforms. No acquisition of resistance occurred in patients taking Macrodantin, and the few breakthrough infections noted were due to sensitive bacteria.

Long-term prophylaxis of urinary infections in women: comparative trial of trimethoprim, methenamine hippurate and topical povidone-iodine.

We randomized 64 patients with a history of recurrent urinary tract infections among 3 regimens of long-term (1 year) prophylactic treatment: 20 were given 100 mg. trimethoprim at night, 25 received 1,000 mg. methenamine hippurate every 12 hours and 19 were asked to cleanse the perineum (especially the periurethral area) twice daily with povidone-iodine solution. The progress of patients in terms of urinary symptoms and/or bacteriuria, changes in periurethral flora, side effects, and hematological and biochemical profiles was followed at regular intervals. All treatments were effective in reducing the incidence of symptomatic attacks when compared to the 12 months immediately before therapy and there was little to choose between the individual regimens on this account. However, trimethoprim was tolerated better than were the other 2 treatment regimens. In the group given trimethoprim most of the breakthrough infections (71.4 per cent) that occurred were caused by trimethoprim-resistant organisms (usually Escherichia coli), while in the other 2 groups the incidence of trimethoprim-resistant organisms causing infection was low (2.7 per cent). Treatment with trimethoprim reduced significantly the periurethral colonization of Escherichia coli.