The effect of bile acids on the growth and adherence of Helicobacter pylori.

Bile reflux gastritis occurs in the absence of Helicobacter pylori (H. pylori). The aim of this study was to see if the bile acids cheno or ursodeoxycholic acid affected the growth or adherence of H. pylori in vitro. Twenty-seven strains growth were inhibited by 0.1% chenodeoxycholic acid whereas only 11 out of the 27 were inhibited by 0.1% ursodeoxycholic acid. Growth was totally inhibited by a combination of 0.05% chenodeoxycholic acid +0.05% ursodeoxycholic acid. Chenodeoxycholic acid was a more effective inhibitor of adherence in that the number inhibited and percentage inhibition were greater than with ursodeoxycholic acid. Bile salts might be useful in the treatment of H. pylori infection.

Nebulized amphotericin B as prophylaxis against invasive aspergillosis in granulocytopenic patients.

The efficacy of inhaled amphotericin B in prevention of invasive aspergillosis in patients with granulocytopenia (granulocytes less than 0.5 X 10(9)/l for greater than 10 days) was investigated over a 12-month period. Amphotericin B prophylaxis was administered twice daily for the period of granulocytopenia to 34 patients who were at risk during 144 episodes of granulocytopenia. The cohort at risk was compared with historical controls. In the 2 years prior to institution of prophylaxis, 14 patients (11.4% of those at risk) developed invasive aspergillosis. All cases occurred whilst the patients were nursed on the open wards. Aspergillosis did not develop in 25 granulocytopenic patients nursed in single rooms with HEPA filtration. Since institution of prophylaxis, there have been no cases of invasive aspergillosis. These data suggest that nebulized amphotericin B may be useful in preventing invasive pulmonary aspergillosis in granulocytopenic patients, especially those nursed on the open wards, and warrants further investigation.

An outbreak of multiply resistant Serratia marcescens: the importance of persistent carriage.

An outbreak of multi-resistant Serratia marcescens involving 24 patients occurred in a bone marrow transplant and oncology unit, from September 1998 to June 1999, of whom 14 developed serious infection. This is the first such outbreak described in a BMT unit. All isolates demonstrated the same antimicrobial susceptibility pattern and were the same unusual serotype O21:K14. The antimicrobial susceptibility profile showed reduced susceptibility to ciprofloxacin, gentamicin and piperacillin-tazobactam. As the latter two antimicrobials are part of our empiric therapy for febrile neutropenia, they were substituted with meropenem and amikacin during the outbreak. Investigation revealed breaches in infection control practices. Subsequently, the outbreak was contained following implementation of strict infection control measures. A prominent feature of the outbreak was prolonged carriage in some patients. These patients may have acted as reservoirs for cross-infection. This report also indicates that patients who become colonised with Serratia marcescens may subsequently develop invasive infection during neutropenic periods.

Small intestinal bacterial overgrowth--an incidental finding?

OBJECTIVES: To assess the prevalence of typical clinical features and need for treatment of small intestinal bacterial overgrowth (SIBO) in the elderly. DESIGN: Random selection of patients, regardless of their nutritional status. SETTING: Acute admissions ward in the Dept. of Medicine for the Elderly. PATIENTS: Thirty elderly patients between 68 and 90 years of age. MEASUREMENTS: Active clinical problems, including the presence of recent weight loss and diarrhea, were recorded. Routine blood tests, including serum vitamin B12, red cell folate, albumin and calcium, and qualitative small bowel bacteriology results, were analyzed. The effect of age on all variables was studied. RESULTS: Twenty of the 30 small bowel aspirates had proven SIBO, and strict anaerobes were isolated in 15. The mean blood test values did not differ significantly between culture-positive and culture-negative patients. There was no significant correlation between those variables and the total bacterial counts. Of the 20 proven SIBO cases, eight had anemia, five had hypoalbuminemia, five had diarrhea, four complained of recent weight loss, and none had B12 deficiency. Alternative causes other than SIBO were identified for many of these abnormalities. Advancing age correlated significantly with rising counts of small bowel strict anaerobes. CONCLUSIONS: These data suggest that age may be a predisposing factor in the development of anaerobic overgrowth but that SIBO is a benign entity in the elderly. Contrary to previous recommendations, treatment of this condition is not routinely indicated.

Coagulase testing compared with commercial kits for routinely identifying Staphylococcus aureus.

Five commercial Staphylococcus aureus identification kits--Staphaurex (Wellcome), Staphylase (Oxoid), Staphyslide (bioMèrieux), Biostaph (Medlabs) and Bacto Latex (Difco)--were evaluated for the routine identification of S aureus from primary plates in the routine microbiology laboratory. Comparison was made with two methods of tube coagulase testing and five slide methods for detecting clumping factor (slide coagulase testing). Performances were assessed for two groups of organisms, staphylococcal species alone and a combined staphylococcal and non-staphylococcal species group. The effects of growth on selective media and storage of isolates at room temperature and 4 degrees C were investigated. Selective media cannot be recommended, nor can storage of isolates before testing. Ranked according to efficiency value with the combined staphylococcal and non-staphylococcal species group, the kits and coagulase methods performed as follows (the figures in parentheses are the efficiency values for the staphylococcal group alone): tube coagulase reference method 100% (100%), tube coagulase SJH method 99% (99%), Staphaurex 94% (97%), Staphylase 93% (96%), slide coagulase method No 4 93% (94%), slide coagulase method No 5 93% (93%), Bacto Latex 92% (95%), Staphyslide 92% (95%), and Biostaph 87% (91%). It is concluded that a commercial S aureus identification kit should not replace tube coagulase testing for the routine identification of the organism from primary plates and that, even the kits with the best performances, have little advantage over a good slide coagulase test method.

New method for detecting slime production by coagulase negative staphylococci.

An alternative method for detecting the production of slime by coagulase negative staphylococci was compared with the routinely used Christensen method on 124 isolates of coagulase negative staphylococci from carriage sites, blood cultures, and infected peritoneal dialysis fluids. The alternative method requires the use of a specially prepared solid medium--brain heart infusion broth, supplemented with 5% sucrose, and Congo red stain. Of the 124 tests, there was complete agreement between methods in 107 and only one strain was clearly negative by Christensen's method while positive on Congo red agar. The Congo red method is rapid, sensitive, and reproducible and has the advantage that colonies remain viable on the medium. It is also not subject ot interbatch variation of media which sometimes affects the reproducibility of the Christensen method.

Cross infection in a surgical ward caused by Pseudomonas aeruginosa with transferable resistance to gentamicin and tobramycin.

An outbreak of gentamicin- and tobramycin-resistant Pseudomonas aeruginosa infection occurred in a surgical ward over a three-month period. Resistant Ps. aeruginosa strains with the same serological, phage, and pyocin type were cultured from the urine of six patients. Identical organisms were found on urine bottles, bedpans, and the hands of attendant staff. Inadequate disinfection played a major role in cross-infection. Isolates of the epidemic strain from each of the patients and of an unrelated but similarly resistant Ps. aeruginosa from one of them could transfer resistance to a recipient strain of Ps. aeruginosa. Resistance to gentamicin, kanamycin, tobramycin, sulphonamides, and mercuric chloride was determined by R factors belonging to Pseudomonas incompatibility group P-3. Aminoglycoside resistance was due to acetylation.

Enhanced cultivation of Helicobacter pylori in liquid media.

AIMS: To evaluate a technique for culture of Helicobacter pylori in large quantities of liquid media and to determine the factors that could influence the results. METHODS: Fifteen clinical isolates of H pylori and a reference strain of H pylori NCTC11637 were used to evaluate a method to cultivate the organism in 100 ml liquid medium comprising brain heart infusion broth with 5% horse serum and 0.25% yeast extract. Tissue culture flasks containing the inoculated liquid medium were placed in a CO2 incubator with 5% CO2 for 2 hours and then incubated in a shaking incubator at 120 rpm. RESULTS: All the clinical isolates and the reference strain grew in the broth, although only a moderate growth of the reference strain occurred. Inoculum size significantly influenced the kinetics of growth of H pylori in the liquid medium. Vancomycin, nalidixic acid, and amphotericin B, used to suppress contamination, did not affect growth of H pylori in the medium. CO2 was essential for H pylori to grow or survive in the liquid medium. Incubation with CO2 in a CO2 incubator for 30 minutes or 2 hours did not affect the results. CONCLUSIONS: H pylori can be cultivated in large quantities of brain heart infusion broth with 5% horse serum and 0.25% yeast extract. Initial inoculum concentrations influence the kinetics of H pylori growth in the liquid medium. Vancomycin, nalidixic acid, and amphotericin B can be used as selective antimicrobial agents. CO2 is essential for initial growth of H pylori in liquid media. The findings in this study may provide a useful, reproducible, and simple method for biochemical, molecular, and physiological studies of H pylori, when those require large quantities of the organism.

Epidemiological information from active and passive pyocine typing of Pseudomonas aeruginosa.

An investigation was carried out to determine the value of active and passive pyocine typing in the study of Pseudomonas aeruginosa infections acquired in hospital. Active typing was a more reliable and reproducible method than passive typing. Both methods were used in studies of nine outbreaks of infection. In six of these episodes there was good agreement between the two methods. Less clear-cut results were achieved in the remaining three episodes. In one of these, active typing gave more valuable information. However, both methods are easy, convenient and of value in epidemiological studies.

The use of RAPD-PCR as a typing method for Serratia marcescens.

Serratia marcescens has emerged in the last few years as an important nosocomial pathogen. Many methods for typing this organism have been described. In this study the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was shown to be a convenient typing method for S. marcescens. Different combinations of primers previously used for typing other gram-negative bacilli were assessed. The combination of primer HLWL-74 and 1254 gave distinguishable patterns for different serotypes and proved to be the most satisfactory. By applying this combination to 175 isolates of S. marcescens, which could be classified into 38 groups on the basis of serotyping and phage typing, 73 different RAPD patterns with good reproducibility were obtained. This is, to our knowledge, the first application of the method to a large collection of S. marcescens representing a wide range of serotypes.

Increased expression of Fc gamma receptors on neutrophils and monocytes may reflect ongoing bacterial infection.

The Fc gammaR receptors for IgG, Fc gammaRI, Fc gammaRII and Fc gammaRIII were measured on neutrophils and monocytes from 36 patients suspected of systemic infection. These results were compared with 30 blood donor controls to assess the level of expression as an early indicator of bacterial infection. Fc gammaRI expression on neutrophils was found to be significantly increased from patients with systemic or localised infections, when compared to non-infected patient group, i.e., patients with no cultural evidence of bacterial infections, (p=0.02, p=0.04) or normal controls (p<0.0001, p=0.0005). Fc gammaRI expression on monocytes was also significantly increased in both of the infected groups compared to normal controls (p<0.0001, p=0.001); however, no significant difference could be seen when compared with the non-infected patients. Fc gammaRIII was found to be significantly increased on a subset of monocytes in patients with systemic or localised infections compared to the non-infected group (p=0.009, p=0.006) and compared to the normal controls (p=0.009, p=0.003). Infections caused by gram-negative bacilli induced a higher Fc gammaR response than infection with either streptococci or staphylococci. These data suggest that the measurement of Fc gammaRI on neutrophils and Fc gammaRIII on monocytes may be a useful rapid indicator of bacterial infection.

Determination of the optimal transport system for Helicobacter pylori cultures.

A range of solid and liquid media was evaluated for the ability to maintain survival of Helicobacter pylori strains under different conditions. Chocolate agar slopes maintained survival of most strains for longer than 3 days, some strains surviving for up to 9 days, despite a decreased number of viable cells. Temperature and atmosphere did not significantly influence the performance of these slopes. The BBL Campy Pouch system also achieved a considerable recovery rate of H. pylori after storage for 3 days at the same range of temperatures. Brain-heart infusion broth with horse serum was superior among the liquid media tested, maintaining the viability of H. pylori for c. 3 days at temperatures ranging from -4 degrees C to 21 degrees C. Chocolate agar slopes are recommended as suitable for transport of H. pylori strains.

Pre-formed urease activity of Helicobacter pylori as determined by a viable cell count technique--clinical implications.

The pre-formed urease activity of three NCTC reference strains and five clinical isolates of Helicobacter pylori was determined at room temperature (21 degrees C) and 37 degrees C by a viable cell count technique with a conventional urea slope test (Christensen's agar) as well as the commercial CLO-test. The urease activity of two gastroduodenal commensals, Proteus mirabilis and Klebsiella pneumoniae, was also tested. H. pylori strains produced positive reactions with viable cell counts of 10(6)-10(8) cfu within 30 min and with counts of 10(3)-10(6) cfu within 2 h. For some strains, smaller numbers of organisms were needed with the CLO-test than with the conventional test, and incubation of the CLO-test strips at 37 degrees C slightly decreased the number of organisms required for positive results. P. mirabilis produced a positive result on urea slopes with an initial inoculum of 10(7)-10(8) cfu at 2 h, but no positive reaction occurred for K. pneumoniae at 12 h, even with an initial inoculum of 10(11) cfu. However, both P. mirabilis and K. pneumoniae gave a positive result after incubation for 24 h with initial inocula of < 10(1) cfu and 10(3)-10(4) cfu respectively. Incubation at 37 degrees C significantly reduced the inoculum size of these organisms required for a positive result after incubation for 4 h when tested with the slopes, but not with the CLO-test. These findings indicate that H. pylori possesses much greater pre-formed urease activity than P. mirabilis and K. pneumoniae. False negative results for clinical detection of H. pylori in gastroduodenal biopsies may be due to small numbers of organisms, especially after treatment with antimicrobial agents, and false positive results may arise from gastroduodenal commensals or contaminants.

Antibiogram-resistogram typing scheme for methicillin-resistant Staphylococcus aureus.

An antibiogram-resistogram (AR) typing scheme that can simply and rapidly differentiate methicillin-resistant Staphylococcus aureus (MRSA) isolates has been devised. Susceptibility to antibiotics and chemicals was determined by disk diffusion testing. Three disk diffusion methods and three control S. aureus strains were evaluated. A modified Stokes' technique in which S. aureus ATCC 25923 replaced S. aureus NCTC 6571 as the control organism was chosen. Susceptibility patterns against 18 antibiotics and four chemicals were used to determine AR types. AR subtypes were determined with reference to knowledge of the local MRSA population so that plasmid loss would not result in misclassification. AR typing was compared with phage typing and plasmid profiling and found to be more discriminatory than either of these typing methods. Representative isolates of the most frequently occurring AR patterns were further characterised by investigating enterotoxin production, MICs of gentamicin and amikacin, and restriction endonuclease analysis of plasma DNA, to determine whether apparently different strains could have the same AR pattern and to devise confirmatory tests for any such similar patterns. One pattern was shared by two strains but isolates could be differentiated by susceptibility to minocycline. This typing scheme can be used in the diagnostic laboratory and results may be obtained within 24 h.

Evaluation of an antibiogram-resistogram typing scheme for methicillin-resistant Staphylococcus aureus.

Between Dec. 1992 and Aug. 1993, the MRSA population in the Federated Dublin Voluntary Hospitals and St James's Hospital group was studied with an antibiogram-resistogram (AR) typing scheme in which AR patterns were determined by testing susceptibility to 22 antibiotics and chemicals by a modified Stokes' disk diffusion technique. The typing scheme divided this MRSA population into 31 AR types but 90% of isolates belonged to seven types. Isolates belonging to the most frequently occurring types (AR types 13 and 14) differed only in their reaction to lincomycin (or clindamycin) and could not be distinguished by phage typing, plasmid profiling or restriction endonuclease analysis. The AR typing scheme showed that the incidence of different AR types varied in different hospitals and changed during the study period. This typing method differentiated a strain of MRSA responsible for a nosocomial outbreak in an intensive care unit from other MRSA isolated in the unit, and has distinguished imported strains from local ones. In one hospital, AR typing showed that, although a major outbreak occurred with one AR type, there was also a series of smaller outbreaks with other AR types. The technique can be performed in the diagnostic laboratory and results were available within 24 h.

Differentiation of fluorescent pseudomonads by their effect on milk agar.

Eighty-six clinical isolates of fluorescent pseudomonads that did not produce pyocyanin on Diagnostic Sensitivity Test Agar or Cetrimide Agar were identified on the basis of their antibiotic sensitivity, production of pigment on King's "A" medium, growth at 42 degrees C, production of lecithinase and hydrolysis of gelatin. The identity of the strains was confirmed in tests with the ammonium salt sugars ethanol, glucose and mannitol. These tests were adequate for distinguishing between the three important fluorescent pseudomonads. The detection of casein hydrolysis on milk agar was assessed as a rapid method of distinguishing P. aeruginosa from the other species of fluorescent pseudomonads but proved unhelpful when compared with, or included in, a small set of tests. Most strains of P. aeruginosa and P. fluorescens hydrolysed casein.

Incidence and detection of multi-drug-resistant enterococci in Dublin hospitals.

In February 1994, an outbreak of vancomycin-resistant Enterococcus faecium (VREM) occurred in the oncology unit of a Dublin hospital. Between February and July 1994, VREM was isolated from 18 patients, one staff member and 14 environmental sites within the unit. The isolates also had high-level aminoglycoside and penicillin resistance. Three pulsed-field gel electrophoresis (PFGE) types were identified, two of them from multiple patients and environmental sites. Plasmid typing allowed subdivision of PFGE types. A retrospective study of enterococci isolated from blood cultures between January 1991 and January 1994 showed that, before the outbreak, fewer than 2% of isolates were vancomycin-resistant but that the incidence of high-level gentamicin resistance had increased from 17% to 60% and ampicillin resistance from 22% to 51%. Among clinically significant non-blood-culture enterococci isolated between September and December 1993, fewer than 1% were vancomycin-resistant, 13% were ampicillin-resistant and 44% highly gentamicin-resistant. None produced beta-lactamase. High-content gentamicin disks (120 micrograms) and low-content vancomycin disks (5 micrograms) allowed simple, reliable detection of resistant enterococci. MICs of vancomycin and teicoplanin determined by agar dilution and E-test agreed well, but values tended to be slightly lower by E-test.

Gentamicin and methicillin resistant Staphylococcus aureus in Dublin hospitals: clinical and laboratory studies.

Strains of Staphylococcus aureus resistant to gentamicin and methicillin first appeared in Dublin hospitals in 1976, and rapidly became widely disseminated. The number of patients infected or colonised increased throughout the period of study, especially in 1979 and 1980. Most isolates were from burns, surgical wounds and traumatic skin lesions. During the 12 months after first isolation of these multiply antibiotic resistant strains, colonisation or minor infection was the usual event. Invasive infection such as bacteraemia, deep wound sepsis and osteomyelitis was rarely seen. Subsequently, as the number of patients from whom these organisms were isolated increased, bacteraemia and other severe infection became more common. The predominant phage type of S. aureus changed with the progression of the outbreak. Isolates of different phage type were sometimes found in a single lesion, or in different sites in one patient. By the second half of 1980, most isolates were untypable or typed only with an experimental phage.

Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals.

Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains.

Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers.

In a prospective study, 52 Staphylococcus aureus isolates from individual patients with septicaemia and 27 nasal strains from separate, healthy carriers were compared for production of a range of extracellular proteins and toxins. Whereas there was no difference (p greater than 0.05) between septicaemic and nasal isolates with respect to incidence of alpha, beta, gamma and delta haemolysins, toxic shock syndrome toxin-1 or staphylokinase production, the incidence of enterotoxin A, B, and C production was higher among isolates from septicaemia (p less than 0.01). Of the isolates from septicaemia, 33 (63%) produced enterotoxins A, B, C or D alone or in combination. Only three (11%) of the nasal isolates produced a single enterotoxin, enterotoxin D. Of the isolates from septicaemia, 67% were hospital-acquired and greater than 25% of these were endemic, methicillin-resistant (MRSA) strains. All MRSA strains produced either enterotoxin A, or enterotoxin B, or both. These findings suggest a possible role for enterotoxins in the pathogenesis of S. aureus disease other than food poisoning.