Beta-D-Glucuronidase activity among prototrophic and auxotrophic variants of Escherichia coli and other Enterobacteriaceae commonly implicated in urinary tract infections.

Glucuronidase (GUD) activity of 102 prototrophic, 91 cysteine-requiring, and 19 thymidine-requiring strains of Escherichia coli was examined using growth from MacConkey, CLED, and enriched brain heart infusion (BHI) agars. After 24 h incubation, GUD activity was detected in 92%-96% of prototrophic strains and a similar proportion of thymidine-requiring strains with most reactions detectable in shorter incubation periods. GUD activity among strains requiring cysteine was significantly less than that found amongst prototrophic strains. The effects of different sources of inocula were evident in the shorter incubation periods. Other strains of the Enterobacteriaceae and oxidative strains frequently implicated in urinary tract infection were also tested. Here, positive reactions were detected among Citrobacter and Enterobacter spp. and a strain of Klebsiella oxytoca, but only after 24 h incubation. GUD activity was not detected among the oxidative strains tested under the same conditions. Although an incubation time of 24 h is necessary to detect activity in a small number of "slow hydrolyzing" E. coli, the increased sensitivity thus attained compromises the specificity of the test for this organism by simultaneously enhancing detection of the enzyme in other enterobacteria.

Characterisation of two new gene cassettes, aadA5 and dfrA17.

Escherichia coli INS33 was isolated from the urinary tract of an infected patient. It was resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfafurazole, tetracycline and trimethoprim. PCR screening revealed the presence of a class 1 integron that harboured two new gene cassettes, designated dfrA17 and aadA5. The new dfrA17 cassette was 91% identical to the known dfrA7 cassette. The aadA5 cassette was 95% identical over the first 830 bp to aadA4, but lacked the IS26 element found at the 3' end of this truncated cassette. Cloning and expression of the cassette region demonstrated that dfrA17 conferred high level resistance to trimethoprim but aadA5 conferred resistance to spectinomycin but not to streptomycin.

Characteristics of cysteine-requiring strains of Klebsiella isolated from urinary tract infections.

Clinical and bacteriological findings in seven cases of urinary tract infection with cysteine-requiring strains of Klebsiella are described. The organisms were isolated from patients with long-standing urinary tract abnormalities and grew as small (c. 1 mm) colonies on MacConkey agar. The organisms failed to grow in a minimal medium supplemented with sodium sulphate but grew when the medium was supplemented with cysteine sulphinic acid, sodium sulphide or L-cysteine. The smallest amount of cysteine required for optimal growth in a chemically defined medium was 20 mg/L. Cysteine-requiring strains of Escherichia coli had previously been shown to require a similar amount of cysteine and to be unable to reduce sulphate to sulphite; this suggests a common influence in the selection of cysteine auxotrophs in vivo. However, the amino acid inhibited the growth of E. coli at concentrations which only slightly altered growth of the Klebsiella strains. Problems with the isolation, identification and sensitivity testing of cysteine-requiring Klebsiella were also observed and methods by which these may be minimised are suggested.

Virulence and other phenotypic characteristics of urinary isolates of cysteine-requiring Escherichia coli.

Urinary isolates of cysteine-requiring Escherichia coli were found to be generally lacking in virulence factors commonly associated with uropathogenic strains. The proportion of auxotrophic strains showing type-1 fimbriation, haemolysin production, motility and sensitivity to normal human serum was significantly less than that of a comparable number of urinary isolates of prototrophic E. coli, although the proportion in both groups possessing K1 antigen was similar. Furthermore, the biotyping and serogrouping of these and other strains from systemic infections demonstrated a high degree of phenotypic diversity. This is further evidence that infection with these auxotrophs results from a combination of decreased host resistance and a physiological condition conducive to the random selection of these auxotrophs in vivo.

Septicaemia caused by cysteine-requiring isolates of Escherichia coli.

The clinical and bacteriological findings in five cases of septicaemia with cysteine-requiring isolates of Escherichia coli are reported. Infections with these nutritionally-dependent organisms have been found previously in the urinary tract only, associated usually with chronic rather than acute conditions. The urinary tract was considered to be the source of the septicaemia in our patients and that site should be investigated when such strains are isolated from blood cultures. When first isolated the organisms characteristically form small translucent colonies on media deficient in appropriate growth factors. Their nutritional requirement for cysteine can be determined by a simple auxanographic technique, thereby enabling the appropriate supplementation of media necessary for reliable identification and antibiotic-sensitivity testing.

Further studies of clinical isolates of cysteine-requiring Escherichia coli and Klebsiella and possible mechanisms for their selection in vivo.

Cysteine-dependent (cys-) Escherichia coli and Klebsiella spp., defective in sulphate assimilation, were isolated from urine and stool samples of infected patients. These isolates reverted to prototrophy under conditions of cysteine deprivation but the revertant strains and a prototrophic wild-type E. coli strain became auxotrophic for cysteine in a cysteine-enriched medium. This suggested that excess cysteine acts as a repressor of the cys HIJ operon known to control aspects of cysteine biosynthesis. A group of mostly elderly patients infected with cys- strains suffered a disproportionate amount of renal impairment as compared with a control group. In renal impairment, sulphur compounds, including cysteine, are retained. This raises the possibility that these raised levels of cysteine and related compounds may enhance the selection of cys- strains in vivo.

Detection of astrovirus gastroenteritis in children.

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.

Cysteine requirements of naturally occurring cysteine auxotrophs of Escherichia coli.

The requirements for cysteine of naturally occurring cysteine auxotrophs of Escherichia coli were determined in a defined liquid medium. Maximal growth was obtained in the presence of cysteine concentrations between 20 and 250 mg/l. At concentrations below 20 mg/l growth of the auxotrophs, but not the prototrophic control, was suboptimal in this system. In the presence of cysteine concentrations in excess of 250 mg/l, growth of both auxotrophic and prototrophic E. coli was inhibited with lower growth yields, a decreased specific growth rate and an extended lag phase being observed. These effects were minimised in the presence of 2 mM L-leucine, L-isoleucine and L-valine.

Diagnosis of enteric pathogens in children with gastroenteritis.

The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12-month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Children's Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1 %). Giardia-specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients (P = 0.23). Amongst this subset, Norwalk-like viruses (NLVs) were detected by RT-PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral-like particles (SVLPs), and three of 126 EM-negative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary. The cost-effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.

Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury.

BACKGROUND/AIMS: Toll-like receptors (TLR's) are critical receptors that promote innate immune responses to pathogen-associated molecular patterns. Activation of TLR's leads to production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha. This study investigates whether peripheral blood monocyte expression of TLR's is disturbed in patients with chronic hepatitis C and whether levels of expression of these molecules are significantly correlated with hepatitis C virus (HCV) genotype, viral load, hepatic necroinflammatory activity, histological stage and circulating TNF-alpha concentrations. METHODS: In 18 non-cirrhotic patients with biopsy-proven, virologically-confirmed chronic hepatitis C and 32 controls, we measured expression of TLR2 and TLR4 on peripheral blood monocytes. HCV genotype, viral load, serum alanine aminotransferase (ALT) levels, histological stage of disease and circulating TNF-alpha and endotoxin levels were also determined. RESULTS: Peripheral blood monocyte expression of TLR2 and TLR4 were significantly increased in patients with chronic hepatitis C compared to controls, irrespective of HCV genotype or histological stage of disease. Circulating levels of TNF-alpha were also significantly increased in patients with chronic hepatitis C. In both the overall study cohort and patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, correlated significantly with serum TNF-alpha levels. In patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, also correlated significantly with serum ALT levels. Expression of TLR's was not significantly correlated with viral load. CONCLUSIONS: Up-regulation of peripheral blood monocyte expression of TLR2 and TLR4 occurs in patients with chronic hepatitis C. Increased monocyte expression of TLR2, but not of TLR4, correlates significantly with both increased circulating TNF-alpha levels and hepatic necroinflammatory activity in this disorder.

Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7.

Vertical transmission of viruses is an important cause of morbidity in the fetus and neonate. Placental viral infection indicates risk of vertical transmission, but not always transmission to, or disease of the fetus. Specimens from mothers and babies from three groups-two prospective and one retrospective cohort-were tested for pathogens of teratogenic potential using multiplex PCR. Placental infection was present in 13% of the 105 samples collected. Assessment of the prospective cohorts showed cytomegalovirus (CMV) detected in 4% of placentae from unselected women, parvovirus B19 in 1% and Ureaplasma parvum in 1% of placentae. In a retrospective cohort of women at high risk of transmitting congenital infection due to seroconversion during pregnancy, miscarriage or stillbirth, CMV was detected in 64% and human herpes virus type 7 in 9% of placentae. Of 14 PCR-positive placentae, two were associated with the birth of a living symptomatic infant, two with stillbirth, one with miscarriage, and two with elective terminations of pregnancy. Directed laboratory assessment of women at high risk of transmitting congenital infection, on the basis of clinical or laboratory markers, is important for accurate diagnosis of adverse outcomes of pregnancy. However, routine screening for viruses in the placentae from women with a low-risk serological profile for transmitting congenital infection is unlikely to result in significant numbers of additional diagnoses and is confounded by inadequacy of current diagnostic methods. The major pathogen detected in all cases of placental infection associated with fetal death was human CMV.

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

In vitro susceptibilities of clinical isolates of cysteine-requiring Escherichia coli to 12 antimicrobial agents.

The MICs of 12 antimicrobial agents for 42 cysteine-requiring strains of Escherichia coli showed a high concordance when determined on three different media, one of which was supplemented with cysteine. Differences in the MICs of several agents were detected between 18 prototrophic revertants and their parent auxotrophs. A total of 64.7% of the isolates were fully susceptible to all agents, and no particular resistance pattern was evident.

Assessment of conventional and commercial methods for identification of clinical isolates of cysteine-requiring strains of Escherichia coli and Klebsiella species.

Cysteine-requiring strains of the family Enterobacteriaceae that are auxotrophic for this amino acid because of defects in the sulfur assimilatory pathway account for about 1.5% of urinary tract isolates of Escherichia coli and Klebsiella species. Forty Escherichia and eight Klebsiella cysteine-requiring strains were used to test the ease with which various test systems identified clinical isolates of cysteine auxotrophs. In a preliminary experiment, the growth yield of 10 cysteine-requiring E. coli in 10 solutions of commercially available peptones was in each instance less than that of prototrophic control and showed that these sources of nutrients were suboptimal for these strains. A significant proportion of the cysteine-requiring strains were not adequately identified by growth-dependent tests which used various peptones as a nutrient source. Problems were encountered with all test systems examined, which were as follows: conventional methods; the API 20E, Microbact, and Vitek systems; and two rapid methods for the identification of E. coli, the Rapidec coli and the beta-D-glucuronidase tests. The performance of the test systems was only partly improved when inocula were derived from appropriately supplemented media. However, the problems of the growth-dependent tests were resolved when a cysteine-supplemented suspension was used to inoculate each test system.

Study of growth requirements other than cysteine of naturally occurring Escherichia coli and Klebsiella spp. auxotrophic for cysteine.

Cysteine remains the preferred supplement for cultivation of Cys- auxotrophs in vitro. Methionine, which reduced cysteine requirements, and branched-chain amino acids, which decreased cysteine toxicity, were identified as the components of casein hydrolysate responsible for growth enhancement by this additive. Glutathione and DL-homocysteine can be substituted for cysteine. Accumulation of these compounds in patients with renal impairment may favor selection of Cys- strains in vivo.

Integrons and gene cassettes in the enterobacteriaceae.

Integrons were detected in 59 of 120 (49%) urinary isolates of Enterobacteriaceae by PCR using degenerate primers targeted to conserved regions of class 1, 2, and 3 integrase genes. PCR sequencing analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to the aminoglycosides and trimethoprim.

Liver damage in human small intestinal bacterial overgrowth.

OBJECTIVE: Some rodent strains with experimental small intestinal bacterial overgrowth (SIBO) unrelated to jejunoileal bypass are susceptible to hepatic damage, possibly because of increased small intestinal permeability to proinflammatory bacterial polymers. However, data on the prevalence of hepatic damage in human subjects with SIBO in this setting are lacking. This study addressed this issue. METHODS: Seventy adult subjects were investigated for possible SIBO and hepatic damage with bacteriological analysis of small intestinal aspirates and measurement of serum concentrations of alkaline phosphatase, gamma-glutamyl transpeptidase, aspartate aminotransferase, and alanine aminotransferase. Nutritional indices (serum albumin and anthropometry) and the urinary lactulose/mannitol ratio, an index of small intestinal permeability, were measured in all subjects with SIBO and liver damage. RESULTS: SIBO was present in 40 of 70 subjects (57.1%). Overgrowth flora included salivary-type bacteria alone in 11 subjects and colonic-type bacteria in 29 subjects (facultative anaerobes [Enterobacteriaceae] alone in 21 subjects and both facultative and obligate anaerobes [Enterobacteriaceae and Bacteroides spp] in eight subjects). Biochemical evidence of liver damage was found in zero of 30 subjects without SIBO, zero of 11 subjects with SIBO with salivary-type bacteria alone, zero of 21 subjects with SIBO with facultative but not obligate anaerobic colonic-type bacteria, and in one of eight subjects (12.5%) with SIBO with obligate anaerobic colonic-type bacteria, in whom serum alkaline phosphatase and gamma-glutamyl transpeptidase levels were elevated. Nutritional indices were normal in this patient. Small intestinal permeability was increased and, along with liver enzyme abnormalities, normalized after eradication of SIBO. Small intestinal permeability was also increased in three of six patients (50.0%) with SIBO with obligate anaerobic colonic-type bacteria who had no evidence of liver damage. CONCLUSIONS: SIBO per se is not a major risk factor for liver damage in humans, even when the overgrowth flora includes obligate anaerobes. Liver damage is not a necessary consequence of increased small intestinal permeability in this setting.

An appraisal of a 'string test' for the detection of small bowel bacterial overgrowth.

The efficacy of a string test for the detection of small bowel bacterial overgrowth (SBBO) was determined by comparison with a sterile endoscopic method for sampling small bowel secretions in 15 subjects investigated for SBBO. Clinical value was found to be limited by poor sensitivity, specificity and positive predictive value. The string test is not an adequate substitute for oro-duodenal intubation for the detection of SBBO.

Fasting breath hydrogen concentrations in gastric and small-intestinal bacterial overgrowth.

BACKGROUND: Although elevated fasting breath hydrogen concentrations have been reported in small-intestinal bacterial overgrowth, this diagnosis has been presumptive or based on definitions that vary from study to study. The influence of gastric bacterial overgrowth and gastroduodenal pH has not been documented. Conflicting evidence exists as to the reproducibility of breath hydrogen measurements. METHODS: Forty-two subjects underwent culture of gastric and duodenal aspirates. The pH was measured by indicator paper. Paired fasting breath hydrogen concentrations were measured by gas chromatography within 7 days of endoscopy. RESULTS: Paired fasting breath hydrogen concentrations differed in terms of normality or abnormality in 21% of subjects. Paired concentrations correlated significantly in overgrowth but not in culture-negative subjects. Sensitivity for bacterial overgrowth was 4-29%, and specificity 71-100%. No correlation with gastroduodenal pH was found. CONCLUSIONS: The clinical relevance of a single fasting breath hydrogen concentration is limited. The efficacy of paired measurements for gastric or small-intestinal bacterial overgrowth is poor.