Rapid presumptive identification of enteropathogenic Escherichia coli in faecal smears by means of fluorescent antibody: 3. Field evaluation

This paper reports on the evaluation of the fluorescent antibody technique for detection of enteropathogenic Escherichia coli in faecal smears from children both with and without diarrhoea. In field studies involving 315 children in Puerto Rico, it was demonstrated that presumptive diagnosis of infection with enteropathogenic E. coli could be made more rapidly and with greater sensitivity by immunofluorescence than by isolation and slide agglutination. The specificity of the fluorescent antibody test was of the same order as that of slide agglutination tests with OB antisera. The incidence of salmonellae, shigellae, coagulasepositive staphylococci and Candida in the diarrhoeal specimens was studied, but no relationship appeared to exist between these organisms and the enteropathogenic E. coli.     

Rapid presumptive identification of enteropathogenic Escherichia coli in faecal smears by means of fluorescent antibody: 1. Preparation and testing of reagents

In outbreaks of infantile diarrhoea caused by enteropathogenic Escherichia coli, rapid detection of the etiological agent is essential for specific and adequate therapy. Fluorescent antibody techniques have proved advantageous for rapid identification of many microorganisms. The development of fluorescein-labelled antibodies specific for nine serogroups of E. coli and the testing of these reagents for specificity are described in this paper. Results of testing pure cultures of various Enterobacteriaceae as well as faecal specimens from cases of diarrhoea are reported. These data show excellent correlation between the fluorescent antibody technique and conventional serological methods for identifying enteropathogenic types of E. coli. The immunofluorescent technique is more rapid than the usual bacteriological and serological methods in that smears from faecal specimens can be stained directly with the labelled antibodies. Details of the procedures for carrying out the technique are given in annexes.     

Sampling considerations for herd-level measurement of faecal Escherichia coli antimicrobial resistance in finisher pigs.

The objective of this study was to determine the most efficient means of sampling faeces of finisher pigs for accurate and precise farm-level estimates of antimicrobial resistance among faecal Escherichia coli. Resistance to tetracycline and gentamicin of 8250 isolates of E. coli from 55 finisher pigs on one farm was measured with a hydrophobic grid membrane filter method. The between-pig, within-pen component of variance in resistance was large (97.5%), while between-pen, within-room and between-room components were small (2.5% and 0%, respectively). Using these resistance data, the abilities of two sampling strategies to estimate prevalence were modelled with a Monte Carlo 'bootstrap' procedure. Compositing faecal samples from several pigs before testing produced unbiased and precise estimates of prevalence and is simpler technically than individual animal testing.     

Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates

Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances.     

A medium for the rapid enumeration of Escherichia coli in the presence of other faecal coliforms in tropical waters.

A selective membrane filtration medium is described for use in the rapid assessment of water quality in tropical countries where the incidence of faecal coliforms other than E. coli presents problems in the interpretation of results. The medium gives comparable results to MPN values obtained in the multiple tube dilution test using modified Gray''s glutamate medium, and to membrane filtration counts obtained using M-FC broth and membrane-enriched Teepol broth, whilst differentiation of E. coli is enhanced.     

Correlation between electrophoretic types B1 and B2 of carboxylesterase B and host-dependent factors in Escherichia coli septicaemia.

Electrophoretic types B1 and B2 of carboxylesterase B produced by strains of Escherichia coli isolated from 100 septicaemia cases were correlated with alpha-haemolysin and mannose resistant haemagglutinin (MRHA) production and with clinical data including eventual underlying diseases, origin of septicaemia and evolution. Electrophoretic type B2 was phenotypically linked with alpha-haemolysin and MRHA production. The proportion of type B2 isolates varied significantly with occurrence of an underlying illness (45% for patients without an underlying disease and 22% for compromised patients) and with the site of origin of the septicaemia (40% for those of urinary origin and 18% for infection of digestive origin). In the former infections, type B2 isolates were obtained in the majority from male patients while type B1 isolates predominated in women. The septicaemias associated with type B1 were characterized by a lower proportion of isolates producing alpha-haemolysin and MRHA and by a greater frequency of septic shock and death than those associated with type B2. These facts emphasize the importance of host-dependent factors in E. coli septicaemia.     

Hepatitis B core antigen synthesised in Escherichia coli: its use for antibody screening in patients attending a clinic for sexually transmitted diseases.

Hepatitis B core antigen (HBcAg) synthesised in Escherichia coli by recombinant DNA techniques was compared with HBcAg prepared from infected liver tissue. The two antigens were used in radioimmunoassays (RIA) to detect antibody to HBcAg (anti-HBc) in sera from patients attending a clinic for sexually transmitted diseases. Out of 2151 sera tested, 260 were anti-HBc positive with both HBcAg preparations but seven were positive with the liver-derived antigen alone. Reasons for these discrepant results are discussed. The slight loss of sensitivity of the anti-HBc RIA using E. coli HBcAg was not considered significant when compared with the potential advantages of a synthetic antigen. The presence of other hepatitis B markers in the 267 anti-HBc positive sera was determined: 25 contained HBsAg, 220 anti-HBs and, of the 22 that were HBsAg/anti-HBs negative, 12 contained anti-HBe. In the 10 remaining sera, anti-HBc was the only hepatitis B marker that could be found.     

Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.

Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.     

Clonal diversity of Shiga toxin-producing Escherichia coli O103:H2/H(-) in Germany.

Shiga toxin producing Escherichia coli O103:H2/H(-) belong to the third most frequently isolated EHEC serotypes in Germany following isolates of O157:H7/H(-) and O26:H11/H(-). A total of 145 respective E. coli 103 isolates from single cases of diarrhoea and haemolytic uremic syndrome (HUS) in 1997-2000 were characterised by a range of molecular subtyping methods (PFGE, P-gene profiling, ribotyping, electrotyping) and phage typing in order to analyse their genetic relatedness and the practicability for new epidemiological tracing back. All isolates cluster into a distinct EHEC subgroup and reveal a high clonal diversity together with a considerable stability. Since strains of this serotype rank up to the third most frequently isolated EHEC in Germany a large population of this serotype, and therefore, a great supply of such strains may exist in this country.     

A 2-year trend of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Thailand: an alert for infection control

A variety of gram-negative bacteria, particularly Enterobacteriaceae, produce extended-spectrum beta-lactamase (ESBL) resulting in resistance to several beta-lactam agents. These bacteria play a major role in nosocomial infections. While the prevalence of ESBL producers appear to be rapidly increasing worldwide, data from Thailand are rarely reported. We analysed the recent trends in prevalence and antimicrobial resistance among ESBL-producing Escherichia coli and Klebsiella pneumoniae over the 2-year period of August 2005 to July 2007 at a major university hospital in Thailand. The results showed that the prevalence of ESBL producers was very high (up to 65.9% among sputum isolates) and continues to increase. These bacteria also demonstrated a significant increase in resistance rates to several non-beta-lactam antibiotics and expressed a multidrug resistance phenotype at a high rate. This study poses a serious concern for infection control in Thailand and indicates the need for immediate action to prevent further spread of these resistant bacteria.     

Bacteriostasis of Escherichia coli by milk. V. The bacteriostatic properties of milk of West African mothers in the Gambia: in-vitro studies.

Bacteriostatic activity was measured in 244 specimens of milk collected during 1977 throughout lactation of up to one year from 78 mothers; the activity varied from very good to fair and only seven were inactive. There was a wider range of activity than was found previously in milk from English mothers. Activity usually fell slowly during lactation but some of the Gambian mothers produced milk of very high activity, like that of colostrum into the second week of lactation, and two mothers did so at six and nine months; other mothers produced good-activity milk throughout lactation. The bacteriostatic activity varied little with the season but slight decreases from that expected were found after the high incidence of infant diarrhoea towards the end of the rainy season. The bacteriostatic activity of most of the milk tested could be prevented by iron salts but that of colostrum and some of the milks with high activity could not. Only these highly active colostra and milks were inhibitory in vitro when the inoculum was increased from 10(4) to 10(6) organisms per ml. These and less active milks were able to inhibit the smaller, standard inoculum for longer than 3 h with the addition of bicarbonate and extra iron-binding protein at the concentrations likely to be present in vivo. Both commensal and pathogenic E. coli were inhibited to a similar degree by these milks and there was no evidence of serotype specificity.     

Removal of Escherichia coli and faecal coliforms from surface water and groundwater by household water treatment devices/systems: a sustainable solution for improving water quality in rural communities of the Southern African development community region

There is significant evidence that household water treatment devices/systems (HWTS) are capable of dramatically improving microbially contaminated water quality. The purpose of this study was to examine five filters [(biosand filter-standard (BSF-S); biosand filter-zeolite (BSF-Z); bucket filter (BF); ceramic candle filter (CCF); and silver-impregnated porous pot (SIPP)] and evaluate their ability to improve the quality of drinking water at the household level. These HWTS were manufactured in the workshop of the Tshwane University of Technology and evaluated for efficiency to remove turbidity, faecal coliforms and Escherichia coli from multiple water source samples, using standard methods. The flow rates ranged from 0.05 L/h to 2.49 L/h for SIPP, 1 L/h to 4 L/h for CCF, 0.81 L/h to 6.84 L/h for BSF-S, 1.74 L/h to 19.2 L/h and 106.5 L/h to 160.5 L/h for BF The turbidity of the raw water samples ranged between 2.17 and 40.4 NTU. The average turbidity obtained after filtration ranged from 0.6 to 8 NTU (BSF-S), 1 to 4 NTU (BSF-Z), 2 to 11 NTU (BF), and from 0.6 to 7 NTU (CCF) and 0.7 to 1 NTU for SIPP. The BSF-S, BSF-Z and CCF removed 2 to 4 log(10) (99% to 100%) of coliform bacteria, while the BF removed 1 to 3 log (90% to 99.9%) of these bacteria. The performance of the SIPP in removing turbidity and indicator bacteria (>5 log(10), 100%) was significantly higher compared to that of the other HWTS (p < 0.05). The findings of this study indicate that the SIPP can be an effective and sustainable HWTS for the Southern African Development Community (SADC) rural communities, as it removed the total concentration of bacteria from test water, can be manufactured using locally available materials, and is easy to operate and to maintain.     

Evaluation of hha and hha sepB mutant strains of Escherichia coli O157:H7 as bacterins for reducing E. coli O157:H7 shedding in cattle

Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 10⁹ heat-killed cells of the hha⁺ wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha⁺ wild-type bacterins. Following oral inoculations with 10¹⁰ CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha⁺ wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce.     

Serological studies in cholera: 2. The vibriocidal antibody response of cholera patients determined by a microtechnique*

A microtechnique is described for the determination of vibriocidal antibodies to Vibrio cholerae, using 0.025 ml of fingertip blood or venous serum per test. This test could be used in epidemiological surveys, or as a routine test on patients admitted to hospital.     

Epidemiology of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp.: a nested case-control study from a tertiary hospital in London

Information on risk factors for acquisition of extended-spectrum ss-lactamase (ESBL)-producing organisms and their outcomes in patients with invasive infections is scant. The objectives of this study were to evaluate risk factors and all-cause mortality associated with infection due to ESBL-producing organisms using a nested case-control design, and to document transmission within a hospital employing molecular and conventional epidemiological methods. From December 2003 to April 2005, 50 patients with bloodstream infections (BSIs) due to ESBL-producing E. coli and Klebsiella spp. were recruited. Controls (N=50) were chosen, within the same period, from patients with non-ESBL-producing BSIs by simple random sampling; account was taken of potential confounding factors. Cases and controls were followed-up until November 2005, and outcomes were recorded as discharged or deceased. Molecular methods, supported by conventional epidemiology, were used to study the transmission of organisms. Logistic regression analyses showed prior ss-lactam antibiotics [odds ratio (OR) 11.57; 95% confidence intervals (CI) 2.31-51.15; P=0.003], hospital stay >15 days (OR 2.63; 95% CI 1.01-6.89; P=0.04) and prior admission to the intensive care unit (OR 13.98; 95% CI 1.88-19.15; P=0.006) to be independent risk factors for the acquisition of ESBL-producing organisms. In the first 15 days of follow-up, a significant proportion of patients with ESBL-producing organisms died; however, there was no difference in mortality between cases and controls at the end of the follow-up period. Molecular epidemiology identified five clusters amongst the ESBL-producing isolates. Conventional epidemiological analyses supported the evidence of transmission in three of these clusters.     

Immunofluorescent staining of trypsinized formalin-fixed brain smears for rabies antigen: results compared with those obtained by standard methods for 221 suspect animal cases in Nigeria.

Formalin-fixed samples from 221 animal brains received for rabies diagnosis in Nigeria were digested in 0.1% trypsin in phosphate buffered saline, pH 7.4, and smears stained for rabies antigen by direct immunofluorescence (IF). The results were compared with those obtained using fresh material from the same animals for Negri body staining, mouse inoculation (MI) and occasionally immunofluorescent staining. From 191 specimens examined for Negri bodies and by mouse inoculation 51 and 64 respectively proved positive. The IF smear technique under investigation failed to detect 5 of these but showed up as positive 30 which had been recorded as Negri-negative and 19 that had gone undetected by MI too. In a direct comparison with IF staining of fresh tissue from 23 known rabies positive animals the similar staining of trypsin-digested formalized smears failed to give a positive result in 2 out of 23 cases. Some problems were encountered with smears not sticking to slides. When gelatinized slides were used fluorescence was sometimes poorer. Where transport and refrigeration are difficult and section-cutting equipment is lacking the technique of IF staining of smears prepared from formalized brain tissue after treatment with trypsin can be a useful adjunct to other diagnostic methods. It also makes for safer working where special facilities are absent.