Multiple typing for the epidemiological study of the contamination of broilers with Salmonella from the hatchery to the slaughterhouse.

Eighteen Belgian broiler flocks were followed from the hatchery to the slaughterhouse by a multiple typing approach (sero-, geno-, and phage types) for the investigation of the transmission of Salmonella and its subtypes. For 12 of the 18 flocks, there was no correlation between the serotypes found preharvest and those isolated from the feces in the transport crates and on the carcasses in the slaughterhouse. Serotypes found in the crates were usually also found on the carcasses. In 5 of the 10 flocks with Salmonella-positive broilers, complex contamination patterns with the involvement of different serotypes, genotypes, or both were revealed. In two of these flocks (flocks 8 and 9), the Salmonella Enteritidis contamination of the broilers could be traced to the hatchery. In flock 9, evidence was found for the acquisition, during rearing, of a megaplasmid in the Salmonella Enteritidis strain. In the other three positive flocks (flocks 6, 7, and 10), the environment and movable material (e.g., footwear) played a determining role in the infection and shedding pattern of the broilers. For flocks 6 and 7, reared consecutively in the same broiler house, a persistent Salmonella Hadar geno/phage type predominated in the preharvest period, while another Salmonella Hadar geno/phage type was found in the house or the environment but never in the broilers. Only for the above-mentioned five flocks were the same strains that were found preharvest also recovered from the carcasses, although these strains were not predominant on the carcasses, with the exception of one flock (flock 10). In conclusion, it can be said that most of the time, Salmonella strains that contaminate Belgian broiler carcasses do not predominate in the preharvest environment.

Arcobacter species in humans.

During an 8-year study period, Arcobacter butzleri was the fourth most common Campylobacter-like organism isolated from 67,599 stool specimens. Our observations suggest that A. butzleri displays microbiologic and clinical features similar to those of Campylobacter jejuni; however, A. butzleri is more frequently associated with a persistent, watery diarrhea.

Campylobacter, from obscurity to celebrity.

After its successful isolation from stools in the 1970s, Campylobacter jejuni has rapidly become the most commonly recognised cause of bacterial gastroenteritis in man. Reported cases of human campylobacteriosis represent only a small fraction of the actual number. In industrialised countries, the incidence of C. jejuni/Campylobacter coli infections peaks during infancy, and again in young adults aged 15-44 years. Acute self-limited gastrointestinal illness, characterised by diarrhoea, fever and abdominal cramps, is the most common presentation of C. jejuni/C. coli infection. The introduction of selective media has made the diagnosis of Campylobacter enteritis a simple procedure. In general, Campylobacter enteritis is a self-limiting disease which seldom requires antimicrobial therapy, although one in 1000 infections may lead to the Guillain-Barré syndrome. In industrialised countries, most infections are acquired through the handling and consumption of poultry meat. In developing countries, where the disease is confined to young children, inadequately treated water and contact with farm animals are the most important risk factors. Many infections are acquired during travel. Fluoroquinolone resistance has been reported in C. jejuni since the late 1980s in Europe and Asia, and since 1995 in the USA. The use of fluoroquinolones to treat animals used for food has accelerated this trend of resistance. In Australia, where fluoroquinolones have not been licensed for use in food production animals, C. jejuni remains susceptible to fluoroquinolones. The public health burden of Campylobacter spp. other than C. jejuni/C. coli remains unmeasured. Better diagnostic methods may reveal the true health burden of these organisms.

Evaluation of the ProSpecT Microplate Assay for detection of Campylobacter: a routine laboratory perspective.

OBJECTIVES: To evaluate the use of the new enzyme-linked immunosorbent assay, the ProSpecT Campylobacter Microplate Assay (Alexon-Trend, Minneapolis, MN, USA), which allows 2-h detection of both Campylobacter jejuni and Campylobacter coli antigen directly in stool specimens. METHODS: Over 4 months, all stool samples preserved in Cary-Blair medium, or fresh specimens, from non-hospitalized children and HIV-infected patients (adults and children), submitted to our laboratory were evaluated with the ProSpecT Campylobacter Microplate Assay. Results were compared with those obtained by routine culture methods using both a specific medium and a filtration method for the recovery of Campylobacter spp. RESULTS: Of the 1205 stool specimens cultured, 101 were found to be positive for either C. jejuni or C. coli, giving an overall recovery rate of 8.38%. Ninety samples were positive by both culture and ProSpecT Campylobacter Microplate Assay, and 11 were positive by culture only, giving a sensitivity of 89.1%. In addition, of 1104 samples negative by culture, 25 were initially positive by ProSpecT Campylobacter Microplate Assay. We found no cross-reaction with other bacterial enteropathogens isolated from stool specimens. These results thus confirm a high specificity (97.7%) for both C. jejuni and C. coli. The positive and negative predictive values found were 78.3% and 99%, respectively. There was no statistically significant difference in sensitivity and specificity if the stool was fresh or preserved with Cary-Blair medium. CONCLUSION: These data suggest that the ProSpecT Campylobacter Microplate Assay is a rapid and easy-to-use test for the detection of both C. jejuni and C. coli in stool specimens. It could be used for patients for whom early antibiotic therapy is needed or for epidemiologic studies.

Twelve year observation of primary and secondary antibiotic-resistant Helicobacter pylori strains in children.

BACKGROUND: The effectiveness of Helicobacter pylori eradication regimens is influenced by antibiotic susceptibility of infecting strains. Data concerning antibiotic resistance in children are limited. We report the evolution of primary and secondary resistance in a series of Belgian children during the last 12 years. PATIENTS AND METHODS: From 1989 through 2000, H. pylori gastritis was diagnosed in 569 children, and antibiotic susceptibility tests were performed in 555. Eradication, using different schemes, failed in 128 of 457 treated children. After eradication failure antibiotic susceptibility determination was performed in 87 of 128. Comparison of antibiotic susceptibility of strains isolated from the gastric body and from the antrum was performed in 238 samples. RESULTS: Resistance to amoxicillin was not observed. The rate of primary resistance to nitroimidazole derivatives was 18.0% (101 of 555) and remained constant throughout this period, whereas primary resistance to macrolides increased from an average of 6.0% (range, 0 to 10%) before 1995 to 16.6% (range, 10 to 25%, P < 0.001) thereafter. Antibiotic consumption in Belgium, especially macrolides, did not show important fluctuations during the study period. Secondary resistance developed in 39 of 87 patients (46%). Strains isolated from different gastric locations show identical susceptibility testing in all but 5 of 238. CONCLUSIONS: Resistance of H. pylori to macrolides increased in our pediatric population which did not appear to correlate with macrolides prescription habits in our country. After eradication failure acquired secondary resistance was observed in one-half of the patients.

Community-acquired bacteremia among hospitalized children in rural central Africa.

OBJECTIVE: To describe the epidemiology of community-acquired bacteremia in children admitted to a rural hospital in central Africa and to identify useful diagnostic signs or symptoms. METHODS: On admission, a blood culture was obtained from all children admitted to Children's Hospital of Lwiro between 1989 and 1990. Clinical and biologic signs of infection and nutritional status were recorded. RESULTS: Among the 779 children included in the study, 15.9% were bacteremic on admission. The rate of bacteremia was the highest among children with jaundice (20/56; 35.7%) and fever (119/487; 24.4%). In contrast, children with severe malnutrition had a lower rate of bacteremia (13.2%) than weight growth retarded or well-nourished children (19.5%) (P = 0.046). Fever was the most useful diagnostic criteria (sensitivity and negative predictive value of 96.0% and 97.8%, respectively) even in severely malnourished children (sensitivity and negative predictive value of 96.4% and 99.1%, respectively). Enterobacteriacea, mostly Salmonella spp, caused 73% of the bacteremia. There was a high rate of resistance to ampicillin and chloramphenicol among the responsible organisms. Only 31 (47.7%) of 65 bacteremic children responded to the combination of ampicillin and gentamicin. The presence of bacteremia on admission did not significantly increase the risk of morality during hospitalization (19.4% compared with 13.5%; P = 0.088). Age less than 12 months and jaundice were independent risk factors for deaths in bacteremic children. CONCLUSIONS: Community-acquired bacteremia caused by multiresistant Enterobacteriacea is an important problem of hospitalized well-nourished and malnourished children in central Africa. Fever on admission is a sensitive diagnostic sign, even in malnourished children.

A Campylobacter coli foodborne outbreak in Belgium.

In May 1995, the Scientific Institute of Public Health was informed of an outbreak of gastrointestinal illness in a congregational school in the Brussels area. The field investigation identified 24 cases with mild to severe gastrointestinal and general symptoms of acute bacterial enterocolitis. Campylobacter coli was detected in the stools of 5 patients. A retrospective cohort study suggested that a mixed salad (containing ham and feta cheese) was the probable source of infection, but the route of contamination remained unknown. The rapid investigation of such episodes of collective foodborne infections is essential for the implementation of adequate control measures.

Evaluation of ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens.

A new automated amplification method, Ligase Chain Reaction (LCx MTB), was evaluated for direct detection of Mycobacterium tuberculosis in respiratory specimens from 208 patients and its performance was compared with culture and direct smears. Out of 226 specimens, 28 LCx MTB and 15 cultures were found positive for M. tuberculosis. After resolution of clinical history, the sensitivity of LCx MTB and culture was respectively 89.3% and 53.6% with a specificity of 98.5% and 100%. However, samples coming from untreated patients presented similar results between culture and LCx MTB (sensitivity 75% and 83.3% for culture and LCx MTB).

Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates in a Belgian hospital.

In 1995, the rate of isolation of Enterobacter aerogenes in the Saint-Pierre University Hospital in Brussels, Belgium, was higher than that in the preceding years. A total of 45 nosocomial E. aerogenes strains were collected from 33 patients of different units during that year, and they were isolated from 19 respiratory specimens, 13 pus specimens, 7 blood specimens, 4 urinary specimens, 1 catheter specimen, and 1 heparin vial. The strains were analyzed to determine their epidemiological relatedness and were characterized by their antibiotic resistance pattern determination, plasmid profiling, and genomic fingerprinting by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The majority of the strains (82%) were multiply resistant to different commonly used antibiotics. Two major plasmid profiles were found: most strains (64%) harbored two plasmids of different sizes, whereas the others (20%) contained a single plasmid. PFGE with SpeI and/or XbaI restriction enzymes revealed that a single clone (80%) was responsible for causing infections or colonizations throughout the year, and this result was concordant with those obtained by plasmid profiling, with slight variations. By comparing the results of these three methods, PFGE and plasmid profiling were found to be the techniques best suited for investigating the epidemiological relatedness of E. aerogenes strains, and they are therefore proposed as useful tools for the investigation of nosocomial outbreaks caused by this organism.

Evaluation of a new Epstein-Barr virus Combi Test for rapid serologic diagnosis of infectious mononucleosis.

We evaluated the EBV Combi Test (Virion) in serum samples from 574 children with a clinical presentation suggestive of infectious mononucleosis (IM) and compared its performance with several other EBV serological tests. Out of 574 sera 66 gave an acute IM pattern, 406 gave a past infection pattern and 102 were found negative in the EBV Combi Test. Positive VCA IgM and VCA IgG IFA results, in the absence of EBNA antibodies, were found in 62 cases in which the EBV Combi Test gave an acute IM pattern. In addition, 4 to the 574 tested sera gave an acute positive result in the EBV Combi Test (two of them were VCA IgM positive and the other two VCA IgM negative but also EBNA negative). None of these four sera were CMV IgM or Toxoplasma gondii IgM positive. The heterophile antibody test was positive in only 28, and VCA IgM EIA positive in 44 of the 62 IM cases. These data confirm the necessity for an EBV serological diagnosis in children where the clinical diagnosis of EBV infectious mononucleosis must be confirmed or ruled our.

Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens.

A PCR assay for the detection of Helicobacter pylori in gastric biopsy specimens with specific primers for ureC gene amplification (herein referred to as ureC PCR) was compared with other routine invasive methods (culture, the rapid-urease test, and Giemsa staining of histological sections) with samples from a group of 104 consecutive dyspeptic patients. Bacteria were found in 40 (38.5%), 38 (36.5%), 36 (34.6%), and 35 (33.7%) of the patients by ureC PCR, culture, the rapid-urease test, and Giemsa stain, respectively. Sixty-three patients had negative cultures, negative histological examinations, and negative rapid-urease test results, and 61 of these patients were also negative by ureC PCR. ureC PCR detected H. pylori in two culture-negative patients. In parallel, a PCR-based assay to detect the H. pylori cytotoxin-associated antigen (cagA) gene, a putative virulence gene, was also developed. To assess the likelihood of detection of H. pylori genes directly from gastric biopsy samples and from the corresponding H. pylori isolates, specimens from 31 patients were subjected to PCR with ureC- and cagA-targeting primers. All 31 biopsy specimens and the corresponding H. pylori isolates were positive in the ureC PCR. H. pylori strains that were cagA positive also gave positive cagA PCR fragments with biopsy specimens from the same patients. All ureC PCR-positive patients were examined; biopsy specimens from 10 of 11 (91.7%) duodenal ulcer patients harbored H. pylori cagA-positive strains, whereas 19 of26 (73%) of those from patients with chronic gastritis only were found to be cagA positive. These findings indicate first that ureC PCR is at least as sensitive as culture for diagnosing H. pylori infection and second that the presence of the H. pylori cagA gene can also be detected directly in biopsy specimens by PCR amplification.

Detection of 4-quinolone resistance mutation in gyrA gene of Shigella dysenteriae type 1 by PCR.

To study 4-quinolone resistance, the N-terminal coding region of gyrA from nalidixic acid-susceptible and -resistant isolates of Shigella dysenteriae type 1 was amplified by PCR, cloned, and sequenced. DNA sequence analysis of gyrA from nalidixic acid-resistant isolates revealed a C-to-T transition at nucleotide position 248 leading to a Ser-83-to-Leu substitution which was absent in susceptible clinical isolates. Direct HinfI digestion of PCR-amplified DNA detected similar mutations. Thus, DNA gyrase A subunit mutation Ser-83 to Leu is implicated in 4-quinolone resistance in S. dysenteriae type 1.

Iron-regulated proteins in outer membranes of Campylobacter jejuni diarrhoea isolates and immune response to the proteins in patients.

The outer membrane protein (OMP) profiles from 8 Campylobacter jejuni and 5 Campylobacter coli fecal isolates grown under various conditions were compared by SDS-PAGE. The bacteria were grown under usual conditions, in iron-deficient medium (Dip) and on iron-supplemented medium (Fe). The OMP profiles of most bacterial strains grown under usual conditions, or in the Fe-supplemented medium, contained four major bands of approximately 31, 45, 63-66 and 97 kDa, and in addition, a number of minor bands. It was found that OMP from 10 of 13 strains tested and grown on iron deficient medium contained an intensive band of a protein in the molecular weight region of 76 kDa which was lacking in the OMP of bacteria grown in the presence of iron (iron-regulated protein). Sera from 11 children with C. jejuni infection analyzed by Western blot recognized the 76 kDa bands, in contrast to only one out of 10 control sera from healthy children. The Western-blot experiments demonstrated also various bands of other OMP components, both in OMP-Dip and OMP-Fe. The 45 kDa (porin protein) was recognized by all 11 serum samples from C. jejuni-infected patients and in 8 out of 10 control sera. The data suggested that the 76 kDa iron-regulated protein was expressed by bacteria during infection and it stimulated the immune response in children infected with C. jejuni.

Discrimination of epidemic and sporadic isolates of Arcobacter butzleri by polymerase chain reaction-mediated DNA fingerprinting.

DNA polymorphisms of Arcobacter butzleri outbreak-related strains and Arcobacter reference strains were determined by use of the polymerase chain reaction with primers aimed at repetitive sequences. The epidemiological relationship among 14 outbreak-related strains was substantiated, as they showed virtually no genomic variations. Their DNA amplification patterns were, however, clearly different from those of all Arcobacter reference strains studied; each reference strain was characterized by a unique DNA fingerprint.

Roles of leukotriene B4, prostaglandin E2, and cyclic AMP in Campylobacter jejuni-induced intestinal fluid secretion.

Infection of rabbit ileal loops with inflammatory Campylobacter jejuni strains caused elevation of cyclic AMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. Incubation of cultured Caco-2 cells with loop fluids caused elevated cellular cyclic AMP levels, an effect which was inhibited by antiserum against prostaglandin E2.

Neutralising antibodies to the vacuolating toxin of Helicobacter pylori in gastritis only and peptic ulcer patients.

Sera from 36 patients with gastritis only and 26 patients with peptic ulcer were tested for the presence of neutralising antibodies against H. pylori vacuolating toxin. The frequency with which sera did neutralise the vacuolating toxin was not significantly different among the groups of patients with gastroduodenal ulcer infected either with toxigenic or nontoxigenic H. pylori strains (9/14 vs 4/12; ns) or in patients with gastritis only harbouring toxigenic H. pylori strains (8/12). By contrast, none of 24 sera obtained from patients with gastritis and harbouring non-toxigenic H. pylori strains did neutralise the vacuolating toxin. It is suggested that H. pylori vacuolating toxin is involved in the pathogenesis of peptic ulcer disease.

Plasmid profiles and antimicrobial susceptibility of Campylobacter jejuni isolated from Israeli children with diarrhea.

Thirty Campylobacter jejuni (C. jejuni) strains isolated from stools of Israeli children with enteritis were tested for sensitivity to eight antimicrobial agents (MIC) and the presence of plasmids. It was found that all the isolates were sensitive to ciprofloxacin, ofloxacin, furazolidone and erythromycin. Of the 30 strains tested, 21 (70%) were found to be tetracycline-resistant, a relatively high resistance rate as compared with data from other countries and previous reports from Israel. Plasmids were detected in 17 out of 30 C. jejuni isolates (55.6%). A total of nine different plasmid profiles could be distinguished; six profiles were represented by one strain each. Of the 21 tetracycline-resistant strains, plasmids were found in 17 isolates (80%) carrying from 1-2 to 5 plasmids of various sizes. No plasmids were found in tetracycline-sensitive strains, with the exception of one isolate which contained a 24.4 MDa plasmid and was co-trimoxazole-resistant. Our studies indicate a relatively high percentage of tetracycline-resistant C. jejuni isolates in the Tel Aviv area. In 80% of these strains, various plasmid profiles were detected.

Molecular characterization of resistance plasmids in epidemiologically unrelated strains of multiresistant Haemophilus influenzae.

Thirty-three epidemiologically unrelated strains of ampicillin-chloramphenicol-resistant isolates of Haemophilus influenzae (22 type b, 11 unencapsulated), isolated over 10 years in Belgium, were compared with 53 ampicillin-resistant chloramphenicol-susceptible isolates (22 type b, 31 unencapsulated). All ampicillin-chloramphenicol-resistant and 76% of ampicillin-resistant chloramphenicol-susceptible strains were resistant to tetracycline, kanamycin, or both. Resistance to these antibiotics was specified by a 37- to 44-MDa conjugative plasmid. The genetic relatedness of these plasmids and of those in multiresistance strains from Spain was investigated. Plasmids specifying ampicillin-chloramphenicol-tetracycline-kanamycin resistance in Belgium or in Spain had highly related restriction fragment patterns. By homoduplex analysis, they had similar molecular organization and contained a structure identical to Tn10-TnCm, a transposon previously identified in chloramphenicol-tetracycline-resistant H. influenzae. Plasmids coding for different resistance phenotypes had less resemblance by restriction endonuclease analysis; however, study of heteroduplex molecules indicated they shared a high proportion of core sequences. These findings support the hypothesis of independent transposition events resulting in resistance plasmids of close molecular organization.