MALDI-TOF MS contribution to the diagnosis of Campylobacter rectus multiple skull base and brain abscesses.

Campylobacter rectus is rarely associated with invasive infection. Both the isolation and the identification requirements of C. rectus are fastidious, probably contributing to an underestimation of its burden. We report the case of a 66-year-old man who developed several skull base and intracerebral abscesses after dental intervention. Campylobacter rectus was isolated from the brain biopsy. Within 45 minutes of reading the bacterial plate, the strain was accurately identified by MALDI-TOF MS. This rapid identification avoided the extra costs and delays present with 16S rRNA gene sequencing and allowed for a rapid confirmation of the adequacy of the empirical antibiotic treatment.

Accuracy of the API Campy system, the Vitek 2 Neisseria-Haemophilus card and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Campylobacter and related organisms.

Biochemical identification of Campylobacter and related organisms is not always specific, and may lead to diagnostic errors. The API Campy, the Vitek 2 system and matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are commercially available methods that are routinely used for the identification of these microorganisms. In the present study, we used 224 clinical isolates and ten reference strains previously identified by multiple PCR assays, whole cell protein profiling and either DNA-DNA hybridization or sequencing analysis to compare the reliability of these three methods for the identification of Campylobacter and related pathogens. The API Campy accurately identified 94.4% of Campylobacter jejuni ssp. jejuni and 73.8% of Campylobacter coli, but failed to correctly identify 52.3% of other Epsilobacteria. The Vitek 2 Neisseria-Haemophilus card correctly identified most C. jejuni ssp.jejuni (89.6%) and C. coli (87.7%) strains, which account for the majority of campylobacterioses reported in humans, but it failed in the identification of all of the other species. Despite a good identification rate for both C. jejuni ssp. jejuni and C. coli, both methods showed poor sensitivity in the identification of related organisms, and additional tests were frequently needed. In contrast to API Campy and Vitek, MALDI-TOF MS correctly identified 100% of C. coli and C. jejuni strains tested. With an overall sensitivity of 98.3% and a short response time, this technology appears to be a reliable and promising method for the routine identification of Campylobacter and other Epsilobacteria.

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.

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.

Investigation of an outbreak of Campylobacter upsaliensis in day care centers in Brussels: analysis of relationships among isolates by phenotypic and genotypic typing methods.

An outbreak of Campylobacter upsaliensis in four Brussels day care centers (A, B-1, B-2, and C) affected 44 children. Diarrhea was the major symptom. From January 1991 to June 1992, the outbreak strain was isolated from 3, 1, and 21 (of 68) children in centers A, B-1, and B-2, respectively, and from 19 of 22 children in center C, IgG, IgM, and IgA antibodies were detected by Western blotting of serum specimens of 9 of 10 and 13 of 16 children in centers B-2 and C, respectively. Strains were typed by biotyping, DNA restriction-based and antibiotic susceptibility typing, whole cell protein and plasmid analysis, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR). On the basis of RFLP and PCR typing, the strains could be divided into two strongly related clonal variants: One was isolated only from the children of center A and the second only from children in the other day care centers.

Outbreak of recurrent abdominal cramps associated with Arcobacter butzleri in an Italian school.

In the autumn of 1983, an outbreak of recurrent abdominal cramps occurred in a nursery and primary school in the Rovigo area in Italy. None of the 10 affected children had diarrhea. An atypical Campylobacter-like organism was isolated from feces in all cases. Conventional enteropathogens were searched for but not detected. The Campylobacter-like organism was identified as Arcobacter butzleri by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and cellular fatty acid analysis. Its identity was confirmed by DNA-DNA hybridizations versus Arcobacter reference strains. All of the preserved outbreak strains have identical protein profiles and phenotypic characteristics and belong to serogroup 1 of the Lior serotyping scheme on the basis of slide agglutination of crude and absorbed antisera of A. butzleri reference strains versus heat-labile antigens of live bacteria. These data point to an epidemiological relationship. The successive timing of the cases suggests person-to-person transmission.

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.

Campylobacter enteritis in Portugal: epidemiological features and biological markers.

From 1984 to 1989, stool samples from 2811 gastroenteritis cases were examined for the presence of Campylobacter jejuni and C. Coli, Salmonella, Shigella and Yersinia species. Isolation rates were: Campylobacter jejuni and C. Coli, 5.3%, Salmonella spp., 14.8%, Shigella spp., 4.6% and Yersinia enterocolitica, 1.1%. Age group distribution analysis shows a higher Campylobacter isolation rate in children under one year of age. Seasonal distribution revealed a peak incidence in winter as in other Mediterranean countries. Predominant biotypes were C. jejuni I (51%), C. jejuni II (21.5%) and C. coli I (18.8%). Antimicrobial susceptibility testing did not reveal resistance to erythromycin. Thirty of the strains harboured plasmids with 7 different profiles.

Restriction fragment length polymorphism for the identification of Campylobacter jejuni-isolates.

Companion animals ("pets") are occasionally carriers of organisms pathogenic for man. In the present, study fecal samples of clinically inapparent animals with direct contact to 204 patients, suffering from campylobacter enteritis, were investigated for C. jejuni or C. coli (CJC). CJC positive animals were seen in the environment of only five patients (= 2.4%). By comparison of biotypes and serotypes of thermostable and thermolabile antigens from human and animal isolates no clear epidemiological relationship could be deduced. Using chromosomal DNA of the strains, genetic identity of the isolates was studied for HaeIII-restriction fragment length polymorphism (RFLP), applying a biotinylated commercial CJ probe. The probe was found to be specific for most CJ strains and revealed a pattern of one to four bands. In contrast to biotyping no identity of patient strains and animal isolates was seen in three cases; one case with different biotypes had identical RFLP patterns; one patient CJ strain did not show any pattern with the CJ probe. Serotypes were identical for a larger number of animal strains but differed in HaeIII RFLP and vice versa. Comparing the results from the different technological approaches it seems impossible to give a clear statement on the epidemiology of campylobacter infections or carrier state by biotyping alone. It is concluded that DNA RFLP patterns are a useful additional tool, but for epidemiological analysis a set of different methods should be used.

Characterization and description of "Campylobacter upsaliensis" isolated from human feces.

During a 3-year period, "Campylobacter upsaliensis" was isolated from 99 patients. Phenotypic characterization and numerical analysis of protein electrophoregrams showed evidence that "C. upsaliensis" is a distinct Campylobacter species with unique characteristics. The MBCs of 13 antibiotics were determined. In general, these organisms were highly susceptible to drugs that were present in the selective isolation media, making none of the available selective media suitable for the isolation of "C. upsaliensis." Ten strains were found to be resistant to erythromycin (MBCs, greater than or equal to 12.50 mg/liter). Plasmid DNA was detectable in 89 of the 99 strains; 16 plasmid profiles could be identified. Plasmid pattern 16, containing four plasmids of 52, 32, 5.5, and 2.6 megadaltons, represented 60.7% of the plasmid-containing strains. None of the "C. upsaliensis" strains could be agglutinated with antisera against heat-labile antigens from C. jejuni, C. coli, or C. laridis. "C. upsaliensis" was found to be susceptible to serum killing and was readily phagocytized by human polymorphonuclear cells.

Is "Campylobacter upsaliensis" an unrecognised cause of human diarrhoea?

For 3 years a filtration system for the isolation of "new" campylobacter was included in the culture protocol of 15,185 stool specimens. "C upsaliensis" was isolated in 99 patients, C jejuni subsp doylei in 4, and C hyointestinalis in 2. "C upsaliensis" was the only organism isolated in 83 patients. Clinical information was available for 77 out of these 83 patients. 92% of the patients had diarrhoea; vomiting and fever were rare (14% and 7%, respectively); the onset was mostly sudden; and the symptoms usually lasted for less than a week. Gross or occult blood was present in a quarter of cases and neutrophils were detected in faecal smears in about a fifth. "C upsaliensis" may be an unrecognised and frequent cause of diarrhoea in man, and selective isolation media should be combined with non-selective isolation systems.

In-vitro evaluation of antibiotic combinations against Campylobacter fetus.

We evaluated the in-vitro activity of 16 antibiotics against 22 clinical isolates of Campylobacter fetus. The interaction of these antibiotics was also tested by chequer-board titration and time-kill assay. Eight antibiotic combinations were evaluated. Antagonism was not found with any of the combinations tested. The highest synergistic effect was obtained with a combination of ampicillin and cefazolin or ampicillin and gentamicin.

Semisolid blood-free selective-motility medium for the isolation of campylobacters from stool specimens.

Isolation of Campylobacter jejuni and C. coli from stool specimens is done by growing campylobacter colonies on solid selective media with or without blood. However, recognition of these colonies can be difficult. Therefore, we decided to evaluate an isolation procedure based on the swarming of campylobacters through a semisolid medium. We developed a semisolid blood-free selective motility (SSM) medium which is composed of Mueller-Hinton broth with 0.4% agar and supplemented with cefoperazone (30 micrograms/ml) and trimethoprim (50 micrograms/ml). The SSM medium was compared with our previously described Butzler Medium Virion (Goossens et al., J. Clin. Microbiol. 24:840-843, 1986) and blood-free medium (Bolton and Coates, J. Appl. Bacteriol. 54:115-125, 1983) with cefoperazone (32 micrograms/ml) (Bolton et al., J. Clin. Pathol. 37:956-957, 1986). Of 1,890 routine stool specimens tested, 100 were found to be positive for campylobacters: 95 were recovered with the SSM medium, 94 with the Virion medium, and 90 with the blood-free medium. The SSM medium performed equally well whether it was incubated in the special incubator or the candle jar. Only 4.4 and 7.3% of the plates grew contaminating fecal flora when incubated in the special incubator and the candle jar, respectively. Clearly the SSM medium is easy, quick, cheap, sensitive, and more selective than any other medium which has been developed so far and does not require the addition of blood. We believe that this medium has a future in the routine microbiology laboratory in developed as well as in developing countries.

Modified selective medium for isolation of Campylobacter spp. from feces: comparison with Preston medium, a blood-free medium, and a filtration system.

Our previously described (H. Goossens, M. De Boeck, and J. P. Butzler, Eur. J. Clin. Microbiol. 2:389-393, 1983) selective medium, consisting of cefoperazone (15 mg/liter), rifampin (10 mg/liter), colistin (10,000 IU/liter), and amphotericin B (2 mg/liter) (medium M1), for the isolation of Campylobacter jejuni and Campylobacter coli from stool specimens was modified as follows: cefoperazone (30 mg/liter), rifampin (10 mg/liter), and amphotericin B (2 mg/liter) (medium M2). A comparative study of the isolation of Campylobacter spp. from stool specimens was carried out with medium M1; medium M2; a selective blood-free medium consisting (per liter) of charcoal (4 g), ferrous sulfate (0.25 g), sodium pyruvate (0.25 g), casein hydrolysate (3 g), sodium deoxycholate (1 g), nutrient broth no. 2 (25 g), agar (12 g), and cefoperazone (32 mg) (medium M3); and Preston medium containing (per liter) trimethoprim (10 mg), rifampin (10 mg), polymyxin B (5,000 IU), and cycloheximide (100 mg) (medium M4). We also included a filtration system in which membrane filters were applied directly to the surface of the nonselective blood-free medium distributed in small petri dishes. A total of 5,276 stool specimens were tested: 2,788 stool specimens were tested on M1 and M3 in study 1; 2,488 stool specimens were inoculated on the four selective media in study 2, and the last 986 specimens of the 2,488 were tested in parallel with the filtration system. In study 2, 128 Campylobacter strains were isolated from 126 different patients; 85.0, 88.3, 82.5, and 66.6% of these strains were isolated on M1, M2, M3, and M4, respectively. No contaminating fecal flora was found on 65.4, 70.7, 62.4, and 40.3% of the M1, M2, M3, and M4 plates, respectively. Furthermore, C. coli was found to be more susceptible to antibiotics present in the selective media, particularly colistin and polymyxin B, than was C. jejuni. We therefore recommend M2 for the isolation of Campylobacter spp. Finally, the filtration method was found to be easy and cheap; although the sensitivity was low, this method allowed the isolation of new Campylobacter spp. which seem to be associated with diarrhea.

Nosocomial outbreak of Campylobacter jejuni meningitis in newborn infants.

In a nosocomial outbreak of Campylobacter jejuni infection 11 newborn infants (7 female, 4 male) had meningitis. The outbreak was caused by a single strain of C jejuni, as demonstrated by biotyping (biotype I), serotyping (LAU 7/PEN 18 on heat-stable antigens, a new serotype on heat-labile antigens), and the identical susceptibility pattern and outer-membrane-protein profile on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Specific antibodies against the outbreak strain (enzyme-linked immunosorbent assay and Western blot) developed in all the babies. They were treated with gentamicin and ampicillin. All but one baby, who had a moderately dilated left lateral ventricle after the meningitis, recovered well. The source of infection could not be clearly determined. Thus, C jejuni can cause serious nosocomial infection; it should be considered as a possible agent of meningitis of unknown origin, particularly in newborn infants and other compromised hosts.