Kurt Houf

Development of a new protocol for the isolation and quantification of Arcobacter species from poultry products

None of the presently available selective supplements for the specific isolation of Arcobacter species allows the growth of Arcobacter butzleri, A. cryaerophilus and A. skirrowii and at the same time fully suppresses the accompanying flora present in poultry and poultry products. Furthermore, little is known about the contamination levels of poultry with Arcobacter species. In this study, a new selective supplement comprising amphotericin B (10 mg/l), cefoperazone (16 mg/l), 5-fluorouracil (100 mg/l), novobiocin (32 mg/l) and trimethoprim (64 mg/l) was developed. With a new isolation procedure, including enrichment in Arcobacter broth with the selective supplement, incubated for 24 to 48 h at 28 degrees C under microaerobic conditions, arcobacters were isolated from 100% (n = 34) of neck skin of laying hens and from 90% (n = 71) of similar samples from broilers. Of the broiler breast meat samples examined (n = 52), 65% were found to be contaminated with these bacteria. In 64% of the samples, A. butzleri was the only Arcobacter species isolated. In 9% of the samples, A. cryaerophilus was the only species present, while 11% of the samples were positive for both species simultaneously. Using direct isolation on the selective agar medium developed in this study, incubated for 24 to 48 h under microaerobic conditions at 28 degrees C. 32 out of 45 broiler carcasses and 6 out of 25 broiler breast meat samples carried a bacterial load of arcobacters of 10(2) to 10(3) cfu/g. The prevalence of Arcobacter in Belgian poultry was found higher than the prevalence of thermophilic Campylobacter species in each of the poultry categories examined. The enrichment procedure and the direct plating method were validated for the isolation of A. skirrowii. For this species, growth performance was less than the other two Arcobacter species and it was not isolated nor detected by m-PCR from the naturally contaminated poultry samples examined. This new protocol provides a fast and reliable method for the isolation of Arcobacter species from poultry and can contribute to more comprehensive epidemiological investigations.

Isolation of Arcobacter skirrowii from a Patient with Chronic Diarrhea

The genus Arcobacter currently includes four species ([7][1]). Two species, Arcobacter cryaerophilus and Arcobacter butzleri , have been associated with human disease. They were mainly isolated from patients with diarrhea and bacteremia ([2][2], [3][3], [6][4]). Arcobacter nitrofigilis has been

Assessment of the Genetic Diversity among Arcobacters Isolated from Poultry Products by Using Two PCR-Based Typing Methods

ABSTRACT In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.

Comparison of Five Repetitive-Sequence-Based PCR Typing Methods for Molecular Discrimination of Salmonella enterica Isolates

ABSTRACT Five repetitive-element PCR (rep-PCR) techniques [primer sets ERIC1R-ERIC2 and REP1R-REP2I and primers ERIC2, BOXA1R, and (GTG)5] were evaluated for the discrimination of Salmonella enterica isolates at the serotype level. On the basis of number, even distribution over the whole fingerprint, and clarity of bands in the fingerprints, the enterobacterial repetitive intergenic consensus (ERIC) primer set and the (GTG)5 primer were chosen for use in the following experiments. For these two primer sets, reproducibility was tested on different lysates of five selected serotypes of Salmonella in the same PCR by using three different PCR runs. Reproducibility was poor between different PCR runs but high within the same PCR run. Furthermore, 80 different serotypes and five isolates which were not typeable by serotyping were fingerprinted. All strains were typeable by the ERIC primer set and the (GTG)5 primer and generated unique fingerprints, except for some strains with incomplete antigenic codes. Finally, 55 genetically different strains belonging to 10 serotypes were fingerprinted to examine the genetic diversity of the rep-PCR within serotypes. This experiment showed that one serotype did not always correlate to only one ERIC or (GTG)5 fingerprint but that the fingerprint heterogeneity within a serotype was limited. In epidemiological studies, ERIC- and/or (GTG)5-PCR can be used to limit the number of strains that have to be serotyped. The reproducibility of isolates in one PCR run, the discriminatory power, and the genetic diversity (stability) of the fingerprint were similar for the Eric primer set and the (GTG)5 primer, so both are equally able to discriminate Salmonella serotypes.

Occurrence and distribution of Arcobacter species in poultry processing.

A total of 16 broiler flocks slaughtered in the morning in eight Belgian poultry slaughterhouses were examined for the presence of Campylobacteraceae. In samples collected before and after chilling, the prevalence of arcobacters was found to be higher than the prevalence of thermophilic campylobacters, with the slaughter procedure used having no clear effect. Two slaughterhouses were selected for a detailed investigation of the occurrence and distribution of arcobacters. Sampling carried out before slaughter revealed that both Arcobacter butzleri and Arcobacter cryaerophilus were commonly present on the slaughter equipment in both plants. These findings indicate inadequate decontamination of the slaughterhouse environment and suggest potential Arcobacter contamination of broiler carcasses through the slaughter equipment. Even before evisceration, contamination levels of hundreds to several thousands of arcobacters per gram of neck skin were detected. It appears unlikely that contamination through slaughter equipment alone explains the high contamination levels found for poultry products. Arcobacters were not isolated from the 30 intestinal tracts sampled for each broiler flock examined. A. cryaerophilus was the only Arcobacter species recovered from the transport crate samples collected before and after washing. Arcobacter contamination during slaughter, either direct (from chicken intestinal content or feces) or indirect (from equipment), was not confirmed. The origin and the precise routes of contamination remain to be determined.

Identification of five human and mammal associated Arcobacter species by a novel multiplex-PCR assay.

A multiplex-PCR assay with seven primers was developed for the identification of the five human and mammal related species of the emerging foodborne pathogen Arcobacter. The assay was validated using 58 reference and 358 collection strains isolated from humans and mammals. The selected primers on the 23 S RNA gene amplify a 2061 bp fragment from A. butzleri, a 1590 bp fragment from A. thereuis, a 1125 bp fragment from A. cibarius and an A. skirrowii specific fragment of 198 bp. For A. cryaerophilus, a primer set on the gyrA gene amplified a specific fragment of 395 bp. No PCR product was generated for closely related bacteria including Campylobacter and Helicobacter species. Furthermore, examination of the 23 S RNA gene of A. cryaerophilus revealed, besides large heterogeneity, the presence of intervening sequences ranging from 87 to 196 bp.

Arcobacter thereius sp. nov., isolated from pigs and ducks.

During a Danish study on the prevalence of campylobacteria in pig abortions and food of animal origin, eight Gram-negative, slightly curved, rod-shaped, non-spore-forming bacteria were clustered by using amplified fragment length polymorphism analysis in a distinct phenon within the genus Arcobacter. In the present study, numerical analysis of whole-cell protein profiles also showed that all isolates clustered in a single group distinct from other recognized Arcobacter species. DNA-DNA hybridization among two representative strains exhibited a mean DNA-DNA relatedness value of 79 %. DNA-DNA hybridization with the type strains of recognized Arcobacter species revealed levels of DNA-DNA relatedness of 41 % or less. The DNA G+C content of the type strain was 28.5 mol%. Pairwise comparison of the 16S rRNA gene sequences with those of the type strains of established species identified Arcobacter cryaerophilus (97.9 %), Arcobacter cibarius (97.5 %) and Arcobacter skirrowii (97.2 %) as the nearest phylogenetic neighbours. The isolates could be distinguished from other Arcobacter species by means of the following biochemical tests: activities of catalase and urease, reduction of nitrate and growth on minimal medium, lack of growth at 37 degrees C under standardized aerobic and microaerobic conditions, in 4 % NaCl and 1 % glycine media. Finally, DNA fingerprints obtained by using enterobacterial repetitive intergenic consenus-PCR showed that the eight isolates represent eight strains of a single novel Arcobacter species, for which the name Arcobacter thereius sp. nov. is proposed. The type strain is LMG 24486(T) (=CCUG 56902(T)).

Detection and characterization of Salmonella in lairage, on pig carcasses and intestines in five slaughterhouses

In this study, conducted at five slaughterhouses, individual pigs were sampled and followed up from stunning to cooling down of the carcasses. In this way, Salmonella prevalence and possible risk points were described. At the lairage area, pens were sampled using overshoes. At stunning and bleeding, pigs were individually identified and subsequently swabs were taken of the oral cavity and the carcass after polishing, splitting and forced chilling. Additionally, duodenum, ileum, rectum and mesenteric lymph nodes were extracted and samples were taken of the scalding water. All samples were submitted to Salmonella isolation and Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). Of all samples taken (n = 1953), 14.1% were Salmonella positive. The prevalence of S. in the lairage area varied widely (from 0 to 100%) between the slaughterhouses. Of the sampled pigs (n = 226), 48.2% were positive in at least one sample. Statistical analysis revealed that the contamination of the lairage area was related to a higher amount of positive carcasses after polishing. Furthermore, the contamination of the carcasses after splitting and forced chilling was related to the contamination level of the carcass after polishing. A relation between the outer (carcass) contamination and the inner (gut content and lymph nodes) contamination of a pig could not be established. The predominant serotypes were S. Typhimurium (58.7%) and S. Derby (17.4%). Genotyping revealed 46 different PFGE profiles among the 276 Salmonella isolates. The same genotype at the lairage area as in the oral cavity of the pigs was found in 95%. The results indicate that the lairage area is a primary source of Salmonella in slaughter pigs and that carcass contamination originates from the environment rather than from the pig (inner contamination) itself. It further shows that slaughterhouses vary in their capability of dealing with Salmonella positive pigs. A slaughterhouse specific approach is needed, however, general guidelines should be provided to decrease the contamination level of the lairage area and the slaughter environment.

Molecular Characterization of Arcobacter Isolates Collected in a Poultry Slaughterhouse

In a poultry slaughterhouse, Arcobacter contamination was examined over a period of 1 week to establish possible routes of contamination. Samples were collected from the slaughter equipment and from processing water before the onset of slaughter and from the first broiler flock slaughtered on each sampling day. Characterization of 1,079 isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction and a random amplified polymorphic DNA assay resulted in the delineation of 159 Arcobacter butzleri and 139 Arcobacter cryaerophilus types. From almost all 140 neck skin samples collected before and after evisceration, A. butzleri and A. cryaerophilus were isolated simultaneously at contamination levels ranging from 10(1) to 10(4) CFU/g. Only six A. butzleri types present in the slaughterhouse environment were also present on the broiler carcasses. None of the A. cryaerophilus genotypes were detected in both the neck skin and the environmental samples. All A. butzleri types isolated from the feather samples were also isolated from broiler neck skin samples. The slaughter equipment was contaminated with arcobacters before the onset of slaughter, but it appeared unlikely that contamination through the slaughter equipment alone explained the high contamination levels on poultry products. Arcobacters were also present in processing water, but types present in water and poultry products were different. Characterization of the Arcobacter isolates did not clarify the routes of transmission, probably because of the extreme heterogeneity among Arcobacter isolates. However, the results obtained in this study brought to light insufficient decontamination at the processing plant involved in the study and confirmed the survival capacity of certain A. butzleri strains.

The prevalence of Arcobacter spp. on chicken carcasses sold in retail markets in Turkey, and identification of the isolates using SDS-PAGE

In this study, the prevalence of Arcobacter spp. on chicken carcasses sold in various retail markets in Turkey was investigated. The isolates were characterized and identified using various phenotypic and molecular tests. The membrane filtration technique employing 0.45-microm pore size membrane filters laid onto a nonselective blood agar was used after enrichment in Oxoid Arcobacter Enrichment Broth (AEB) to examine a total of 75 chicken carcasses (44 fresh and 31 frozen). Species level identification was performed using SDS-PAGE of whole-cell proteins and a recently developed multiplex-PCR assay. All isolates were identified as Arcobacter butzleri. Of the 44 fresh chicken carcasses examined, 42 (95%) were positive for A. butzleri. A. butzleri was also recovered from seven (23%) of the 31 frozen carcasses examined.