From September 2005 to December 2006, in order to define the prevalence of Helicobacter
pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of
animals reared in 76 different farms were collected at the slaughterhouse. A caecum content
of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H.
pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University
Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott
membrane filter method was used. Gram-negative curved rod bacteria were preliminary
identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR
assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was
randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE
analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for
seven different antibiotics were also determined by agar dilution method. Moreover, to
examine the intraspecific genomic variability, two strains isolated from 17 different farms
were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess
the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP
104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining
Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin
and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of
366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted
infected. All positive samples showed a high number of colonies (>50) phenotipically
consistent with H. pullorum on the first isolation media, which suggests that this
microrganism, when present, colonizes the poultry caecum at an elevate load. No human
sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis
showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP
104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin,
chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for
erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these
antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H.
pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability
observed in this study confirm that this species don’t have a clonal population structure, as
motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open
Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time
sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA
(Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of
the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was
functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry
isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating
that poultry species are the reservoir of this potential zoonotic microorganisms. In order to
understand the potential role as food-borne human pathogen of H. pullorum, further studies
must be carried on.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:817 |
| Date | 07 May 2008 |
| Creators | Rossi, Mirko <1977> |
| Contributors | Zanoni, Renato Giulio |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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