Veterinary Microbiology 149 (2011) 492–496

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Short communication

Occurrence of Helicobacter pullorum in turkeys Renato Giulio Zanoni a,*, Silvia Piva a, Mirko Rossi b, Fre´de´rique Pasquali c, Alex Lucchi c, Alessandra De Cesare c, Gerardo Manfreda c a

Department of Veterinary Public Health and Animal Pathology, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano Emilia, Bologna, Italy b Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Agnes Sjo¨bergin Katu 2, Helsinki, Finland c Department of Food Science, Alma Mater Studiorum - University of Bologna, Via Del Florio 2, 40064, Ozzano Emilia, Bologna, Italy

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 May 2010 Received in revised form 28 September 2010 Accepted 8 November 2010

In order to investigate the occurrence of Helicobacter pullorum in turkeys, caecum contents collected at the slaughterhouse from 55 animals intensively reared in 11 farms were sampled. Gram-negative curved rod bacteria were isolated by a modified Steele and McDermott filter technique and further identified as H. pullorum by polymerase chain reaction (PCR). Eleven and 31 isolates, randomly selected from each positive farm, underwent phenotypic (biochemical and antibiotic susceptibility tests) and genotypic characterization (PFGE and AFLP analysis), respectively. Forty-two out of 55 animals (76.4%) and all the 11 farms sampled were positive for H. pullorum. Isolates showed similar biochemical characteristics and whole cell protein profiles but showed a high degree of genetic heterogeneity. Ten out of 11 isolates were resistant to one or more antibiotics with erythromycin, ciprofloxacin and nalidixic acid resistance being the most frequently detected. This is the first description of H. pullorum in turkeys. H. pullorum is a frequent intestinal colonizer in turkeys; therefore, attention should be given to clarify the foodborne risk linked to carcass contamination. Antibiotic resistance is a concern since high values of resistance rates were observed. Published by Elsevier B.V.

Keywords: Helicobacter pullorum Turkey PFGE AFLP Antibiotic resistance

1. Introduction Helicobacter pullorum was classified as a new species by Stanley et al. in 1994. This organism, or its DNA, has been detected in the intestinal contents of chickens (Atabay et al., 1998; Ceelen et al., 2006a; Zanoni et al., 2007), guinea fowl (Nebbia et al., 2007) and a psittacine bird (Ceelen et al., 2006b). Furthermore, H. pullorum has been isolated in human patients with gastroenteritis (Burnens et al., 1994; Steinbrueckner et al., 1997; Melito et al., 2000; Ceelen et al., 2005a). The role of H. pullorum in human infections is still unclear. However recent findings on the pathogenicity of H. pullorum could give

* Corresponding author. Tel.: +39 051 2097066; fax: +39 051 2097039. E-mail address: [email protected] (R.G. Zanoni). 0378-1135/$ – see front matter . Published by Elsevier B.V. doi:10.1016/j.vetmic.2010.11.013

an insight to the mechanism of H. pullorum infection in humans (Young et al., 2000; Hynes et al., 2004; Ceelen et al., 2006c). In particular, a direct proinflammatory effect of H. pullorum, due to the bacterial adherence to human epithelial cells has been described, suggesting the possible involvement of this microorganism in inflammatory bowel disease (IBD) onset and perpetuation (Varon et al., 2009). Since there are no data regarding the presence of H. pullorum in turkeys and only few data about H. pullorum antibiotic resistance are available (Ceelen et al., 2005b; Pasquali et al., 2007; Zanoni et al., 2007), the aims of this study were (i) to investigate the occurrence of H. pullorum in commercial turkey farms, (ii) to characterize the isolates from a phenotypic and genotypic point of view and (iii) to investigate the susceptibility of the isolates to different antibiotic agents.

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and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole cell proteins as previously described (Zanoni et al., 2007). The profiles underwent comparative numerical analysis using the Diversity Database (Bio-rad). Similarity among protein profiles was calculated by the Pearson product moment correlation coefficient using UPGMA as a clustering method (Costas, 1992).

2. Materials and methods 2.1. Sampling and isolation Between September 2005 and December 2006, a total of 55 caeca were collected in a slaughterhouse from healthy turkeys coming from 11 different farms (5 caeca per farm) located in northern Italy, and were labeled as 22, 23, 25, 26, 43, 44, 45, 83, 84, 85, 86. Within 5 h after sampling, the caecal contents were processed using a modified Steele and McDermott filter technique as previously described by Zanoni et al. (2007).

2.4. Genotypic characterization Thirty-one H. pullorum isolates randomly selected from the 11 farms, inclusive of the 11 strains selected for phenotypic characterization, underwent genotypic characterization by macrorestriction analysis – pulsed field gel eletrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE was carried out according to the PulseNet protocol for C. jejuni (Ribot et al., 2001) using SacII (Fermentas, St. Leon-Rot, Germany) as restriction enzyme (Gibson et al., 1999). H. pullorum CIP 104787 and C. jejuni LMG 8842 were used as positive control strains. AFLP was carried out as previously described by Parisi et al. (2010), using HindIII and HhaI as restriction enzymes. PFGE and AFLP profiles were imported into Bionumerics 6.0 software (Applied Maths, Saint-Martens-Latem, Belgium). Normalized profiles were compared using the discriminatory index (D) (Hunter and Gaston, 1988) and clustered by the unweighted pair group method with arithmetic mean (UPGMA). The profiles showing a similarity level >95% were assigned to the same AFLP or PFGE type, assuming that they were closely genetically correlated.

2.2. Identification When available, five suspected colonies from each sample, labeled as A, B, C, D and E, were preliminarily identified as H. pullorum by a polymerase chain reaction (PCR) assay as described by Stanley et al. (1994). Since this PCR is also able to amplify the 16S rDNA gene of the closely related species Helicobacter canadensis, in order to distinguish these two species, a 1200 bp PCR product of the 16S rDNA gene was amplified and subsequently underwent ApaLI (Fermentas, International INC, Burlington, ON, Canada) digestion as described by Fox et al. (2000). H. pullorum CIP 104787T was used as a positive control strain for both PCRs while Campylobacter jejuni ATCC 33560 and H. canadensis CCUG 47163T were used as negative control strains for the first and the second PCR, respectively. 2.3. Phenotypic characterization One isolate of H. pullorum randomly selected from each farm (11) underwent several biochemical tests (Table 1)

Table 1 Comparison of phenotypic characteristics of Helicobacter pullorum isolates from turkeys (this study) and chickens (Zanoni et al., 2007; Atabay et al., 1998). Phenotypic tests

Turkeys (this study) (%)

Chickens (Zanoni et al., 2007) (%)

Chickens (Atabay et al., 1998) (%)

Number of strains Gram-negative Oxidase Catalase Urease Hippurate hydrolysis g-Glutamyltranspeptidase Alkaline phosphatase Indoxyl acetate hydrolysis Trace H2S in TSI Nitrate reduction Growth on special media MacConkey Growth on media containing 1% (w/v) glycine 1% (w/v) bile Growth at 37 8C (mO2) 37 8C (mO2 without H2) 37 8C (O2) 37 8C (AnO2) 42 8C (mO2) 25 8C (mO2) Resistant to Nalidixic acid (30 mg) Cephalotin (30 mg)

11 100 100 73 0 0 0 0 0 27 100

15 100 100 93 0 0 0 40 0 87 100

18 100 100 89 0 0 0 11 0 100 83

0

0

0

0 100

0 100

0 72

100 36 0 100 100 0

100 93 0 100 100 0

100 0 0 94 78 0

73 100

7 100

28 89

494

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2.5. Antibiotic susceptibility testing The 11 strains selected for phenotypic assays were tested for the minimum inhibitory concentration (MIC) value of 7 different antibiotic agents according to the agar dilution method described by the Clinical and Laboratory Standard Institute (CLSI, formerly NCCLS) for C. jejuni and related species (NCCLS, 2002) modified as suggested by Zanoni et al. (2007). All antibiotic agents were purchased from Sigma, except for the ciprofloxacin which was obtained from Bayer AG (Leverkusen, Germany). C. jejuni ATCC 33560T was used as a quality control strain for ciprofloxacin, erythromycin, nalidixic acid, gentamycin and tetracycline (NCCLS, 2002) whereas Escherichia coli ATCC 25922T and Staphylococcus aureus ATCC 29213T were used as quality control strains for ampicillin and chloramphenicol (NCCLS, 2004). 3. Results According to the PCR results, all the 11 farms examined were positive for H. pullorum and 42 out of 55 animals (76.4%) were infected. Moreover, all positive samples showed a high number of colonies (>50 CFU) phenotypically referable to H. pullorum in the first isolation plates. The biochemical test results of turkey isolates are reported in Table 1 and compared to previously described chicken isolates (Atabay et al., 1998; Zanoni et al., 2007). The protein profiles of the isolates tested did not show any significant difference in comparison to the protein profile of the type strain H. pullorum CIP 104787T. Overall, the similarity level among the protein profiles of the isolates tested was 88%. In contrast with phenotypic characterization results, both PFGE and AFLP analyses revealed a considerable degree of genomic heterogeneity among the H. pullorum isolates among the various farms, as shown in the dendrograms (Figs. 1 and 2). From 31 isolates undergoing genotypic characterization, isolates 3 and 9 were untypable by PFGE and AFLP, respectively, and were omitted from further comparisons. Regarding PFGE analysis, the genome DNA of the isolates tested showed a number of recognition sites yielding between 5 and 17 DNA bands with a molecular weight up to approximately 570 kb. Considering a cut off of 90%, 20 different PFGE types were identified (Fig. 1). Only isolates from farms 23 and 85 showed a within-farm similarity of 90% whereas isolates from farms 22, 25, 43, 44, 45, 83 and 84 showed PFGE profiles with similarities ranging from 46.3% to 88.9%. The D value achieved using PFGE was 0.976. Using a 90% similarity level cut-off, 13 different AFLP types were identified (Fig. 2). Only isolates from farms 23, 85 and 84 showed a within-farm similarity of 90% whereas isolates from farms 22, 25, 44 and 43 showed AFLP profiles with similarities of 54%, 61%, 61% and 74.5%, respectively. The D value achieved using AFLP was 0.920. Regarding antibiotic resistance, all the isolates of H. pullorum tested were sensitive to gentamycin and chloramphenicol. Nine isolates were resistant to erythromycin, 8 each to nalidixic acid and ciprofloxacin, 4 to tetracycline and 1 to ampicillin. Regarding multiresistance, 9 out of 11

Fig. 1. Dendrogram resulting from the analysis of Helicobacter pullorum PFGE profiles. The numbers on the horizontal axis indicate the percentage similarities as determined by the Dice correlation coefficient and UPGMA clustering (optimization 1% and tolerance 1.8%).

isolates were resistant to two or more antibiotics with multiresistance to ciprofloxacin, nalidixic acid and erythromycin as the most frequently identified antibiotic resistance profile (Table 2).

4. Discussion This is the first report of occurrence of H. pullorum in turkeys. The high rate of H. pullorum observed in turkeys was similar to other descriptions in poultry (Nebbia et al., 2007; Zanoni et al., 2007).

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60

70

80

90 100 96.3

growth at 37 8C in microaerophilic conditions without H2, the isolation of strains which are not dependent on hydrogen has already been described by Steinbrueckner et al. (1997) who isolated H. pullorum after 48 h of incubation under these conditions. The results of the biochemical tests and whole cell protein profiles showed a high degree of phenotypic similarity for all the H. pullorum isolates of this study. Unlike the phenotyping results, genotyping characterization indicated a high degree of genomic diversity. Comparing the two genotyping techniques, PFGE by SacII showed a higher discriminatory power than AFLP by HindIII and HhaI following the calculated D values. The data presented in this study are in accordance with previous reports on genetic diversity in Helicobacter spp. (Taylor et al., 1992; Ha¨nninen and Hirvi, 1999; Gibson et al., 1999), probably related to high rates of intraspecies recombination and/or horizontal gene transfer which have been well described in related species, such as C. jejuni (Suerbaum et al., 2001) and Helicobacter pylori (Suerbaum and Josenhans, 2007). The antibiotic resistance rates observed in turkey H. pullorum isolates were generally higher than in chickens (Ceelen et al., 2005b; Zanoni et al., 2007). Those differences were also described for other pathogenic species (Wallmann et al., 2007) and they might be due to the greater selective pressure caused by a more common use of antibiotics in turkey farming than chicken farming (Van den Bogaard et al., 2001). In this study, the majority of turkey isolates were resistant to ciprofloxacin and erythromycin. Antibiotic resistance, particularly toward fluoroquinolones and macrolides, has now emerged globally in C. jejuni and Campylobacter coli isolated from poultry (Moore et al., 2006), giving rise to concerns about how these organisms have acquired such resistance characteristics as well as consequences for human and animal treatments. In conclusion, this study shows that H. pullorum is a frequent intestinal colonizer of commercial turkeys. If the role of H. pullorum as a disease-causing agent transmitted by food is confirmed, the high frequency of isolation accompanied by the elevated intestinal concentration and the antibiotic resistance rates could represent a serious con-

25/06 A 25/06 C

66.1

44/06 B

62.4

44/06 E CIP 104787 97.2

61.0

43/06 B 43/06 C

80.3 74.5

25/06 B 43/06 D

69.1 77.8

94.6

22/06 B 22/06 C 44/06 A

58.7 66.4

98.7 98.1

76.7

23/06 B 23/06 E 23/06 D 83/06 A

98.6 92.3

54.3

85/06 A 85/06 C 85/06 D 84/06 A

93.8 92.2

84/06 B 84/06 C

60.3

495

22/06 D

Fig. 2. Dendrogram resulting from the analysis of Helicobacter pullorum AFLP profiles. The numbers on the horizontal axis indicate the percentage similarities as determined by the Dice correlation coefficient and UPGMA clustering (optimization 5% and tolerance 0.5%).

In relation to biochemical tests, our results on turkey isolates are in accordance with the data on chicken isolates (Zanoni et al., 2007; Atabay et al., 1998) except for alkaline phosphatase production, traces of H2S in TSI, growth at 37 8C mO2 without H2, and resistance to nalidixic acid which was also variable among chicken isolates (Zanoni et al., 2007; Atabay et al., 1998). Regarding the test of

Table 2 MIC values (mg/mL) of 11 H. pullorum turkey isolates. Isolate

H. pullorum CIP 104787 22/06B 23/06B 24/06D 25/06A 26/06C 43/06B 44/06B 45/06A 83/06A 84/06A 85/06A a b

MIC (mg/mL)a AMP

CIP

NAL

CHL

ERY

GEN

TET

16 32b 4 8 16 4 4 16 2 2 2 2

0.12 32 64 128 0.25 128 16 0.25 32 64 0.25 64

8 128 32 64 32 64 64 16 8 64 8 64

8 16 8 8 16 16 16 8 4 8 16 8

1 >128 >128 >128 2 >128 >128 4 8 >128 >128 >128

0.5 0.5 0.25 0.25 0.25 0.5 0.25 0.5 0.25 0.12 0.25 0.25

2 >128 1 4 2 2 1 1 2 128 64 128

AMP: ampicillin; CIP: ciprofloxacin, NAL: nalidixic acid, CHL: chloramphenicol, ERY: erythromycin, GEN: gentamycin. Resistant isolates, on the basis of the clinical breakpoints indicated by the CLSI (formerly NCCLS, 2004) are represented in bold.

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tamination risk of the carcasses as has already been observed in chickens (Atabay et al., 1998; Zanoni et al., 2007). Acknowledgements We thank Prof. Sanguinetti for her helpful advice and Antonio Parisi for his critical evaluation of the AFLP results. This research study was supported by European Project FOOD-CT-200x-007076 named POULTRYFLORGUT. References Atabay, H.I., Corry, J.E., On, S.L.W., 1998. Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum. J. Appl. Microbiol. 84, 1017–1024. Burnens, A.P., Stanley, J., Morgenstern, R., Nicolet, J., 1994. Gastroenteritis associated with Helicobacter pullorum. Lancet 344, 1569–1570. Ceelen, L., Decostere, A., Verschraegen, G., Ducatelle, R., Haesebrouck, F., 2005. Prevalence of Helicobacter pullorum among patients with gastrointestinal disease and clinically healthy persons. J. Clin. Microbiol. 43, 2984–2986. Ceelen, L., Decostere, A., Devriese, L.A., Ducatelle, R., Haesebrouck, F., 2005. In vitro susceptibility of Helicobacter pullorum strains to different antimicrobial agents. Microb. Drug Resist. 11, 122–126. Ceelen, L.M., Decostere, A., Van den Bulck, K., On, S.L., Baele, M., Ducatelle, R., Haesebrouck, F., 2006. Helicobacter pullorum in chickens, Belgium. Emerg. Infect. Dis. 12, 263–267. Ceelen, L., Decostere, A., Martel, A., Pasmans, F., Haesebrouck, F., 2006. First report of H. pullorum in the faeces of diarrhoeic psittacine bird (Psephotus haematogaster). Vet. Rec. 159, 389–390. Ceelen, L.M., Haesebrouck, F., Favoreel, H., Ducatelle, R., Decostere, A., 2006. The cytolethal distending toxin among Helicobacter pullorum strains from human and poultry origin. Vet. Microbiol. 113, 45–53. Costas, M., 1992. Classification, identification and typing of bacteria by the analysis of their one-dimensional polyacrylamide gel electrophoretic protein patterns. In: Chambrach, A., Dunn, M.J., Rodola, B.J. (Eds.), Advances in Electrophoresis, vol. 5. VCH Verlagsgesellschaft, Weinheim, pp. 351–408. Fox, J.G., Chien, C.C., Dewhirst, F.E., Paster, B.J., Shen, Z., Melito, P.L., Woodward, D.L., Rodgers, F.G., 2000. Helicobacter canadensis sp. nov. isolated from humans with diarrhea as an example of an emerging pathogen. J. Clin. Microbiol. 38, 2546–2549. Gibson, J.R., Ferrus, M.A., Woodward, D., Xerry, J., Owen, R.J., 1999. Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis. J. Appl. Microbiol. 87, 602–610. Ha¨nninen, M.L., Hirvi, U., 1999. Genetic diversity of canine gastric helicobacters Helicobacter bizzozeronii and H. salomonis studied by pulsed-field gel electrophoresis. J. Med. Microbiol. 48, 341–347. Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J. Clin. Microbiol. 26 (11), 2465–2466. Hynes, S.O., Ferris, J.A., Szponar, B., Wadstro¨m, T., Fox, J.G., O’Rourke, J., Larsson, L., Yaquian, E., Ljungh, A., Clyne, M., Andersen, L.P., Moran, A.P., 2004. Comparative chemical and biological characterization of the lipopolysaccharides of gastric and enterohepatic helicobacters. Helicobacter 9, 313–323. Melito, P.L., Woodward, D.L., Bernard, K.A., Price, L., Khakhria, R., Johnson, W.M., Rodgers, F.G., 2000. Differentiation of clinical Helicobacter pullorum isolates from related Helicobacter and Campylobacter species. Helicobacter 5, 142–147.

Moore, J.E., Barton, M.D., Blair, I.S., Corcoran, D., Dooley, J.S., Fanning, S., Kempf, I., Lastovica, A.J., Lowery, C.J., Matsuda, M., McDowell, D.A., McMahon, A., Millar, B.C., Rao, J.R., Rooney, P.J., Seal, B.S., Snelling, W.J., Tolba, O., 2006. The epidemiology of antibiotic resistance in Campylobacter. Microbes Infect. 8, 1955–1966. National Committee for Clinical Laboratory Standards (NCCLS), 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals, Second Edition: Approved Standard M31-A2. NCCLS, Wayne, PA, 80 pp. National Committee for Clinical Laboratory Standards (NCCLS), 2004. Performance Standards for Antimicrobial Susceptibility Testing. Fourteenth Informational Supplement: Approved Standard M100-S14. NCCLS, Wayne, PA, 156 pp. Nebbia, P., Tramuta, C., Ortoffi, M., Bert, E., Cerruti Sola, S., Robino, P., 2007. Identification of enteric Helicobacter in avian species. Schweiz. Arch. Tierheilkd. 149, 403–407. Parisi, A., Latorre, L., Normanno, G., Miccolupo, A., Fraccalvieri, R., Lo russo, V., Santagada, G., 2010. Amplified fragment length polymorphism and multi locus sequence typing for high resolution genotyping of Listeria monocytogenes from foods and the environment. Food Microbiol. 27, 101–108. Pasquali, F., Rossi, M., Manfreda, G., Zanoni, R., 2007. Complete nucleotide sequence of the gyrA of Helicobacter pullorum and identification of point mutations leading to ciprofloxacin resistance in poultry isolates. Int. J. Antimicrob. Agents 30, 222–228. Ribot, E.M., Fitzgerald, C., Kubota, K., Swaminathan, B., Barrett, T.J., 2001. Rapid pulsed-field gel electrophoresis protocol for subtyping of Campylobacter jejuni. J. Clin. Microbiol. 39, 1889–1894. Stanley, J., Linton, D., Burnens, A.P., Dewhirst, F.E., On, S.L., Porter, A., Owen, R.J., Costas, M., 1994. Helicobacter pullorum sp. nov.—genotype and phenotype of new species isolated from poultry and from human patients with gastroenteritis. Microbiology 140, 3441–3449. Steinbrueckner, B., Haerter, G., Pelz, K., Weiner, S., Rump, J.A., Deissler, W., Bereswill, S., Kist, M., 1997. Isolation of Helicobacter pullorum from patients with enteritis. Scand. J. Infect. Dis. 29, 315–318. Suerbaum, S., Lohrengel, M., Sonnrvend, A., Ruberg, F., Kist, M., 2001. Allelic diversity and recombination in Campylobacter jejuni. J. Bacteriol. 183, 2553–2559. Suerbaum, S., Josenhans, C., 2007. Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat. Rev. Microbiol. 5, 441– 452. Taylor, D.E., Eaton, M., Chang, N., Salama, S.M., 1992. Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level. J. Bacteriol. 174, 6800–6806. Van den Bogaard, A.E., London, N., Driessen, C., Stobberingh, E.E., 2001. Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother. 47, 763–771. Varon, C., Duriez, A., Lehours, P., Me´nard, A., Laye´, S., Zerbib, F., Me´graud, F., Laharie, D., 2009. Study of Helicobacter pullorum proinflammatory properties on human epithelial cells in vitro. Gut 58, 629–635. Wallmann, J., Schro¨er, U., Kaspar, H., 2007. Quantitative resistance level of bacterial pathogens (Escherichia coli, Pasteurella multocida, Pseudomonas aeruginosa, Salmonella sp., Staphylococcus aureus) isolated from chickens and turkeys: national resistance monitoring by the BVL 2004/2005. Berl. Munch. Tierarztl. Wochenschr. 120, 452–463. Young, V.B., Chien, C.C., Knox, K.A., Taylor, N.S., Schauer, D.B., Fox, J.G., 2000. Cytolethal distending toxin in avian and human isolates of Helicobacter pullorum. J. Infect. Dis. 182, 620–623. Zanoni, R.G., Rossi, M., Giacomucci, D., Sanguinetti, V., Manfreda, G., 2007. Occurrence and antibiotic susceptibility of Helicobacter pullorum from broiler chickens and commercial laying hens in Italy. Int. J. Food Microbiol. 116, 168–173.