Molecular Characterization of the 16S rRNA Gene of Helicobacter fennelliae Isolated from Stools and Blood Cultures from Paediatric Patients in South Africa
Forty strains of H. fennelliae collected from paediatric blood and stool samples over an 18 year period at a children's hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes of H. fennelliae were identified based on the phylogenetic analysis. This was confirmed by sequencing a portion of the beta subunit of the RNA polymerase (rpoB) gene. All isolates from South Africa clustered with a proposed novel Helicobacter strain (accession number AF237612) isolated in Australia, while three H. fennelliae type strains from the northern hemisphere, NCTC 11612, LMG 7546 and CCUG 18820, formed a separate branch. A large (355bp) highly conserved intervening sequence (IVS) in the 16S rRNA was found in all isolates. Predicted secondary structures of the IVS from the 16S rRNA and 23S rRNA were characterised by a primary stem structure formed by base pairing of the 3′ and 5′ ends and internal loops and stems. This phylogenetic analysis is the largest undertaken of H. fennelliae. The South African H. fennelliae isolates are closely related to an Australian isolate previously reported to be a possible novel species of Helicobacter. This study suggests that the latter is strain of H. fennelliae. 1. Introduction Since the discovery of Helicobacter pylori (H. pylori) by Warren and Marshall in 1983 [1], more than 30 non-pylori-Helicobacter species have been described [2, 3]. To date, H. bizzozeronnii, H. canadensis, H. canis, H. cinaedi, H. fennelliae, H. felis, H. heilmannii, H. pullorum, H. rappini, H. salomonis, H. winghamensis, and H. westmeadii have been found in humans with gastritis, enteritis, and septicaemia [3–8]. H. fennelliae was first described in 1985 as a new Campylobacter species isolated from asymptomatic homosexual men with enteritis and proctitis [9]. This organism was subsequently reclassified as a Helicobacter species based on 23S rRNA hybridisation studies [10]. H. fennelliae is a fastidious organism and difficult to culture; thus, there are very few reports of the clinical relevance of the organism. In 2000, Tee et al. [11] described a novel species of Helicobacter isolated from the blood of a young aboriginal child with diarrhoea and vomiting which was most closely related to H. fennelliae. The authors proposed, this may be a new species of Helicobacter. From 1977 to 1990, the routine microbiological laboratories at Red Cross War Memorial Children’s and Groote Schuur Hospitals in Cape Town, South Africa used a variety of antibiotic-containing media plates and standard microaerophilic atmospheric growth
References
[1]
J. R. Warren and B. J. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,” The Lancet, vol. 1, no. 8336, pp. 1273–1275, 1983.
[2]
C. Seymour, R. G. Lewis, M. Kim et al., “Isolation of Helicobacter strains from wild bird and swine feces,” Applied and Environmental Microbiology, vol. 60, no. 3, pp. 1025–1028, 1994.
[3]
J. G. Fox, “The non-H pylori helicobacters: their expanding role in gastrointestinal and systemic diseases,” Gut, vol. 50, no. 2, pp. 273–283, 2002.
[4]
J. Stanley, D. Linton, A. P. Burnens et al., “Helicobacter pullorum sp. nov.—genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis,” Microbiology, vol. 140, no. 12, pp. 3441–3449, 1994.
[5]
J. L. O'Rourke, A. Grehan, and M. Lee, “Non-pylori helicobacter species in humans,” Gut, vol. 49, no. 5, pp. 601–606, 2001.
[6]
J. V. Solnick, “Clinical significance of Helicobacter species other than Helicobacter pylori,” Clinical Infectious Diseases, vol. 36, no. 3, pp. 349–354, 2003.
[7]
K. Van Den Bulck, A. Decostere, M. Baele et al., “Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats,” Journal of Clinical Microbiology, vol. 43, no. 5, pp. 2256–2260, 2005.
[8]
A. J. Lastovica and B. M. Allos, “Clinical significance of Campylobacter and related species other than Campylobacter jejuni and Campylobacter coli,” in Campylobacter, I. Nachamkin, C. M. Szymanski, and M. J. Blaser, Eds., pp. 123–149, ASM Press, Washington, DC, USA, 3rd edition, 2008.
[9]
P. A. Totten, C. L. Fennell, F. C. Tenover, et al., “Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men,” Journal of Infectious Diseases, vol. 151, no. 1, pp. 131–139, 1985.
[10]
P. Vandamme, E. Galsen, R. Rossau et al., “Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov,” International Journal of Systematic Bacteriology, vol. 41, no. 1, pp. 88–103, 1991.
[11]
W. Tee, S. Hinds, J. Montgomery, and M. L. Dyall-Smith, “A probable new Helicobacter species isolated from a patient with bacteremia,” Journal of Clinical Microbiology, vol. 38, no. 10, pp. 3846–3848, 2000.
[12]
A. J. Lastovica, “Emerging Campylobacter spp.: the tip of the iceberg,” Clinical Microbiology Newsletter, vol. 28, no. 7, pp. 49–56, 2006.
[13]
K. Wilson, “Preparation of genomic DNA from bacteria,” in Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, et al., Eds., supplement 24, pp. 2.4.1–2.4.5, John Wiley & Sons, New York, NY, USA, 1990.
[14]
S. M. Marshall, P. L. Melito, D. L. Woodward, W. M. Johnson, F. G. Rodgers, and M. R. Mulvey, “Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene,” Journal of Clinical Microbiology, vol. 37, no. 12, pp. 4158–4160, 1999.
[15]
C.-Y. Lim, K.-H. Lee, M.-J. Cho et al., “Detection of Helicobacter pylori in gastric mucosa of patients with gastroduodenal diseases by PCR-restriction analysis using the RNA polymerase gene (rpoB),” Journal of Clinical Microbiology, vol. 41, no. 7, pp. 3387–3391, 2003.
[16]
P. Kuhnert, A. P. Burnens, S. L. W. On, and P. A. R. Vandamme, “Misidentifying Helicobacter cinaedi,” Journal of Clinical Microbiology, vol. 39, no. 7, pp. 2751–2752, 2001.
[17]
J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins, “The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools,” Nucleic Acids Research, vol. 25, no. 24, pp. 4876–4882, 1997.
[18]
Y. Van de Peer and R. De Wachter, “TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment,” Computer Applications in the Biosciences, vol. 10, no. 5, pp. 569–570, 1994.
[19]
D. H. Mathews, J. Sabina, M. Zuker, and D. H. Turner, “Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure,” Journal of Molecular Biology, vol. 288, no. 5, pp. 911–940, 1999.
[20]
M. Zuker, “Mfold web server for nucleic acid folding and hybridization prediction,” Nucleic Acids Research, vol. 31, no. 13, pp. 3406–3415, 2003.
[21]
F. E. Dewhirst, Z. Shen, M. S. Scimeca et al., “Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics,” Journal of Bacteriology, vol. 187, no. 17, pp. 6106–6118, 2005.
[22]
E. Stackebrandt and B. M. Goebel, “A place for DNA-DNA re-association and 16S rRNA sequence analysis in the present species definition in bacteriology,” International Journal of Systematic and Evolutionary Microbiology, vol. 44, pp. 846–849, 1994.
[23]
F. E. Dewhirst and B. J. Paster, “Helicobacter fennelliae (CCUG 18820) partial 16S ribosomal RNA,” 1993, http://www.ncbi.nlm.nih.gov/nuccore/174807.
[24]
T. Adékambi, M. Drancourt, and D. Raoult, “The rpoB gene as a tool for clinical microbiologists,” Trends in Microbiology, vol. 17, no. 1, pp. 37–45, 2009.
[25]
L. Ait Tayeb, E. Ageron, F. Grimont, and P. A. D. Grimont, “Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates,” Research in Microbiology, vol. 156, no. 5-6, pp. 763–773, 2005.
[26]
J.-S. Ki, W. Zhang, and P.-Y. Qian, “Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification,” Journal of Microbiological Methods, vol. 77, no. 1, pp. 48–57, 2009.
[27]
J.-S. Ki, R. Zhang, W. Zhang, Y.-L. Huang, and P.-Y. Qian, “Analysis of RNA polymerase beta subunit (rpoB) gene sequences for the discriminative power of marine vibrio species,” Microbial Ecology, vol. 58, pp. 679–691, 2009.
[28]
E. Evguenieva-Hackenberg, “Bacterial ribosomal RNA in pieces,” Molecular Microbiology, vol. 57, no. 2, pp. 318–325, 2005.
[29]
M. E. Konkel, R. T. Marconi, D. J. Mead, and W. Cieplak Jr., “Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni,” Molecular Microbiology, vol. 14, no. 2, pp. 235–241, 1994.
[30]
D. Linton, J. P. Clewley, A. Burnens, R. J. Owen, and J. Stanley, “An intervening sequence (IVS) in the 16S rRNA gene of the eubacterium Helicobacter canis,” Nucleic Acids Research, vol. 22, no. 11, pp. 1954–1958, 1994.
[31]
D. Linton, F. E. Dewhirst, J. P. Clewley, R. J. Owen, A. P. Burnens, and J. Stanley, “Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains,” Microbiology, vol. 140, no. 4, pp. 847–855, 1994.
[32]
T. J. Trust, S. M. Logan, C. E. Gustafson et al., “Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp,” Journal of Bacteriology, vol. 176, no. 15, pp. 4597–4609, 1994.
[33]
J. G. Fox, L. L. Yan, F. E. Dewhirst et al., “Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice,” Journal of Clinical Microbiology, vol. 33, no. 2, pp. 445–454, 1995.
[34]
A. Hurtado, J. P. Clewley, D. Linton, R. J. Owena, and J. Stanley, “Sequence similarities between large subunit ribosomal RNA gene intervening sequences from different Helicobacter species,” Gene, vol. 194, no. 1, pp. 69–75, 1997.
[35]
Y. Etoh, A. Yamamoto, and N. Goto, “Intervening sequences in 16s rRNA genes of Campylobacter sp.: diversity of nucleotide sequences and uniformity of location,” Microbiology and Immunology, vol. 42, no. 3, pp. 241–243, 1998.
[36]
C. S. Harrington and S. L. W. On, “Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications,” International Journal of Systematic Bacteriology, vol. 49, no. 3, pp. 1171–1175, 1999.
[37]
C. A. Kemper, P. Mickelsen, A. Morton, B. Walton, and S. C. Deresinski, “Helicobacter (Campylobacter) fennelliae-like organisms as an important but occult cause of bacteraemia in a patient with AIDS,” Journal of Infection, vol. 26, no. 1, pp. 97–101, 1993.
[38]
V. L. Ng, W. K. Hadley, and C. Fennell, “Successive bacteremias with “Campylobacter cinaedi” and “Campylobacter fennelliae” in a bisexual male,” Journal of Clinical Microbiology, vol. 25, no. 10, pp. 2008–2009, 1987.
[39]
S. L. Orlicek, D. F. Welch, and T. L. Kuhls, “Helicobacter fennelliae bacteremia in a child with leukemia,” Infectious Diseases in Clinical Practice, vol. 3, no. 6, pp. 450–451, 1994.
[40]
P.-R. Hsueh, L.-J. Teng, C.-C. Hung et al., “Septic shock due to Helicobacter fennelliae in a non-human immunodeficiency virus-infected heterosexual patient,” Journal of Clinical Microbiology, vol. 37, no. 6, pp. 2084–2086, 1999.