Detection and Isolation of Campylobacter Species - Literature review Example

I - While recent studies claim that inflammatory bowel diseases (IBD, are primarily caused by bacteria, there is yet a study that pinpoints the exact organism that result to, for instance, Crohn’s disease (CD). Using molecular and cultural methods, Z. Li et al. (2009) attempt to locate Campylobacter species other than Campylobacter jejuni and Campylobacter concisus – the role of which, despite being presently considered human intestinal pathogens, is little known viz. human IBD -- in intestinal biopsy specimens of children with CD. Z. Li et al. (2009) collected intestinal biopsy specimens from diagnostic-colonoscopy-undergoing eighty-five (85) children – fifty-one (51) of which are male, with ages from two (2) to sixteen (16). Three (3) biopsy specimens were collected from the cecum of children with endoscopically normal mucosae; three (3) biopsy specimens were collected from the inflamed region of children with endoscopic abnormalities. To ascertain the extent of the effect of inflammation on account of Campylobacter species, three (3) additional biopsy specimens from endoscopically normal areas near the inflamed region were collected from thirteen (13) children. The three different biopsy specimens were later on made to serve different functions: from one, DNA was extracted; another group of specimen served for bacterial cultivation; and the third set of specimen was used for histological examination. The diagnosis of CD was realized through standard endoscopic, histologic and radiological investigations, and eventually became the basis of grouping of children-patients into a CD group (with thirty-three [33] members) and a control group (of fifty-two [52] members). From the extracted DNA, the so-called 16S rRNA gene of Campylobacter species was amplified with the use of Campylobacter PCR. The amplification involved 400 ng of DNA in a 50-μl PCR mixture, and with number of thermal cycles pegged to forty (40). The PCR products were studied on agarose gels, sequenced, and the sequence results were compared to gene sequences of knowing identities using the BLAST search program. There yielded five samples whose sequencing results revealed mixed sequences, and these were subjected to C. concisus-specified PCR designed to group Campylobacter species into two (2) genotypes. Z. Li et al. (2009), however, considered every sample positive for either of the genotypes positive for C. concisus. The set of biopsy specimens that was subjected for bacterial cultivation was set against blood agar base no. 2 supplemented with six per cent (6%) sterile defibrinated horse blood, trimethorpim (10 μg/ml), and vancomycin (10 μg/ml). Then, it was incubated under microaerophilic conditions generated by a Campylobacter gas generating system. The bacterial colonies were labeled and sequenced by the use of Oxoid biochemical identification system. C. concisus specific antibodies were determined in the sera from eight (8) children of CD group and twelve (12) children of control group. It was done using enzyme-linked immunosorbent assay, with the isolated whole-cell lysate of C. concisus used as the antigen. The above-mentioned methods resulted to significant research findings. Firstly, it was established that the Campylobacter PCR positivity rate for children with CD was significantly higher than for children of control (82% to 23%). The sequence of PCR products was found to be not vacillating from those of various Campylobacter species. And, C. concisus detection was higher, too, in children with CD than in children of control (51% to 2%); however, the prevalence of other Campylobacter species in CD children was found to be not significantly different from those in control. Secondly, it is worth noting that from the thirteen (13) children from whom an extra set of biopsy specimens were collected, seventy-six per cent (76%) and forty-six per cent (46%) of the non-inflamed and inflamed tissues respectively were Campylobacter PCR positive. Z. Li et al. (2009) however held that the difference was never significant. Finally, from this study, four different Campylobacter species were isolated based on sequencing of the nearly complete 16S rRna gene. These were C. concisus, C. hominis, C. showae, and Bacteroides ureolyticus. These isolates were detected by Campylobacter PCR. This constituted the first report of successful isolation of these organisms from CD patients. Now, Z. Li et al. (2009) closes their paper by pointing out future research directions. Noting that the level of immunoglobulin G (IgG) antibodies specific to C. concisus was higher in children with CD than those in the control (0.991 ± 0.447 to 0.320 ± 0.303 respectively), they were candid in stating that the significance of this information is never established in their study. They nevertheless pointed out that in other related studies C. concisus is believed to be an emerging human pathogen of intestinal infectious diseases. Similarly, our researchers wish future studies to examine mucus-associated bacteria, such as Campylobacter species, biopsy samples from the edges of the inflamed areas where the mucus is more likely to be intact may be useful. This followed from their finding that, with the Campylobacter PCR positivity rate for normal biopsy specimens higher than for biopsy specimens from inflamed areas, there may be a depletion of mucous – potentially reducing the bacterial load -- in the inflamed areas. Lastly, the researchers propose that future studies should include as research materials fecal samples collected prior to bowel preparation to culture Campylobacter species following among others their observation that osmotic changes due to bowel preparation may affect the viability of Campylobacter species. - II - Specified by the apt title of their study, the originality and specific contribution of the study by Z. Li et al. (2009) is appreciated more should one consider its context. Researches in the past have established that CD is attributable to a variety of pathogenic bacteria. There are a lot of suggestions as to what strains of bacteria are indeed responsible for IBD (in particular, CD). Among others, we have Sartor (2006), who suggested that Mycobacterium avium subsp. paratuberculosis causes CD, and Baumgart (2007), who links the specific strains of enteroadherent E. coli to CD. More particularly, the importance of C. jejuni and C. coli in causing diarrheal is very well established (Aabenhaus et al. 2008); however, what was yet to be established was a possible enteric pathogenic role for C. species other than C. jejuni and C. coli. Expectedly, then, Z. Li et al. (2009) take pride in asserting that there is the first report of successful determination of at least four different C. species from CD patients. There is no doubt that this study is indeed innovative. For the study is not only able to provide an answer to a former query or concern, but it also does chart future research directions that they generously shared to their readers right before they close their written presentation of their study. Admittedly, the brief introduction provided by Z. Li et al. (2009) provides a succinct “situationer”, so to speak. It briefly but intelligently sketches the context of the study and, in general wordings, state the research question(s). However, starting from here, the report has revealed its nature -- that is, it is meant primarily for those who have medical background – as evidenced by its heavy use of jargons. The details of the materials and methods are sufficiently described so that another microbiologist can repeat the whole process. Even the measurements are matter-of-factly indicated. It appears obvious too that the processes and technologies – e.g., the sequencing of C. species -- leading to the isolation of the C. species are not novel in themselves. Close scrutiny of the reference citations of the report would show very clearly that the methods were previously done by other researchers. Nevertheless, there are technical (writing) matters that should not be taken for granted as one critically reviews the report. Foremost of all, the abstract of the report is too economical to include the essentials. It is understandable that an abstract is just an outline of the aims of the study, the main methods employed by the researchers, the specific results and the conclusion of the study. But, one may argue that, since the paper takes pride in detecting and isolating at least four (4) C. species from CD patients, it is deemed that its abstract should have contained the names of the isolates. As a scientific writing, majority of its readers are anticipated to have “scientific slant”, which will make them focus on the process and the method not the content or the end result. And, thus, a mere mention of the names of the isolates may even arouse curiosity and inflame desire to read on and pay attention to the details of how the study was done. Another fair critique on this report concerns its presentation of its results. While it may be true that it’s not completely a bad idea to make use of written words to fully present a fact, it is similarly a given fact that making use of tables would increase the readability, so to speak, of the results. As one reads the report, one may have wished that, for instance, the groupings of the patients viz. the collection of the specimens was plotted on tables. In this case, the table would have helped in making the items more intelligible. Over-all, the research goes down to the history of microbiological studies as that which pioneered the determination and isolation of four C. species from CD patients. In itself, it is an important way forward towards understanding the pathogenesis of such malady as IBD. References: Aabenhus, R., U. Stenram, L.P. Andersen, H. Permin, & A. Ljungh. 2008. First attempt to produce experimental Campylobacter concisus infection in mice. World J Gastroenterol 14(45): 6954-6959. Baumgart, M. 2007. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn’s disease involving the ileum. The ISME Journal 1:403. Li, Z., M.M. Si, AA. Day, S.T. Leach, D.A., Lemberg, S. Dutt, M. Stormon, A. Otley, E.V. O’Loughlin, A. Magoffin, P. Ng, & H. Mitchell. 2009. Detection and isolation of Campylobacter species other than C. jejuni from children with Crohn’s Disease. Journal of Clinical Microbiology 47(2): 453-455. Parkhill, J., B.W. Wren, K. Mungall, J.M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R.M. Davies, T.Feltwell, S. Holroyd, K. Jagels, A.V. Karlyshev, S. Moule, M.J. Pallen, C.W. Penn, M.A. Quail, M.A. Rajandream, K.M. Rutherford, A.H.M. van Vliet, S. Whitehead & B.G. Barrell. 2000. The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403: 665-668. Sartor, R. 2006. Mechanisms of disease: Pathogenesis of Crohn’s disease and ulcerative colitis. Nature Clinical Practice Gastroenterology and Hepatology (3): 390-407. Read More