A Study of the Virulence of Campylobacter coli - Research Proposal Example

A Study of the Virulence of Campylobacter coli A Research Proposal Executive Summary The genus Campylobacter is oneof the major causes of infectious intestinal illnesses such as bacterial diarrhoea and gastroenteritis in humans all over the world (Luber et al., 2003). According to Tam et al. (2003), infections particularly from Campylobacter jejuni and C. coli amounts to 420,000 cases of intestinal disease in England alone every year. On the other hand, the said bacteria ranks second in the leading causes of bacterial food poisoning in the United States (Miller, et al., 2006). Found primarily to affect swine, cattle and poultry animals, the strains of Campylobacter proved to be transmissible with humans when meat is contaminated during processing (Silva et al., 2011). However, most of researches are focused on C. jejuni and very little on the impact and virulence of C. coli. Admittedly smaller in terms of human disease case percentage, C. coli remains a huge risk factor in the prevalence of campylobacter infections. This study aims to explore the foodborne pathogen C. coli as limited studies are conducted in this topic and to characterise a number of C. coli isolates from organic chicken in terms of their virulence potential using a range of techniques including association and invasion assays and some PCR screening for known virulence genes. Also, the study intends to stimulate people to eat organic chicken instead of frozen ones that are most likely to be infected with bacterial pathogens such as C. coli. The virulence of isolated C. coli strains will be tested and will be compared to those of known C. jejuni strains. Literature Review The significance of Campylobacter coli in spreading microbial intestinal infections gains worldwide recognition as its properties and virulence are slowly being exposed. Luber, et al. 92003) considered Campylobacter spp. as one of the major causes of infectious intestinal illnesses such as bacterial diarrhoea and gastroenteritis in humans all over the world. A total of 2.4 million reported illnesses and 150 mortalities are caused by the genus Campylobacter every year in the United States (Mead, et al., 1999). Infections particularly from Campylobacter jejuni and C. coli amounts to 420,000 cases of intestinal disease in England alone every year (Tam et al., 2003), while it ranks second in the leading causes of bacterial food poisoning in the United States (Miller, et al., 2006). In addition, the English case-control Infectious Intestinal Disease Study revealed that among the cases in 1993 to 1996, Campylobacter spp. amounts to a percentage of 23% among all detected enteric pathogens from faecal samples (Amar, et al., 2007). There are 11 species of Campylobacter that are pathogenic to humans and animals, with C. jejuni and C. coli as the most frequently isolated species in food products (He, et al., 2010). Gurtler, et al. (2005) emphasized the importance of C. coli in human campylobacter contagions as their research uncovered that it comprises about 18.6% of Campylobacter spp. found in an infected human body. According to the World Health Organization (2011), infections caused by Campylobacter spp. is mild but fatal especially to infants and elderly people. It is generally regarded as the foremost cause of gastroenteritis, diarrhoea and other intestinal diseases in developing and developed countries. The strong impact and high prevalence of Campylobacter foodborne illnesses placed the said bacteria to the list of pathogens of high significance in terms of health and socio-economic viewpoint. The mode of action of Campylobacter involves the colonization of the intestines of live animals such as poultry, cattle, sheep, swine and other animals. Thereafter, the bacteria pollute the meat produces during slaughtering and packaging of products. In turn, humans who consume the contaminated meat that are either raw or undercooked are can be infected with Campylobacter diseases (Kemp, et al. 2005). Aside from food and contaminated meat products, C. jejuni and C. coli are also present in different sources of water (Tam, et al., 2003). The pervasiveness of this microorganism in poultry, cattle and swine is disturbing, given that the growth and survival characteristics of Campylobacter spp. are capable to withstand extreme conditions. Silva, et al. (2011) noted that Campylobacter spp. are thermophiles and can only cease to grow below 30oC. As such, both fresh and frozen meats are likely candidates of contamination of this foodborne pathogen. Although most cases of intestinal illnesses are self-limiting, advanced cases such as “systemic Campylobacter infections, Campylobacter infections in immunosuppressed patients, and severe or long-lasting Campylobacter infections” (Luber, et al., 2003) essentially need antimicrobial treatment like erythromycin or fluoroquinolones. Nevertheless, Gebreyes et al. (2005, p. 766), maintain that C. coli has the “ability to show increased resistance to a greater number of antimicrobials.” This resistance factor against known and effective antibiotics is an imperative concern since the medications that are commonly used to cure such diseases will be put to waste. Luber, et al. (2003) reported that Campylobacter jejuni and C. coli strains were resistant to antibiotics such as ciprofloxacin, tetracycline, ampicillin and trimethoprim-sulfamethoxazole at different levels. Whereas all isolates in the research was proven susceptible to gentamicin, they have also showed resistance to erythromycin at a low percentage of 6.1%. Specifically, Campylobacter sp. isolates from chickens and turkeys exemplified high levels of resistance that are developed over the research scope of 10 years. Hence, Luber, et al. (2003, p. 3825) concluded that the “discrepancies in the antimicrobial resistance rates among Campylobacter isolates originating from poultry and humans support the hypothesis that at least some of the resistant Campylobacter strains causing infection in humans come from sources other than poultry products.” Meanwhile, Vashin, et al. (2009) reported that an abundant amount of Campylobacter spp. were isolated from different parts of packed frozen poultry giblets in Bulgaria, where C. coli and C. jejuni populated 77.8% and 22.2% of the chicken liver, respectively. A similar research work conducted by Wieczorek, et al. (2012) on the prevalence, antimicrobial resistance, and molecular characterization of Campylobacter jejuni and C. coli isolated from retail raw meat in Poland, revealed that all of the meat samples originating from poultry were contaminated with Campylobacter spp. A total of 189 strains of C. coli were recorded amounting to 58.9% of the isolates. Incidentally, C. coli is evident on all samples of swine carcass when tested for prevalence of Campylobacter spp. and its antimicrobial resistance in antimicrobial-free (ABF) swine production systems from the study conducted by Gebreyes, et al. (2005). They concluded that the reason behind this is brought about by the slaughterhouse effect whereas a blast chilling system in the plant is lacking. The research stated that “resistance against ciproflaxin was also detected at the farm level in the antimicrobial free swine production systems. Resistance against ciprofloxin and chloramphenicol in C. coli is striking since both the antimicrobials are not licensed for use in any system production in the USA (Gebreyes, et al., 2005, p. 767).” Nevertheless, the issue of antimicrobial resistance of Campylobacter spp. particularly C. coli raises an ethical concern in consuming fresh and frozen meat products specifically that of poultry, cattle and swine. While it is conventional to purchase these food products in the market from farms that uses antimicrobials for growth advancement and therapeutic purposes for common animal diseases (Gebreyes, et al., 2005), the antibiotic residue that fresh and frozen meat products contain promotes the prevalence of antimicrobial strains of pathogens like C. coli and therefore decreases the ability of medicines to cure advanced stages of Campylobacter diseases in infected humans. Research aims The general research aim is to test the virulence of C. coli in frozen chicken meat. Specific aims of this research paper are: a. to characterise the number of C. coli isolates from organic chicken in terms of their virulence potential b. to stimulate people to prefer organic chicken instead of frozen chicken that are highly susceptible to bacterial contamination Methodology The virulence of C. coli will be tested using the following procedures: 1. Stress assays This association assay will test for the acidic and percentage bile salt tolerance and aerobic stress of the strains. The acidic stress tolerance assay aims to establish the strains’ properties by finding and measuring the acidity limit that the bacteria could thrive in before undergoing the death phase (Requena, 2012). The bile salt percentage tolerance test aims to compare the maximum and minimum percentage salinity that the strain could tolerate under different bile salts as described (Raphael, et al., 2005). Aerobic stress level assays on the other hand will measure the tolerance limit of the strains by measuring cell viability after oxygen exposure (Wiedmann and Zhang, 2011). 2. Virulence assays This invasion assay will test for the motility, autoagglutination, adhesion and invasion of isolated C. coli strains. Motility tests detect the capacity of the strains of self-movement through the use of semisolid motility medium plates (Raphael, et al., 2005). Autoagglutination tests aim to identify the strains through serotyping for heat stable antigens against C. coli, and cell adhesion and invasion tests are used to check the presence of bacterial colonies that will grow attached to eukaryote cells after undergoing several treatments (Konkel and Joens, 1989; Wassenar and Newell, 2000). The ability for self-movement is to be compared among strains of C. coli since motility increases chances of a wider infection among foodborne pathogens, and the adhesion and autoagglutination methods are important since bacteria mostly attach via cell surface sugars interactions and are therefore affected by the class of sugar residues present on host cells (Cheesbrough, 2006; Sharon, et al., 2009). 3. PCR screening PCR screening will be conducted to identify known virulence genes of Campylobacter spp. by the detection of certain virulence and toxin genes, which were already identified in previous studies (Bang, et al., 2003; Hong, et al., 2003). This method of strain detection helps in properly identifying the virulent strain through genotyping, which has higher detection accuracy in comparison with phenotypic levels of identification and thus is integral in confirmatory testing. Time Frame The study will undergo five phases of the research calendar, as shown in the following 2013 Gannt Chart: April May June July August 1 2 2 3 3 3 3 4 4 4 4 5 Legend: 1. Stress assays – April 2013 2. Motility tests –Middle of April to Middle of May 2013 3. Virulence assays – Late April to Middle of June 2013 4. PCR screening – Middle of April to July 2013 5. Research complete – August 2013 Bibliography Amar, C., East, C., Gray, J., Iturriza-Gomara, M., Maclure, A & McLauchin, J. (2007) Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control infectious intestinal disease study (1993-1996). European Journal of Clinical Microbiology & Infectious Diseases, 26 (5), pp. 311-323. Bang, D., Moller Nielsen, E., Scheutz, F., Pedersen K.,Handberg, K., Madsen, M. 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(2010) Simultaneous detection and differentiation of Campylobacter jejuni, . coli and C. lari in chickens using multiplex real-time PCR assay. Food Anal. Methods, DOI: 10.1007/s12161-010-9136-6. Hong, Y., Berrang, M., Liu, T., Hofacre, C., Sanchez, S., Wang, L, & Maurer, J. (2003) Rapid detection of campylobacter coli, c. jejuni, and salmonella enterica on poultry carcasses by using pcr-enzyme-linked immunosorbent assay. Applied and Environmental Microbiology, 69(6) June, pp. 3492-3499. Kemp, R., Leatherbarrow, A., Williams, N., Hart, C., Clough, H., Turner, J., Wright, E. & French, N. (2005) prevalence and genetic diversity of Campylobacter spp. in environmental water samples from a 100-square-kilometer predominantly dairy farming area. Applied Environmental Microbiology, 71, pp. 1876-1879. Konkel, M. & Joens, L. (1989) Adhesion to and invasion of Hep-2 cells by campylobacter spp.. Infection and Immunity, 57(10), pp. 2894-2990. Luber, P., Wagner, J., Hahn, H. & Bartelt, E. (2003) Antimicrobial resistance in Campylobacter jejuni and C. coli strains isolated in1991 and 2001-2002 from poultry and humans in Berlin, Germany. Antimicrobial Agents Chemotherapy, 47 (12), pp. 3825-3830. Mead, P., Slutsker, L. & Dietz, V. (1999) Food-related illness and death in the United States. Emergency Infectious Diseases, 5 (1), pp. 607-625. Miller, W., Englen, M., Kathariou, S., Wesley, I., Wang, G., Pittenger-Alley, L., Siletz, R., Muraoka, W., Fedorka-Cray, P. & Mandrell, R. (2006) Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals. Microbiology, 152 (1) January, pp. 245-255. Raphael, B.H., Pereira, S., Flom, G. A., Zhang, Q., Ketley, J. M., & Konkel, M. (2005) The campylobacter jejuni response regulator, CbrR, modulates sodium deoxycholate resistance and chicken colonization. Journal of Bacteriology. 187 June, pp. 3662-2770. Requena, J. M. (2012) Stress response in microbiology. 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(2003) Antimicrobial resistance in Campylobacter jejuni and C. coli strains isolated in1991 and 2001-2002 from poultry and humans in Berlin, Germany. Antimicrobial Agents Chemotherapy Wieczorek, K., Szewczyk, R. & Osek, J. (2012) Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter jejuni and . coli isolated from retail raw meat in Poland. Veterinari Medicina, 57 (6), pp. 293-299. Wiedmann, M. & Zhang, W. (2011) Genomics of foodborne bacterial pathogens. New York: Springer. World Health Organization (2011) Fact sheet: Campylobacter [Internet], World Health Organization. Available from: < http://www.who.int/mediacentre/factsheets/fs255/ en/index.html> [Accessed 10 February 2013]. Read More