CHAPTER ONE: INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Vibrio cholerae is the causative agent of Cholera disease which has spread globally in seven pandemic waves since 1817 (Tappero & Tauxe, 2011). Vibrios are highly motile, Gram-negative, comma-shaped rods with a single polar flagellum. Though few V. cholerae strains are clinically significant to humans, V. cholerae type O group 1 is the most important as a cause of epidemic Cholera disease. Cholera causes severe dehydrating diarrhea which can lead to death in untreated patients, especially in developing countries thus making cholera an issue of major public health importance in Kenya. This life threatening diarrhoea is attributed to massive luminal secretion of water and electrolytes from enterocytes induced by the cholera toxin (Salim, 2005).
Cholera was first discovered in Kenya in 1971 and since then several outbreaks have been reported thus rating it among the 35 priority diseases in Kenya. Fifteen discrete outbreaks have been reported from 1971 to 2010. These recurrent outbreaks mostly affected Nairobi, Nyanza and Coast regions. In addition, refugee camps, remote arid and semi-arid regions were also affected. The largest outbreak which resulted in 26,901 cases and 1,362 deaths was reported between 1977 and 1999 (Mutonga, et al., 2013). Earlier (1970s to early1990s) V.cholerae strains belonged to serotype Ogawa which showed resistance to a wide range of antibiotics like chloramphenicol, streptomycin, tetracycline, sulfamethaxazole/trimethoprim (cotrimoxazole) and ampicillin mediated by plasmid ca 100mD of incompatibility group C (Finch , et al., 1988). However, in the recent past, serotype Inaba emerged as the main cause of cholera disease with isolates showing reduced sensitivity to chloramphenicol and cotrimoxazole. Isolates collected between 1994-2007, contained strB ,Sull 2, dfrA1 and floR genes coding for resistance to streptomycin, sulfamethoxazole, trimethoprim and chloramphenicol respectively. These isolates were also negative for class 1, 2 and 3 integrons (Materu, et al.,1997 Kiiru, et al.,2009). It appears that the genetic relationship among past vibrio isolates is based on the type of technique used. In a past study for example, isolates studied using the Multilocus-Variable Tandem Repeat Analysis (MLVA), revealed that multiple genetic lineages of V.cholerae were simultaneously infecting persons in Kenya while those studied using pulsed field gel electrophoresis revealed that all isolates were genetically related (Mohamed, et al., 2012).
The standard way of treating severe cases of cholera currently is through administering rehydration fluids. However, the use of antibiotics reduces the duration of diarrhea by 50 – 56 %, reduces stool output by 8 – 92 % and reduces shedding of bacteria by 26 – 83 %. In general, antibiotics reduce the duration of illness by up to 50 % (Sack, et al., 2001).
Despite the advantages associated with the use of antibiotics, it has been compromised by the evolution and spread of strains conferring resistance to multiple antibiotics including those which have been recommended by WHO that include doxycycline, furazolidone, sulfamethoxazole, trimethoprim, chloramphenical and ciprofloxacin. An erratic shift in antibiotic resistance among the V. cholerae strains has also been noted in isolates collected in different regions over the same period of time.
The most common treatment used against bacterial pathogens is the administration of β-lactam antibiotics such as penicillins, monobactams and cephalosporins but the emergence of β-lactamases that mediate resistance to these antibiotics is on the rise (Shaikh, et al., 2015). β-lactamases are rare in Vibrio but recently, the prevalence of these enzymes has been increasing in this Genus. β-lactamases are frequently found in Gram negative bacteria which destroy the β-lactam ring of these antibiotics through hydrolysis thus resulting in ineffective compounds (Pitout, et al., 2005). The β-lactamases are classified based on two schemes namely, the Bush-Jacoby Medeiros functional classification scheme which is based on enzyme properties such as substrate and inhibitor profiles and the Ambler molecular classification scheme which is based on amino acid sequence of enzymes where class B enzymes are metallo-β lactamases which require zinc ions for substrate hydrolysis (Bush & Jacoby, 2010) and class A,C,D which utilize serine for β-lactam hydrolysis (Shaikh, et al., 2015 Bush & Jacoby, 2010). These enzymes have evolved over time through mutations and continuous overproduction which can be attributed to constant exposure of bacteria to a multitude of β-lactams (Shaikh, et al., 2015). This has then given rise to enzymes exhibiting increased activity against newly developed β-lactam antibiotics and these are the extended spectrum β-lactamases. Most β-lactamases are encoded in plasmids that are easily transferred from one bacterium to another (Shaikh, et al., 2015).
Evolution of transposons and integrons has greatly influenced flexibility in the genetic response to a variety of antibiotics (Wozniak, et al., 2009). This has however not been extensively studied in Kenya. Integrons are commonly found in many bacterial genomes. They are flexible gene acquisition systems which have the ability to acquire, express and disperse antibiotic resistant genes thus posing a major threat to antibiotic therapy. They are not capable of self-transposition hence they associate with insertion sequences, transposons and conjugative plasmids which serve as vehicles for transmission (Gillins, 2014). They may also have smaller integrons embedded in mobile genetic elements such as transposons and conjugative plasmids which can disseminate horizontally (Burrus.,et al,2006).Vibrio isolates harbor large chromosomal integrons giving them the capacity to rapidly transfer gene cassettes containing antibiotic resistant genes. Vibrio isolates have also been associated with class 1 integrons in other parts of the world but to date none have been isolated in Kenya. Integrative conjugative elements (ICEs) integrate and replicate within the host chromosome and can excise themselves and transfer between bacteria by conjugation (Wozniak et al., 2009). The ICEs commonly carry several antimicrobial drug resistance genes and play a major role in the spread of antimicrobial drug resistance in V. cholerae, a good example is the SXT element which harbors resistance to trimethoprim and sulphamethaxazole (Burrus et al., 2006). This element belonging to SXT/ R391 family was present in Kenyan isolates collected between 1994 and 2007 (Kiiru, et al., 2009).
Bacteriophages which are abundant in aquatic ecosystem also play an important role in the evolution of bacterial genomes. To date, no phage encoded resistant genes are known, however, they may assist in mobilizing plasmids during transduction (Davinson, 1999).
Resistance to antibiotics poses a global threat to the fight against infections and has been shown to vary with time in V.cholerae. This can be attributed to evolution of mobile genetic elements which has been poorly studied in Kenya. Therefore, it becomes a necessity to study evolution of antibiotic resistance over a period of time. This knowledge will then assist in making decisions with regards to antibiotic use and also in coming up with strategies of controlling the antibiotic resistant strains.
1.1.1 PROBLEM STATEMENT AND JUSTIFICATION
Despite advances made in understanding the cholera disease, it still continues to be a major public health problem in Kenya with the recent outbreak reported in the month of February 2015 (relief web, 2015).
Several studies conducted have shown increased multiple resistance to some of the antibiotics recommended for use in cholera by WHO and these include; tetracycline, doxycycline, furazolidone, sulfamethaxazole, trimethoprim, chloramphenical and ciprofloxacin. In addition, differences have been reported in antibiotic resistant patterns in studies carried among V.cholerae isolates during a given period of time in different regions. For instance, V.cholerae O1 strains isolated between 1994 and 1996 from five countries in the East African region (Kenya, Sudan, Somali, Tanzania and Rwanda) exhibited no uniformity in the antibiotic resistance pattern. However isolates collected in Kenya between 1992 and 2007 had similar resistance patterns (Kiiru.et al., 2009). Erratic shifts in antibiotic resistance among V. cholerae isolates have also been noted and yet the genetic basis of these changes has not been investigated in Kenya.
Over the years, antibiotic pressure in the bacteria’s environment increases the need for them to acquire a resistant gene through mobile genetic elements. However, only a small proportion of these elements have been screened in Kenyan isolates and temporal changes in carriage of these elements is still unknown.
Therefore, in order to understand changes in antimicrobials, it becomes necessary to carry out this study in isolates accumulated over a long period of time. This will guide the health policy makers in formulating policies with regards to antibiotic use and also in coming up with strategies of controlling the antibiotic resistant strains.
1.1.2 HYPOTHESIS
There is no difference in the resistance profile and the genetic determinants of resistance among isolates recovered between 2006 and 2015.
1.1.3 OBJECTIVES
1.1.3.1 GENERAL OBJECTIVE
To determine genetic basis for antibiotic resistance and phylogenetic relationship in V.cholerae clinical isolates collected between 2006 and 2015 in Kenya.
1.1.3.2 SPECIFIC OBJECTIVES
1. To determine shifts in antibiotic resistance profiles of V.cholerae isolates recovered over a period of ten years.
2. To determine changes in carriage of genetic determinants among isolates recovered over a ten year period.
3. To determine change in diversity of selected mobile genetic elements (plasmids, Integrons, Integrative Conjugative elements) among these isolates over a period of ten years.
4. To establish changes in phylogenetic relationship among the antibiotic resistant strains obtained between 2006 to 2015