ANTIBIOFILM FORMATION ACTIVITY, RESISTANT GENES PROFILING AND DETECTION OF VIRULENCE FACTORS OF TOXIGENIC Vibrio cholerae ISOLATES FROM KISUMU COUNTY, KENYA

Introduction Vibrio cholerae can switch between motile and biofilm lifestyles with some of its strains forming biofilms in addition to production of various virulence traits and possessing antimicrobial resistance traits. This study is aim to show antibiofilm formation activity, resistant genes profiling and detection of virulence factors of toxigenic vibrio cholerae isolates from Kisumu County. Methodology A total of 119 Vibrio cholerae O1, biotype El Tor isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on TCBS and PCR assay carried out using standard procedures. Biofilm assay tests and detection of virulence factors were also done by use of standard procedures. Results Of the 101 confirmed vibrio cholerae isolates, 80.2% possessed the cholera toxin gene (ctxA) whereas 19.8% did not. Analysis of the toxR gene revealed that 98.0% harbored the toxR gene and only 2.0% did not. It was also revealed that 80.2% harbored the class I integron (inDS gene) while 19.8% did not, 93.1% were confirmed to possess the SXT integrating conjugative element (ICE) while 7.0% did not. The tetracycline resistance gene was present in 96.0% of the isolates. In 7 isolants strains which were resistance to common used antibiotics were screened for biofilm formation. Three of the strains (04/17-07, 06/17-14, and 05/17-03) failed to form biofilm while four strains namely 03/17-16, 02/17-09, 04/17-13 and P. aeruginosa ATCC 10145 as a positive control formed biofilms. In addition, out of those 7 isolants 71.42% produced protease, 85.71% produced phospholipases, 71.42% of isolates has the ability to produce lipase and 100% were able to produce the haemolysin. Conclusion An understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.


INTRODUCTION
Vibrio cholera is a causative agent of cholera an acute diarrheal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholera that belongs to genus vibrio, family Vibrionaceae Vibrio cholera have shown a number of products important for its virulence, such as cholera toxin (CT), whose action is largely responsible for the host secretory response, and toxin-co-regulated pili (TCP), which greatly enhances colonization of the intestinal epithelium [3]. TCP also serves as a receptor for CTXØ phage so it appears that TCP pathogenicity island is the initial genetic element required for the virulence associated genes in V. Cholerae [4,5]. A toxin-coregulated pilus has been shown to be necessary for colonization in both the classical and El Tor biotypes of V.cholerae O1 [6]. Besides CT and the pilus, other factors [other potential toxins, accessory colonization factors, outer membrane proteins, proteases, hemolysins, hemagglutinins (HAs), and a capsular polysaccharide] including those which are necessary for survival of the bacteria in vivo, penetration of the mucous layer and adherence to the underlying epithelial cells of the intestine, binding and internalization of CT, evasion of the host defense system, etc., may also contribute to the virulence contributing to survival and multiplication of V.
cholerae within the host [7]. TcpI is thought to be a chemo sensor and is proposed to be a sensor protein that negatively regulates tcpA [5]. ToxR dimers, but not monomers will bind to operator region of ctxAB operon and activate its transcription [8]. Transcription of the ctxAB operon is regulated by a number of environmental signals, including temperature, pH, osmorality and certain amino acids. Other V. cholerae genes are corregulated in the same manner including the tcp operon which is concerned with fimbrial synthesis and assembly. The ctx operon and the tcp are part of regulon, the expression of which is controlled by the same environmental signals [9]. The proteins involved in control of this regulon expression have been identified as ToxR, ToxS and ToxT. ToxR is a trans membranous protein with about two thirds of its amino terminal part exposed to the cytoplasm. ToxS is a periplasmic protein. It is thought that ToxS can respond to environmental signals, change conformation and somehow influence dimerization of ToxR which activates transcription of the operon. Expression of ToxT is activated by ToxR, while ToxT in turn activates transcription of tcp genes for synthesis of tcp pili [10]. Besides CT and the pilus, other factors including those which are necessary for survival of the bacteria in vivo, penetration of the mucous layer and adherence to the underlying epithelial cells of the intestine, binding and internalization of CT, evasion of the host defense system, etc., may also contribute to the virulence of this important human pathogen [7]. These factors included other potential toxins, accessory colonization factors, outer membrane proteins, proteases, hemolysins, haemagglutinins (HAs), and in some strains, a capsular polysaccharide, all of which may contribute to survival and multiplication of / within the host [6,11]. The genes that encode the cholera toxin subunits ctxA and ctxB are localized to a CTX genetic element which is made up of a 4.6 kbp central core region 2.4 kbp repititive sequence termed RS2.
Similar RS sequences called RS1 may flank the CTX element [12]. Zonula occludens toxin increases the permeability of enterocytes and is encoded by zot gene which is within core region of CTXф [13]. A third toxin encoded by ace affects intestinal secretion [12]. HylA for haemolysin damages cells by acting as pore forming toxin and a number of studies have shown purified haemolysin is enterotoxic [6]. Outer membrane proteins which include OmpU, OmpT, OmpS, OmpV and others are major cell envelope proteins. OmpU is thought to contribute to bile resistance and functions as a colonizing factor [14]. Bile salts facilitate survival of V. cholerae in the intestine [15]. Toxin co-regulated pilus is clearly required for intestinal colonization [16]. This is because biofilms provides a reservoir for microbial cells, which when dispersed enhances the risk of chronic and persistent infections. It may also promote the reinfection of colonized sites [21,22].
Likewise, the matrix confers a protection against biocides, Immune system activity & drugs andhas environmental promoters that induce biofilm formation that contributes to drug resistance development [23,24]. All these factors contribute to biofilm cells being 1000 fold more resistant to antimicrobial agents than planktonic cells [17]. Equally, the current treatment and control of biofilm is complicated, because antimicrobials have been developed against planktonically grown bacteria cells and other microorganisms in metabolically active stage [25].
Other virulence factors are also produced by different strains of V. cholerae and may be related to the hydrolysis of lipid barrier in intestinal epithelial cells [26]. While Neuraminidase, is secreted to cause an increase in the number of receptors in the gut [27]. Therefore it's against this background that this study was to be carried out from the isolated which were found to be resistance to common used antibiotics [46].

Study design
This was a descriptive cross-sectional study that focussed on the 2017 cholera outbreak previously isolated from diarrhoeal stool specimens. The specimens were obtained from patients presenting with passage of three or more watery stools with or without vomiting during cholera outbreaks.

Study area
The study was performed at the six sub-counties of Kisumu County, including Muhoroni, Nyando, Nyakach, Kisumu West, Kisumu East and Seme.

Study samples
A total of 119 isolates were sourced from KEMRI-Kisumu where they were kept following the 2017 cholera outbreak at Kisumu County. The stool samples were randomly collected from patients with severe diarrhoea who were suspected of having cholera and who were attending to different health facilities within the County in 2017 [47].

Culturing of the V. cholerae isolates
Diarrhoeal stool samples were thawed and cultured on alkaline peptone water followed by 6 h incubation at 37 o C using established procedures as used before [28].The cultures were then plated on thiosulfate citrate bile salts sucrose agar (prepared using manufacturer's guidelines). The plates were incubated at 37 o C for 18-24 h, typical yellow colonies, which were presumed to be V. cholerae isolates were subjected to biochemical, serological and genotypic analyses using well-established protocols and as reported in our previous paper [28].

Biofilm formation inhibition assay
As described by [29], micro titter plate assay was performed to quantify the effect of commonly used antibiotics on the biofilm formation of V. cholerae strains. The test bacteria was first inoculated on LB agar and incubated at right conditions at 37˚C overnight. Then a colony was identified, picked and

Molecular biology techniques/assays -PCR and Gel electrophoresis
A conventional PCR assay was performed to detect the ctxA gene for confirmation of the identification of

Detection of hemolysin
Haemolysin was detected according to well-established protocols as documented by [30]. The βhemolytic activity was tested for on base agar (Himedia, India) supplemented with 7% sheep erythrocytes. A colony of 18-24 hr. isolates cultured on TSA (prepared according to manufacturer's guidelines) was transferred and streaked on blood agar and incubated for 24 hr. at 37 ºC. Zones of haemolysis around the colonies indicated the ability of these bacteria to haemolyse RBCs [31].

Detection of protease
To detect protease on skim milk agar, we followed the protocol described in [30]. In which Solution A was prepared by adding 10 g skim milk to 90 ml of distilled water then volume was completed to 100 ml. gently heated at 50˚C, then autoclaved and cooled to 50-55˚C. And solution B was also prepared by adding 2 g of agar powder to 100 ml of distilled water, mixed thoroughly, then autoclaved and cooled to 50-55˚C. Aseptically, 100 ml of solution A was mixed with 100 ml of solution B Then the mixture was poured into sterile petri dishes, and then stored at 4˚C until to use. This media used to detect the ability of the bacteria to produce protease [30]. The appearance of a cleared hydrolysis zone indicates a positive test

Detection of lipase
Lipase production ability by V. cholerae isolates was determined by methods that have been used before [30]. Briefly a single colony of an overnight growth was cultured on Rhan medium (Supplementary data S1), and then incubated for 1-5 days at 37 °C. The appearance of a turbid zone around colonies indicates a positive result [30].

Detection of lecithinase (phospholipase)
To detect lecithinase, we followed the procedure of [32]. In which a colony was cultured on Medium of Phospholipase Activity Assay (Supplementary data S2) followed by incubation for 1-3 days at 37 o C using established procedures as used before [28]. The appearance of a white to brown color elongated precipitated zone Data analysis All experiments were conducted in triplicate to validate reproducibility. Statistical analysis was performed using Stata software. Data on graphs were analyse by Graph Pad Prism version 6. All values of diameter zones of inhibition were reported as mean ± standard error. Data analysis was performed during and after collection. The study did not involve patients.

Ethical consideration
Confidentiality and privacy were strictly adhered to and no names of individuals were recorded or made known in the collection or reporting of information. The study was granted ethical clearance by the Board

Vibrio cholerae O1 virulence genes
Eighty one (80.2%) isolates possessed the cholera toxin gene (ctxA) whereas 20 (19.8%) did not. Analysis of the toxR gene revealed that 99 (98.0%) harbored the toxR gene and only 2 (2.0%) did not. Table 1 describes the PCR results in regard to detection of pathogenic and antimicrobial resistance genes. It was also revealed that 81 (80.2%) of the isolates harbored the class I integron (encoded by inDS gene) while 20 (19.8%) did not. Majority, 94 (93.1%) were confirmed to possess the SXT integrating conjugative element (ICE) while 7 (7.0%) did not. The tetracycline resistance gene was present in 97 (96.0%) of the isolates. These findings add value to our previous findings on these seven (7) isolates that proved to be resistant against commonly used antibiotics in management of this condition at the study site including tetracycline that had resistance of 97.3% [46]. Based on PCR analysis, ace gene was revealed in all the seven isolates as shown in Plate A ( Figure 1).
Also, inDS, toxR and int genes were revealed to be present as presented Plate B (Figure 1). Additionally, PCR genotyping also did show the presence of ctxA and tcpI genes from clinical V. cholerae isolates as represented in Plate C in Figure 1 above.

Biofilm formation inhibitory effects of selected antibiotics against the V. cholerae strains
The most resistant drugs towards the seven isolants (Tetracycline, Ampicillin, Amoxicillin, Cotrimoxazole, Erythromycin and Nalidixic Acid) were subjected to biofilm formation inhibition assay in a 96-well microtiter plate. Different concentrations of the drugs were prepared using two-fold serial dilution (with dosage ranging from 0.5 to 0.03125mg/ml) and the wells were inoculated with 10 μL of V.
cholerae isolants. P. aeruginosa served as the positive control. The results showed that the biofilm formation inhibitory effects of the various concentrations (0.5mg/mL, 0.25 mg/ml, 0.125mg/ml, and 0.0625 mg/dl up to 0.03125 mg/mL) were significantly lower than that of the positive control, an indication that biofilm formation was inhibited at these concentrations as shown in figures 2 to 7 below.
As much as such inhibitory effects were recorded these findings clearly demonstrate that the 7 isolates that proved to be resistant to commonly used antibiotics do form biofilms. This test isolate was more prone to the inhibitory effects of the various dosages of the various antibiotics as compared to other isolates. Significance difference was also observed almost in each antibiotic used as shown in Figure 5 above when they were compared with the positive control. In Figure 6 antibiofilm formation activity against isolate 05/17-03 of V. cholerae against various antibiotics was also reduced in all the antibiotics. Interestingly, the concentrations of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.0625 mg/mL and 0.03125 mg/mL were able to inhibit biofilm formation much more as compared with the positive control with a significance differences observed at most dosages as represented in Figure 6 above.

Detection of Some Virulence Factors of V. cholerae:
In this study the production of various virulence enzymes like protease, phospholipase, lipase and haemolysin were studied on the 7 isolates which were found to be resistant to all drugs examined from our previous study. It seemed that 5/7 (71.42%) of these isolates of V.cholerae produced protease enzyme (Fig.8-plate A). Also it was deduced that 6 out 7 isolates (85.71%) produce phospholipases ( Fig.8-plate B). Further findings also indicate that out of the 7 isolates 5 isolates (71.42%) had the ability to produce lipase (Fig.8-plate D). Lastly, it was also deduced that all of the 7 isolates (100%) were able to produce the haemolysin by haemolysing the sheep red blood cells causing beta (β) haemolysis as shown in (Fig.8-plate C) above. Haemolysin therefore was the most produced virulence factor by these isolates (100%) and it was followed by phospholipase (85.71%) and lastly lipase and

DISCUSSION
In this study it's clear that the seven isolates that had been discovered from our previous study to be resistant to various commonly used antibiotics in deed possess various factors that enable them either to be resistant or induce various infections in human kind. Some of these traits are controlled at the gene level and as such they can be passed on from one bacteria cell to another [46] Antimicrobial drug resistance in Vibrio species for instance, may arise through mutation or through acquisition of resistance A B .

C. D.
genes on mobile genetic elements like plasmids, transposons integrons, and integrating conjugative elements [33]. Isolates analysed in this study possessed the class I integron and the SXT integrating conjugative element. Genetic elements like the class I integron (inDS) and the integrating conjugative elements such as SXT have been associated with the spread of genetic determinants, encoding for antimicrobial resistance in Vibrio cholerae [34]. The SXT element has been reported to harbor genes encoding for resistance to chloramphenicol (encoded by floR), streptomycin (encoded by strA and strB), trimethoprim (encoded by dfrA18) and sulfamethoxazole (encoded by sul2) [35]. The class 1 integron has also been reported to harbor aminoglycoside resistant gene cassettes in Vibrio cholerae O1 isolates cholerae [36]. As such therefore, resistance to erythromycin in the analysed isolates could be attributed to the presence of the class 1 integron gene that was found to be present in some strains isolated. The results also suggest that erythromycin and Nalidixic Acid do inhibit growth at high concentrations but from our study, it was deduced that they inhibit biofilm formation at lower concentrations. Same scenario was also observed in most antibiotics like tetracycline, Amoxicillin, Cotrimoxazole etc with such "Goldilocks" effect. A possible explanation to the less activity observed at greater doses could be associated to the aggregation effects of the antibiotics at site of entry into bacteria cell especially at high dosages something that is not observed at lower dosages. It is likely that aggregation may favour biofilm formation as antibiotics struggle to reach at the point of action and hence bacteria will continue to thrive hence form more biofilms [39]. This finding agrees with the previous studies on biofilm inhibition by However, isolates 05/17-07 and 05/17-03 of V. cholerae seemed to be very sensitive to the antibiotics screened at various dosages. This clearly demonstrates that the biofilm formation by these isolate can easily be managed by these antibiotics. Since they proved to be resistant when they were subjected to antimicrobial tests from our previous study it's clear that they could be possessing other mechanisms of activity against the antibiotics screened. One of such possible mechanisms could be presence of multi drug efflux pumps presence in them as they have been found to be present in some strains of V. cholerae [30]. This make the bacteria to be resistance to antibacterial agents and other toxic compounds by a mechanism known as active efflux, where the integral membrane transporters known as drug efflux pumps, prevent the accumulation of drugs inside the bacterial cells [40].
In addition to antibiofilm formation determination we also investigated the ability of these test strains to produce various virulence factors which may play role in their pathogenicity. Among the virulence traits we examined include detection of proteases, lipases, haemolysin and phospholipase. We did find that (71.42%) of the isolates of V. cholerae produced protease. This finding concurs to the findings of a previous study done by [42], which shows that (72.7%) of isolates were protease positive and that this enzyme have limited effect on the pathogenesis of this bacteria. Our findings also concurred with [10] study that documented that all isolates in her study were having ability to protease production. Proteases produced by V.cholerae have a very important role in its pathogenicity, due to the hydrolysis of several physiologically important protein such as mucin, fibronectin, and lactoferrin [43]. It could also proteolytically activate cholera toxin A subunit, El Tor cytolycin and haemolysin hence making this pathogen to be more virulent [44].
For phospholipases, 85.71% of the isolates were found to be positive. These findings do concur with the previous study findings that were done by [9], who did report that out of 20 isolates 13 (67%) of isolates were having the ability to phospholipase production. Our findings are also in tandem with [45] study findings that documented that all of his isolates were phospholipase positive. [36] mentioned the role of this enzyme in the cholera disease by the release of Arachidonic acid from the phospholipid found in the cell membranes of the lumen cells, this plays an important role in the prostaglandin E2 (PGE2) production which is responsible for the increase of liquids secretion from the lumen cells and this lead to watery [36]. Therefore, its presence makes V. cholerae isolate to be more virulent in watery diarrhoea production a key symptom of cholera.
Also, out of the 7 isolates 5 (71.42%) had the ability to produce lipase. Our findings also concur with other studies [10], which showed that all isolates in her study were having the ability to lipase production.
Abbas, (2006) Abass also did demonstrate that 65.4% of his isolates had the ability to produce lipases [42]. Lipases enzymes catalyse the hydrolysis of the ester bonds of triacylglycerols and may have a critical role in V.cholerae pathogenicity or nutrition acquisition. The production of excesses amount of lipases allows bacteria to penetrate fatty tissue with the consequent formation of abscesses [3]. The production of these enzymes by the isolates may reflect the presence of genetic organization of a discrete genetic element which encodes three genes responsible for the production of proteases, lipases and phospholipase. This organization could be a possible part of pathogenic island, encoding a product capable of damaging host cells and being involved in nutrient acquisition [43].
In this study 100% of, the isolants were able to produce the haemolysin. A finding that concurs to the findings of a previous study done by Abbass in which 100% of isolates were haemolysin positive.
Halpern and Izhaki stated that the purified haemolysin is capable of causing fluid accumulation, in contrast to the watery fluid produced in response to CT, the accumulated fluid produced in response to haemolysin with invariably bloody with mucous [1].

Conclusion
From this study, we can conclude that most clinical isolates produced virulence factors such as heamolysin, lipase, protease and phospholipase. As much as inhibitory effects were recorded, these findings clearly demonstrate that the isolates proved to be resistant to commonly used antibiotics and they do form biofilms. and these findings therefore, add value to our previous findings on these seven isolates that proved to be resistant against commonly used antibiotics in management of this condition at the study site. To the best of our knowledge, this is the first time such data has been documented from the study site that has had a good number of cholera outbreaks before and we hope it will assist in management of this condition.