Haemolysin and Shigella Toxin Production in Multidrug Resistant Escherichia Coli Pathotypes from Clinical Specimens

Bacterial Isolates

A total of 178 Escherichia coli isolates were screened on 5% sheep blood agar for haemolysin production and inoculated onto Sorbitol MacConkey (SMAC) agar media for phenotypic identification of suspected Shigatoxin producing E. coli (STEC) colonies [12]. The isolates were also selected based on the ability to grow on Luria-Bertani (LB) agar medium containing 100µg/ml Ampicillin. Twelve isolates phenotypically confirmed for these virulence factors (Haemolysin and Shigatoxin) were used for genotypic characterization.

Screening of Isolates for Multidrug Resistance Characteristics

The isolates were screened for antimicrobial

susceptibility pattern, multidrug resistant characteristics on Iso-sensitest agar (ISA) media (Oxoid, UK) according to Kirby-Bauer disc diffusion methods as described by Mahon C, et al. [13]. The following commercial discs were used:Combined discs M13/M14 rings (Mast Diagnostics, UK): Chloramphenicol (5µg), Erythromycin (5µg), and Fusidic acid (10µg), Oxacillin (5µg), Novobiocin (5ugµg), Streptomycin (10µg), Tetracycline (25µg), and M14 rings: Amoxycillin (30 µg), Cephalothin (5µg), Colistin sulphate (25µg), Gentamycin (10µg), Sulphatriad (200µg), and Cotrimoxazole (25 µg). Single discs (Oxoid): Amoxycillin-Clavulanate/Augmentin (30µg), Cefuroxime (30µg), Ceftriaxone (30µg), Cefotaxime (30µg), Ciprofloxacin (10µg) and Imipenem (10µg). The diameter zone of inhibition was measured in millimetre and results were interpreted according to British society for antimicrobial chemotherapy (BSAC) guidelines [14]. Escherichia coli ATCC 35401 were used as positive control; while E. coli K-12 DH5α as negative control.

DNA Extraction

The clinical isolates were grown for 24 hours on Luria- Bertani (LB) agar plates (containing 100µg/ml ampicillin). Chromosomal DNA was isolated from overnight bacterial culture by boiling method as described by British Society for Antimicrobial Chemotherapy [15]. A loopful of cells from a single colony was transferred to 100µl of sterile/double distilled water and the mixture was boiled for 10 min, at 95oC in water bath to lyse the cells. The cell lysate were then centrifuged briefly (10s at 10,000 rpm). The supernatant was carefully pipetted into fresh tube and kept at -20oC for further use. A 5μl of the sample was used for the PCR reaction [16]. Plasmid DNA was isolated from the samples using Qiagen miniprep protocol by alkaline lysis method according to manufacturers’ instructions.

PCR Amplification of Virulence Genes

Virulence genes were amplified using hlyA and Stx1 specific primers (Integrated DNA Technologies, USA). PCR was performed in a final reaction volume of 25 µl, which comprises of 5.5 µl sterile distilled water, 1 µl (each) reverse and forward primers, 12.5 µl of Master mix, Taq DNA polymerase, and 5 µl of bacterial lysate (supernatant with template DNA). Amplifications were performed with the G-Storm thermocycler GS0001 (ThermoFisher Scientific, UK). PCR amplification start with initial denaturation at 94oC for 5 minutes, then 35 cycles of denaturation at 94oC for 30 seconds, annealing at 54oC for 30 seconds, extension at 72oC for 1 minute. A final extension at 72oC for 10 minutes was conducted, as described by Li, et al. [7]. A molecular marker 1 kb DNA Hyperladder (Bioline, UK) was used to assess the PCR product size (Table 1).

The product was separated by gel electrophoresis on 1.0 % agarose, stained with gel red and the image was captured digitally with UV transillumination (Syngene).The product size was estimated using 1kb DNA Ladder (Bioline, UK). The PCR products was purified and stored at 4oC, before sequencing. The characteristics of the primers used and the expected amplicon size are given as shown above.

Sequencing Analysis for Virulence Genes Identification

The purified DNA from the PCR products was finally analyzed by sequencing techniques for the genes identification at the Medical Research Institute, University of Dundee (Scotland), UK.

Statistical Analysis

The data of the study was analyzed by using the SPSS version 21.1. Chi-square test was used to compare the difference in virulence genotypes in relation to MDR profile. Probability level (P<0.05) was considered statistically significant.

Multidrug Resistant Characteristics of the Isolates

Multidrug resistance in bacteria is most commonly associated with the presence of plasmids which contain one or more resistance genes, each encoding a single antibiotic resistance phenotype. It has been shown that antibiotics therapy can select for antibiotic resistant strains in the enterobacterial flora and plasmid-mediated antibiotic resistance can spread in a population subjected to heavy antibiotic therapy [19]. In the present study, (Table 2) all the twelve isolates (100%) are resistant to Amoxycillin, Cephalothin, Erythromycin, Fusidic acid, Novobiocin and Oxacillin. Eleven isolates (91.7%) are resistant to Chloramphenicol, Cotrimoxazole, Streptomycin and Tetracycline. Eight isolates (66.7%) are resistant to Ciprofloxacin. Seven isolates (58.3%) are resistant to Cefotaxime, Ceftriaxone Cefuroxime and Gentamycin. Nine (9) isolates (75%) are sensitive and three (3) isolates (25%) are resistant to Augmentin. However, the isolates were sensitive to Colistin sulphate (100%), Imipenem (100%), and Augmentin (75.0%).The E.coli isolates U03, U04, S05, S06, S07, B09, U10, U11 and S12 were sensitive to Augmentin and at least five of the isolates are also sensitive to Cefotaxime, Ceftriaxone, Cefuroxime and Gentamycin. The result showed higher resistance of the isolates to beta-lactams like ampicillin which alarms us that such drugs should no longer be used as first line of treatments in this area, as it used to be in the previous decades. These results of ampicillin-resistant E.coli are congruent to the report of Farshad, et al. [20], who found 100% resistance of their E.coli isolates to ampicillin.

Table 2: Distribution of bacterial isolates according to Antibiotic susceptibility pattern. N.B: Zones of inhibition was interpreted according to BSAC, guidelines (2013) The greater prevalence of resistance to common antibiotics was reported by Mohajeri, et al. [10] where maximum number of isolates (100%) was resistant to ampicillin, carbenicillin and ceftazidime and the lowest to chloramphenicol (37%). A maximum resistance among E. coli species isolates was observed against cefpodoxime 100% by Sahu, et al. [21]. The presence of multidrug resistance in this study may be related to the dissemination of antibiotic resistance among hospital isolates. Zhao, et al. [22] observed that Escherichia coli are one of the main bacterial pathogens responsible for nosocomial infections especially in immunocompromised patients. A high sensitivity of E. coli strains to Imipenem has been previously reported by Farshad, et al. [20]. It seems this antibiotic can serve as a medication of choice for the treatment of UTI caused by E. coli. However, it should be noted that unlimited use of a chemotherapeutic agents can gradually lead to rising antibiotic resistance.

Genotypic Characteristics of Virulence Factors

The ability of some bacteria like E. coli to cause diseases is determined by harbouring and acquisition of some pathogenic attributes [23]. For example, presence of cytotoxins and haemolysins in these organisms is responsible for established UTI in some women and diarrhoeal cases of medical importance [2]. In the present study (Figures 1 & 2), all the virulence genes, hlyA and stx1 produced were found in isolates S07 and U08. This is followed by U01, U02, S06 and B09 which produced 3 of the genes, while U10 and S12 are M 01 02 03 04 05 06 07 08

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1000bp 800bp 600bp 400bp the least with only one gene each. The results of this study show that virulence genes are more commonly found among gastroiontestinal bacteria (stool isolates) than in other cases such as UTI.

HlyA 1177bp

Figure1: Haemolysin A gene PCR products (chromosomal DNA) agarose gel image. Lane 01, 02, 03, 04, 05, 06, 07 and 08, are isolates that harbours hlyA gene with expected amplicon size of 1177bp [17]. Lane M is molecular marker (1kb DNA ladder, Bioline, UK).

M 01 02 03 04 05 06 07 08 09 10 11

400bp

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The above figure shows that the isolates, U01, U03, S05, S06, S07, U08, B09, U10, U11 and S12, harbours stx1 gene.

N.B: Zones of inhibition was interpreted according to BSAC, guidelines (2013) Birosova, et al. [23] reported that alpha-haemolytic E. coli strains are probably found more frequently in the intestinal tract than urinary tract. But in previous studies Tarchouna, et al. [24] reported frequent occurrence of fimH and haemolysin in patientwith UTI. Haemolysin and adherence through fimbriae are important properties of the uropathogenic E.coli. The degree of severity depends on the virulence of the responsible strains and on the susceptibility of the host [25]. A better cognition of the virulence characteristics of the microorganisms causing infection will permit the clinicians to anticipate the evolution of infection in the host. Many virulence determinants contribute to the pathogenicity of Escherichia coli.

The results indicated that the urine isolates with this high prevalence of virulence factors can be a major causative agent for UTIs in humans in the study area. In spite of the previous studies, our results showed that the urine isolates have a different virulence profile. These differences in prevalence of virulence genes showed that the properties is closely depending on geographic region and even weather conditions of each regions. It seems that the epidemiology and prevalence of virulence factors of urine isolates from patients with UTI is different in this area. It was also confirmed that haemolysin is important in pathogenesis and the pathogenicity of these gene encoding isolates. It also depends greatly on the ability of the gene to switch between conformations and this is dependent on the different alleles that can be expressed by this gene [26, 27, 28]. Another important secreted virulence factor of uropathogenic E. coli also found in our isolates is a lipoprotein, α-haemolysin (HlyA), which is associated with upper UTIs such as pyelonephritis. Haemolysin production is associated with pathogenicity of E.coli, especially in the more severe forms of infection [22] (Table 3).

At high concentrations, HlyA is able to lyse erythrocytes and nucleated host cells, a process that may enable extraintestinal pathogens like UPEC to better cross mucosal barriers, damage effect or immune cells, and gain enhanced access to host nutrients and iron stores [29]. Haemolysin is known to confer selective advantage to the pathogen by releasing iron from lysed erythrocytes and enhances pathogenicity by destroying phagocytic cells and epithelial cells. Haemolysin production has also been shown previously to influence pathogenicity. Haemolysins has been proposed to inflict direct cytotoxic effects on renal epithelium [22, 26].

The hlyA gene prevalence (66.7%) in our studies is significantly associated (p<0.05) with both UTI and gastrointestinal disorders, as the gene was detected in both urine and stool isolates. There was a clear relation between tissue damage and the presence of haemolysin in this study. Prevalence of these genes differs on the basis of phylogenetics, geographical distribution, and clinical presentation. A huge variation in the frequencies of these genes was recorded worldwide [24]. Haemolytic activity in E. coli has been attributed to many types of Haemolysin genes. A new class of haemolysin represents a novel bacterial gene designated hlyF gene that is strongly associated with E. coli isolates from bloodstream infection [30]. Virulence is due to a plasmid such as HlyA that encodes genes required for invasion, cell survival and apoptosis of macrophages [10]. The virulence genes in E. coli are often located on transmissible genetic elements that can be transferred to E. coli recipient strains. These virulence determinants give each pathotype the capacity to cause a clinical syndrome with distinctive epidemiologic and pathologic characteristics [27] and are therefore ideal targets for the determination of the pathogenic potential of any given E. coli variant. Shiga toxin (stx) is one of the major virulence factors involved in E. coli O157:H7 pathogenesis based on immune reactivity, toxins are classified as either Stx1 or Stx2, which damage intestinal epithelial cells and kidneys, causing haemorrhagic colitis and haemolytic uremic syndrome, respectively [9]. The stx gene in E. coli O157:H7 is associated with a prophage, and different subtypes of shiga- toxin are identified [24].

The prevalence of stx genes in our studies was low. However, these virulence genes are usually associated with occurrence of diseases. A study in Egypt by Morsi and Elsaid Tash [31], showed similar distribution of virulence genes. This indicates that some social and environmental factors may contribute in the virulence pattern of E. coli in different communities. The negative isolates for some of the genes in our study may be part of the normal flora that lack these virulent genes or may be due to the possibility of corresponding gene mutations, as negative test results does not always indicate the absence of the corresponding operon while a positive result usually confirms the presence of the virulence genes. When antibiotic resistance was tested among the virulent isolates, a significant relation was observed (P<0.06), where most of the isolates harbouring the virulence genes were multidrug resistant.

Shigella toxin-producing E. coli (STEC) were identified as the cause of diseases in human host due to its association with E. coli O157:H7 involved in diarrhoea, purpura and the haemolytic uremic syndrome. Many water and food- borne disease episodes have been reported from different geographical areas worldwide [30, 31, 32, 33]. Whatever, STEC is associated with a wide range of human diseases such as bloody diarrhoea, haemorrhagic colitis (HC), and haemolytic- uremic syndrome (HUS). The Shiga toxins either 1 (stx1) or 2 (stx2) interfere the binding of aminoacyl tRNA to the ribosomes and preventing the protein synthesis resulting in depurinating specific residues of the host cell ribosomes after internalization. The biological activities of stx1 and stx2, involving cytotoxicity to Vero and HeLa cells, are similar, but the immunological properties are different [33].

Virulence Gene Sequences with Significant Alignment

Escherichia coli are a common and widespread bacterium associated with various infectious diseases of both intestinal and extraintestinal sites. This is due to acquisition and dissemination virulence genes that determined their pathogenicity. The worldwide burden of these diseases is staggering, with hundreds of millions of people affected annually. Eight E. coli pathovars have been well characterized, and each uses a large arsenal of virulence factors to subvert host cellular functions to potentiate its virulence [33, 34]. Haemolysin A (hlyA) gene from isolates S12 shows a high significant sequence alignment at 97% homology with the EPEC strain, E. coli O128:H27 chromosomal DNA, complete genome (CP024243.1). Enteropathogenic Escherichia coli (EPEC) strains were the first E. coli strains recognized as important pathogens in diarrhoeal diseases [6]. Even today, EPEC strains are the major bacterial cause of neonatal and infantile gastroenteritis throughout the world, especially in developing countries [33]. Enteropathogenic Escherichia coli (EPEC) implicated in gastrointestinal infections represents a major causative agent of infant diarrhoea [35]. It has been recognized to exhibit a great pathogen-attributable risk of death in infants eleven months of age and younger [36]. Studies have shown that its morbidity and mortality rates, especially in developing countries, are quite significant [37].

It is conceivable that virulence genetic determinants, if located on the same genetic platform as antimicrobial resistance genes, may be co-mobilized under antimicrobial selective pressure [38]. In this study, isolates S07 and U08 which carry all the virulence genes are resistant to 17 (85%) out of the 20 antibiotics tested, while U01 and S06, which harboured 3 of the virulence genes are resistant to 18 (90%), with U02 (85%) and B09 (65%). The remaining isolates which mainly carry hlyA genes are resistant to 10 to 17 antibiotics. The control of E. coli infections is therefore becoming complicated due to the increasing resistance to antibiotics (Table 4).