Virulence and antimicrobial resistance of Escherichia coli isolated from Tigris River and children diarrhea

Introduction

Escherichia coli is a gram-negative bacteria belonging to the Enterobacteriaceae family, short bacilli, non-spore forming, facultative anaerobic, and it is grown on a simple media.¹ E. coli is a major component of the human normal intestinal flora. Among the intestinal pathogens, there are six well-described categories: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli, enteraggregative E. coli, enteroinvasive E. coli, and diffusely adherent E. coli.² There are several virulence factors that contribute to E. coli pathogenicity, such as pilli, enterotoxins (LT, ST), shiga-like toxins, endotoxin (lipopolysaccharide), hemolysin, aerobactin, cytonecrotizing factor, intimin, and biofilm formation.^3,4 Plasmid profile analysis is useful in determining the epidemical strain in outbreaks caused by multiple species: Escherichia, Klebsiella, Pseudomonas, Serratia, Streptococcus, and so on.⁵ The adherence plasmid of EPEC strain is associated with the ability of EPEC to adhere to tissue culture cells in a pattern called localized adherence.^6,7 EHEC also carry a large plasmid (91.2 kbp) that is associated with the presence of fimbriae.⁸ Plasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera.⁹ The widespread development of resistance to several different antibiotics is generally as a result of lateral or horizontal gene transfer.¹⁰ Many studies have demonstrated that plasmid transfer between bacteria occurs in diverse environments. Various diverse phenotypic characteristics are encoded by plasmids; these include antibiotic and metal resistance, degradation of complex organic compounds, production of enterotoxins and colicins, and the production of restriction enzymes.¹¹ Most of the transfers are described using bacteria from the same group or ecological niche.¹² Plasmids size range from 1 kbp to 2,000 kbp resistance plasmids code for enzymes that can inactivate antibiotics, prevent the uptake of an antibiotic, or pump out the particular antibiotic.¹³ Gram-negative bacilli causing nosocomial infections are usually multiple-antibiotic resistant, resulting in significant problems for treatment.¹⁴ The aim of this study is comparing virulence factors and plasmid content among E. coli isolates from clinical and environmental sources.

Materials and methods

Specimens

In this study, 20 E. coli isolates were analyzed, ten E. coli isolates were obtained of the Tigris River water in Baghdad city and the other ten bacterial isolates were collected from children diarrhea from Children Hospital Center in Baghdad city. Samples collections were carried out from May to July 2013.

Isolation and identification of E. coli

Membrane filter technique

A known volume (30 mL) of water samples collected from swim areas on the Tigris River in Baghdad city was filtered through sterile 47 mm diameter membrane filters with 0.45 μm pores.¹⁵ The membrane filters were placed on the surfaces of eosin methylene blue agar plates for isolation of enteric bacteria and on MacConkey agar plates for isolation of lactose and nonlactose-fermenting bacteria.

E. coli isolation from clinical samples

Ten E. coli isolates were collected from children (aged under 4 years) diarrhea. Fecal samples were cultured on MacConkey agar and eosin methylene blue agar plates.¹⁶

Biochemical tests

Enteric bacteria isolated on respective selective and differential media were identified on the basis of colonial characteristics, Gram stain and biochemical tests, IMViC, Urea, and Kligler Iron Agar.¹⁶

Latex O157:H7

All E. coli isolates were investigated with latex O157:H7 (Well Colex) slide agglutination test.

Detection of hemolysin production

The E. coli isolates tested for blood hemolysis were streaked on blood agar plates containing 5% (v/v) human blood and incubated aerobically at 37°C for 24 hours. The clear zones around the growth colonies indicate a positive reaction.¹⁷

Detection of β-lactamase production

All E. coli isolates were tested for production of β-lactamase enzyme using iodometeric test method.¹⁸

Detection of biofilm formation

The ability of E. coli isolates to colonize abiotic surface was investigated by using Christensen et al’s method.¹⁹ The E. coli isolates were cultivated in tubes with Tryptone soy broth and incubated aerobically at 37°C for 48 hours and thereafter the culture tubes were emptied carefully and stained with 1% crystal violet solution for 30 minutes, and then the tubes were rinsed with distilled water and left to dry at room temperature. Results were compared with negative control, and biofilm formations as a layer at the internal wall of the tubes noted by the naked eye indicate a positive result.

Antibiotic sensitivity test

Antibiotic susceptibility profiles of E. coli isolates were determined by the standard Kirby–Bauer disk diffusion method.²⁰ The antibiotics with their respective disk concentrations are as follows: amoxicillin–clavulanic (30 μg), carbenicillin (100 μg), cefodizime (30 μg), chloramphenicol (30 μg), gentamicin (10 μg), imipenem (10 μg), norfloxacin (10 μg), piperacillin (100 μg), rifampicin (5 μg), and pefloxacin (5 μg). Bacterial cultures suspension equivalent of 0.5 tube McFarland turbidity standards were spread on Mueller-Hinton agar plates using sterile swabs and incubated aerobically at 37°C for 24 hours, and then the diameter of inhibition zones around antibiotic disks were measured. Results were expressed susceptible or resistant according to the criteria recommended by the Clinical and Laboratory Standards Institute.²¹

Plasmid DNA isolated procedure

All E. coli isolates were screened for plasmid content by the alkaline method of Brinboim and Doly²² and separated on a 1% agarose, at 50 vol for 1 hour and 1.5 hours. The DNA bands were visualized and photographed under UV light after the gel had been stained with ethidium bromide.

DNA purity

DNA purity was estimated by NanoDrop method (ACT gene, USA). The absorbance at 260 nm (A₂₆₀) and at 280 nm (A₂₈₀) for DNA was measured to check its purity. The ratio of A₂₆₀/A₂₈₀ was found to be between 1.65 and 1.84.

Results and discussion

In this study, ten E. coli isolates obtained from the Tigris River in Baghdad city and ten E. coli isolates collected from children diarrhea from Children Hospital Center in Baghdad city were tested for hemolysin production, β-lactamase production, biofilm formation, antibiotic sensitivity, and plasmid content.

Latex O157:H7

Results showed that all E. coli isolates investigated with latex O157:H7 slide agglutination test gave a negative reaction. Chigor et al²³ found that rate of E. coli O157 in surface waters was 2.2% and its prevalence in children with diarrhea was 5.4%. Baqir et al²⁴ revealed that E. coli O157:H7 was isolated from four (1.14%) children with diarrhea.

Hemolysin production

The hemolytic toxin α-hemolysin belongs to the RTX toxin family²⁵ and is an important virulence factor produced by several strains of E. coli. It is involved in human diseases such as urinary tract infections (UTIs), peritonitis, meningitis, and septicemia.²⁶ Results illustrated in Figure 1 showed that eight (40%) E. coli isolates (six isolated from river and two isolated from children diarrhea) were producing hemolysin, while the remaining 12 (60%) isolates showed γ-hemolysis (no hemolysis) and were considered as non-hemolysin producers. Martinez-Martinez et al²⁷ found that 56 (27.3%) of clinical E. coli isolates were hemolytic. Al-Chalabi²⁸ recorded that 57.1% of uropathogenic E. coli produced hemolysin and Abdel Rahman²⁹ revealed that 70.5% of E. coli isolated from chronic UTIs were hemolytic. The difference percentage of hemolysin production by E. coli isolates may be due to source of blood, type of hemolysin produced, source of bacteria, and screening method.

Figure 1 Number of E. coli isolates producing hemolysin.

β-lactamase enzyme production

Extended spectrum β-lactamases have emerged as a major threat worldwide with limited treatment options.³⁰ The results showed that eleven (55%) E. coli isolates (seven isolated from river and four isolated from children diarrhea) were β-lactamase enzyme producers (Figure 2). This result disagreed with the result recorded by Panus et al,³¹ who found that 21 (14.66%) aquatic E. coli isolates (seven isolated from drinking water and 14 from sea water) were positive for β-lactamase test. Results illustrated in Figure 3 showed that all E. coli (100%) isolates were resistant to β-lactam drugs, such as carbenicillin, piperacillin, cefodizime, and imipenem. In gram-negative bacteria, a variety of plasmid-determined β-lactamases can hydrolyze ampicillin, carbenicillin, cephalothin, and related drugs.^32,33 The “newer β-lactamases” that consist of plasmid-mediated AmpC β-lactamases (eg, CMY types), extended spectrum β-lactamase (eg, CTX-M types), and carbapenemases (eg, NDM) are important causes of resistance to β-lactam antibiotics among extraintestinal pathogenic E. coli.³⁴

Figure 2 Number of E. coli isolates producing β-lactamase.

Figure 3 Number of E. coli isolates producing biofilm.

Biofilm formation

The biofilm formation is considered to be a two-step process in which the bacteria first adhere to a surface mediated by capsular antigen or flagellar antigen followed by multiplication to form a multilayered biofilm, which is associated with production of exopolysaccharide matrix. The ability of bacteria to form biofilms helps them to survive hostile conditions within host and is considered to be responsible for chronic or persistent infections.³⁵

Five of the ten water E. coli isolates and two of the ten clinical E. coli isolates showed biofilm formation (Figure 3). Al-Chalabi²⁸ found that 90% of E. coli isolated from UTI were producing biofilm. There are complexities of genetic and environmental effectors of the biofilm phenotype within the species E. coli.³⁶

Antibiotic sensitivity

There was a high correlation between water isolates and clinical isolates of different sources in a pattern of antibiotic resistance, which implies that the water sources might have been contaminated with mixed contaminants originating from human and animal excreta.³⁷ All E. coli isolates showed multidrug resistance (Figure 4). Overall resistance was most frequently observed with antibiotics carbenicillin, cefodizime, imipenem, and piperacillin (100%) for both clinical and water E. coli isolates; chloramphenicol showed 100% for clinical isolates and 80% for river isolates; norfloxacin showed 100% for clinical isolates and 90% for water isolates; gentamicin showed 90% for clinical isolates and 80% for water isolates; amoxicillin–calvulanic showed 90% for clinical isolates and 70% for water isolates; and pefloxacin showed 90% for clinical isolates and 50% for water isolates; water isolates showed 90% resistance to rifampicin and clinical isolates revealed 80% resistance to rifampicin. Other researchers revealed that multidrug resistance (MDR) was higher among aquatic isolates than the clinical isolates.³⁸ The presence of antibiotic-resistant bacteria in water and surface water is a subject of public health concern; this leads to a real risk factor of acquiring such bacteria from the environments.³⁹

Figure 4 Number of E. coli isolates resistant for antimicrobial agents.

Plasmid DNA

All E. coli isolates had plasmids bands that ranged from 4.507 kbp to 5.07 kbp (Figure 5). Loss of plasmids (>2.1 kbp) due to treatment with sodium dodecyl-sulfate correlated with loss of resistance to antibiotics, suggesting that the observed MDR was plasmid mediated.³⁸ Our results may be referring to the main β-lactamase production and MDR to plasmids range 4.507–5.07 kbp.

Figure 5 Agarose gel electrophoresis of plasmids extracted from various E. coli isolates. Notes: Numbers 1–11 water isolates, 14 clinical isolates. S-ladder λ DNA (Pst1) Kb; (1% agarose, 50 vol, 1.5 hours).

Clinical isolates (Number 11–20 isolates) showed multiple plasmids more than 1,000 bp (Figure 6). These multiple copies of plasmid bands might have resulted from covalently close circular, open circular, or linear forms of the same plasmid. There are several types of E. coli virulence plasmids, including those essential for the virulence of enterotoxigenic E. coli, enteroinvasive E. coli, EPEC, EHEC, enteroaggregative E. coli, and extraintestinal pathogenic E. coli.⁴⁰ It has been suggested that special pathogenicity is due to small and large plasmids; some researcher revealed that 212.8 kbp plasmid was significantly associated with enteroinvasiveness⁴¹ and EHEC carry a large plasmid (91.2 kbp) that is associated with the presence of fimbriae.⁸

Figure 6 Plasmids comparative results. Notes: (A) Agarose 1%, 50 vol, 1 hour. (B) Agarose (1% agarose, 50 vol, 1.5 hours). Gel electrophoresis of plasmids extracted from various E. coli isolates. Numbers 1–11 water isolates, 14 and 21 clinical isolates. S-DNA ladder (100–1,000 bp).

Disclosure

The authors report no conflicts of interest in this work.

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