Special biochemical profiles of Escherichia coli strains isolated from humans and camels by the VITEK 2 automated system in Al-Ahsa, Saudi Arabia

The VITEK 2 automated system was used for comparing biochemical characteristics of Escherichia coli isolates from human urinary tract infections (UTIs) and camel faecal samples for the first time in the study area. Identification by the system to the species level was accurate. Recovery rate of E. coli from camel specimens was 26% and for human specimens was 33%. Based on biochemical activities, human and animal strains were distributed into 19 profiles. Biochemical profiles 1 and 2, of the classical E. coli activity, comprised 26 camel (50%) and16 human strains (24.2%). The rest human strains (75.8%) were distributed among 10 profiles and 50% camel strains among 7 profiles. E. coli O157 was not confirmed as 6.1% human isolates were β-glucuronidase negative but sorbitol positive whereas, 11.5% camel isolates were sorbitol negative and β-glucuronidase positive. The results showed atypical biochemical reactions but no unique biochemical profile number for E. coli causing community-acquired UTIs in the study area. Phenotypic similarity between camel and human isolates was demonstrated and implication of camel isolates in environmental contamination is discussed.


Escherichia
coli is a member of Family Enterobacteriaceae that live as a commensal in the intestinal tract of humans and animals but occasionally may cause infection in the intestinal tract and other body systems.E. coli is Gram negative, rod-shaped, non-spore forming, motile with peritrichous flagella or non-motile and about 2.0 μm long and 0.25 -1.0 μm in diameter.
They are able to grow under aerobic and anaerobic conditions.Optimal growth occurs at 35-37°C (Koneman et al., 2005).
Traditionally, biochemical reactions are used for identification and confirmation of bacteria to species level.All Enterobacteriaceae are oxidase negative except Pleisomonas shigelloides.Escherichia species are E-mail: alfadlelmula@yahoo.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License positive for indole.It ferments dextrose (D-glucose) by producing mixed acids (e.g.lactic, acetic and formic acids) that can then be made visible with the addition of an indicator sensitive to pH change as phenol red or methyl red.E. coli is catalase positive, oxidase negative and reduces nitrates.There are many other biochemical tests to indicate the presence of E. coli.For instance, Voges and Proskauer found a test to detect acetoin and 2,3-butanediol produced when Klebsiella and Enterobacter ferment glucose.The researchers found that under alkaline conditions, these two compounds oxidize themselves into diacetyl.Diacetyl then reacts with creatine (a guanidine derivative) and appears as a pinkish-red compound, or it reacts with a-naphtol and appears cherry-red in colour (Koneman et al., 2005).
Kingdom of Saudi Arabia (KSA) has a wealth of camel (Camelus dromedaries) population that provide milk, meat, wool, hides and skin.In rural areas, a close relationship occurs between camels and their owners.
Extra-intestinal pathogenic E. coli (ExPEC) is a diverse E. coli pathogenic type with genetic diversity which is reflective of its colonization of widespread ecological niches (Singer, 2015).Among these, urinary tract infections (UTIs) are one of the most common reasons for attendance at primary and secondary healthcare services.There are an estimated 150 million UTIs every year worldwide (Russo and Johnson, 2003).Enterohemorrhagic strains of E. coli, especially E. coli O157, have emerged as important enteric pathogens in recent years.The group produces a toxin almost identical to that of Shigella dysenteriae and this is responsible for the gastroenteritis in man, which ranges in severity from mild to bloody diarrhea and hemorrhagic colitis.Some patients develop hemolytic uremic syndrome (HUS) with anemia and acute renal failure.Some farm animals are infected with E. coli O157 without showing signs of the illness, that is, they are sub-clinically infected.Feces from these animals may contain E. coli O157 in varying numbers.E. coli O157 is generally identified as being a non-sorbitol fermenting, Gram negative rod shaped organism, ranging from 0.7 to 1.5 x 2 to 5 μm in size, oxidase negative, catalase positive and indole positive (ISO, 2001).
Evidence accumulates that E. coli populations in the GI tract of the human host changed frequently over time through clonal replacement, but the ecological and genetic reasons for these changes were never clarified (Caugant et al., 1981).Similar observations of a high rate of E. coli turnover in the GI tract have also been made in animal populations (Hinton, 1986).Natural selection may increase the frequency of new beneficial mutations as standing genetic variation.It is not yet well understood how different features of population biology or different environmental circumstances affect these adaptive processes (Burke, 2012).Multilocus sequence typing (MLST) provides an efficient genotyping tool for molecular epidemiology analysis.E. coli strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes.This enables different ecotypic or pathotypic lifestyles.However, STs are not uniform with regard to genetic properties or ecotypic/pathotypic behaviors (Weissman et al., 2012).
The VITEK 2 Automated System (bioMérieux, Marcy L'Etoile, France) is one of the most widely used systems in clinical microbiology laboratories for the identification of bacteria up to species level.The system uses reagent cards that have 64 wells, each with individual substrate for sugar utilization, enzymatic and biochemical tests.Identification cards are inoculated with microorganism suspensions using an integrated vacuum apparatus.A test tube containing the microorganism suspension is placed into cassette and the identification card was placed in the neighboring slot while inserting the transfer tube into the corresponding suspension tube then the vacuum is applied and air is re-introduced into the station, the organism suspension is forced through the transfer tube into micro-channels that fill all the test wells (Pincus, 2006).Basically, it is a colorimetric reading of biochemical reactions of microorganisms.Based on these readings, an identification profile is established and interpreted according to a specific algorithm.Final profile results are compared with the database, generating identification of the unknown organism.Final results are analyzed using a software which is an Advanced Expert System (AES) specifically designed to evaluate the results generated by the VITEK 2 system.Testing is repeated wherever suggested by the AES.
Many bacterial species can be transmitted between animals and humans, either through the food chain, via the environment, or by physical contact.Transmission of extra-intestinal infections by E. coli from food animals could be responsible for human infection (Bergeron et al., 2012).Normal bacterial flora in the body of camels may benefit the host; occasionally, may be source of infection.From a public health perspective, camel may act as reservoir for E. coli infection.
Studies on the phenotypic characteristics of E. coli living as commensals in the gut of dromedaries camel are very few.Hence, the goal of this study was to determine phenotypic similarities of E. coli isolates from humans and camel.The hypothesis for this investigation is that E. coli recovered from fecal samples of camels is a reservoir for E. coli causing community-acquired UTI in the study area.

Animal specimens
Freshly voided feces (200 samples) were collected from camel farms in Al Ahsa Province in sterile containers and transferred immediately in icebox to the laboratory.To prepare the samples, 1 g of fecal sample was dissolved in 9 mL sterile physiological saline for culturing (Manyi-Loh et al., 2014).

Human specimens
The study population consisted of positive cultures of urine samples (therefore no ethical approval or informed consent was required) from female patients diagnosed with UTIs, who had samples sent to the medical diagnostic laboratory of King Fahad Hospital, Al-Ahsa for culture and sensitivity testing.E. coli isolates from a total of 200 urine samples were randomly selected to be included in the study.

Laboratory procedures
Camel specimens were streaked onto blood agar (Oxoid, Basingstoke, UK) and MacConkey agar plates (Oxoid).The plates were incubated at 37°C for 24 h.Culture characteristics and microscopic features were observed and recorded for presumptive identification, as described by Koneman et al. (2005).
Human specimens were sub-cultured on blood and MacConkey agar, incubated at 37°C for 24 h and prepared for identification as described by Koneman et al. (2005).Sorbitol MacConkey agar (SMA) (Oxoid) was used to type E. coli O157 from the obtained human and animal isolates.

VITEK 2 GN identification procedure
Confirmation of the identification of isolates was performed using the VITEK 2 technique (Valenza et al., 2007).
A bacterial suspension made in 0.45% aqueous NaCl was adjusted to a McFarland standard of 0.5 with a VITEK 2 DensiCheck instrument (bioMérieux).The card for biochemical tests for Gram negative bacterial species which consists of 47 substrates (Table 1) was used.Result interpretation was done by comparing an unknown biochemical pattern to the database of reactions for each taxon and a numerical probability was calculated.Various levels of identification were assigned based on numerical probability calculations as, excellent (% probability 96-99), very good (% probability 93-95), good (% probability 89-92) and acceptable (% probability 85 to 88).

Animal specimens
From camel fresh feces, a total of 200 specimens were examined.E. coli in pure culture was isolated and identified by conventional methods.Cultural characteristics on Blood agar and MacConkey agar were studied together with microscopic examination of microbiological smears stained with Gram's stain.Lactose-fermenting colonies on MacConkey agar were suggestive to be E. coli.

Hemolytic activity
Five (9.6%) camel strains showed β-hemolysis on blood agar plates.All the isolates were confirmed by VITEK 2 technique.E. coli was confirmed from 52 specimens with recovery rate of 26% by the biochemical tests of VITEK 2 technique (Table 2).

Identification of E. coli O157 strains
Testing of E. coli isolates on SMA, showed six isolates (11.5%) with clear colorless colonies after incubation for 24 h at 35°C.The isolates were considered to be nonsorbitol fermenting and presumptive E. coli O157 strains.The rest of the isolates gave pink colonies indicating sorbitol fermentation.
From 200 human UTI specimens, E. coli was isolated and confirmed from 66 cases giving a percentage of 33% by the biochemical tests of Viteck 2 technique (Table 3).On SMA, all human strains were sorbitol fermenters.

Hemolytic activity:
A total of 14 (21.2%)human strains showed β-hemolysis on blood agar plates.Based on biochemical activities, all 118 human and camel E. coli strains were divided into 19 biochemical profiles.Profiles were different at least in one of the reactions tested.

DISCUSSION
In the present study, identification of E. coli by the Vitek 2 technique was excellent or very good.Crowley et al. (2012) in an evaluation study of the technique, concluded that the VITEK 2 GN identification method is an acceptable automated method for the rapid identification of Gram-negative bacteria.
Based on biochemical activities, E. coli human strains were divided into 12 biochemical profiles and camel strains in 9 profiles.P1 and P2 which contain biochemical reactions of classical E. coli isolates, were represented by 24.1% human and 50% camel strains (Table 2).Percentage variation between human and camel strains could be explained by the fact that camel isolates were obtained from apparently healthy animals.However, it has been established that domestic animals are the natural reservoirs of E. coli and the uncontrolled release of faeces results in the presence of these bacteria in the environment (Capriole et al., 2005).Pathogenic E. coli has been recovered from water, sewage, vegetables and sprout (Fremaux et al., 2008;Miko et al., 2013;Scharlach et al., 2013).
All human and camel strains, in the present study, were urease negative although human isolates were obtained from cases of UTI.It needs further work to detect whether this phenotype offers any pathological advantage to isolates of UTI.
E. coli O157 strains are β-glucuronidase and sorbitol negative.β-Glucuronidase appears to be a confirmed character to differentiate between E. coli O157 and non-O157 strains, however, the sorbitol fermentation is more questionable (Leclercq et al., 2001).In the present study, four human isolates (6.1%) were β-glucuronidase negative but sorbitol positive whereas, three camel isolates (11.5%) were sorbitol negative and βglucuronidase positive.It is worth mentioning that Vitek 2 system gives results of testing for 24 h; some strains of E. coli may ferment sorbitol after 48 h of incubation.As for human isolates, being sorbitol positive, throws doubt for confirmation as O157 strains.Furthermore, E. coli O157 has not been reported from UTIs in humans.In another study, Leclercq et al. (2001) reported that negativity of β-glucuronidase was fairly frequent (17.9%) among non-O157 serotypes.
In the present study, P4 was positive for malonate and identified from 4 human strains.This is in disagreement with other studies which reported that all E. coli tested strains were negative for malonate (Farmer et al., 1985;Ewing, 1986;Leclercq et al., 2001;Koneman et al., 2005), however, Krieg and Holt (1984) reported positive reaction in a range of 0 to 1% in the strains.The results of the present study suggest that E. coli of profile number P12 consisted of four human strains which were negative for ornithine decarboxylase and βglucuronidase.Other reports indicated that approximately 30% of E. coli clinical isolates were ornithine decarboxylase negative (Leclercq et al., 2001;Koneman et al., 2005).P18 and P19 of camel strains did not ferment D-maltose and D-mannose.It was reported that about 2% E. coli isolates are D-maltose and D-mannose negative (Koneman et al., 2005).
In the present study, it was demonstrated that 9.6% camel and 21.2% human strains were β-hemolytic.This indicates that some camel isolates could be potential pathogens.Other investigators reported that 10% of E. coli isolates from UTI in humans were hemolytic (Bhattacharyya et al., 2015).Results of the current study show that 50% of camel strains and 25% of human strains displayed biochemical activities of classical E. coli.The rest of the strains could not be assigned to a single profile, half of camel isolates were distributed into 7 profiles and three-quarters of human isolates were distributed into 10 profiles.This is interesting as there are no previous studies on biochemical characterization of E. coli camel isolates in the study area.All the tested strains should be considered as non-O157 E. coli.More work is needed to investigate more E. coli strains from sick camels and human UTIs for phenotypic and genotypic characteristics from the study area.

Conclusion
E. coli isolates from community-acquired UTIs in the study area belongs to the group, non-O157 E. coli which showed similarity with camel faecal isolates.Biochemical activities indicated that half camel and quarter human strains were classical as non-O157 E. coli, the remaing strains displayed deviation in some biochemical reactions.
The rest of the strains could not be assigned to a single profile, half of camel isolates were distributed into 7 biochemical profiles and threequarters of human isolates were distributed into 10 profiles.VITEK 2 identification system for Gram negative bacterial species is helpful in biochemical confirmation of E. coli isolates especially for differentiation of E. coli O157 and non-O157.

Table 1 .
VITEK 2 biochemical test substrates and amount per well on the card for Gram negative bacterial species.

Table 2 .
Biochemical profiles of E coli isolates of profiles 1 and 2 from camel faecal samples and human urinary tract infection specimens from Al Ahsa Province, KSA.