Biolog identification of non-sorbitol fermenting bacteria isolated on E . coli O 157 selective CT-SMAC agar

E. coli O157:H7 is recognised as an important human pathogen world-wide and has been associated with diseases such as haemorrhagic colitis (HC), haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP). Accurate laboratory detection of E. coli O157:H7 is important for diagnostic purposes and to justify epidemiological data on E. coli O157:H7. A well-known phenotypic characteristic of E. coli O157:H7 bacteria is their inability to ferment sorbitol. This characteristic is often used to isolate these organisms from food and water using selective agar medium such as SMAC. However, the high number of false positive results obtained by a number of researchers when selectively screening for E. coli O157: H7 on CT-SMAC has prompted an investigation to determine which other sorbitol-negative bacteria also grow on CT-SMAC. The agar medium used for the investigation consisted of Sorbitol MacConkey agar (SMAC) supplemented with Cefiximetellurite (CT). All sorbitol-negative colonies obtained from CT-SMAC, after selective enrichment and IMS were identified using the Biolog microbial identification system. The majority of sorbitol-negative isolates identified were Burkholderia, Pseudomonas, Vibrio and Aeromonas spp. Only two E. coli O157:H7 isolates were identified with Biolog and confirmed with a polymerase chain reaction (PCR) specific for the shiga toxin 1 (Stx1) genes and with O157 and H7 antisera. The inability of the CT-SMAC agar medium to specifically select for E.coli O157:H7 was confirmed by the results of this study. These observations call for further improvement of affordable methods for the selective isolation of E. coli O157:H7 in the presence of large numbers of interfering bacteria capable of growing on CT-SMAC.


Introduction
Escherichia coli (E.coli) is the species most commonly isolated from human faecal samples and is part of the normal intestinal flora of healthy individuals (Nataro and Kaper, 1998).E. coli O157:H7 is the classical serotype linked to serious outbreaks and sporadic cases of enterohaemorrhagic diseases such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) (Nataro and Kaper, 1998;Verweyen et al., 2000).Animals such as cattle are the main reservoir for E. coli O157:H7 but they do occur in other animal species such as sheep, goats, pigs, cats, dogs, chickens and gulls (Johnson et al., 1990;Griffin and Tauxe, 1991;Beutin et al., 1994;Wallace et al., 1997).Water-borne transmission of E. coli O157:H7 has been well documented and reported from both recreational water and contaminated drinking water (Swerdlow et al., 1992;Keene et al., 1994;O'Connor, 2002).These strains of E. coli produce shiga toxins (Stx), which are potent cytotoxins that are similar to the toxin produced by Shigella dysenteriae Type 1 (Scotland et al., 1983;O'Brien et al., 1984).
Immunomagnetic separation (IMS) has been proposed as a sensitive method for the isolation of E. coli O157:H7 bacteria from clinical, food and environmental samples (Wright et al., 1994;Bennet et al., 1996;Chapman and Siddons, 1996;Cubbon et al., 1996;Vernozy-Rozand et al., 1997;Tomoyasu, 1998;Müller et al., 2003).A variety of selective and differential plating media have been developed for the isolation of E. coli O157:H7 (Nataro and Kaper, 1998).The most common E. coli O157 isolation media are Sorbitol MacConkey (SMAC) agar supplemented with Cefixime and potassium-tellurite (CT-SMAC) (March and Ratnam, 1986;Nataro and Kaper, 1998;Müller et al., 2001;Fujisawa et al., 2002;Müller et al., 2002).SMAC contains sorbitol, which replaces the lactose of the standard MacConkey agar medium (SMAC Oxoid product brochure, 2003).Unlike typical E. coli, E. coli O157:H7 do not ferment or produce acid from Dsorbitol within 24 h and lack glucoronidase activity (Manafi and Kremsmaier, 2001).E. coli O157:H7 bacteria present as colourless colonies on SMAC media (Manafi and Kremsmaier, 2001).SMAC agar is not suited for the isolation of non-O157 EHEC bacteria because there is no genetic link between Stx production and sorbitol fermentation (Nataro and Kaper, 1998).However, some non-O157 EHEC strains have proven to be sorbitol-negative (Ojeda et al., 1995).The addition of Cefixime and tellurite to SMAC agar permits the selective growth of E. coli O157:H7 and Shigella sonnei strains but inhibits the growth of most of the other E. coli strains (Zadik et al., 1993).Pseudomonas spp.do not ferment sorbitol and also present as colourless colonies on CT-SMAC (Cousin, 2000).Except for the addition of Cefixime and potassium-tellurite (CT) to SMAC (Zadik et al., 1993), researchers have modified SMAC agar by adding other agents to increase the selectivity of the media such as rhamnose and chromogenic enzyme substrates (Szabo et al., 1986;Chapman et al., 1991;Fujisawa et al., 2000;Okrend et al., 2000).Müller et al. (2001) found CT-SMAC to lack in selectivity and suggested further research to improve available methods for the selective cultivation of E. coli O157:H7 in the presence of large numbers of wild type E. coli and other bacteria capable of growing on the selective media.Resistance to the antibiotics used for the suppression of bacteria other than E. coli O157:H7 may largely be accountable for the problem.One solution may be to find an-timicrobial agents, which more efficiently suppress background growth and strains of E. coli other than E. coli O157:H7 (Szabo et al., 1986;Okrend et al., 1990;Chapman et al., 1991).
Automated microbial identification systems, such as the MicroLog/Biolog identification system (Biolog Inc., Hayward, Calif.), have become widely used in both food, clinical and research laboratories (Odumeru et al., 1999).The analysis of carbon-source utilisation is a simple and rapid method to identify bacterial isolates from various sources (Odumeru et al., 1999).This system establishes identifications based on the exchange of electrons generated during bacterial respiration (Truu et al., 1999).
In this study Biolog identification was used to determine the efficacy of CT-SMAC agar as a selective agar medium for the presumptive isolation of sorbitol-negative E. coli O157:H7.

Samples
Settled sewage samples (28) were obtained from 13 sewage purification plants (SPPs) in the Gauteng region of South Africa.Additional isolates (7) were obtained from 5 river water sampling points.

Immunomagnetic separation
Twenty microlitres of the Dynabead ® suspensions were incubated in 1.5 mℓ Eppendorf tubes with 1 mℓ aliquots of the pre-enriched samples at room temperature for 10 min with continuous mixing by hand.This step was performed to allow the O157-specific antibodies coated onto the beads to bind to the target bacteria.The bead-bacteria complexes were separated using a magnetic particle concentrator, Dynal MPC-M for 3 min (Dynal, Oslo).After discarding the supernatants and washing the bead-particles with PBS-Tween (Sigma), the complete immunomagnetic separation and washing procedure was repeated 4 times (Dynal ® product  brochure, 1995).The final bead-bacteria complexes were resuspended in 100 μℓ washing buffer (PBS-Tween).After IMS, 10 μℓ of the IMS concentrates were transferred to E. coli O157 selective media by the spread plate method.

Escherichia coli O157 selective media
The media used in this study were Cefixime-tellurite Sorbitol-MacConkey agar (CT-SMAC) (Oxoid).E. coli O157:H7 strains produced typical colourless colonies on CT-SMAC after 24 h of incubation at 37 o C. Suspect E. coli O157:H7 colonies were individually tested for agglutination using a commercial E. coli O157 slide agglutination kit with antisera against E. coli O157 (Mast Assure, Mono Factor O157, code: M12030).All E. coli colonies were biochemically confirmed by their ability to produce indole from tryptophan using Kovac's reagent (ISO, 2001).

Biolog bacterial identification
Gram-stains were performed on all the sorbitol-negative isolates to determine which Biolog micro-plate (GN2/GP2) (Biolog, Hayward, Calif.) to use in the bacterial identification procedure.Bacterial cell morphology was also documented (results not shown).Biolog GN2 and GP2 micro-plates were respectively used to identify Gram-negative and Gram-positive bacterial isolates.Appropriate cell densities (OD = ± 0.65 at 560 nm) (Spectro 22, Digital Spectrophotometer, Labomed, USA) of sorbitol-negative microbial suspensions were inoculated into the micro-well plates as described in the GN2/GP2 Biolog Micro-Plate system manuals and incubated at 37 o C for 24 h.Colour development was automatically recorded using a micro-plate reader (ICN Flow Titertek ® Multiscan Plus, Version 2.03, Lab-Systems, Finland) with a 590 nm wavelength filter.A similarity index of 0.500 or higher was used for all micro-plates after 24 h.The results were interpreted with the Biolog Microlog 3.70 database and software (Biolog, Hayward, Calif.).

Bacterial DNA extraction
Suspect colonies were suspended in distilled water and heated (99 o C) in a thermocycler (Pharmacia LKB Thermocycler) in 0.5 mℓ Eppendorf tubes for 10 min.The samples were centrifuged at 10 000 x g (Eppendorf Centrifuge 5402) for 1 min and 10 μℓ volumes of the supernatant containing genomic DNA particles were used for DNA amplification without further treatment.Suspect E. coli O157:H7 colonies were examined for the presence of the phage-integrated genes coding for Stx1 and Stx2.

Results and discussion
A high number of bacterial isolates, which included both sorbitol-positive and sorbitol-negative isolates, were detected in 10 µℓ of IMS-concentrate, which were plated on CT-SMAC.This observation could be due to non-specific binding of other bacteria to the O157-antibody-coated dynabeads or possibly on account of bacteria remaining in suspension during washing of the dynabead suspensions.These bacteria were resistant to the cefixime-tellurite antibiotic solution present in the SMAC media.Gram-staining of 81 Gram-negative, sorbitol-negative isolates revealed that the majority of the bacteria were bacilli (82.5%) followed by 17.5% with cocco-bacilli features.CT-SMAC was able to suppress the growth of all Gram-positive sorbitol-negative bacteria.
The Biolog bacterial identification system was used to identify the 81 Gram-negative isolates (using GN2 micro-plates) from 33 samples isolated after IMS and CT-SMAC.A number of reference strains (Gram-negative and Gram-positive) were included as controls and identified using GN2 and GP2 microplates to determine the specificity of the Microlog/Biolog microbial identification system.All reference strains were correctly identified with Biolog at genotype level and species level except Streptococcus pyogenes, which was identified as species S. intestinalis.E. coli O157:H7 was identified up to E. coli species level.
Shortcomings of media for the selective cultivation of E. coli O157:H7 in test samples with heavy background growth such as sewage and river water have been reported by other researchers (Bettelheim, 1998).False-positive laboratory reports can result in delayed consideration of alternative diagnoses or therapeutic interventions.Resistance of non-O157 bacteria to the antibiotics used in SMAC may largely be accountable for the problem.It could be beneficial to incorporate other antimicrobial agents into the current CT-SMAC protocol, which could more efficiently suppress other bacteria in the VCC-BPSW and background growth on CT-SMAC.Another solution may be to develop affordable alternative media based on the chromogenic/fluorogenic properties of E. coli O157.
Only two strains isolated from 2 different sewage samples were positively identified as E. coli O157 (2.47% of 81 isolates) using Biolog.These two strains agglutinated with O157 antisera and possessed the gene coding for Stx1 according to the PCR results.This implies a false positive E. coli O157 detection rate of 97.68%.Other researchers have observed false positive detection rates for E. coli O157:H7 of between 37% and 57% in beef and milk samples using CT-SMAC agar (Manafi and Kremsmaier, 1999;Manafi and Kremsmaier, 2001).
A total of 13/81 (16%) of all sorbitol-negative isolates was unidentifiable with the Biolog microbial identification system.A possible explanation is that the isolates from this study were isolated from environmental samples still unknown and not included in the Biolog database.All the sorbitol-negative bacteria identified were able to multiply in VCC-BPSW and grow on CT-SMAC.Additional or alternative antibiotics might be useful in improving the selective enrichment of E. coli O157:H7 in BPSW and SMAC.The addition of Cefixime-tellurite was not sufficient in suppressing all interfering bacteria.Other researchers have suggested adding Cefixime-rhamnose (CR-SMAC), 5bromo-4-chloro-3-indoxyl-ß-D-glucuronide (BCIG-SMAC) or 4-methylumbelliferyl-ß-D-glucuronide (MSA-MUG) to SMAC agar to improve selectivity (Szabo et al., 1986;Okrend et al. 1990;Chapman et al., 1991).Stein and Bochner (1998) suggested adding novobiocin (10 mg.ℓ -1 ) in selective agar for the inhibition of background flora.Non-specific binding and/or bacterial clumping of other bacteria to the O157-antibody-coated dynabeads during IMS posed another problem when aiming to selectively isolate E. coli O157:H7 from sources such as sewage and polluted water.Multiple washing steps (4 or more washing steps) of the IMS solutions with PBS-Tween and careful resuspension and/or slow vortexing after washing may increase the sensitivity of the method.It is thus important to consider all these problems to improve the selective capabilities of the IMS / CT-SMAC method.

Conclusions
The selective media used in this study had shortcomings with regard to the selective cultivation of E. coli O157 bacteria.Consequently the isolation of E. coli O157:H7 among suspect colonies was rather low (2.4%).It is therefore possible that E. coli O157:H7 colonies were missed, and that the actual prevalence is higher than reflected by the two isolates obtained from two different sewage samples.However, it can also be possible that the prevalence of E. coli O157:H7 in the samples investigated in this study was indeed low.Unfortunately it was difficult to assess the potential presence of these pathogens in the environmental water concerned because no meaningful data are available on the prevalence of E coli O157:H7 infections in the communities from which these wastes originated.In addition, there were no meaningful data in the rest of the world to which these results can be compared.Although E. coli O157:H7 has been recovered from sewage in countries such as Germany (Höller et al., 1999), no quantitative data on the prevalence of the pathogens in the samples under investigation were available.The apparently low prevalence of E. coli O157:H7 in the environmental water investigated may be explained by data, which suggested that the prevalence of E coli O157:H7 infections is lower among humans in the Southern Hemisphere, such as South Africa, than in countries of the Northern Hemisphere (WHO, 1997).
According to the results in this study it is clear that the presence of these interfering bacteria obstructs the isolation of E. coli O157:H7 from sources with high bacterial counts.New selective media have been developed to compensate for the number of false positive results of CT-SMAC and to increase the effectiveness of E. coli O157:H7 isolation (Manafi and Kremsmaier, 2001).Other E. coli O157:H7 isolation media include Rainbow agar O157 (Biolog, Hayward, USA), CHROMagar O157 (CHROMagar, Paris, France), Biosynth Culture media O157:H7 (Biosynth, Staad, Switzerland) and Fluorocult E. coli O157:H7 (Merck, Darmstadt, Germany).A major consideration when using alternative selective media is the cost.These E. coli O157 selective media are too expensive for the routine analyses of contaminated water samples.Sorbitol-MacConkey agar with the addition of alternative antimicrobial, chromogenic or fluorogenic agents will still be the most cost-effective E. coli O157 selective media available on market.Therefore, the modification of the selective CT-SMAC E. coli O157:H7 cultivation media is recommended with regards to E. coli O157 isolation from environmental samples with high bacterial content.