The main findings of this study were; (1) Tellurite resistant diarrhoeic E. coli were obtained from 5% (33/733) of the stool specimens and included; five STEC, twelve EPEC, one DAEC, and fifteen EAggEC strains, (2) All the diarrhoeic E. coli isolated in this study were resistant to trimethoprim-sulphamethoxazole while 55% (18/33) were resistant to ampicillin.
Detection of diarrhoeic E. coli in stool
The inhibitory action of potassium tellurite on coliforms was first reported by Fleming in 1940 [18]. Tellurite-containing media have been routinely used to screen for STEC [19], but not the other E. coli pathotypes. However, Hirvonen et al., 2012 reported the formation of mauve colonies by other tellurite resistant diarrhoeic E. coli on this medium [8]. Of the 733 stool samples processed in this study, 33 (5%) contained tellurite-resistant diarrhoeic E. coli. However, tellurite-containing media have limited ability to select for STEC. In a study conducted on STEC from human, animals, and food in Austria, the prevalence of tellurite resistance amongst STEC was 26% - therefore 74% of the STEC would not be detected by the tellurite-containing medium. [20]
Tellurite resistance and antimicrobial resistance are often observed in highly pathogenic diarrhoeic E. coli strains [20]. Commercial selective media such as CHROMagar™STEC and Sorbitol MacConkey with cefixime and potassium - tellurite use this property to select STEC O157: H7 and other pathogenic STEC. There was a relatively low prevalence (5%, 33/733) of tellurite-resistant diarrhoeic E. coli in stool samples submitted to NHLS, GSH. We were unable to determine the prevalence of tellurite susceptible STEC in this study; this is because these would not grow on CHROMagar™STEC.
STEC
Five STEC were detected in this study. All the five were detected after enrichment in TSB, and none was obtained by directly streaking on CHROMagar™STEC. Since the other diarrheic E. coli pathotypes were detected with and without enrichment, it can, therefore, be speculated that TSB enrichment is more beneficial for STEC isolation relative to the other diarrheic E. coli pathotypes. Overall, the number of diarrheic E. coli obtained after enrichment in TSB was significantly higher than the number obtained without (p = 0.004). This may suggest that enrichment in TSB may be a beneficial step as regards recovering STEC (and other pathotypes) from a stool sample. The STEC diagnostic strategy routinely employed by the NHLS clinical laboratory at the GSH targets only O157 STEC and is based on the non-sorbitol fermenting attribute of STEC O157: H7. Therefore, this strategy would miss the non-O157 STEC and sorbitol-fermenting E. coli O157. Of the five STEC reported in this study, only one was non-sorbitol fermenting and belonged to serotype O101. The five non- O157 STEC that we report in this study would have been missed (four because they were sorbitol fermenting and one because it was not O157) given that the routinely used method is based on absence of sorbitol fermentation and targets only E. coli O157:H7. Only two of the five STEC tested positive for Shiga toxin production by immunoassay because the other three did not express the stx genes.
The relatively low rate of STEC isolation in this study could be due to several reasons: (1) The stx primers may not have been suitable for detection of all the stx gene variants [21]. (2) The tellurite susceptible STEC could not be isolated on CHROMagar™STEC. (3) This study had a short sampling frame and not all children presenting with diarrhoea may have had a stool specimen taken. (4) There could have been a low prevalence of STEC in stool samples processed, especially given that the stool specimens were not from patients presenting with bloody diarrhoea, or HUS. There are higher chances of recovering STEC from the stool of patients with bloody diarrhoea or HUS as opposed to those without [22].
In South Africa, as in other African countries, there may be a lower prevalence of STEC as compared to America and Europe. This may be related to the type of diet given to ruminants in America and Europe that favour the proliferation of STEC in cattle-the primary ruminant reservoirs for STEC [23].
It is important to detect STEC in stool because the use of antibiotics such as the quinolones leads to bacterial lysis and toxin release which increases the chances of HUS in infected patients [23]. Four of the five STEC detected in this study carried the stx
1
genes. Possession of stx
2
genes has been associated with the more severe form of illness [24]. Also, we detected eae in three of the five STEC, while two did not possess the eae gene. The eae gene which is located in the locus of enterocyte effacement (LEE) codes for the intimin protein which is necessary for the formation of attaching-effacing lesions in the intestinal tract [25]. STEC that carry eae (LEE-positive STEC) have been shown to cause more severe disease.
Per the manufacturer of CHROMagar™STEC, the commonly encountered STEC serotypes should form mauve coloured colonies on this medium. However, the serotypes were categorised as “common” (O157, O26, O45, O145, O111, O121, and O103) based on outbreaks that occurred in developed countries. In this study, STEC belonged to serotypes O101 and O186. These serotypes were not detected in an earlier study conducted at the NICD (2006 to 2009) which screened 2378 diarrhoeic E. coli isolates. In that study, STEC in stool had not been purposively targeted (and thus CHROMagar™STEC was not used) but was an incidental finding among EPEC (the strains carried the eae gene) that had been sent to the central public health laboratory for serotyping. The 14 STEC identified in that study belonged to serotypes O4, O5, O21, O26, O84, O111, and O157 [26]. Other related studies reported STEC serotypes O8 and O9 in pigs in South Africa [27]. We did not detect E. coli O157: H7 despite high sensitivity of CHROMagar™STEC for this serotype [3].
Clinical relevance of STEC carriage
On review of the clinical records of the five patients that carried STEC, none presented with typical Hemolytic Uremic Syndrome (HUS). However, the assessment of clinical relevance of STEC carriage was not possible due to the protracted time lag between the onset of sickness in the community and reporting to primary health care and eventual referral to tertiary health care. Earlier research has shown that only a small percentage of acute diarrhea cases in South Africa report to the health centres [28].
EAggEC, EPEC, and DAEC
At the GSH, stool from diarrhoea patients is not routinely screened for diarrhoeic E. coli other than non-sorbitol fermenting O157. Of all the 33 diarrhoeic E. coli strains isolated on CHROMagar™STEC, 45% (15/33) were EAggEC, 18% (6/33) were atypical EPEC, 15% (5/33) were typical EPEC, and 3% (1/33) were DAEC. No enteroinvasive E. coli (E. coli) were detected. This finding is in agreement with an earlier study which showed that EAggEC were most likely to be cultured from stool since they cause a more persistent form of diarrhea [14].
Serotypes
Of the 16 diarrhoeic E. coli serotypes that were isolated using CHROMagar™STEC in this study, only serotypes O111, O104 and O26 were previously reported to be detectable on this medium by studies conducted in Europe [3, 8]. In this study, we report the detection of the other tellurite resistant serotypes including O16, O175, O182, O186, O25, O3, O33, O175, O8, O9, O55, and O101. The dominant tellurite resistant diarrhoeic E. coli serotypes we identified in this study were O104 (15%), and O55 (18%). The cluster of six serotype O55 EPEC had clearly distinct antimicrobial resistance patterns and so was not an outbreak cluster. Since the cluster of five serotype O104 EAggEC strains were noted within a collection period of 38 days and had the same antimicrobial susceptibility pattern, they are possibly an outbreak cluster. However, we were unable to perform an epidemiological investigation to support this hypothesis.
Antibiograms
Even though diarrhoea is not normally treated with antibiotics (except if accompanied with invasive disease), E. coli is used for sentinel surveillance of antimicrobial resistance. The STEC detected in this study were only resistant to trimethoprim-sulphamethoxazole (SXT). This finding is similar to a 2011 study that was conducted in Kenya that reported a high level of resistance to SXT among STEC [29]. EAggEC, EPEC, and DAEC in this study showed resistance to SXT (100%, 28/28) and ampicillin (64%, 18/28). These findings are similar to reports from Kenya which showed a high prevalence of resistance to sulfamethoxazole among intestinal E. coli [30]. There are increasing reports of resistance to multiple antibiotics among EAggEC [31]. We identified multidrug resistance in one EAggEC (resistant to six antibiotics), one atypical EPEC strain (resistant to four antibiotics), and one typical EPEC strain (resistant to three antibiotics).
Sorbitol fermentation
Of the 33 diarrhoeic E. coli isolated on CHROMagar™STEC, 15 (45%) were non-sorbitol fermenting. A similar study conducted in Tanzania reported a 14% prevalence of non-sorbitol fermenting E. coli in 1049 human stool and non-human samples [32]. This is higher than the 2% (15/733) prevalence of non-sorbitol fermenting E. coli reported in this study. The high number of non-sorbitol fermenting EAggEC can be explained by the fact that serotype O104 is one of the prevalent non-sorbitol fermenting serotypes globally [3].
Limitations of this study
CHROMagar™STEC only permits the growth of tellurite resistant STEC and not the tellurite susceptible strains. This study did not have a long sampling timeframe, and not all children that presented with diarrhoea may have had stool specimens taken. Only diarrhoeic E. coli that possessed virulence genes were characterised; therefore, we might have missed strains that lost the virulence genes.