INTRODUCTION:

E. coli remains commensally in animal and human intestine, however pathogenic strains in men and animals can cause a variety of infections with varying severity which include self limiting diarrhea to bloody diarrhea, urinary tract infections (UTI), hemorrhagic uremic syndrome (HUS), hemorrhagic colitis (HC), systemic infections^1,2. Usual route of E. coli transmission in feco-oral through contaminated water and food, however role of other potential agents cannot be ignored³. The pathogenic E. coli strains are diverse in terms of expression of virulence and epidemiological markers^4-7.

Over decades serotyping has been widely used as one of the most frequently used epidemiological markers and thus has got a very special place in characterization of pathogenic E. coli ^8-11 Three commonly used antigens detected in serotyping are cell wall “O”, flagellar “H” and capsular “K” antigens. Over 184 serogroups of pathogenic E. coli are known on the basis of “O” antigen detection alone^12,13,14. Pathogenic E. coli strains are represented by very few serotypes and these may vary from place to place, serotype O157, O26, O45, O103, O111, O121, and O145 are considered the most pathogenic strains in developed countries however these serotypes are reported with very low frequency in isolated studies in developing countries. Geographically, India is a diverse country; however there is lack of sufficient studies on prevalence of E. coli serotypes in different geographical area of the country. In the present study the distribution of “O”serogroups in intestinal and extra-intestinal clinical isolates from different geographical locations of the country was made and data is presented here.

MATERIAL AND METHODS:

Samples

The study samples in the present study were clinical isolates received during January 2016 to January 2018 from different parts of the country at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh, India. A total of 783 clinical isolates suspected to be E. coli comprising of 437 Intestinal (human diarrheal-DEC) and 346 extra-intestinal isolates (human UTI) were studied

Media and reagents:

Media and reagents used for Biotyping the isolates was procured from reputed manufacturers viz. Microexpress, SRL P. Ltd., BDH, E-Merck and Hi-media Pvt Ltd.Mumbai, India. Polyvalent and monovalent Anti “O” E. coli antisera for serotyping was provided by National Salmonella and Escherichia Center, Central Research Institute, Kasauli, Himachal Pradesh, India.

Biotyping:

Isolates were confirmed as E. coli on the basis of biotyping performed as described by Edward and Ewing ¹⁵. Briefly, growth characteristics on MacConkey agar, nutrient agar and nutrient broth were studied. Bacterial motility was observed by hanging drop experimentation and bacterial morphology was examined microscopically after Gram’s staining. All the isolates were bio-typed using set of biochemical including sugars fermentation (lactose, glucose, sucrose), indole production, methyl red reaction, voges proskauer test, citrate utilization (IMViC), urea utilization, Triple Sugar Iron test, nitrate reduction assay, oxidase test, catalase test , and ONPG test. Isolates giving characteristic reaction consistent to E. coli were considered as confirmed E. coli.

Serotyping:

All confirmed E. coli isolates were typed using standard anti “O” E. coli antiserum as described by Orskov and orskov ¹².Briefly, the organism to be typed were grown in nutrient broth overnight at 35+2^OC. The bacterial growth was boiled for 1 hour and formalin was added to final concentration of 0.25%. Suspension was diluted to concentration of 3X10⁹ organisms/ml using Brown’s opacity set. This suspension was used as antigenic suspension. Antigenic suspension was allowed to react first with pooled antisera in a U-shaped microtiter plate and monovalent antisera of the pool that gave clear agglutination were then tested again with the test suspension for agglutination to finalize the “O” serogroup. Normal saline was used in place of antiserum in negative control. Isolates exhibiting agglutination with normal saline were referred as auto-agglutinating and those showing no agglutination with any of the anti-E. coli antisera were referred as Un-typeable.

Statistical Analysis:

Frequencies, percentages and associations using Mann-Whitney U Test (95% confidence level) and Kruskal-Wallis Test (95% confidence level)were calculated using SPSS software version 22.0.

RESULTS:

Biotyping and serotyping are two widely used tools for detection and characterization of Escherichia coli isolates and plays very important role in epidemiological studies. In present study out of total 783 E. coli suspected isolates, characterized morphologically and biochemically, only 534 (302 Intestinal and 232 extra-intestinal) isolates that gave morphological and biochemical characteristics consistent to Escherichia coli were confirmed as E. coli after Biotyping (Results not shown). These confirmed E. coli isolates were from different regions of the country i.e. 170 from southern, 134 eastern, 148 northern, 59 central and 23 from western India. These confirmed 534 E. coli isolates were further subjected for serotyping analysis.

On “O” serotyping these 534 E. coli isolates were found to be clustered over 36 different groups; including 96 isolates that could not be typed using the serotyping and were designated as UT (untypeable). Ten most frequent “O” serogroups detected were O8 (46 isolates; 8.6%), O11 (36 isolates; 6.7%), O22 (33 isolates; 6.2%), O88 (33 isolates; 6.2%), O126 (28 isolates; 5.2%), O83 (25 isolates; 4.7%), O35 (25 isolates; 4.7%), O141 (23 isolates; 4.3%), O149 (22 isolates; 4.1%) and O7 (21 isolates; 3.9%) (Figue1).

Figure 1: Distribution of "O" serogroups in Intestinal (DEC) & Extraintestinal E. coli (ExPEC)

Here: UT- untypeable, O1 to O157- different “O” serogroups detected

Expression of “O” serogroups among E. coli isolates from different regions was not uniform (Kurskal-Wallis test p<0.05) as some of the serogroups were found among isolates from one or two regions only i.e. O1, O4, O5, O16, O89, O98, O101, O114, O116, O117, O118 were detected in isolates from mainly eastern and southern regions. Some “O” serogroups were frequently detected from different geographical regions i.e. Southern India: O8 (21/170; 12.3%), O88 (17/170; 10%), O11 (14/170;8.2%), O20 (12/170; 7.06%), O83 (10/170; 5.9%); Northern India: O83 (14/148; 9.5%), O149 (14/148; 9.5%), O126 (12/148; 8.1%), O35 (10/148; 6.7%); Eastern India: O22 (19/134; 14.2%), O126 (12/134; 9%), O141(12/134; 9%), O8 (11/134; 8.2%), O2 (10/134; 7.5%); Central India: O11 (12/59; 20.3%), O7 (7/59; 12%),O149 (4/59; 7%), and Western India: O88 (5/23;22%), O22 (3/23; 13.04%) [Table 1 & Figure 2]. Untypeable isolates were frequently encountered isolates from all the geographical locations.

Figure 2: Distribution of “O” Serogroup in different geographical regions across India.

Table 1: Distribution of “O” serogroups in different geographical locations of India

“O” Serogroup	Frequency of expression of “O” Serogroup in different Geographical Regions (n)					Total
“O” Serogroup	Central India	Eastern India	Northern India	Southern India	Western India	Total
UT	13	30	24	28	1	96
O1	0	4	0	1	0	5
O2	1	10	2	3	0	16
O4	0	3	0	0	0	3
O5	0	1	0	0	0	1
O7	7	2	3	9	0	21
O8	3	11	9	21	2	46
O9	0	2	1	3	0	6
O11	12	4	7	14	1	38
O16	0	2	0	0	0	2
O17	0	1	5	7	0	13
O20	0	2	6	12	0	20
O22	2	19	6	3	3	33
O34	1	1	1	2	0	5
O35	3	3	10	8	1	25
O49	0	0	3	1	1	5
O63	0	0	1	0	2	3
O83	0	1	14	10	0	25
O84	0	0	6	1	0	7
O86	0	0	2	0	0	2
O88	3	1	7	17	5	33
O89	0	0	0	7	0	7
O98	0	0	0	2	0	2
O101	0	4	0	2	2	8
O114	0	0	0	1	0	1
O116	0	2	0	0	0	2
O117	0	1	0	0	0	1
O118	0	1	0	1	0	2
O119	0	1	7	2	0	10
O120	1	0	0	1	2	4
O126	3	12	12	1	0	28
O128	0	4	4	0	0	8
O141	2	12	1	6	2	23
O149	4	0	14	4	0	22
O157	3	0	1	2	1	7
Auto-agglutinating	1	0	2	1	0	4
TOTAL	59	134	148	170	23	534

Here: UT-untypeable, Auto..-Autoagglutinating, O1 to O157- different “O” serogroups detected

Majority of serogroups were detected in both intestinal as well as extra-intestinal isolates except few which were detected in either of the source (intestinal or extra-intestinal only) as serogroups O4, O89 and O117 were detected only in extra-intestinal isolates while O5, O84, O86, O98, O114, O126, O128 and auto-agglutinating were detected only in intestinal isolates. Among the serogroups detected in both intestinal as well as extra-intestinal isolates some of the “O” serogroups were significantly found to be associated with its source i.e. intestinal: O2, O22, O119,O149,O157 or extra-intestinal: O7, O11, O35 indicating that distribution of serotypes is not uniform among the source of isolates i.e. intestinal (DEC) and extra-intestinal (ExPEC) (Mann-Whitney U test P<0.05) (Figure 3) .

Figure 3: Percentile Distribution of “O” serogroups among intestinal and Extra-intestinal E. coli

Here: UT-untypeable, Auto..-Autoagglutinating, O1 to O157- different “O” serogroups detected.

DISCUSSION:

E. coli is one of the most common infectious agents of human and animals, especially in developing countries due to dense population and poor socio economical reasons ^16,17,18. Disease presentations in E. coli infections are diverse and include self limiting to diarrhea to systemic infections which usually involve infection with multidrug resistant strains ^19,20. E. coli is one of the most diverse microorganisms in terms of its antigenic structure, pathogenesis and virulence profiles ^4-7. Association of some of the serotypes frequently with specific E. coli pathotypes makes serotyping one of the most widely used epidemiological and virulence marker⁸. Sometimes serotyping is solely used to assign pathotype to E. coli isolates ^9,21. Though various newer molecular techniques have emerged for characterization and typing of E. coli i.e. PCR for detection of pathotype specific gene targets, RFLP, whole genome sequencing etc.; serotyping is still an indispensable tool in characterization of E. coli and is the most dependent technique to characterize certain highly pathogenic E. coli strains like O157:H7 and the big six group associated with food borne outbreaks²².

In the present study 534 (302 Intestinal and 232 extra-intestinal) E. coli isolates received from various parts of India were typed using specific anti “O” E. coli antisera to evaluate the prevalent “O” serogroups in India and geographical variability of “O” serogroups if any in the country. E. coli isolates in the present study were found to be diverse in terms of “O” serogroups as these strains were clustered over 36 different “O” serogrougs. Among the total 534 isolates serotyped, 96 (18%) isolates were found to be non-typeable. In other similar studies also non-typeable E. coli isolates were detected in large numbers which were considered to be probably representing emerging serogroups/serotypes associated with pathogenic E. coli strains^{23, 24}. We found non-typeable isolates from all the geographical regions; however only one isolate was found to be non-typeable from Western India. Among type-able isolates the most predominant “O” serogroups in this study were O8, O11, O22, O126, O88, O83, O35, O7, O149 and O141. A difference in the serogroup distribution in human isolates was observed in the present study and a similar report during 2012-2013 on isolates from various parts of country. Serogroups O163, O90, O10, O133, O166, O113, O18, O153, O6, O41, O149 and O172 exclusively detected in human isolates during 2012-2013, however of these serogroups only O149 was detected in the present study; whereas serogroups O5, O20, O35, O86, O11, O101 and O118 which were detected in the present study were not detected during 2012-2013 study²⁵.

An association between E. coli serotype and pathotype has been shown by Kauffman as early as 1947 and thereafter various studies in support has been published and very few serogroups are known to be associated with different E. coli pathotypes^8,11,26-28. In the present study some of the serotypes were detected among extra-intestinal samples only (O89, O4, O117) while some others were detected only among intestinal isolates (O126, O128, O84, O86, O98, O5). Certain serotypes were found to be frequently associated with either of the source (intestinal: O2, O8, O22, O84, O126, O149, O157; extra-intestinal: O7, O8, O11, O89).

A non-uniform distribution pattern of “O” serogroups in the isolates studied from different parts of the country was detected in the present study as few serogroups were detected exclusively in isolates from certain regions i.e. southern region: O89, O98, O114; eastern region: O4, O5, O16, O116, O117; northern region: O86, while some serogroups were most frequently detected in isolates from some of the regions (O2-Eastern, O7- Southern and Central, O8, O9-Southern and Eastern). This non-uniform distribution of “O” serogroups was found to be statistically significant as tested by Kurskal Walis test (p value < 0.05). Various workers have reported other “O” serogroups than those detected in the present study associated with intestinal and extra-intestinal Escherichia coli isolates from various geographical locations in India ^{10, 21, 29-36}. These findings indicate that E. coli isolates in India are very diverse in terms of serotypes as wide range of “O” serogroups is in circulation among intestinal and extra-intestinal isolates in the country.

In the present study serogroup O157 which is associated with the most severe type of E. coli infections that leads to bloody diarrhea and HUC were detected in very less number (1.3%) as only 7 isolates (6 intestinal and 1 extra-intestinal) were detected in the present study as O157. Serogroup O157 was not detected among isolates received from eastern India in the present study. The six most prominently associated STEC non-O157 serotypes i.e., O26, O45, O103, O111, O121, and O145 that are considered as serious threat to human health especially in developed countries, were not detected from any of the geographical regions in the present study ^{37, 38}. Another important serogroup i.e. O104 which was associated with HUS in adults in Germany and US was also not detected in the present study in any of the isolate from any of the geographical regions in the country ³⁹. Of the five STEC seropathotypes A to E described by Karmali et al. on the basis of incidence rates, frequency of outbreaks and association with HUS/HC only O157 placed in the most severe class A (high incidence rate, frequently involved in outbreaks, associated with HUS/HC) and O5 placed in class C (low incidence rate, rare involvement in outbreaks, associated with HUS/HC) were detected but with very insignificant percentage i.e. 1.3% and 0.2% respectively. Serogroups placed in other seropathotypes associated with human STEC i.e. class B (moderate incidence rate, uncommon involvement in outbreaks, associated with HUS/HC) were not detected in the present study^{40, 41}. While few serogroups associated with class D (low incidence rate, rarely involved in outbreaks, not associated with HUS/HC) were detected i.e. O7; 21/534 (3.9%), O119; 10/534 (1.9%), O117;1/534 (0.2%). Studies carried out in different parts of India on E. coli isolates from human has rarely reported occurrence of E. coli O157 and big six non-O157 STEC serogroups, thus indicating very few incidences of human STEC infections in India ^42-47, however frequent isolation of STEC and O157 serogroups among STEC strains from animal and food isolates is considered as posing serious threats of outbreaks of this pathotype in India.

CONCLUSION:

It can be concluded that very diverse E. coli serogroups are in circulation in India. E. coli serogroups frequently associated with EHEC/STEC the most severe type of E. coli infectious agent are not so common in human isolates in India; however their frequent reporting from animal and food isolates always pose a threat of outbreaks.

ACKNOWLEDGEMENT:

All laboratories who submitted Escherichia coli isolates to National Salmonella and Escherichia Center (NSEC) and support extended by staff of NSEC during this study is thankfully acknowledged.

CONFLICT OF INTEREST:

None.

AUTHORS CONTRIBUTION:

Investigation, data collection and writing of original draft was carried out by Gulshan Kumar. All authors equally contributed in conceptualization, data analysis and finalization of the manuscript.

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Received on 21.05.2021 Modified on 11.01.2022

Research J. Pharm. and Tech 2022; 15(11):5239-5244.

DOI: 10.52711/0974-360X.2022.00882