IAETSD JOURNAL FOR ADVANCED RESEARCH IN APPLIED SCIENCES, VOLUME 4, ISSUE 4, SEPT /2017

ISSN (ONLINE): 2394-8442

Laboratory Approaches for the Prevalence and Antibiotic Susceptibility of

Bacterial Pathogens from Stool Samples in Different Provinces of
Kanyakumari District, Tamilnadu

KARTHICK. S1, SRINIVASA KANNAN, S.R2. THANGA MARIAPPAN, K3. VIJAY, M.,
Dr. K.THANGA MARIAPPAN*
1 & 4 Student of M.L.T; 2. Director, Vivek Laboratories; 3. Research coordinator
Vivek Institute of Laboratory Medicine, Nagercoil 629 003

(Affiliated to Tamilnadu Dr. M.G.R. University, Chennai, India)

www.vivekinstitution.com | vivekinstitute2011@gmail.com
Corresponding Author*: Research coordinator, micro_appan@rediffmail.com 091591 22733

ABSTRACT.

Gastroenteritis is a common infection among all age groups of people. It accounts for a
significant proportion of morbidity and mortality cases, especially in the age group of 1 to 20 years in
developing countries. Clinical observation of an etiological agent like stool culture examination is
essential for the management of patients with severe or prolonged diarrhea, symptoms consistent with
invasive disease, or other complicated diseases. Biochemical evaluation of bacterial pathogens from stool
cultures in clinical laboratories is one of the major commitment for identifying and tracking the
outbreaks of bacterial gastroenteritis This research approach provides a guidance for investigating the
prevalent enteric pathogens from stool samples namely Escherichia coli, Salmonella sp., Klebsiella sp.,
Pseudomonas aeruginosa, Citrobacter sp., and to understand their antibiotic susceptibility pattern against
various antibiotics via., Gentamycin, Tobramycin, Cefazolin, Ampicillin, Meropenum, Amikacin,
Ampicillin, Imipenum, Co-trimozole, Cefuroxime, Piperacillin and Tozobactum, mixture of Ticarcillin
and Clavulanic acid, mixture of Amoxycillin and Clavulanic acid, Cephalothin, Levofloxin, Tetracycline,
Ceftazidime and Aztreonam.

Key words: Stool samples, Bacterial pathogens, Antibiotic resistance and sensitivity

INTRODUCTION
Clinical Microbiology Laboratories are the fundamental source of laboratory based public health surveillance for infectious diseases,
including recognition of outbreaks and clarification of disease trends over time [1, 2, 3, 4, and 5]. CML plays a pivotal role in the recognition of
infectious disease outbreaks and their epidemiological understanding through isolation, identification and notification of culture based infections
to public health authorities. The interpretation of results obtained in laboratory-based surveillance data must therefore consider, among other
things, laboratory testing procedures (Andrew et al., 2004).

To Cite This Article: KARTHICK. S, SRINIVASA KANNAN, S.R. THANGA MARIAPPAN, K. VIJAY, M. AND Dr. K.THANGA
MARIAPPAN,. LABORATORY APPROACHES FOR THE PREVALENCE AND ANTIBIOTIC SUSCEPTIBILITY OF
BACTERIAL PATHOGENS FROM STOOL SAMPLES IN DIFFERENT PROVINCES OF KANYAKUMARI DISTRICT,
TAMILNADU. Journal for Advanced Research in Applied Sciences. Volume 4, Issue 4, Sept-2017; Pages: 64-68
65. KARTHICK. S, SRINIVASA KANNAN, S.R. THANGA MARIAPPAN, K. VIJAY, M. AND Dr. K.THANGA MARIAPPAN,.
Laboratory Approaches for the Prevalence and Antibiotic Susceptibility of Bacterial Pathogens from Stool Samples in Different
Provinces of Kanyakumari District, Tamilnadu. Journal for Advanced Research in Applied Sciences. Volume 4, Issue 4, Sept-
2017; Pages: 64-68

The human habitat in some developing countries is insanitary likely unprotected water supplies, inadequate waste disposal and a close
association between man and domestic animals. Observing the pattern of prevalence of microbial pathogens in such countries is essential to
apply adequate measures to control them [6,7]. Most bacterial pathogens potentially transmitted by water infect the gastrointestinal tract and are
excreted in the faeces of infected humans and other animals. Stool (faeces) is an important body substance which has to be checked for the
presence of disease-causing microorganisms (Ayrikim et al., 2015). Diarrhoea is defined as an increase in the frequency, fluidity or the volume
of the bowel movement, which is relative to the usual habit of an individual. The passage of three or more motions a day is considered as
diarrhoea. Dysentery is defined as the passage of blood and mucus stained stools, which is often associated with abdominal cramps and
tenesmus. Gastroenteritis is defined as an inflammation of the mucus membranes of the stomach and the intestines, resulting in diarrhoea which
is associated with vomiting.

Several studies revealed that the incidence of some common pathogens including the enteric and non enterics including Salmonella
sp., Shigella sp., E.coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter aerogenes and Pseudomonas aeruginosa have been reported
often from stool samples. Some of these are gut commensal organisms, which have also been reported as a cause of neonatal enterocolitis and a
variety of infections in hospital patients.

In addition, emergence of these bacterial pathogens from stool samples have shown resistance to several antibiotics and has become a
notable public health concern worldwide and especially in India due to their overuse and misuse [8]. Consequently, antibiotic resistance presents
a great challenge to the treatment of infections. The major aim of this laboratory guideline was to identify the more common bacterial organisms
associated with gastroenteritis and screens their antimicrobial susceptibility.

MATERIALS & METHODS

Sample Collection

A total of 107 stool samples from patients with various symptoms of gastroenteritis were received from clinics and diagnostic
laboratories located in different Provinces of Kanyakumari District, Tamilnadu for microbiological examinations. As part of enhancing the value
of this surveillance study, stool samples were categorized with different age and sex groups and the study period (October 2016 to December
2016). All patients had received antibiotic treatments on previous occasions, but no one was receiving antibiotic administration at the time of
sampling [9]. The collected samples were inoculated into Cary-Blair transport media and brought to the Microbiology Laboratory of Vivek
Laboratories, Nagercoil, Kanyakumari District, and Tamilnadu and bacteriologically analyzed within 24 hours as per the standard procedures
(Vandepitte et al., 2003).

Samples were subjected to the preliminary test for macroscopic observations of various organoleptic parameters such as cysts,
trophozoites, blood, mucus or pus, worms and color of stools (Church, 2007). The samples were also subjected to microscopic observations for
the identification of cellular exudates such as the clumps of pus cells and leucocytes and wet mount process for the observation of motile
bacteria in stool samples (Ayrikim et al., 2015).

Stool samples were then routine plated on Nutrient agar, MacConkeys agar, Xylose Lysine Deoxycholate (XLD) agar and sub
cultured on to nutrient agar following growth after 24 hours (Church, 2007). After 24 hours of incubation [10,11,12], tentative colonies from XLD
for suspected Salmonella sp. and Shigella sp; from lactose MacConkey agar for different E. coli; from TCBS for Vibrio cholera; from Sorbital
MacConkey to detect entero pathogenic E. coli were harvested onto individual slants and were identified by using the standard procedure for
cultural characteristics and biochemical methods (NCCLS, 2002;

Andrew et al., 2004). Isolated bacterial colonies were grown later on appropriated media. Three well isolated colonies were placed into
10 ml of sterile deionized water and adjusted to a 0.5 McFarland standard. Antimicrobial susceptibilities of all pathogenic isolates were
determined by disc diffusion method of Bauer et al. (1966) and the breakpoints used were those recommended by the CLSI (Clinical and
Laboratory Standard Institute) on guidelines for susceptibility testing.

All these isolates were examined for antibiotic susceptibility to the various antibiotics used such as Gentamycin (10mcg), Tobramycin
(10mcg), Cefazolin (30mcg), Ampicillin (10mcg), Meropenum (10mcg), Amikacin (30mcg), Ampicillin (10mcg), Imipenum (10mcg), Co-
trimozole (25mcg), Cefuroxime (30mcg), mixture of Piperacillin and Tozobactum (10mcg), mixture of Ticarcillin and Clavulanic acid (10mcg),
mixture of Amoxycillin and Clavulanic acid (30mcg), Cephalothin (10mcg), Levofloxin (5mcg), Tetracycline (30mcg), Ceftazidime (30mcg)
and Aztreonam (30mcg).

Standard E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 of known susceptibility were used as control organisms.
Statistical analysis was performed using SPSS (Version 9) by p value

Results and Discussion

Stool samples were analysed from 10 different study sites of Kanyakumari District that were collected between December 2016 and
March 2017 from a total of 107 patients belonging to various age and sex groups.
66. KARTHICK. S, SRINIVASA KANNAN, S.R. THANGA MARIAPPAN, K. VIJAY, M. AND Dr. K.THANGA MARIAPPAN,.
Laboratory Approaches for the Prevalence and Antibiotic Susceptibility of Bacterial Pathogens from Stool Samples in Different
Provinces of Kanyakumari District, Tamilnadu. Journal for Advanced Research in Applied Sciences. Volume 4, Issue 4, Sept-
2017; Pages: 64-68

Table 1: Frequency of positive culture with respect to age groups

Number of No. of Positive Pattern of Positive Cultures
Age Groups Sex (n)
S.No. Samples Cultures (n=97)
(Years)
(n) Monomicrobial Polymicrobial
Male 23 (22) 22.7% 22 -
1. 1- 20 -
Female 37 (35) 36.1% 35
Male 13 (12) 12.4% 12 -
2. 21-40
Female 4 (4) 4.1% 4 -
Male 14 (11) 11.3% 10 1
3. 41-60
Female 6 (6) 6.2% 6 -
Male 6 (4) 4.1% 4 -
4. 61-80 -
Female 4 (3) 3.1% 3
Mean Age: 40.523.23 Total: 107 100% 96 1

A total of 107 patients with the mean age of 40.523.23 months were evaluated. Of these, 90.6% patients had stool culture positive for
bacteria from which 97 pathogens were isolated. Ten specimens yielded no microbial growth. The frequencies of culture positive cases with
respect to different age groups are exhibited in Table1.

The results also indicated that the predominant age group of patients who were positive for bacterial pathogens was between 1 year to
20 years and the least frequent affected age group was below 60 years. Intestinal enteropathogens which cause gastroenteritis play a vital role in
causing major public health problems in developing countries, especially among all age groups. The present study exhibited that the
enteropathogenic bacteria were almost equally distributed in all categories. However, the majority of bacterial enteropathogens isolated were
from patients of 1 to 20 years age group. This result was found to be in agreement with recent outbreaks of diarrheal diseases which reporting
adults of age 15 to 44 years with equal impact on males and females (Palpasa et al., 2011). According to recent reports of WHO, diarrheal
diseases caused by viruses, parasites and enteropathogenic bacteria are a leading cause of morbidity and mortality in developing countries
(Abdelraouf et al., 2012).

The stool samples were tested by conventional methods for biochemical, microbiological and antimicrobial susceptibility tests. Table
2 presents the various types of bacterial pathogens screened from stool samples. Of the total 97 isolates, E. coli were 58.8% followed by
Salmonella sp. (24.7%), Klebsiella sp. (9.3%), P. aeruginosa (6.2%) and Citrobacter sp. (1%). Escherichia coli (58.8%) was found to be the
most predominant pathogen followed by Salmonella sp. (24.7%).

Table 2. Prevalence of bacterial pathogens in stool samples

Total Pathogens Isolated

S.No. Bacterial Isolates (n =97)
(%)
1. Escherichia coli 58.8%
2. Salmonella sp., 24.7%
3. Klebsiella sp. 9.3%
4. Pseudomonas aeruginosa 6.2%
5. Citrobacter sp. 1%

Table 3. Frequencies of bacterial pathogens isolated from blood cultures by age groups

Age Pathogens Isolated (n)

S.No. Group
(Years)
E. coli Salmonella sp. P. aeruginosa K. pneumoniae Citrobacter spp.
1 1- 20 31 (54.4%) 16 (28.0%) 5 (8.8%) 5 (8.8%) -
2 21 40 9 (56.25%) 4 (25.0%) 1 (6.25%) 1 (6.25%) 1 (6.25%)
3 41- 60 12 (70.6%) 4 (23.5%) - 1 (5.9%) -
4. 61 80 5 (71.4%) - - 2 (28.6%) -
67. KARTHICK. S, SRINIVASA KANNAN, S.R. THANGA MARIAPPAN, K. VIJAY, M. AND Dr. K.THANGA MARIAPPAN,.
Laboratory Approaches for the Prevalence and Antibiotic Susceptibility of Bacterial Pathogens from Stool Samples in Different
Provinces of Kanyakumari District, Tamilnadu. Journal for Advanced Research in Applied Sciences. Volume 4, Issue 4, Sept-
2017; Pages: 64-68

The present data illustrate clearly that E. coli and Salmonella sp., were mainly responsible for causing gastrointestinal infections. The
present results mainly pertain to the screening of the bacterial causative agents of gastrointestinal tract infections similar to the works conducted
by Samie et al. (2007). Enteric bacterial pathogens are the major cause of food borne gastroenteritis among humans, and remains as an important
health problem worldwide (Teshale et al., 2016)

All bacterial isolates were examined for their susceptibility to the commonly used antimicrobials. All bacterial isolates namely E. coli,
Salmonella sp., Klebsiella sp., P. aeruginosa, and Citrobacter sp. showed a high degree of sensitiveness to Gentamycin followed by
Tobramycin, Imipenum, Meropenum, Amikacin, Ampicillin + Sulbactum, Chloramphenicol, Ticarcillin + Clavulanic acid, Piperacillin +
Tozobactum, Levofloxin, Cefazidime, Tetracycline, Aztreonam and Cefuroxime commercial drugs used commonly in the management of
enteric pathogens (Table 4). Earlier studies conducted with stool samples in India and Kenya also showed high sensitivity to Gentamycin
(Wariso et al., 2006; Patil et al., 2013)

E. coli were observed to be present in maximum stool samples collected for microbiological examinations. It was on expected lines
because E.coli is a normal inhabitant of the gastrointestinal tract of these patients (Fluckey et al., 2006). Increased resistance of E.coli and
Salmonella sp., for various antibiotics was observed namely Ampicillin (69.1%), Cefazolin (54.2%), Amoxycillin + Clavulanic acid (48.1%),
Co-trimozole (38.3%), Cefuroxime (35.7%), Levofloxin (29.6%), Tetracycline (29.6%), Aztreonam (21.1%),Cephalothin (14.8) , Cefazidime
(12.4%), Piperacillin +Tozobactum (11.1%), and less than 10 percentage of resistance was observed with Tobramycin, Meropenum, Amikacin,
Imipenum, Choloramphenicol, Ampicillin + Sulbactum and Ticarcillin +. The resistance to these antibiotic was varied highly among the isolates
of Klebsiella sp. Cefazolin (71.4%), Ampicillin (96.9%), Amikacin (14.3%), Co-trimozole (71.4%), Cefuroxime (42.9%), Piperacillin
+Tozobactum (14.3%), Amoxycillin + Clavulanic acid (71.4 %) and Levofloxin (28.6%).

However, the isolates E.coli and Salmonella sp., were resistant to more antibiotics than Klebsiella sp. (Table 3). On the other hand,
other isolates from stool samples namely P.aeruginosa and Citrobacter sp. were resistant to lesser number of antibiotics that included Ticarcillin
+ Clavulanic acid as compared to E.coli and Klebsiella sp. Antimicrobial resistance in human pathogens has become a major cause of public
health concern. Several studies have indicated the high resistance of Salmonella sp., to various antibiotics such as Ampicillin (67.2%),
Cefazolin (60.3%), Amoxycillin + Clavulanic acid (50%), Co-trimozole (50%), Cefuroxime (38.3%), Levofloxin (35.7%), Tetracycline (67%),
Aztreonam (25%), Cephalothin (20%) , Cefazidime (14.3%), Piperacillin +Tozobactum (14.8%), Ampicillin + Sulbactum (12.4%) and less
than 10 percentage of resistance was observed with Tobramycin, Meropenum, Amikacin, Imipenum, Choloramphenicol and Ticarcillin +
Clavulanic acid which is in agreement with our study (Farid et al., 2007).

In the current study, most E.coli and Salmonella sp., isolates were resistant to Ampicillin, Cefazolin, Amoxycillin + Clavulanic acid,
Co-trimozole, Cefuroxime, Levofloxin, Tetracycline, Aztreonam , Cephalothin, Piperacillin +Tozobactum, Ampicillin + Sulbactum and
Cefazidime which are in accordance with previous findings from India (Mridul Malakar, 2014). Co-trimoxazole is recognized as an empirical
therapy for curing diarrheal diseases. The wide use of this drug has led to the emergence of resistant E.coli. As for Ampicillin, Tetracycline,
Chloramphenicol and Gentamycin, these antibiotics are inexpensive, broad-spectrum, and are used widely for prophylaxis and treating bacterial
infections (Pourakbari, 2010; Ayrikim et al., 2015).
 68. KARTHICK. S, SRINIVASA KANNAN, S.R. THANGA MARIAPPAN, K. VIJAY, M. AND Dr. K.THANGA MARIAPPAN,.
Laboratory Approaches for the Prevalence and Antibiotic Susceptibility of Bacterial Pathogens from Stool Samples in Different
Provinces of Kanyakumari District, Tamilnadu. Journal for Advanced Research in Applied Sciences. Volume 4, Issue 4, Sept-
2017; Pages: 64-68

No resistance was observed against Gentamycin in this study. The findings underlines and corroborates with the results of other
studies from India, in which all of the E.coli and Salmonella sp., isolates showed susceptibility to Gentamycin (Seema et al., 2013; Mridul
Malakar, 2014). The present study reveal that the isolated E.coli and Salmonella sp., resistance to other antibiotics might be due to routine
indiscriminate use of those antibacterial agents in study areas and/or rapid chromosomal mutation and presence of specific plasmid DNA. Thus,
the provided information may aid and help the physicians to select appropriate antibiotics to reduce economic loss through selecting the sensitive
antibiotics prior to detailed medication.

CONCLUSION
The present study on the screening of entero pathogens from stool samples of the patients with gastrointestinal tract infectious
exhibited that E.coli and Salmonella sp., are currently the predominant species followed by Klebsiella sp., P. aeruginosa and Citrobacter sp. The
antimicrobial resistance patterns suggested widespread resistance of E.coli and Salmonella sp., to Ampicillin, Cefazolin, Amoxycillin +
Clavulanic acid, Tetracycline, Co-trimozole, Cefuroxime, Levofloxin, Tetracycline, Aztreonam , Cephalothin, Piperacillin +Tozobactum,
Ampicillin + Sulbactum and Cefazidime . As resistance to antibiotics changes constantly, these strains should be under surveillance in order to
monitor local susceptibility and subsequently formulate policies for the use of antimicrobial drug administration only after preliminary testing. It
is also advocated that self use of antibiotics or through medical counters unauthorizodly should be brought under control through medical
awareness.

ACKNOWLEDGEMENTS
The author would like to thank Mrs. Shabeena Merin, M.Sc., M.Phil., (Principal), Mrs. S. Lalitha Srinivasan M.Sc., M.Phil. (Microbiologist),
Mrs. P. Jagatha M.Sc., M.Phil. (Quality Manager), Mrs.R.Vigila Christy, M.Sc., M.Phil., (Microbiologist), Prof. Dr. S. Ramesh, Ph.D., Mrs. A.
Padma, B.Sc. MLT (Lab Technician) and other technicians of our institution for their help in the manuscript preparation, data collection and
sampling

REFERENCES
1. Abdelraouf, A., Elmanama, Nahed, A. Antimicrobial Resistance of Enteric Pathogens Isolated from Acute Gastroenteritis Patients in Gaza strip,
Palestine. The International Arabic Journal of Antimicrobial Agents. 2012; 2 (4): 1-5.
2. Andrew C., Voetsch, Frederick J., Angulo, Terry R., Sue S., Maureen C., Stephanie M., Thomas, Ellen S., Shelley M., Zansky, Marguerite A.,
Hawkins, Timothy F., Jones, Pamela J., Shillam, Thomas J., Van G., Joy G. Laboratory Practices for Stool-Specimen Culture for Bacterial
Pathogens, Including Escherichia coli O157:H7, in the Food Net Sites, 19952000. Clinical Infectious Diseases 2004; 38 (3):190197
3. Farid H., Abu Elamreen A., Abdalla A., Abed B., Fadel A., Sharif C. Detection and identification of bacterial enteropathogens by polymerase
chain reaction and conventional techniques in childhood acute gastroenteritis in Gaza, Palestine. International Journal of Infectious
Diseases.2007: 11; 501- 507.
4. Ayrikim A., Mulugeta K., Bayeh A., Endalkachew N., Melaku A. Antibiogram of E. coli serotypes isolated from children aged under five with
acute diarrhea in Bahir Dar town. African Health Sciences.2015; 15 (2): 656-664.
5. Seema, K., Sanjeev Kumar, A. Isolation of microorganism from stool sample of diarrhea patient and effect of antibiotics and herbal extract. CIB
Tech Journal of Microbiology. 2013; 2 (2):24-32.
6. Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol.
1966; 45: 493-496.
7. Church, L.D. Aerobic bacteriology. Up dated March 2007; 3.8.2.1 -3.8.2.19.
8. Fluckey, W.M., Loneragan, G.H., Warner, R., Brashears, M.M. Antimicrobial drug resistance of Salmonella and Escherichia coli isolates from
Cattle feces, Hides and Carcasses. Journal of Food Protection. 2007:70 (3); 551-556.
9. Mridul Malakar. Antimicrobial susceptibility patterns of Escherichia coli isolated from urine and stool samples of infected patients in
Lakhimpur district of Assam, India. International Journal of Innovative Science, Engineering & Technology.2014: 1 (2); 184-189.
10. National Committee for Clinical Laboratory Standards (2002) Performance standards for antimicrobial disk and dilution susceptibility tests for
bacteria isolated from animals, 2nd ed. Approved standard M31-A2. National Committee for Clinical Laboratory Standards, Wayne. 2002; 96
124.
11. Palpasa K., Pankaj B., Sarala M. , Gokarna R.G. Antimicrobial susceptibilities of enteric bacterial pathogens isolated in Kathmandu, Nepal,
during 2002-2004. J. Infect. Dev. Ctries. 2011:5(3):163-168.
12. Patil, A, Patil, K, Pawar, P, Maheshwari, V. Isolation and survey of antibiotic sensitivity in nosocomial infections in north Maharashtra region.
J Assoc Physicians India. 2013; 61(7): 454-458.
13. Pourakbari B, Mamishi S, Mashoori N, Mahboobi N, Ashtiani MH, Afsharpaiman S. Frequency and antimicrobial susceptibility of Shigella
species isolated in Children Medical Center Hospital, Tehran, Iran, 2001-2006. Braz J Infect Dis. 2010; 14(2):1537.
14. Samie, A., Guerrant, R.L., Barrett, L., P.O. Bessong, P.O., Igumbor, E.O., Obi, C.L. Prevalence of Intestinal Parasitic and Bacterial Pathogens in
Diarrhoeal and Non-diarroeal Human Stools from Vhembe District, South Africa. J. Health Popul. Nutr. 2009;27(6):739-745.
15. Teshale, W, Ayalew J., Subbaram K., Beyene, W. Isolation and Antimicrobial Sensitivity Patterns of Enteric Bacterial Pathogens from
Asymptomatic Food Handlers, Jimma, Ethiopia. American Journal of Health Research. 2015; 3(6): 399-406.
16. Vandepitte, J., Verhaegen, J., Engbaek, K., Rohner, P., Piot, P., Heuck, C.C. Basic laboratory procedures in clinical bacteriology, 2 nd ed. World
Health Organization Geneva. 2003.2-10.
17. Wariso, B.A., Ibe, S.N. Bacteriology of chronic discharging ears in Port Harcourt, Nigeria. West Afr J Med. 2006; 25(3): 219-222.