European Journal of Experimental Biology Open Access

Keywords

Antibiotics; Clinical; Escherichia coli; Urinary tract infection; Susceptibility pattern

Introduction

Antimicrobial resistance has become an issue of public health concern and is a major factor contributing to mortality and morbidity in settings with limited diagnostic facilities and treatment options. Escherichia coli accounts for 17.3% of clinical infections requiring hospitalization and is the second most common source of infection next to Staphylococcus aurous among out-patient infections.

Studies on commensal bacteria in developing countries have shown high resistance rates to diverse antimicrobial agents [1]. In developing countries, because of diarrhea associated infection, 2.2 million people die every year and annually, 130-175 million patients suffer uncomplicated Urinary Tract Infection (UTI) worldwide and more than 80% of them are due to E. coli [2-4].

Despite concerning trend in antimicrobial resistance among E. coli isolates worldwide, a growing armamentarium of antimicrobial agents provides multiple options for treating E. coli infections [5]. In general, monotherapy with trimethoprimsulfamethoxazole, aminoglycosides, cephalosporin or fluoroquinolones is recommended as the treatment of choice for most known infections with E. coli, though many broad spectrum agents (such as beta-lactam/beta lactamase inhibitor combinations and the carbapenems) remain highly active.

Pathogenic E. coli strains use a multi-step scheme of pathogenesis that is similar to that used by other mucosal pathogens, which consists of colonization of a mucosal site, evasion of host defenses, multiplication and host damage [6,7]. There is an alternate side to E. coli afforded through gene gain and loss that enables it to become a highly diverse and adapted pathogen [8]. Pathogenic E. coli can cause a broad range of human diseases that span from the gastrointestinal tract to extraintestinal sites such as the urinary tract, bloodstream and central nervous system [6].

In this study we aimed at screening and assessing the antibiotic susceptibility pattern of E. coli isolated from clinical specimens in the Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria.

Materials and Methods

Study area

The clinical samples used were obtained from the Microbiology and Parasitology Department of the Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile- Ife, Nigeria in October, 2015.

Sample population

A total of 41 samples comprising 19 stool samples and 22 urine samples were taken from the Microbiology and Parasitology Department, Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), Ile-Ife, Nigeria. The samples were transferred to the Department of Microbiology Laboratory, Obafemi Awolowo University, Ile-Ife, Nigeria for subsequent culturing and biochemical tests [9,10].

Bacterial isolation and identification

Culture plates from Deoxycholate agar (Oxoid, UK), MacConkey agar (Oxoid), nutrient agar, blood agar were used. The swab sticks used for the collection of the samples were streaked directly on the labelled agar plate and incubated at 37°C for 24 h. After incubation, cultures were examined for significant growth. Biochemical test were performed to identify microbes that could not be characterized by morphology. Biochemical tests applied were standard catalase test, citrate utilization, oxidase, Voges- Prokauer, indole production, motility, sucrose, maltose, lactose, nitrate reduction and mannitol [11].

Antibiotic susceptibility

In vitro susceptibility of the isolates against microbial agents was determined by the standard disc diffusion procedure (Bauer et al., 1996). The following antibiotics were used Augmentin (30 μg), Gentamycin (10 μg), Oflaxacin (10 μg), Streptomycin (30 μg), Tetracycline (30 μg), Chloramphenicol (30 μg), Sparfloxacin (10 μg), Ciprofloxacin (5 μg), Amoxycillin (30 μg), Pefloxacin (30 μg). Identified isolates of E. coli were inoculated into nutrient broth at 37°C for 24 hours. The zones of inhibition were measured, recorded and interpreted according to the Clinical Laboratory Standard Institute provided [12].

Results and Discussion

In this study, eleven (11) isolates of Escherichia coli were obtained from 41 samples. Table 1 shows the percentage distribution of E. coli in relation to age and sex. Female individuals falling within the age range 21-30, 31-40, 41-50 constituted higher E. coli infection rates of 18.18% each. The rest of the individuals were 9.09% each.

Age range (years)	Isolate code	Total patient samples	Percentage distribution
			Males	Females
0-10	-	-	-	-
11-20	U3	2	9.09%	9.09%
	U12	-	-	-
	S2	-	-	-
21-30	S10	3	9.09%	18.18%
	S17
31-40	S11	2	0%	18.18%
	S12	-	-	-
41-50	S4	-	-	-
	U9b	3	9.09%	18.18%
	U17c	-	-	-
51-60	U7b	1	9.09%	0%
61-70	-	-	-	-
71-80	-	-	-	-
81-90	-	-	-	-
91-100	-	-	-	-

Table 1 Distribution of E. coli infections in relation to age and sex.

Table 2 shows the distribution of E. coli infection in polymicrobial association and variable medical diagnosis. Urinary Tract Infection (27.27%) was predominantly associated with E. coli followed by other implicated clinical conditions ranging from Gastroenteritis, RVDX, BAA to CAP which constituted 9.09% each of the mixed infection.

Case Histories	Isolate Codes	Total number %
Gastroenteritis	S2	9.09%
Typhoid	S4	9.09%
Medical Test	S10	-
Medical Test	S11	-
Medical Test	S17	36.36%
Medical test	U9b	-
Urinary Tract Infection	U3	-
Urinary Tract Infection	U12	27.27%
Urinary Tract Infection	U17c	-
Retroviral Disease (RVDS)	S12	9.09%
BAA to CAP	U7b	9.09%
Total		100%

Table 2 Distribution of E. coli infection case histories.

Morphological characteristics of colonies were recorded on the media used (MacConkey, Sugars, EMB) for primary identification of E. coli. A total of 11 isolates were retrieved from 4 males and 7 females. All the isolates were Gram’s negative showing pinkish colonies on MacConkey agar and greenish metallic sheen on Eosin Methylene Blue agar (Table 3). Table 4 shows the biochemical characteristics of the isolates.

Sources of isolation	Sex	Diagnosis	Isolate code	Gram stain	Growth on Mac	Growth on EMB
Stool	Male	Gastroenteritis	S2	Negative	Positive	Positive
Stool	Male	Typhoid	S4	Negative	Positive	Positive
Stool	Female	Medical Test	S10	Negative	Positive	Positive
Stool	Female	Medical Test	S11	Negative	Positive	Positive
Stool	Female	RVDX	S12	Negative	Positive	Positive
Stool	Female	Medical Test	S17	Negative	Positive	Positive
Urine	Male	Urinary Tract Infection	U3	Negative	Positive	Positive
Urine	Female	Urinary Tract Infection	U12	Negative	Positive	Positive
Urine	Male	BAA to CAP	U7b	Negative	Positive	Positive
Urine	Female	Medical test	U9b	Negative	Positive	Positive
Urine	Female	Urinary Tract Infection	U17c	Negative	Positive	Positive

Table 3: Cultural morphology of E. coli isolated from clinical sources.

Isolates	S2	S4	S10	S11	S12	S17	U3	U12	U7b	U9b	U17c
Hydrogen Sulphide	-	-	-	-	-	-	-	-	-	-	-
Indole	+	+	+	-	-	+	+	-	-	-	-
Motility	+	+	+	+	-	-	+	-	+	+	+
Methyl Red	+	+	+	+	+	+	+	+	+	+	+
VogesProskauer	+	+	+	+	+	+	+	+	+	+	+
Urease	-	-	-	-	-	-	-	-	-	-	-
Lactose	+	+	+	+	+	-	+	-	+	+	-
De-Mannitol	+	+	+	+	-	-	+	-	+	+	+
Sucrose	+	-	+	+	-	-	-	-	+	+	+

Table 4 Biochemical characterization of isolates from clinical source.

The chart showing the susceptibility of the eleven isolates of E. coli is presented in Table 5. In Table 6, the frequency of the antibiotic susceptibility of the isolates is presented. 100% of the E. coli isolates were resistant to augmentin, gentamycin, Streptomycin, chloramphenicol and tetracycline [13].

Code sample	Augmentin	Gentamycin	Pefloxacin	Oflaxacin	Streptomycin	Tetracycline	Chloramphenicol	Sparfloxacin	Ciprofloxacin	Amoxycillin
S2	R	R	R	R	R	R	R	R	R	R
S4	R	R	R	R	R	R	R	R	I	R
S10	R	R	I	R	R	R	R	I	I	S
S11	R	R	S	R	R	R	R	R	I	R
S12	R	R	S	S	R	R	R	R	I	R
S17	R	R	R	R	R	R	R	R	R	R
U3	R	R	R	R	R	R	R	R	R	R
U12	R	R	R	R	R	R	R	R	R	R
U7b	R	R	S	R	R	R	R	R	R	R
U9b	R	R	S	R	R	R	R	R	R	R
U17c	R	R	R	R	R	R	R	R	R	R
Key: R-Resistance; I-Intermediate; S-Susceptible

Table 5 Antibiotic susceptibility profile of Escherichia coli.

Antibiotics with disc potency	Frequency %
	Resistance	Intermediate	Susceptible	Total
Augmentin 30 µg	100%	0	0	100%
Gentamycin 30 µg	100%	0	0	100%
Pefloxacin 30 µg	54.54%	9.09%	36.36%	100%
Oflaxacin 10 µg	90.90%	0	9.09%	100%
Streptomycin 30 µg	100%	0	0	100%
Tetracycline 30 µg	100%	0	0	100%
Chloramphenicol 30 µg	100%	0	0	100%
Sparfloxacin 10 µg	90.9.%	9.09%	0	100%
Ciprofloxacin 5 µg	63.63%	36.36%	0	100%
Amoxycillin 30 µg	90.90%	0	9.09%	100%

Table 6 Percentage distribution of antibiotic susceptibility of E. coli isolated.

The intermediate and susceptibility rates of pefloxacin were 9.09% and 36.36% respectively. Ciproflaxin had a resistance rate of 63.63% and susceptibility rate of 36.36%. Table 7 shows the multiple resistance patterns of the isolates. 54.54% were resistant to four classes of the five classes of antibiotics used (beta lactam, aminoglycoside, sulphonamide, trimethoprim and macrolide). 46.46% were resistant to all the five classes of antibiotics used (aminoglycoside, sulphonamide, trimethoprim and fluoroquinolone) [14].

No of Antibiotics (Classes)	Antibiotic resistance pattern	Frequency (%)	Total number (%)
4	AUG/GEN/TET/CHL	6 (54.54%)	6
5	AUG/GEN/PRF/TET/CHL	5 (46.46%)	5
Total		11(100%)	11

Table 7 Multiple Antibiotic Resistance pattern of the E. coli isolates

Of the 41 samples collected, only 11 isolates were obtained (15.94%). This is slightly higher than the results reported by Kibret and Abera on antimicrobial susceptibility patterns of E. coli from clinical sources in northeast Ethiopia. E. coli was not isolated from some stool samples because the individuals had been on antibiotics prior sample collection and this may have caused an imbalance in the normal flora of the body. This is in agreement with the reports of Reynolds [13].

In this study, E. coli infection was more prominent among females compared to male subjects with ratios 1:1, 1:2, 0:1, 1:3, 1:0, and of ages 11-20, 21-30, 31-40, 41-50, 61-60 respectively. This is in agreement with the findings of Harper and George who researched on female urinary tract infection [14,15]. The highest number of infections in this study was linked to females with age ranges between 21-30 years and 41-50 years.

It could therefore be inferred that E. coli infections are not limited to the very young and very old but spread across every age bracket. This further agrees with the reports of Kaper who reviewed the pathogenesis, epidemiology, diagnosis and clinical aspects of diarrheagenic E. coli [16].

Antimicrobial resistance in E. coli has increased worldwide. In this study, the overall resistance of E. coli to the selected antimicrobials was high. This result is consistent with the findings of previous studies of Kibret and Abera [7,17].

Based on this study, E. coli showed the greatest resistance (100%) to augmentin, gentamycin, streptomycin, tetracycline, and chloramphenicol. This doesn’t corroborate the work done by Waseem et al. [18] but is slightly lesser compared to the work of Ogidi and Oyetayo [12].

The isolates were 100% resistant to augmentin, gentamycin, streptomycin, tetracycline, and chloramphenicol. Less resistance (90.9%) was shown to oflaxin, sparfloxin and amoxycillin used. The least resistance was shown to Ciprofloxacin (63.6%) and Pefloxacin (54.5%). These antibiotics seem effective against the isolated E. coli. This corroborates the work done by Onuoha [14] on antibiotics susceptibility pattern of Escherichia coli isolated from well water in Afikpo, south eastern Nigeria.

Additionally, Mydryk [9]; Toth et al. [17]; Cox and Wright [4] have all previously reported bacterial resistance against ampicillin, gentamicin, erythromycin, tetracycline and ciprofloxacin at different times. The reason why some of these E. coli isolates showed high level of resistance to the antimicrobial agents used is an indication that these antibiotics have been abused, hence the possibility they have acquired resistance. It has been reported that bacteria acquire resistance by horizontal gene transfer of mobile genetic element and that gross usage of the antibiotics influences the selection of existing resistance mechanisms (Stoke and Gillings, 2011).

References

1. Bartoloni A, Pallacchi L, Benedetti M (2006) Multidrug-resistant commensal E. coli in Children, Peru and Bolivia. Emerg Infect Dis 12: 907-913.
2. Bora A, Ahmed GU, Hazarika NK (2012) Antibiotic resistance Pattern and prevalence of ESBL producing Escherichia coli isolates in urinary tract infection from a tertiary care hospital in East India. J Med Microbiol 31: 250-256.
3. CLSI (2014) Consensus-based Standard and Guideline. Clinical and Laboratory Standards Institute.
4. Cox G, Wright GD (2013) Intrinsic antibiotic resistance: mechanisms, origin, challenges and solutions. Int J Med Microbiol303: 287-292.
5. Gootz TD (2010) The global problem of antibiotic resistance. Crit Rev Immunol30: 79-93.
6. Kaper JB,NataroJP, MobleyHL (2004)PathogenicEscherichia coli.Nat Rev Immunol 2: 123-140.
7. Kibret M, Abera B (2011) Antimicrobial susceptibility patterns of E. coli from clinical sources in northeast Ethiopia. Afr Health Sci 11: s40-s45.
8. Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, et al. (2013). Recent Advances in Understanding Enteric Pathogenic Escherichia coli. ClinMicrobiol Rev 26: 822-880.
9. Morita Y, Tomida J, Kawamura Y (2012) MexXY multidrug efflux system of Pseudomonas aeruginosa. Front Microbiol3: 408
10. Mydrk ZZ (2002) Antibiotic resistance among bacteria inhabiting surface and subsurface water layers in estuarines Lake Gardno. Polish Journal of Environmental Studies 11: 401-406.
11. Olutiola PO, Famurewa O, Sonntag HG (1991) An Introduction to General Microbiology: A Practical Approach. Heidelberger Verlagsanstalt and Druckerei GmbH Heilderberg, Federal Republic of Germany.
12. Ogidi CO,Oyetayo VO (2013) Antibiotic sensitivity of microorganisms. J Res Sci 9: 209-216.
13. Reynolds C, Quan V, Kim D, Peterson E, Dunn J, et al. (2011). Methicillin Resistant S. aureus(MRSA) Carriage in 10 Nursing Homes in Orange County. Infect Control HospEpidemiol32: 91-93.
14. OnuohaSC (2015) Antibiotics Susceptibility Pattern of Escherichia coli Isolated from Well Water in Afikpo, South Eastern Nigeria. AASCIT Journal of Biology 1: 38-42.
15. Stokes HW, Gillings MR (2011) Gene flow, mobile genetic elements and the recruitment of antibiotic resistance genes into Gram-negative pathogens.FEMS Microbiol Rev 35: 790-819.
16. TarunMadappa (2014) Escherichia coli InfectionsMedication. Medscape.
17. Toth M, Smith C, Frase H, Mobashery S,Vakulenko S (2010) An antibiotic resistance enzyme from a deep-sea bacterium. J Am ChemSoc 132:816-823.
18. Ahmad W, Jamshed F, Ahmad W(2015) Frequency of Escherichia coli in patients with community acquired urinary tract infection and their resistance pattern against some commonly used anti-bacterial. J Ayub Med Coll Abbottabad 27: 333-337.