Antibiotic Resistance and Phynotypic Detection of AmpC Beta-Lactamase Producing Escherichia coli from Urine of Students Attending Fulafia Clinic ()
1. Background of the Study
Escherichia coli (E. coli) is gram-negative, facultative anaerobic, rod-shaped, Coliform bacteria belonging to the genus Escherichia that is commonly found in the lower intestine of both humans and animals (Tenaillon et al. 2010) [1]. Most Escherichia coli exist as normal flora in the intestine (harmless), but some serotypes when consumed with food cause food poisoning and sometimes call for product recall as a result of food contamination (CDC, 2012) [2]. The virulent strains can cause gastroenteritis, Urinary tract infection, and Neonatal meningitis.
The non-virulent strains form part of the normal flora of the gut, and are beneficial to their host organisms by producing vitamin k2 (Bentley and Meganathan, 1982) [3] and also prevent colonization of the intestine with pathogenic bacteria (Hudault et al. 2001) [4].
Escherichia coli are expelled into the environment within fecal matter. The bacterium grows massively in the fresh fecal matter under aerobic conditions for 3 days, but its number decline slowly afterward (Russell and Javis, 2001) [5], Escherichia coli and other facultative anaerobes constitute about 0.1% of gut flora (Eckburg et al. 2005) [6] and fecal-oral transmission is the major route through which pathogenic strains of the bacterium cause disease. Cells are able to survive outside for a limited amount of time, which makes them a potential indicator organism to test environmental samples for fecal contamination (Thompson and Andrea, 2007) [7]. A growing body of research, though, has examined environmentally persistent E. coli which can survive for an extended period outside of a host (Ishii and Sadowsky, 2007) [8].
The bacterium can be cultured easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years. Escherichia coli are a chemoheterotroph whose chemically defined medium must include a source of carbon and energy (Tortora and Gerard, 2010) [9].
The mainstay of treatment is the assessment of the dehydration and replacement of fluid and electrolytes. Administration of antibiotics has been shown to shorten the course of illness and duration of excretion of enterotoxigenic E. coli (ETEC) in adults in endemic areas and traveler’s diarrhea, though the rate of resistance to commonly used antibiotics is increasing and they are generally not recommended (CDC, 2016) [10]. Antibiotics use depends upon the susceptibility patterns in the particular geographical region. Currently, the antibiotics of choice are Fluoroquinolones or Azithromycin, with an emerging role of Rifaximin.
E. coli that vaccine development efforts have been focused on are the ETEC. Other proven prevention methods for E. coli transmission include hand washing and improving sanitation and drinking water, as transmission occurs through fecal contamination of food and water.
2. Materials and Methods
2.1. Materials
Materials used include: Media, Chemicals/Reagents, Glassware, Equipment, and Antibiotic Disks.
2.2. Methods
2.2.1. Study Area
This study was carried out at Federal University Lafia, Nasarawa State, Nigeria.
2.2.2. Ethical Approval
Ethical approval was obtained from Federal University Lafia, Health centre, Lafia, Nasarawa State, Nigeria.
2.2.3. Sample Collection
Urine samples were collected from the laboratory department of Federal University Lafia, Health centre, Lafia. The samples were transported in an ice box to Microbiology Laboratory, Federal University Lafia, for microbiological analysis.
2.2.4. Media Preparation and Sterilization
Media preparation was done according to the manufacturers specifications. Appropriate gram of the powder agar was weighed and dissolved in appropriate amount of water, followed by shacking and/or heat boil to dissolve completely.
2.2.5. Preparation of McFarland Standard
The McFarland’s standard was prepared as follows: 0.5 ml of 1.172% (w/v) BaCl2.H2O was added to 99.5 ml of 1% (v/v) H2SO4.
2.2.6. Sterilization
Media and other materials such as Petri dishes, conical flask, and other glass wares are sterilized by autoclaving (moist heat sterilization at 121˚C for 15 minutes) or by Hot air Oven (Dry heat sterilization) for the plastics.
2.2.7. Isolation of E. coli
Escherichia coli (E. coli) were isolated from urine samples of students attending Federal University Lafia, Health centre, as follows, A loopful of the urine was streaked on McConkey agar plates and incubated at 37˚C for 24 hours, pinkish colonies suspected to be E. coli on McConkey agar were subculture onto Eosine Methylene Blue (EMB) agar, and incubated at 37˚C for 24 hours. Colonies growing with Greenish metallic sheen on EMB agar plates, after 24 hours incubation was selected to be suspected E. coli.
2.2.8. Identification of Escherichia coli
Cultural identification: E. coli grows as a pinkish colony on McConkey agar while on Eosine Methylene Blue agar; E. coli has a greenish metallic sheen colony. Morphological identification: E. coli is a gram negative rod shaped bacteria as seen under the light microscope during the gram staining. Biochemical test used for the identification of E. coli include: Gram staining, Indole test, Methyl-red/Voges proskeur test and Citrate utilization test.
2.2.9. Antibiotic Susceptibility Test
The antibiotic susceptibility testing was carried out using Kirby-Bauer disc diffusion method modified by CLSI (2014) [11]. Exactly, four (4) variants colonies of E. coli isolates were inoculated into 5 ml of sterile normal saline in a test-tube, and the turbidity of the bacteria suspension were adjusted equivalents to turbidity of 0.5 McFarland’s standard. Using sterile swab stick soaked in the adjusted E. coli suspension, streak on Mueller Hinton agar (MHA) plates and antibiotic disc were placed aseptically at equidistance on Mueller-Hinton agar plates inoculated with the E. coli isolates. The plates were allowed to stand for 1 hour for pre-diffusion at room temperature before they were incubated at 37˚C for 24 hours. The diameter zone of inhibition (mm) was determined using meter rule and the results were interpreted in accordance with CLSI (2014) [11]. Antibiotics disks used include: gentamicin (10 µg), cefuroxime (30 µg), amoxicillin-clavulanate (30 µg), perfloxacin (10 µg), streptomycin (30 µg), ampicillin (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), sulphonamide/sulphomethoxazole (30 µg), and cefoxitin (30 µg).
2.2.10. Phenotypic Detection of AmpC Beta-Lactamase Production
A lawn culture of E. coli was prepared on Mueller Hinton Agar plate. Sterile disk (6 mm) were moistened with sterile saline (20 µl) and inoculated with several colonies of test organism. The inoculated disk was then placed beside a cefoxitin disk (almost touching) on the inoculated plate. The plates were incubated overnight at 35˚C. A positive test appears as a flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disk. A negative test was undistorted zone (Black et al. 2003) [12].
3. Result
3.1. Isolation and Identification of Escherichia coli
The cultural, morphological and biochemical characteristics of E. coli isolated from Urine of students attending Federal university of Lafia, Health Centre is as given in Table 1.
3.2. Rate of Occurrence of Escherichia coli
The occurrence of Escherichia coli isolated from the urineof students attending Federal University of Lafia, Health centre, shows that out of the 22 urine samples
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Table 1. Cultural, Morphological and Biochemical characteristics of Escherichia coli isolated from urine of students attending Federal Unversity of Lafia, Health centre.
Key: CT = Citrate Test; IND = Indole Test; MR = Methyl Red Test; VP = Voges-Proskauer Test.
obtained from students, the frequency of occurrence of E. coli in the urine was 11 (50%) as given in Table 2.
3.3. Antibiotics Susceptibility
The antibiotic susceptibility of Escherichia coli from urine of students attending Federal University of Lafia, Health centre, is as shown in Table 3. The E. coli isolated from the urine were less susceptible to gentamicin (27%), streptomycin (22.7%), chloramphenicol (18%), sparfloxacin (13.6%), ofloxacin (9%), augmentin and septrin (4.5%). But none was susceptible to amoxicillin/clavulanic acid, pefloxacin and ciprofloxacin respectively. The differences in the susceptibility of E. coli isolates from urine of students attending Federal University of Lafia, Health centre, Lafia, Nigeria were statistically insignificant (p > 0.05).
3.4. Antibiotic Resistance Phenotypes
The antibiotic resistance phenotypes of E. coli isolated from urine of students attending Federal University of Lafia, health centre is as shown in Table 4. The E. coli isolates are distributed into different antibiotic resistant phenotype and the most common antibiotic resistance phenotype is SXT-CH-SP-CPX-AM-AU-CN-PEF-OFX-S the percentage occurrence was 27.3% as shown in Table 4. The differences in the occurrence of antibiotic phenotypes of E. coli from urine were statistically significant.
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Table 2. Rate of occurrence of Escherichia coli isolated from urine of students attending Federal University of Lafia, Health centre.
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Table 3. Antibiotics Susceptibility of Escherichia coli isolated from urine of students attending Federal Univesity of Lafia, Health centre, Lafia.
3.5. Multiply Antibiotics Resistance (MAR) Index
The MAR index of the E. coli isolates were MAR isolates with MAR Index of ≥0.4 as given in Table 5. The commonest MAR index in the urine isolates was 0.9 and the frequency of it occurrence was 11 (50%) as given in Table 5.
3.6. AmpC Beta-Lactamase Production
The result of phenotypic confirmatory of AmpC beta-lactamase producing E. coli isolated from the urine of students attending Federal University of Lafia, Health centre is as given in Table 6. From the 11 E. coli isolates from the urine,
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Table 4. Antibiotics resistance phenotype of E. coli isolates from urine of students attending Federal University of Lafia, Health centre.
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Table 5. Multiply antibiotics resistance (MAR) index of E. coli isolated from urine of students attending Federal University of Lafia, Health centre.
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Table 6. Phenotypic confirmatory of AmpC beta-lactamase producing E. coli isolated from urine of students attending Federal University of Lafia, Health centre.
5 (45.4%) were resistance to cefoxitin. While, 3 (60.0%) of the resistance E. coli isolates from the urine were confirmed to be AmpC beta-lactamase producing E. coli.
4. Discussion
The growing frequency of AmpC beta-lactamase producing bacteria in clinical settings is causing treatment failure and greater Hospital costs due to infection caused by this bacterium (Tschudin-Sutter et al. 2010) [13]. The presence of AmpC beta-lactmase production in many E. coli strains are of serious concern, since these organism are the most common cause of different human infection (Nathisuwan et al. 2001) [14]. AmpC beta-lactamase producing E. coli are becoming a great challenge and an increasing problem for hospitals worldwide (Nathisuwan et al. 2001) [14].
Study on AmpC β-lactamas production in E. coli isolated from Urine of Students attending FuLafia Clinic was carried out. From this study we observed that the percentage occurrence of E. coli from the urine was high and this is in agreement with the study earlier reported by CDC (2013) [15], Islam et al. (2015) [16] and Ngwai et al. (2014) [17]. Though, the occurrence of E. coli from urine was not surprising and is in agreement with the study earlier described by Ajayi and Ekozien, (2014) [18] that E. coli is one of the common etiological agents of UTIs. The percentage occurrence of E. coli from urine of the students observed in this study (52.0%) was higher than the study earlier reported by Ngwai et al. (2014) [17].
The less susceptibility of E. coli to gentamycin, streptomycin, tarivid and chloronpheniocal observed in this study was expected and this may be due to indiscriminate use of this antibiotic without prescription by physicians (Ngwai et al. 2011 [19]; Okeke et al. 2007 [20]). The susceptibility of E. coli isolated from urine to gentamycin, ofloxacin and streptomycin contradicts the study’s high susceptibility of E. coli to gentamycin, streptomycin, and ofloxacin as earlier described by Ngwai et al. (2014) [17]. The low susceptibility of E. coli to antibiotics mentioned may be due to misuse or abuse of the antibiotics (Ngawi et al. 2011 [19]; Okeke et al. 2007 [20]). The low susceptibility of E. coli to antibiotics such as ofloxacin, ciprofloxacin, amoxicillin, pefloxacin, and augmentin observed in this study seem to be in agreement with the study earlier reported by Ngwai et al. (2011) [19]. The production of AmpC β-lactamase by E. coli isolates resistant to cefoxitin observed in this study was not surprising and this finding is in agreement with the study earlier described by Wiegand et al. (2007) [21] and Jacobsy (2009) [22]. The percentage occurrence of AmpC β-lactamase producing E. coli isolates observed in this study was higher than the study as earlier described by Ogbolu et al. (2013) [23] and Al-Agamy et al. (2016) [24]. The high occurrence of AmpC β-lactamase producing E. coli in the urine of students is an indication of indiscriminate use of cefoxitin antibiotics in the clinical setup.
5. Conclusion
The E. coli isolates from the urine of the students were less susceptibility to gentamicin, streptomycin, ofloxacin, and chloronpheniocal. Most of the E. coli isolates were Multiple Antibiotic Resistance (MAR) isolates. In addition, some of the E. coli isolated from the urine of students attending FuLafia Clinic were AmpC β-lactamase producers.
6. Recommendations
Based on the findings of this study, the following are recommended:
There should be mass education and public awareness programmes on the importance of proper personal hygiene and good environmental sanitation habits. Since antimicrobial-resistant patterns are constantly evolving, and present global public health problems there is a need for constant antimicrobial susceptibility surveillance. This will help clinicians provide safe and effective empiric therapies. There is a need for controlling the spread of multidrug-resistant organisms by providing sufficient personnel and resources for infection control in all healthcare facilities.
Judicious use of antimicrobials i.e. using the appropriate antibiotics at the appropriate dosage and for the appropriate duration through the appropriate route of administration is an important means of reducing the selective pressure that helps resistant organisms emerge.