International Journal of Research in Pharmacy and Biosciences Volume 2, Issue 4, May 2015, PP 1-4 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)

Biochemical Characterization and Antibiotic Susceptibility Patterns of Three Gram Negative Bacteria of Clinical Origin

Akachukwu Amara, Gugu Thaddeus, Malachy Ugwu* Department of Pharmaceutical Microbiology& Biotechnology, Nnamdi Azikiwe University, Awka Anambra, Nigeria

ABSTRACT Information from surveillance studies, coupled with an increased awareness about emerging resistance patterns, are helpful for the development of empirical measures for the treatment of serious bacterial infections. This study was designed to screen the biochemical characters of three Gram negative uropathogens and their antibiotic sensitivity pattern against the commonly used antibiotics. The isolated organisms were biochemically characterize and their antibiogram was evaluated using agar diffusion method. The MIC of four selected antibiotics against the organisms was done using agar dilution. The order of sensitivity of the organisms to antibiotics is: Klebsiella pneumonia>Salmonella typhi > E. coli. The MIC of the antibiotics against the organisms ranged from 0.78-50 g/ml. The organisms were multidrug resistant to most commonly used antibacterial drugs. Keywords: Antibiotic, resistance, uropathogens, bacteria, Gram-negative.

INTRODUCTION Characterization of pathogenic organisms for epidemiological purposes is important in combating outbreaks of infection caused by these organisms. This is of particular importance especially in developing countries where bacterial infections feature very prominently as causes of morbidity and mortality [1]. It is desirable to characterize Gram negative bacterial isolates because of the ubiquity of these organisms and their proven capacity to cause various types of infections which may occur in epidemic form. Multidrug-resistant Gram-negative organisms have evolved as a major threat to hospitalized patients and have been associated with mortality rates of about 30 to 70% [2-4]. Researches have shown that the abundant, widespread, and inappropriate use of broad-spectrum antibiotics contribute to the emergence of multidrug resistant Gram negative organisms [2, 5-6]. Geographical variation has been reported in antibiotic sensitivity profiling due to differences in infection control practices and level of antibiotic usage [6-7]. Surveillance, monitoring studies and programs are very useful in the development of empirical approaches for the prevention, control and treatment of infections caused by these resistant Gram negative organisms[ 7-9]. Thus this study was designed to biochemically characterize and evaluate the antibiogram of three (3) Gram negative bacteria from clinical origin. We hope that these data could be utilized locally, in conjunction with other related studies, to properly interpret significant sensitivity patterns and choose the most appropriate antimicrobial regimens and policies for empirical therapy. METHODS The spectrum of study was limited to the most common gram-negative uropathogenic bacteria. Patient Specimens The study population was limited to patients with complaints of urinary infections. All the included patients consented to the collection of specimens from them before the study was initiated. Microbial Identification Gram-negative bacteria were identified using standard biochemical tests. Several biochemical tests were performed. These included Indole test, citrate test, oxidase test, sugar fermentation test and urease test as described by Cheesbrough 2004 [10]

*Address for correspondence [email protected] International Journal of Research in Pharmacy and Biosciences V2 ● I4 ● May 2015 1 Akachukwu Amara et al.” Biochemical Characterization and Antibiotic Susceptibility Patterns of three Gram negative bacteria of Clinical Origin” Antimicrobial Susceptibility Testing Antimicrobial susceptibility test was performed by disc diffusion technique according to Clinical and Laboratory Standards Institute guidelines. The ABTEK disc (India) containing the following antibiotics: Augmentin(AUG), tetracycline(TET), cotrimoxazole(COT), gentamicin (GEN), chloramphenicol(CHL), streptomycin(STR), erythromycin(ERY), and cloxacillin(CXC), was used and the susceptibility patterns were interpreted according to the Clinical and Laboratory Standards Institute guidelines [11-12]. Determination of the Minimum Inhibitory Concentration The minimum inhibitory concentration of the some antibiotics against the organism was determined using Agar dilution method as described by Okorie [13]. Briefly these antibiotics: erythromycin, streptomycin, chloramphenicol and gentamicin were used for this assay. Stock solution (500 μg/ml) of each antibiotic was made with distilled water. Adopting the agar dilution method, five serial dilutions of each stock solution were made using 2 – fold dilution. Exactly 1ml from each serial dilution was incorporated into 10 ml of molten nutrient agar and allowed to solidify. Each of the solidified plate was divided into seven sectors and labeled. A loopful of each suspension of the test organisms was streaked on the plates according to their numbering and incubated at 370C for 24 h. The MIC of each antibiotic for each organism was recorded after overnight incubation at 370C as the lowest concentration yielding no visible growth. RESULTS AND DISCUSSIONS From table 1 E.coli tested positive to the indole test by production of a red reagent layer. The red colur was as a result of the reaction of the reagent with the indole to produce the red product rosindole. This means that E.coli splits the amino acid tryptophan to form the compound indole. However the other two organisms Salmonella typhi and klebsiella pneumonia tested negative to the indole test. The KIA test equally showed that E.coli has glucose fermenting ability [ 10] .Also the Gram character of all the organism were negative and they were all singly dispersed rods except for the K. pneumonia which was a clustered rods and this is typical of all the organism. Table 2 shows the antibiogram of the test organisms. The order of sensitivity is Klebsiella pneumonia>Salmonella typhi > E. coli. It showed that E.coli had the highest resistance profile among the three Gram negative bacteria. This could be due to antibiotic misuse. It has been reported severally that indiscriminate use antibiotics encourages resistance as this overuse will always kill the sensitive normal flora and pave way for the proliferation of the resistant species[5-6, 14]. E.coli is a popular member of the normal flora and antibiotic usage can cause the emergence of a great number of resistance markers in normal flora, and some of them can be preserved for sometimes [15]. The observed pattern also could be as a result of the potential emergence of extended spectrum beta lactamase -producing organisms and the indiscriminate use of the third generation cephalosporins may be responsible for the selection of extended spectrum ß- lactamase producing multidrug resistant strains. Ibrahim et.al [16] reported that the Gram-negatives quickly acquire resistance to broad spectrum cephalosporins by acquisition of plasmid encoded extended-spectrum β-lactamases (ESBLs), cephamycinases, or carbapenemases. It is interesting to note (Table 2) that the penicilins (Augmentin and cloxacillin) exhited the highest profile of resistance by the test organisms. Penicillins are known to exert their antibacterial activity by inhibiting the synthesis of peptidoglycan, a heteropolymeric component of the cell wall, which provides a rigid mechanical stability by virtue of its highly cross-linked lattice wall structure. This observed resistance pattern is not unusual, as the Gram negatives are known to have a thin peptidoglycan and an external outer membrane. It has been reported that this outer membrane has hydrophobic lipopolysacharides that limit the diffusion /entry of penicillins across the cell memebrane. The organisms were also observed to be more sensitive to protein synthesis inhibitors. On this note we evaluated the MICs of four protein synthesis inhibitors: Streptomycin, gentamycin (50 S), chloramphenicol and erythromycin (30S). From Table 3 it is observed that the E.coli equally manifested high level of resistance to the four antibiotics. No MIC was determined except for erythromycin within the tested concentration range (0.79 -50 g/ml). Chloramphenicol followed by gentamicin and erythromycin appear to be the agent of choice for a nonspecific clinical treatment of infections caused by the isolated Gram negatives within the examined locality. This is because the possession of lower MICs by an antibiotic is suggestive of a higher inherent antibacterial property [17]. 2 International Journal of Research in Pharmacy and Biosciences V2 ● I4 ● May 2015 Akachukwu Amara et al.” Biochemical Characterization and Antibiotic Susceptibility Patterns of three Gram negative bacteria of Clinical Origin” Conclusively we reported that the studied organisms are multidrug resistant especially to most commonly used antimicrobial drugs. Thus adequate efforts should be made to propagate judicious antibiotic use so as to control emergence and spread of antibiotic resistance. TABLE LEGENDS Table1. Biochemical characterization of the bacterial Isolates Biochemical/sugar fermentation test KIA medium Indole Lactose Mannitol Glucose Oxidase Citrate Urea Butt Gas Test organism + + + + - - - yellow + E. coli + + + + - - - yellow + E. coli - - + + - - - yellow - S. typhi - - + + - - - yellow - S. typhi - - + + - - - yellow - S. typhi - + + + - + + yellow + K. pneumoniae. - + + + - + + yellow + K. pneumoniae. Table2. Antibiogram of the Isolates Test COT ERY AUG TET GEN CXC CHL STR Organisms (25µg) (5µg) (30µg) (10µg) (10µg) (5µg) (10µg) (10µg) E .C(T) 0 0 0 0 0 0 0 0 E.C(B) 12 0 0 0 0 0 0 0 SAL (S) 0 0 0 13.5 28.5 0 15 16.5 SAL(T) 0 0 0 0 18.5 0 13.5 15 SAL(B) 0 17.5 9 9 22 0 37 0 KLEB(T) 13 28.5 13 30 33 0 24.5 25 KLEB(B) 25.5 25 11.5 26 31 0 17 26 IZDs ( mm) produced by the antibiotics Table3. Minimum inhibitory concentration (MIC) in microgram per ml Organisms MIC (µg/ml) Chloramphenicol Streptomycin Erythromycin Gentamicin E.C(T) ND ND 50.0 ND E.C(B) ND ND ND ND SAL(S) 3.13 12.5 3.13 6.25 SAL(T) 12.5 ND 3.13 6.25 SAL(B) 3.13 50 ND 3.13 KLEB(T) 12.5 12.5 ND 50 KLEB(B) 0.78 ND 50.0 3.13 ND = Not determined within the tested concentration REFERENCES

[1] K. Ako-nai, A. D. Ogunniyi, A. Lamikanra" and S. E. A. Torimiro (1991). The characterisation of clinical isolates of Staphylococcus aureus in Ile-lfe, Nigeria. J. Med. Microbiol. 34 :109-112 [2] Pranita D. Tamma,a Sara E. Cosgrove,b and Lisa L. Maragakisb (2012 ). Combination Therapy for Treatment of Infections with Gram-Negative Bacteria. Clinical Microbiology Reviews 25(3): 450–470 [3] Bratu S, et al. (2005). Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch. Intern. Med. 165:1430 –1435 [4] Mouloudi E, et al. (2010). Bloodstream infections caused by metallo-betalactamase/ Klebsiella pneumoniae carbapenemase-producing K. pneumonia among intensive care unit patients in Greece: risk factors for infection and impact of type of resistance on outcomes. Infect. Control Hosp. Epidemiol. 31:1250 –1256. [5] Rice LB. (2009). The clinical consequences of antimicrobial resistance.Curr. Opin. Microbiol. 12:476–481. [6] Carlos Kiffer1, Andre Hsiung, Carmen Oplusti Jorge Sampaio, Elsa Sakagami, Philip Turner et al (2005). Antimicrobial Susceptibility of Gram-Negative Bacteria in Brazilian Hospitals: The MYSTIC Program Brazil 2003. The Brazilian Journal of Infectious Diseases;9(3):216-224 International Journal of Research in Pharmacy and Biosciences V2 ● I4 ● May 2015 3 Akachukwu Amara et al.” Biochemical Characterization and Antibiotic Susceptibility Patterns of three Gram negative bacteria of Clinical Origin” [7] Atfit H.Asgbar and Hani S. Faridah (2009). Frequency and antimicrobial susceptibility of Gram negative bacteria isolated from 2 hospitals in Makkah Saudi Arabia. [8] Masterton R.G. (2000). Surveillance studies: how can they help the management of infection? J Antimicrob Chemother;46(T2):53-8. [9] Jones R.N. (2000) .Detection of emerging resistance patterns within longitudinal surveillance systems: data sensitivity and microbial susceptibility. J Antimicrob Chemother;46 1-8. [10] Cheesbrough M. (2004). District laboratory practice in tropical countries. Part 2. Cambridge University press U.K. pp 136-142 [11] Clinical and Laboratory Standards Institute (CLSI): Performance standard for antimicrobial susceptibility testing; 20th Ed. Document M100-S20. Wayne, PA: CLSI; 2010. [12] Clinical and Laboratory Standards Institute (CLSI): Performance standard forantimicrobial susceptibility testing, 20th Ed (June 2010 update). Document M100- S20U. Wayne, PA: CLSI; 2010. [13] Okore VC.( 2005). Evaluation of chemical Antimicrobial agents. Bacterial resistance to antimicrobial agents, Pharmaceutical microbiology, pp. 55-120. [14] Van den Bogaard, A. E. & Stobberingh, E. (2000). Epidemiology of resistance to antibiotics. Links between animals and humans. International Journal of Antimicrobial Agents 14, 327–35 [15] Zora Jelesić, Vera Gusman, Mira Mihajlović Ukropina, Marija Kulauzov and Deana Medić (2011). Med Pregl (7-8): 397-402. [16] Mohamed K. Ibrahim, Abdel-Moneim M. Galal, Idriss M. Al-Turk and Khalid D. Al- Zhrany(2010). Antibiotic resistance in Gram-negative pathogenic bacteria in hospitals' drain in Al-Madina Al-Munnawara. JTUSCI 3: 14-22 [17] Malachy C Ugwu, Damian C Odimegwu, Emmanuel C Ibezim, and Charles O Esimone (2009). Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Nostrils of Healthy Human Subjects in a Southeastern Nigeria Locality. Maced J Med Sci. 15:2(4): 294-300

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