Department of Biology, Microbiology and Biotechnology, Faculty of Science, Federal University, Ndufu-Alike, Ikwo, Nigeria

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1 SciFed Journal of Applied Microbiology Research Article Open Access Frequency and Antibiogram of Urinary Isolates of Klebsiella Pneumoniae Isolated from Urine Samples of Apparently Healthy School Children 1 Okonkwo E C, 1 Orji JO, 2 Nworie O, * 1 Ejikeugwu PC Ejikeugwu PC,, 2018, 1:1 *1 Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, P.M.B.053, Ebonyi State, Nigeria 2 Department of Biology, Microbiology and Biotechnology, Faculty of Science, Federal University, Ndufu-Alike, Ikwo, Nigeria Abstract Antimicrobial resistance is a natural biological phenomenon of bacterial adaption to harsh environment, but this development compromises the efficacy of some available antimicrobials due to the emergence and spread of drug resistant bacteria in both the community and hospital environment. In this study, the prevalence and antimicrobial susceptibility profile of Klebsiella pneumoniae isolated from urine samples of apparently healthy school children in a local primary school in Abakaliki, Nigeria was bacteriologically investigated. A total of 25 urine samples were collected from the school children using sterile urine containers. The isolation of Klebsiella pneumoniae was carried out on eosin methylene blue (EMB) and MacConkey (MAC) agar plates. Culture plates showing significant bacterial growth (>10 CFU/ml) were processed further for the identification of Klebsiella pneumoniae using standard microbiology techniques. Antibiogram was determined using the modified Kirby-Bauer disk diffusion technique as per the CLSI criteria on Mueller-Hinton agar plates. The rate of isolation of Klebsiella pneumoniae from the urine samples of the school children used in this study was high (60%; n=15). The result of the antimicrobial susceptibility testing showed that more than 70% of the Klebsiella pneumoniae isolated were highly resistant or intermediately resistant to ampicillin, chloramphenicol, and clarithromycin and augmentin. This was closely followed by resistance to perfloxacin, ciprofloxacin, ceftriaxone, gentamicin and ofloxacin. Nitrofurantoin had no inhibitory activity against the Klebsiella pneumoniaeisolates recovered in this study. The data provided could help to step up surveillance and monitoring measures geared towards the proper detection and reporting of antimicrobial resistance in the non-hospital environment. Keywords Antimicrobial Resistance; Enterobacteriaceae; Antimicrobial Susceptibility Testing; Gram Negative Bacteria; Nigeria Introduction Antimicrobial resistance is the ability of microorganisms, such as bacteria to grow in the presence of an antimicrobial agent that would normally kill it or inhibit its growth. It is one of the biggest current challenges facing the healthcare sector globally especially in the area of infection control and disease containment. Most Gramnegative bacteria including but not limited to Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are fast becoming resistant to the onslaught of antimicrobial agents because they harbour and transmit drug resistance genes including but not limited to metallo-β-lactamases (MBLs), AmpC enzymes and extended spectrum β-lactamases known as ESBLs [1, 2]. It is now more *Corresponding author: Ejikeugwu PC, Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, P.M.B.053, Ebonyi State, Nigeria. ejikeugwu_chika@yahoo.com Tel: Received January 04, 2018; Accepted February 13, 2018; Published February 20, 2018 Citation: Ejikeugwu PC (2018) Frequency and Antibiogram of Urinary Isolates of Klebsiella Pneumoniae Isolated from Urine Samples of Apparently Healthy School Children. 1:1. Copyright: 2017 Ejikeugwu PC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. page 1 of 6

2 Healthy School Children. 1:1. worrisome that drug resistance in bacteria are not restricted to the hospital environment alone but bacterial pathogens including Klebsiella pneumoniae that are multidrug resistant in nature are now found even in the non-hospital environment which is not known for heavy antibiotic usage. Measuring the prevalence and antibiogram of drug resistant organisms coupled with proper use of available drugs is critical to reducing the possible risks associated with infections due to these organisms [3]. Klebsiella is a genus of non-motile, Gram-negative, oxidase-negative, rod-shaped bacteria with a prominent polysaccharidebased capsule; and they are ubiquitous in the environment [4, 5]. The members of the genus Klebsiella are a part of the normal flora of the gastrointestinal tract (GIT) of man and animals. Infections with Klebsiella pneumoniae can lead to a wide range of disease states, notably pneumonia, urinary tract infections, septicemia, meningitis, diarrhea, and soft tissue infections [4, 5, 6]. The majority of human Klebsiella infections are caused by K. pneumoniae, followed by K. oxytoca. Klebsiella species are sometimes regarded as coliforms as is applicable to E. coli and other enteric bacteria [4, 5]. The antimicrobial resistance nature of Klebsiella species is thought to be attributable mainly to the multidrug efflux pumps of the organism, which allows it to actively pump out antimicrobial agents from the cell [7]. The ability of K. pneumoniae to colonize the hospital environment, including carpeting, sinks, flowers, and various surfaces, as well as the skin of patients and hospital staff, has been identified as a major factor in the spread of hospital-acquired infections including drug resistance genes [8]. Klebsiella species is one of the major causes of both hospital-acquired and community-acquired infections, and some strains of this organism are notoriously resistant to some available antimicrobial agents [2, 6, 8]. It is in view of this that this present study presumptively evaluated the prevalence and antibiogram of Klebsiella pneumoniae isolates recovered from the urine samples of healthy school children in the community. Materials and Methods Study Area and Population The study area of this research was a local primary school located in Abakaliki metropolis of Ebonyi State Nigeria. Both male and female school children were recruited for this study. This study was carried out over a two (2) months period from July to August Sample Collection and Processing A total of 25 school children including male and female were used for this study. The school children were apparently healthy children who presented with no clinical signs and symptoms of any disease. The urine samples were obtained by informed consent of the school children used for this study. Urine samples were aseptically collected in sterile urine bottles containing boric acid from a total of 25 school children. All collected samples were labelled and transported to the Microbiology laboratory unit of Ebonyi State University, Abakaliki within one hour of collection for bacteriological analysis. Culture and identification of Klebsiella pneumoniae A loopful of the urine samples were each plated onto freshly prepared MacConkey (MAC) agar and eosin methylene blue (EMB) agar plates, and incubated at 37 o C for h [9]. All culture plates showing significant bacterial growth (>10 CFU/ml) were further processed by sub culturing onto freshly prepared MAC and EMB agar plates for isolation of pure cultures, and for the identification of Klebsiella pneumoniae. The isolated colonies growing on the culture media plate was identified based on their colonial characteristics, microscopy, and biochemical testing. Klebsiella pneumoniae produces mucoid colonies on MacConkey agar and non-metallic green sheen colonies on EMB agar; and they are Gram negative, citrate positive and urease positive [9]. Antibiogram The antimicrobial susceptibility testing was carried out on Mueller-Hinton (MH) agar plates as per the guidelines of the Clinical and Laboratory Standard Institute (CLSI) using the modified Kirby-Bauer disk diffusion technique as was previously described [1, 2]. The following antibiotics were used: ampicillin (30 mcg), nitrofurantoin (100 mcg), ciprofloxacin (10 mcg), gentamicin (10 mcg), ofloxacin (10 mcg), ceftriaxone (30 mcg), chloramphenicol (10 mcg), augmentin (30 mcg), clarithromycin (30 mcg) and perfloxacin (30 mcg). All susceptibility test plates were incubated at 37 o C for h; and inhibition zone diameter(s) were measured, recorded and interpreted using the standard antibiotic breakpoints recommended by the CLSI [10]. The degree of susceptibility of the isolated Klebsiella pneumoniae to the tested antibiotics were interpreted as either sensitive (S) or resistant (R) by measuring their respective inhibition page 2 of 6

3 Healthy School Children. 1:1. zone diameter (IZD) and comparing it to the standard breakpoints of the CLSI. Results Table 1 shows the colonial features, morphology and biochemical reaction of the isolated Klebsiella pneumoniae from the urine samples. Klebsiella pneumoniae produces mucoid colonies on MacConkey agar and nonmetallic green sheen colonies on eosin methylene blue agar (Table 1). A total of 15 (60%) isolates of Klebsiella pneumoniae was bacteriologically recovered from the urine samples. The prevalence of Klebsiella pneumoniae according to the sex of the school children used in this study is shown in (Table 2). It was observed in our study that more of the Klebsiella pneumoniae was isolated from females (69.2%) than males (50.0%). Statistical analysis however shows that there was no significant difference in the rate of isolation of Klebsiella pneumoniae between the male and female school children (p>0.05). Table 3 shows the distribution of the isolated Klebsiella pneumoniae based on the age of the school children used in this study. In relation to their age, Klebsiella pneumoniae was isolated more from school children in the age range of 8-10 years followed by school children in the age range of >12 (Table 3). The isolation of Klebsiella pneumoniae from the school children shows possible contamination from the school environment. Statistically, the occurrence of Klebsiella pneumoniae shows no significant difference as per the age of the school children (p>0.05). Table 1: Isolation of Klebsiella Pneumoniae from Urine Samples Bacteria Sample (n) Isolates n (%) Klebsiella pneumoniae Urine (25) 15 (60) Morphological appearance Mucoid colonies on MAC; nonmetallic green sheen colonies on EMB Gram reaction Gram negative Biochemical reaction Citrate positive Urease positive n-number, %-Percentage, EMB-eosin Methylene Blue, MAC-Mac Conkey Table 2: Prevalence of Klebsiella Pneumoniae in Urine Samples in Relation to sex Sex Number screened of number infected % Male Female Total Table 3: Distribution of Klebsiella Pneumoniae in Urine Sample in Relation to Age Age Number screened % isolation of Klebsiella pneumonia % of number infected > Aug > Total page 3 of 6

4 Healthy School Children. 1:1. The antimicrobial susceptibility pattern of the isolated Klebsiella pneumoniae is shown in Table 4. All the isolated Klebsiella pneumoniae showed varying levels of susceptibility and resistance to the tested antibiotics used in this study. However, the isolated Klebsiella pneumoniae were highly resistant or intermediately resistant to ampicillin, clarithromycin, chloramphenicol, ceftriaxone and ofloxacin. Moreover, this was closely followed by perfloxacin and augmentin (Table 4). None of the isolated Klebsiella pneumoniae was inhibited by the antimicrobial onslaught of nitrofurantoin. Table 4: Antibiogram of the Isolated Klebsiella Pneumoniae Isolates GN OF AMP N PF CT C AU CIP CM K11 S S R R S S R R R R K2 R S S R S S R R S R K3 S R R R S R R R R R K14 S S R R S S R S R R K5 R R R R R R R R R R K6 S R R R R R R R R S K10 S R R R S S R R S R K12 S R R R R R R S S R K13 S S R R S R R R R R K9 S S S R S R R S R R K15 S S R R R R S S S R K16 S S R R S R R R S S K17 S S R R R R S R R R K18 R R R R S R R S S R K1 R S R R R S R R R R Keys: K = Klebsiella Pneumoniae, R = Resistant, S = Susceptible, GN = Gentamicin (R= 12, S= 15) OF = Ofloxacin (R= 12, S= 16), AMP = Ampicillin (R= 13, S= 17) N = Nitrofurantoin (R= 14, S= 17), PF = Perfloxacin (R= 12, S= 17) CT = Ceftriaxone (R= 19, S= 23), C = Chloramphenicol (R= 12, S= 18) AU = Augmentin (R= 13, S= 18) CIP = Ciprofloxacin (R= 15, S= 21) CM = Clarithromycin (R= 13, S= 18) Discussion Klebsiella pneumoniae is a nosocomial pathogen as well as a normal microflora of the gut of humans and animals; and this organism is implicated as a causative agent in human pneumonia infections as well as in other upper respiratory tract infections [4, 5, 6]. Antimicrobial resistance is a natural phenomenon but it can be contained and averted through proper drug prescription and appropriate detection of their emergence and spread in environmental microbiota. The economic cost of antimicrobial resistance goes beyond the morbidity and mortality associated with it; it also includes the loss of efficacy or ineffectiveness of some available antimicrobial agents [6, 8, 10, 11]. In this study, the prevalence and antimicrobial susceptibility profile of Klebsiella pneumoniae isolates bacteriologically recovered from the urine samples of school children in a local primary school in Abakaliki, Nigeria was analyzed. Out of the 25 urine samples analyzed in this study, a total of 15 (60%) isolates of Klebsiella pneumoniae was bacteriologically recovered, and this indicates a possible bacterial infection of the school children in this school used in our study. According to Podschun et al. [6], Klebsiella pneumoniae is a bacterial organism that is ubiquitously found in the environment including surface waters; and the bacterium is found in animals and humans as an important community/nosocomial pathogen that may harbour antibiotic resistance traits [6, 7, 11]. The prevalence of Klebsiella pneumoniae in our study compares favorably page 4 of 6

5 Healthy School Children. 1:1. with a previous report by Omonigho et al. [12] and Ebie et al. [13] who reported that Klebsiella pneumoniae was the most occurring Enterobacteriaceae found in cases of urinary tract infections (UTI) of urine samples of patients analyzed from both the community and hospital environment. The result of the antimicrobial susceptibility testing shows that the Klebsiella pneumoniae were highly resistant to ampicillin, chloramphenicol, clarithromycin, ceftriaxone, augmentin and ciprofloxacin. This is similar to the reports of Adeyemo et al. [14] who also reported in their study that enteric organisms particularly Klebsiella pneumoniae are multidrug resistant in nature. Most notably, all the Klebsiella pneumoniae isolates were completely resistant to nitrofurantoin (100%), which is used clinically for the treatment of urinary tract infections (UTIs). This is in contrast to the work of Chikere et al. [15] who reported sensitivities of enteric organisms including Klebsiella pneumoniae to nitrofurantoin. In their study, Okonko et al. [16] reported sensitivities of enteric organisms including Klebsiella pneumoniae to nitrofurantoin; and they also recommended the use of nitrofurantoin for the treatment of UTIs especially when treatment is necessary. However, the Klebsiella pneumoniae isolates recovered in this study showed appreciable sensitivity to gentamicin and ofloxacin, which are found in the aminoglycoside and fluoroquinolone families of antibiotics respectively. Conclusively, our study shows that Klebsiella pneumoniae isolates from community samples are notoriously resistant to some commonly used antibiotics especially to nitrofurantoin to which all the Klebsiella pneumoniae isolates showed reduced susceptibility. We recommend the continuous screening of samples from the nonhospital environment for possible emergence and spread of drug resistant bacteria that may be responsible for some community acquired infections. Further molecular characterization of the Klebsiella pneumoniae isolated in this study is required for deciphering the genetic determinants responsible for the multidrug resistant nature of the isolates. References 1. Ejikeugwu C, Esimone C, Iroha I, et al. (2017) Molecular Identification of MBL Genes blaimp-1 and blavim-1 in Escherichia coli Strains Isolated from Abattoir by Multiplex PCR Technique. Res J Microbiol 12: Ejikeugwu C, Ikegbunam M, Ugwu C, et al. (2013) Phenotypic Detection of Klebsiella pneumoniae Strains-Producing Extended Spectrum β-lactamase (ESBL) Enzymes. Scholars Acad J Biosci 1: Ejikeugwu C, Esimone C, Iroha I, et al. (2016) Phenotypic detection of AmpC beta-lactamase among anal Pseudomonas aeruginosa isolates in a Nigerian abattoir. Arch Clin Microbiol 7: Brooks GF, Butel JS, Morse SA (2004) Medical Microbiology, 23rd edition, McGraw Hill Publishers. USA Madigan MT, Martinko JM, Dunlap PV et al. (2009) Brock Biology of microorganisms, 12th edition, Pearson Benjamin Cummings Publishers. USA Podschun R, Pietsch S, Holler C, et al. (2001) Incidence of Klebsiella species in surface waters and their expression of virulence factors. Appl Environ Microbiol 67: Ogawa W, Li Dai-Wei, Yu P, et al. (2005) Multidrug resistance in Klebsiella pneumoniae MGH78578 and cloning of genes responsible for the resistance. Biol Pharm Bull 28: Jadhau S, Rabindranath M, Nageshawari G, et al. (2012) Increasing incidence of multidrug resistance Klebsiella pneumoniae infections in hospital and community settings. Int J Microbiol Res 4: Cheesbrough M (2006) Biochemical tests to identify bacteria. In: Laboratory practice in tropical countries. Cambridge Press Jean B. Patel, Robin Patel, Melvin P. W, et al. (2011) Clinical Laboratory Standard Institute (CLSI) Performance standards for antimicrobial disk susceptibility test. Fifteenth informational supplement. CLSI document, Wayne, USA M100-S Bush K, Jacoby GA (2010) Updated functional classification of β-lactamases. Antimicrob Agents Chemother 54: Omonigho SE, Obasi EE, Akukalia RN (2001) In-vitro Resistance of Urinary Isolates of Escherichia coli and Klebsiella species to Nalidixic Acid. Nigeria J Microbiol 15: Ebie MY, Kandakai-Olukemi YT, Ayanbadejo J, et al. (2001) Urinary Tract Infections in a Nigerian Military Hospital. Nigerian J Microbiol 15: Adeyemo AA, Gbadegesin RA, Onyemenen IN, et al. (1994) Urinary Tract Pathogens and anti-microbial sensitivity in children in Ibadan. Nigeria Annals of Trop Paediatrics 14: page 5 of 6

6 Healthy School Children. 1: Chikere CB, Chikere BO, Omoni VT (2008) Antibiogram of clinical isolates from a hospital in Nigeria. African J Biotechnol 7: Okonko IO, Soleye FA, Amusan TA, et al. (2009) Incidence of Multidrug resistance (MDR) organisms in Abeokuta, Southwestern Nigeria. Global Journal of Pharmacology 3: Citation: Ejikeugwu PC (2017) Frequency and Antibiogram of Urinary Isolates of Klebsiella Pneumoniae Isolated from Urine Samples of Apparently Healthy School Children. 1:1. page 6 of 6