Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2011, 1 (3): 57-65 (http://scholarsresearchlibrary.com/archive.html) ISSN : 2231 3168 CODEN (USA) : JMBRB4 The antibiogram types of Escherichia Coli isolated from suspected urinary tract infection samples Momoh, A.R.M 1 ; Orhue, P.O 2 ; Idonije, O.B 3 ; Oaikhena A.G 4 ; Nwoke E.O 5 and Momoh, A.A 6 Department of 1 medical microbiology, 2,6 microbiology 3 chemical Pathology 4 anatomy, 5 physiology, Ambrose Alli University, Ekpoma, Edo State, Nigeria ABSTRACT Escherichia coli is a common cause of Urinary Tract Infection (UTI) affecting both male and female individuals however data on the antibiogram types of the various strains of E. coli isolated from UTI samples is scanty hence this present study. In this study a total of 407 urine samples were collected from both in and out patients with suspected cases of UTI in University of Benin Teaching Hospital (UBTH), Benin, Edo State, Nigeria.162 (39.8%) and 245 (60.2%) were male and female samples respectively. 97(59.9%) of the male samples returned positive cultures while 103 (42%) of the female samples returned positive cultures. The result of the returned positive cultures showed that the most prevalent gram negative organism was Escherichia coli (35.5%) while; Staphylococcus aureus (46%) was the most prevalent gram positive organism. Worthy of note is the fact that Staphylococcus saprophyticus, a nonagglutinating cocci, hitherto, considered to be non- pathogenic, had 6(3%) isolates; suggesting a likely pathogenic nature for this organism. The Ajumali s mnemonic coding used has a very high resolving power as it showed that no two strains of Escherichia coli are the same. Key words: UTI, Antibiogram types, Ajumali s Mnemonic Coding, Escherichia coli. INTRODUCTION Microbial colonization of the urinary tact as well as tissue invasion of an organ of the urinary system by pathogenic microbes is referred to as urinary tract infection (UTI). Implicated pathogenic microorganisms, could be bacteria, fungi, protozoans or viruses. Usually bacterial are more, prevalent and invasive. 57
UTI is a common presenting complaint in out patient s clinics daily with majority of patients falling into the active reproductive ages (18-37 years), especially young men and women. A patient is said to have a urinary tract infection, when there is the presence of over 100,000 organisms per ml in the midstream sample of urine [1]. The resulting infection may be symptomatic or asymptomatic; with the later usually detected on routine examination. The human urinary tract is a collecting and emptying system, which comprises the kidneys, ureters, bladder and urethra. Infections in any of these anatomical sites are referred to as UTI. Infections extending to the bladder leads to cystitis while those involving the kidneys leads to pyelonephristis [2]. Escherichia coli is the most common cause of urinary tract infection [3, 4] and accounts for approximately 90% of first urinary tract infection in young women [4]. The symptoms and signs include urinary frequency, dysuria, hematuria and pyuria. Flank pain is associated with upper tract infections. None of these symptoms or signs is specific for Escherichia coli infection [5]. Urinary tract infection can result in bacteriuria with clinical signs of sepsis [6]. Nephropathogenic E. coli typically produce a hemolysin. Most of the infections are caused by Escherichia coli of a small number of O antigen types. K antigen appears to be important in the pathogensis of upper tract infection while pyelonephritis is associated with a specific types of pilus, i.e, P pilus, which binds to the P blood group substances [7]. Suffice to add here that, while the kidney and urine in the bladder is normally sterile, the lower urethra in the female and to a lesser extent in the male, may have a detectable bacteria flora, which may include coliforms and Staphylococci species, with the number of microbes diminishing, upwards, as the bladder is approached. However, male infants are reported to have a higher rate of UTI than their female counterparts due to their being prone to congenital genitourinary disorder [8]. Experimentally, a hundred thousand bacterial counts per milliliter of urine is indicative of a urinary tract infection, though lesser counts may be strongly suggestive in some instances, especially, among pregnant women, where asymptomatic UTI could predispose then to greater risk of developing symptomatic UTI and its attendant obstetric complication [9]. Some antibiotics under study Ciprofloxacin: This drug is a fluoroquinolone and acts by inhibiting DNA topoisomerases (gyrases); thereby, inhibiting bacterial DNA synthesis [10]. Erythromycin: This drug belong to the class of macrolide, it is bacteriostatic, binding to the 23-RNA of the 50s ribosomal subunit to inhibit peptide chain elongation during protein synthesis [11]. 58
Augmentin: This is a combination of amoxycillin and clavulinc acid. The clavulinic acid helps protect the amoxycillin from being inactivated by the enzyme beta-lactamsae, an enzyme produced by pathogenic bacteria. Gentamycin: This drug is an aminoglycoside which binds to small ribosome subunits and interfere with protein synthesis by directly inhibiting protein synthesis [4]. Cefuroxime: This is a broad spectum antibiotic of the cephalosporin class. It is an alternate drug of choice when patients are allergic to the penicillins or when there is a need to overcome beta-lactamase inactivation [11]. MATERIALS AND METHODS Specimen: A total of 407 clinical specimen comprising of Mid-Stream urine (MSU), Super-public urine (SPU) and catheter specimen were collected from in-and out patients in UBTH, between July, 2009 and December, 2009 for this study. These samples were taken to the laboratory for standard microbiological analysis with 30 minutes of collection. Isolation and identification: The specimen was inoculated onto nutrient agar, blood agar and MacConkey agar plates by streaking. Inoculated plates were then incubated aerobically at 37 o C for 24 hours. After 24 of incubation, discrete colonies were picked up and Gram stained and further subculturing was done to obtain pure cultures and biochemical tests carried out. Antibiotics susceptibility testing (Antibiogram) This was done by the multi-discs diffusion using 21 different antibiotics. The multi discs were placed on the plates which were previously inoculated, few minutes earlier, then the plates were incubated at 37 o C for 24 hours, thereafter, the plates were examined for zones of inhibition around the different antibiotic disc. Staphylococcus aureus Oxford stain NTC 6751 was used as control for Gram positive organisms, while Escherichia coli strain NCTC 10 418 was used as control for Gram negative Organism. Mnemonic coding: The Ajumali s mnemonic coding method as earlier described by Joghi et al., [12], was adopted as a typing scheme to re-arrange the nominal antibiotics into arbitrary numeric values, making it easy for the differentiation of strains. Using this pneumonic coding scheme, a sensitive result was scored as (+), while a resistance was scored as (-). Also, the 21 different antibiotics were divided into a group of 3 antibiotics each, following their mechanisms of action as well as; their clinical applications; and these 3 antibiotics were given numerical values of 1, 2 and 4. Thus, a perfect sensitivity to the 3 antibiotics will give a summation 0f 1+2+4 =7.While complete resistance to the 3 antibiotics will give a summation of 0+0+0 = 0. 59
The other values as obtained by adding up these numerical values thus, an isolate can receive a score of 0-7 in each triplet segment, which, when the seven triplet segments are combined together, gives a seven (7) digit numerical value as the antibiogram types [13]. Results: Male samples have 59.9% positive cultures while female samples though larger in numbers has 42% positive cultures as shown in table 1. Staph. aureus hasthe highest number of isolates with 92 (46%) isolates while E. coli has 71 (35.5%) isolates giving the organisms high prevalence in UTI, although regarded as non pathogenic Staphylococcus saprophyticus has 6(3%) isolates (see table 2). The antibiogram types is as shown below. Table 1: Percentage Frequency of Positive culture According to sex Sex samples % freq Nos. of positive cultures Male 162 39.8% 97 (59.9%) Female 245 60.2% 103(42.0%) Table 2: Prevalence of Isolated Bacterial Stains from Urine Samples Organisms Number Percentage(%) of Isolates Staphylococcus aureus 92 46% Staphylococcus saprophyticus 6 3% Staphylococcus pyogenes 3 1.5% Escherichia coli 71 35.5% Klebsiella spp 11 5.5% Pseudomonas aeruginosa 10 5% Proteus mirabilis 7 3.5% 60
AMOXICILLIN AMPLICILLIN CLOXACILLIN AUGMENTIN AMPICLOX FLUCLOXACILLIN GENTAMYCIN STREPTOMYCIN NEOMYCIN CO-TRIMOXAZOLE CHLORAMPHENIC OL TETRACYCLINE LINCOCIN ERYTHROMYCIN AZITHROMYCIN CEPHALEXIN RIFAMPIN CEFUROXIME OFLOXACINE NORELOXCINE CIPROFLOXACIN Mg/disc 10 10 5 10 10 10 10 10 10 25 10 10 5 10 5 10 10 10 2 5 5 Bacteria 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 Strain 1 - - - - - - + - - - - - - - - - - - - - + 0010004 2 - - - - - - - - - - - - - - + - - + - - - 0000440 3 - - - - - - - - - + + + - - - - - - - - - 0007000 4 + - - - - - - - - - - - - - - - - - + - - 1000001 5 - - - - - + - - - - - - - - - + - - - - - 0400010 6 - - - - - - - - - - - - - - - + - - + + + 0000017 7 - - - - - - - - - - - - - - - - - + - + - 0000042 8 - - - + - - - - - - - - - + - - - - - - + 0100204 9 - - - + - - - - - - - - - - - - - - - + + 0100006 10 - - - - - - - - - - + - - - + - - - - - - 0002400 11 - - - - - - - - - - - - + - - - - - - - - 0000100 12 - - - - - - - - - - - - + - - - + - - + - 0000122 13 + - - - - - + + - - - - - - - + - - - + - 1030012 14 - - - - - - - - - - - - - - + - - - - - + 0000404 15 - - - - - - - - - - - - - - - - - - + + + 0000007 16 - - - - - - - - - - - - - + + - - + - - - 0000640 17 - + - - - - - - - - - - - - - - - - - - - 2000000 18 - + - - - - - - - - - - - - - + - - - - - 2000010 19 - - - - - - + - - - - - - - - - - - - - + 0010004 20 - - + - - - - - - - - - - + - - + - - + - 4000222 21 + - - - - - - - - - - - - - - - - - - - - 1000000 22 - - - - - - - - + - - - - - - - - - - - - 0040000 61
23 - - - - + - - - - - - - - - - - - - - - - 0200000 24 - - - - - - - - - - - - + - - + - - - - + 0000114 25 - - - - - - - - - - - - - - - - + - - - - 0000020 26 - - - - - - - - - - - + - - + - - - - - - 0004400 27 - - - - - - + - - - - - - - - - - - - - - 0010000 28 - - - - - - + + - - - - - - - - - - - - - 0030000 29 + - - - - - - - - - - - - - - - - + - + - 1000042 30 - - - - - - - + - - - - - - - + - - - - - 0020010 31 - - - - - - + - - - - - - - - - - - - + - 0010002 32 - - - + - - - - - - - + - - - - - - + - - 0104001 33 - - - - - - - - - - - - + - - - - - - + + 0000106 34 - - - - - - - - - - - - - - - - - - - + - 0000002 35 - - - - - - - - - - - - - - - - - - + - - 0000001 36 - - - - - - - - - - + - - - - - + - - + - 0002022 37 - - - - - - + - - - - - - - + + - - - - - 0010410 38 - - + - - - - - - - - - - - - - + - - - + 4000024 39 - - - - - - - - - - - + - - - - - - - - - 0004000 40 - - - - - - - - - - - - - - - - + - + - - 0000021 41 - - - - - - - - - - - - - - - - - - - - + 0000004 42 - + - - - - - - - - - - - - - - - - - + - 2000002 43 - - - - - - + - - - - + - - - + - - - + - 0014012 44 - - - - - + - - - - - - - - + - - - - + - 0400402 45 - - - - - - - - - - - - - - - - - - + + - 0000003 46 - - - - - - - - - - - - - - - - - - + - + 0000005 47 - - - - - - - - - - - - - - - - + - - - - 0000020 48 - - - - - - - - - - - - - - - - - - - - - 0000000 62
49 - + - - - - - - - + - - + - - - - - - - - 2001100 50 - - - - - + - - - - - - - - - - - - - - - 0400000 51 - - - - - - - + - - - - - - - - - - - - - 0020000 52 - - - - - - - - - - - - - + - - - - - + - 0000202 53 - - - - - - - - - - - - + - - - - - - - - 0000100 54 + - + - - - - - - - - - + - - - - - - - - 5000100 55 - - - - - - - - + - - - - - - - - - + + + 0040007 56 - - - + - - - + - - - - - - - - + - - - - 0120020 57 - - - - - + - - - - + - - - - - - - - + - 0402002 58 - - - - - - - - - - - - - + - - - - - - - 0000200 59 - - - - - - - - - - - - + - - - - - + - + 0000105 60 - - - - - - - - + - - + - - + - - - + - + 0044405 61 - - - - - - - - - - - - - - - - - - - + + 0000006 62 - - - - - + - - - - - + - - - - - - + + + 0404007 63 + - - - - - - - - - - - - - - - - + + + + 1000047 64 - - + - - - - - - - - - - - - - - - + + + 4000007 65 - - - - + - - - - - - - - - - - - - - + + 0200006 66 - - - - - - - - - - - - - - - + - - - - + 0000014 67 + - - + - - + + - - - - - - - - - - + + + 1130007 68 - - - - - - - - + - - - - - - - - + - - - 0040040 69 - - - - - - - - - - - + - - - - - - + - - 0004001 70 - - - - - - + - - - - - - - - - - - + + + 0010007 71 - - - - - - - - - + - - - + - - - - + - + 0001205 Frequency 8.4 5.6 5.6 5.6 2.8. 7.0 12. 8.4 7.0 4.2 5.6 11.3 9.9 9.9 11.3 12.7 11.3 8.5 25.4 36.6 31.0 7 Group cumulative frequency 6.5 5.1 9.4 7.0 10.4 10.8 31.0 63
DISCUSSION Among hospital acquired infections, UTI is the most common [14]. Various reports indicate that Escherichia coli is arguably the most prevalent causes of Urinary tract infection [5, 15]. It is pertinent to add that Escherichia coli has several strains, some of these strains may cause infections that may prove difficult to eradicate with commonly used antibiotics. In this study, a high percentage of positive cultures were obtained from samples taken from both males and females. Though Staphylococcus aureus (46%) had a higher prevalence compared to Escherichia coli (35.5%), confirming previous works[3, 15] and other pathogens were equally isolated. Staphylococcus saprophyticus, a non agglutinating cocci, hitherto, presumed not to be pathogenic [4] was also isolated, suggesting a possible pathogenic nature for the organism when isolated from the genitourinary tract. Interestingly, the pneumonic coding (Ajumali s) employed for this work as described by Joghi et al.,[12], has been able to show a very higher resolving strain differentiation. It is pertinent to note that all 71 isolated Escherichia coli strains have different antibiogram type, making them phenotypically different from one another, even though they are of the same species. This typing method is so specific that it can easily pass off as a phenotypic DNA antibiogram typing method. Though it has its own short coming, in this work, Escherichia coli, Ajumali s strain number 48, may prove a handful for medical personnel saddled with managing UTI caused by it. This is because this strain was totally resistant to the antibiotics employed for the antibiogram typing. Various other strains were also highly resistant, being sensitive to only one or two antibiotics. CONCLUSION This study has shown that an appropriate pneumonic coding can be able to resolve strains of the same microorganisms into their different and specific antibiogram types. This makes it easy for laboratory physician with knowledge of the various strain distribution and differentiation, to tackle multi-drug resistant (MDR) strains effectively and also in record times. Though, an Escherichia coli strain from this study was totally resistant to all the antibiotic used, it is heart warming to note that compared to the over 200 antimicrobial agents known and employed in the management of infections all over the world, the 21 antibiotics used in the course of our study is negligible as this particular MDR strain may be susceptible to other antimicrobial agents not cover in this study. This work is therefore, a base line for further work in this new field of pneumonic coding, using larger number of isolates and antibiotics to establish a broad data base for MDR bacterial pathogens and easy management for such micro-organisms. Acknowledgement The authors appreciate sincerely efforts of miss Nkechi and mr. Omon of Nkechi Computer Centre, Ujoelen, Ekpoma, Edo state, Nigeria for their secretariat assistance and also our staffs who in one way or the other assisted in ensuring the success of this research. 64
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