1Research and Development Centre, Bharathiar University, Coimbatore 641046. Tamil Nadu, India, 2Assistant Professor and Head, Department of Microbiology, NKR Government Arts College for Women, Namakkal 637001. Tamil Nadu, India.
Email: muralee1931@gmail.com
Received: 13 Sep 2014 Revised and Accepted: 10 Oct 2014
ABSTRACT
Objective: Urinary tract infection (UTI) is the most common serious infection during infancy, adult male and female, as well as at the time of pregnancy. UTI is also the most common nosocomial infection in many hospitals and accounts for approximately 35% of all hospital acquired infections. Hence the present study was aimed to screen the presence of multi-drug resistant bacterial pathogens among the urine samples collected from in and out patients of multi-speciality hospital.
Methods: Standard microbiological laboratory protocols were followed and about 152 samples were processed and screened. Among them, 49 reported positive for the presence of urinary bacterial pathogens.
Results: Among 49 isolates, Escherichia coli registered its prevalence in about 44 samples followed by Klebsiella spp. (4) and Pseudomones spp. (1). The gender wise distribution was found to be more among female patients (42%) than male patients (24%). The age wise distribution of infection among male and female patients was also noticed. Further, the multi-drug resistance of the isolates was done by using 8 antibiotics.
Conclusion: All the isolates exhibited the multiple antibiotic resistance and the isolates showed 27 different antibiotic resistance patterns. This confirms the prevalence of ESBL producers among the urinary pathogens.
Keywords: Uropathogens, E. coli, ESBL, Hospital acquired infection.
INTRODUCTION
Urinary tract infection (UTI) is projected to be about 150 million incidences per annum worldwide [1]. More than 8 million patients with urinary tract infections visit urology or gynecology clinics per year in the United States [3]. UTIs develop by either ascending or descending bacterial invasion into the urinary tract. The most common mode of infection is the ascending pathway, where fecal flora gain access to the urinary tract via colonization of the urethra [2]. Descending infections are the result of hematogenous spread of bacteria from a primary source located elsewhere in the body and it’s rare. The lower urinary tract infection is known as a simple cystitis (Bladder infection) and the infection in the upper urinary tract or in kidney is known as pyelonephrites [16].
UTI is one of the most recurrent infectious diseases among hospital-acquired group [8]. It also reported to occur by community acquired group. The major causal agents of urinary tract infections by bacterial pathogens were found to be Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens, and Pseudomonas aeruginosa [11].
Depending on whether the infection is occurring for the first time or a repeated event, the UTI is classified into two types, uncomplicated and complicated infections. Uncomplicated UTI occurs due to bacterial infection, most often by E. coli. Women are frequently affected by uncomplicated UTI than men [26]. Complicated infections, which occur in men and women of any age, are also caused by bacteria, but they tend to be more severe and more difficult to treat [22].
In order to achieve a satisfactory therapeutic effect, it is suggested that local information regarding the antimicrobial resistance of frequent pathogens should be established as a reference for the selection of empirical antimicrobial therapy [10]. Higher multidrug resistance rates were found to be common among hospital acquired UTI pathogens [7]. β-lactam antimicrobial agents are among the most widely used antibiotics to treat those communities and hospital acquired infections [14].
Few authors have studied the risk factors associated with the UTI due to ESBL-producing bacteria in hospitalized patients. Extended-spectrum β-lactamases (ESBLs) are a group of β-lactamases enzymes belongs to group 2, produced by Gram negative Enterobacteriaceae [4]. Due to rapid emergence of ESBL producing uropathogens over the last decade, the antimicrobial susceptibility profile has changed dramatically [19, 28]. Hence, the aim of the present study was to determine the prevalence and antibiotic resistant patterns of ESBL-producing bacterial uropathogens isolated from urine sample of both in and out patients in multi-specialty hospitals around Coimbatore.
MATERIALS AND METHODS
Isolation and identification of uropathogenic bacteria
Collection of samples
With prior ethical clearance from the hospitals for this study, a total of 152 urine samples from multi-specialty hospitals in and around Coimbatore were collected for the isolation of uropathogenic bacteria. One millilitre of the sample was inoculated in Luria Bertani (LB) broth and incubated at 37°C for overnight. After incubation for about 24 h, one loopful of the culture was streaked on selective agar medium. The selective agar medium those were used for the isolation of enteric pathogens is tabulated (Table 1). Further, the isolates were identified by Gram’s staining and series of biochemical tests.
Table 1: Selective medium for isolation of pathogens
| Selective media | Bacterial genera |
| Eosine Methylene Blue agar (EMB) | E. coli |
| Mac Conkey Agar | Klebsiella spp. |
| Nutrient Agar (NA) | Proteus spp. |
| King’s B medium | Pseudomonas spp. |
Maintenance of uropathogens
Nutrient agar slants were prepared in the test tubes and pure culture of the uropathogen grown on the selective medium were streaked on it. The tubes were incubated at 37oC for 24 h and refrigerated for preservation. The cultures inoculated on to the Luria Bertani broth tubes were used for further use.
Antibiotic susceptibility testing
Antibiotic sensitivity was tested for all the 49 isolates. The antibiotics that are commonly used for the infection control were selected for the present study.
Test procedure
Antibiotic sensitivity test was carried out by disc diffusion method. Muller Hinton agar (HiMedia, India) were prepared, sterilized and poured onto sterile petriplates. The medium was allowed to solidify. Pure cultures grown in nutrient broth for 6-8 h were swabbed over MHA using sterile cotton swabs. Using antibiotic disc dispenser, discs were placed on the agar surface with sufficient space so as to avoid overlapping of inhibition zones. After 30 min of pre-diffusion time, the plates were incubated at 37oC for 18-24 h. After the incubation period, the diameter of the inhibition zone was measured and compared with the interpretative chart provided by the manufacturer and classified as resistant, intermediate and sensitive.
Multiple antibiotic resistance index
Multiple antibiotic resistance (MAR) index of an isolate is the number of antibiotics to which the test isolate displayed resistance divided by the total number of antibiotics to which the test organism has been evaluated for sensitivity. MAR index value higher than 0.2 is considered that the isolate have originated from high risk source of contamination like human, commercial poultry farms, swine and dairy cattle where antibiotics are very often used. MAR index value less than or equal to 0.2 is considered that the isolate have originated from animals in which antibiotics are used very rarely or never used.
RESULTS
Isolation and identification of uropathogenic bacteria
The urine samples were collected from in and out patient in multispecialty hospitals around Coimbatore from both genders irrespective of age group. Among 152 samples, 133 samples were from in patients and 19 from out patients. The isolates were subjected to gram’s staining and streaked on to selective medium. As none of the isolates were found to be gram positive, the screening of gram positive bacteria on selective medium was neglected. A total of 44 isolates exhibited metallic sheen colonies on to EMB agar plates which confirms the presence of E. coli in the samples, 4 isolates showed pink colour colonies on Mac Conkey agar confirms the presence of Klebsiella spp. And one isolate which produced pyocyanin was further confirmed the King’s B medium confirms the presence of Pseudomonas spp. None of the isolates exhibited swarming motility on the nutrient agar plates, which shows the absence of Proteus spp. Among the samples (Table 2). The isolates obtained from selective medium were further confirmed based on their biochemical profile. The typical biochemical profile of E. coli, Klebsiella spp. And Pseudomonas spp. Were tabulated (Table 3).
Table 2: Percentage of uropathogenic bacteria
S. No. |
Positive samples |
Organism isolated |
Total no. of |
Percentage % |
|
Male |
Female |
||||
1. |
16 |
28 |
E. coli |
44 |
29% |
2. |
2 |
2 |
Klebsiella spp. |
4 |
3% |
3. |
1 |
- |
Pseudomonas spp. |
1 |
0.7% |
Table 3: Typical biochemical profile of E. coli, Klebsiella spp. And Pseudomonas spp
| Gram’s staining | Indole | MR | VP | Urease | TSI | Catalase | Glucose | Lactose | Maltose | Sucrose | motility | Oxidase | Suspected organism |
| G-ve Rod | - | - | + | - | + | + | - | + | - | - | + | - | E. coli |
| G-ve Rod | - | + | - | + | + | + | + | + | + | + | - | - | Klebsiella sp. |
| G-ve Rod | - | - | - | + | + | + | + | - | + | + | + | + | Pseudomonas sp. |
(-) Negative (+) Positive
Table 4: Demograhic data of UTI among patients
| Category | No. of samples | Male | Female | No. of Positive samples | Percentage
% |
| IP | 133 | 73 | 60 | 40 | 30% |
| OP | 19 | 8 | 11 | 9 | 47% |
Table 5: Gender wise distribution of uropathogenic bacteria in UTI samples
| Gender | Total no. of samples | No. of Positive samples | Percentage % |
| Male | 81 | 19 | 24% |
| Female | 71 | 30 | 42% |
Prevalence of uropathogenic bacteria
The percentages of prevalence of uropathogenic isolates were screened. The prevalence was found to be high among out patients (47%) whereas 30% of incidence was noticed among the in patients (Table 4). The gender wise incidence was noticed among the patients.
The highest percentage was noticed among female patients (42%) than male patients (24%), the results are tabulated (Table 5). The age wise prevalence of the pathogens had also registered. The highest prevalence of the UTI was found among the age of 60-69 years of male patients (33%) and 100% of samples from female patients of the age 40-79 showed positive for the presence of bacterial pathogens.
Antibiogram of the isolates
All the 49 isolates obtained were screened for the antibiotic sensitivity against 8 commercially available antibiotics. The highest percentage (100%) of resistance was found against cefotoxime and ceftazidime and least percentage of resistance was found against amikacin (14%). About 98% of isolates showed resistance against cefazolin and cefixime, 96% resistance towards vancomycin, 88% against nalidixic acid and 43% towards gentamicin, respectively. The isolates exhibited about 27 different antibiotic resistance patterns against 8 antibiotics used in this study. Klebsiella spp. and Pseudomona spp. shares the same antibiotic resistance pattern shown by E. coli. This shows that these organisms acquiesced the resistance through transferable elements like ‘R’ plasmids as these were closely related in their phylogeny.
Multiple antibiotic resistance index
The MAR index value of all the isolates ranged from 0.5 to 1. MAR index of 0.91 to 1 and 0.80 to 0.89 was exhibited by 0.04% and 55% of the isolates. MAR index of 0.70 to 0.79 and 0.60 to 0.69 was exhibited by 35% and 0.04% of the isolates respectively. About, 0.02% isolates exhibited a MAR index value of 0.50 to 0.59. None of the isolates exhibited MAR index less than 0.5. The results serves as an evident that all the isolates were procured from the area were antibiotics are intensively used.
DISCUSSION
Various bacterial pathogens were reported to be involved in UTI. Many reporters had marked their conclusion that E. coli and Klebsiella spp. were found to be predominant in causing the UTI among both hospital-acquired and community-acquired patients. There are many evidences that these pathogens harbour resistance towards multiple antibiotics by producing varying β-lactamases. Hence the present study was conducted to screen the epidemiology of various bacterial pathogens in the UTI and to determine their antibiotic resistance.
Tankhiwale et al. [29] screened a total 217 uropathogens and found that E. coli (49.8%) was the most common organisms followed by Klebsiella pneumoniae (37.8%), Pseudomonas aeruginosa (6.5%). This report strongly supports the present study, as E. coli (29%) was found to be more prevail among the patients irrespective of sex and gender followed by Klebsiella spp. (3%) and 0.7% of Pseudomonas spp. This result also strongly coincides with Jain et al. [12].
A large spectrum of organisms has been reported by Jain et al. [13] from patients of UTI with E. coli (65.95 %) and Klebsiella spp. (12.41 %) being the most common in their study. Hasan et al. [9] has reported a high incidence of E. coli (50.7%) followed by Klebsiella spp. (27.6%) in 2436 uropathogens from patients with UTI.
It is stated that UTI is predominantly a disease of the females due to a short urethra and proximity to the anal opening. The present study also marks its concordance for the early reports as the UTI prevalence was found to be more among female (42%) whereas, in male it’s about 24%. In a study, female preponderance for this infection was noted by Jain et al. [13]. They had also reported that the E. coli and Klebsiella spp. Were found to be the common isolates in females. This gives the strong evidence for the present study since, Pseudomonas spp. Was found only from male patient.
Jain et al. [12] found that 17.3% in females patients with UTI whereas 13.3% in males. In coherence with prior studies, a higher prevalence of UTI among female was noted owing to the presence of anatomical and physical factors showing their increased vulnerability towards UTI was confirmed. The occurrence of UTI predominantly in patients with age ranging between 18-60 years in our study is similar to the findings of studies done in Kuwait and Nigeria [6, 23]. Women in the age group 16-45 years are most likely to suffer from UTI. Among the elderly, after 45 years males became more prone to UTI. The number of male patients increased significantly in the age group 46 years as prostatic gland enlargement and decrease of bacteriostatic prostatic secretions might account for such infections [30]. But in the present study evident for the occurrence was high at the age 40-49 among males and 40-79 among females.
As other several reports, our study has also revealed that E. coli is the most predominant pathogen causing UTI in both sexes. On contrary to the findings of various other previous studies which documented Staphylococcus spp. as the second most common isolates [18,21], our results showed Klebsiella spp. Which is in concurrence with the findings of various authors [1,17,27].
Resistance to several antimicrobial agents was prevalent among the isolates recovered not alone from the hospital and also found in environmental sources. These reports were noticed in many of the research works done world-wide. ESBL-producing Enterobacteriaceae is now evident in the community, especially among adults and is no longer confined only to the healthcare environment. Qi et al. [24] described an increase of over 10-fold in the community acquired ESBL E. coli incidence among the general population in Chicago, Illinois over a period of only 5 years while screening for urinary pathogens. In the current study, the incidence of uropathogen was noticed high among out patients (47%) than in patients (30%). This might be resulted due to age factor and risk of exposure of a patient.
In the 1970s, multidrug resistance was practically nonexistent and the cause was restricted to mutation of chromosomal genes. However, during the last two decades bacterial resistance mediated by plasmids, which carry resistant gene to a large number of antibiotics, which are rapidly transferred, has worsened the scenario [25]. Due to high levels of resistance found to first choice drugs, it is important to know the resistance aspects of the pathogens in order to help in use of appropriate therapy or else, the empirical therapy became a difficult clinical decision.
In the present study, all most all the isolates showed multidrug resistance. The least percentage of resistance (7%) was noticed against amikacin. Klebsiella spp. and Pseudomonas spp. Were found to be sensitive towards amikacin. Only E. coli was found to be resistant to amikacin. This result coincides with the result of Khan et al. [15] who recorded 7.1% of E. coli was resistant against amikacin. About 98% of isolates showed resistance against cefazolin in the present study. Moraes et al. [20] reported that 99% of E. coli was found to be resistant towards cefazolin. But Wu et al. [31] reported that cefazolin can be used as an alternate drug for treating urinary pathogens. This difference in susceptibility might be resulted due to non-exposure of the test isolates used by Wu and their co-workers.
The resistance against cefixime and cefotoxime was found to be 90%, 84.3%, 80% and 84.3% according to the reports of Jain et al. [13] and Noor et al. [30], whereas in the current study it was noticed as 98% and 100%. Jain et al. [12] reported that 57% of isolates were found to be resistance towards gentamicin. Dayan et al. [5] recorded 50% of resistance towards gentamycin. The result of the current study also more or less coincides with the early reports since, the resistance against gentamicin was found to be 43% in our study.
About 100% of isolates showed resistance towards ceftazidime in the present study, Jain et al. [13] noticed 82% of resistance. Against nalidixic acid and vancomycin, the test isolates showed 88% and 96% of resistance. This strongly coincides with the report of Jain et al. [13] and Noor et al. [30] as they reported that their 94% and 82.9% of test isolates showed resistance towards nalidixic acid. In contray, Jain et al. [12] has reported only 12% of their test isolates were resistant to vancomycin. These variations in the antibiotic resistance may occur due to the frequency of usage of particular antibiotics.
CONCLUSION
From the present study, we can conclude that E. coli was found to be most predominant bacterial uropathogen in UTI cases of the study area. The results of our study give brief information about the multidrug resistance, and also the prevalence of ESBL producing bacteria among the patients reported having community acquired UTI. Further, we also conclude that amikacin can be used in the empirical antibiotic therapy for achieving the better treatment.
CONFLICT OF INTERESTS
Declared None
REFERENCES