|Year : 2014 | Volume
| Issue : 1 | Page : 20-27
Mixed flora in the urine of hospitalized and elderly patients: Contamination or True infection?
Trupti Bajpai1, Maneesha Pandey2, Meena Varma3, Ganesh S Bhatambare1
1 Department of Microbiology; Sri Aurobindo Institute of Medical Sciences Medical College and Post Graduate Institute, Indore, Madhya Pradesh, India
2 Department of Biochemistry; Indira Gandhi National Open University, New Delhi, India
3 Sri Aurobindo Institute of Medical Sciences Medical College and Post Graduate Institute, Indore, Madhya Pradesh; Department of Biochemistry, Indira Gandhi National Open University, New Delhi, India
|Date of Web Publication||1-Jul-2014|
Department of Microbiology, Sri Aurobindo Institute of Medical Sciences Medical College and Post Graduate Institute, MR-10 Crossing, Indore-Ujjain Highway, Indore, Madhya Pradesh - 453 111
Source of Support: None, Conflict of Interest: None
Background: Bacteriuria in potentially complex group of patients (those including geriatric population and patients with an anatomically abnormal urinary tract or with significant medical or surgical co morbidities) needs to be managed appropriately. In such clinical settings, polymicrobial bacteriuria is frequently considered remarkable especially due to its adverse effects on the subject. Aim: The aim of the study was to report the common pathogens and to evaluate the clinical significance of polymicrobial bacteriuria (mixed flora) from urine samples of elderly and hospitalized (catheterized) patients and study the drug resistance mechanisms revealed by the isolates from mixed flora and possibly reassessing the current diagnostic standard procedure. Materials and Methods: The present study was conducted in the Department of Microbiology of a teaching tertiary care hospital located in central India, from July 2013 to December 2013. All the uropathogenic isolates were identified up to species level by conventional and automated methods. The samples with mixed flora were picked up as significant based on patient's age, clinical conditions, hospitalization status, pyuria, and significant bacteriuria. Drug resistance mechanisms were detected in all the isolates. Results and Discussion: Urine samples from 1471 male and female, inpatients and outpatients were studied. Six hundred and eight (41.3%) samples were found to be positive with 59 (9.7%) confirmed with mixed flora (two pathogens). Among the 59 samples with mixed flora, 44 patients were grouped as catheterized (18 patients were elderly and catheterized both), one was elderly but not catheterized, eight patients had urological while six had gynaecological problems. Study revealed that Escherichia coli were frequently replaced by other organisms (other Gram negative bacilli, Gram positive cocci, and Candida) in urine samples from male patients. The isolates exhibited considerable ranges of antibiotic resistance. Conclusion: The pathogens present in the urine samples with mixed flora that are frequently dismissed as contaminants may constitute important drug resistant mechanisms. As a result, the patients with the mixed flora in their urine may be left untreated or inadequately treated. Therefore, there is an urgent need to modify the guidelines for urinary tract infection (UTI) diagnosis with respect to high risk patients.
Keywords: Catheter-associated infections, drug resistance, polymicrobial UTI, uropathogens
|How to cite this article:|
Bajpai T, Pandey M, Varma M, Bhatambare GS. Mixed flora in the urine of hospitalized and elderly patients: Contamination or True infection?. Niger J Exp Clin Biosci 2014;2:20-7
|How to cite this URL:|
Bajpai T, Pandey M, Varma M, Bhatambare GS. Mixed flora in the urine of hospitalized and elderly patients: Contamination or True infection?. Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 Jul 18];2:20-7. Available from: http://www.njecbonline.org/text.asp?2014/2/1/20/135722
| Introduction|| |
Urinary tract infection (UTI) is the prevalence of bacteria or yeast in the urine, which is normally sterile.  Such infections occur in the lining of urinary tract, when microorganisms cling at the opening of urethra. Patient usually presents with dysuria, frequency, and urgency along with chills, fever, and lower back pain.  It is one of the most common diseases seen in nephrology practice.  Historically, it is defined by presence of pyuria and the term generally denotes significant bacteriuria isolated from urine specimen that meet acceptable standards for infection. , The current diagnostic standard procedure outlined by the health protection agency (HPA) for UTIs in clinical laboratories does not report bacteria isolated from sample containing multiple bacterial species.  The isolation of more than one microorganism from a single urine specimen; mixed flora is known as polymicrobial bacteriuria and the associated condition is polymicrobial UTI. The frequency with which the isolation of more than one bacterial species from urine signifies treatable mixed infection versus contamination or colonization is not known.  Surprisingly, only few studies have evaluated the clinical significance of polymicrobial growth from urine. Such significance can only be demonstrated if the same combination of microorganisms is recovered from both blood and urine in cases of urosepsis or by reproducibility of the same mixture of organisms from follow-up urine cultures. It can also be considered as significant if the sample is obtained by suprapubic aspiration or by urethral catheterization or if there is significant yield of each isolate in a single sample associated with pyuria. , UTIs are common conditions among aged and debilitated population.  It increases with age but is not considered as a part of the normal ageing process.  Polymicrobial UTIs may pose a heightened threat to the health and well-being of the elderly people. Due to the complexities involved in the diagnosis and treatment in this infection, many patients may receive inadequate antibiotic treatment or indeed a lack of treatment altogether.  Bacteriuria associated with long-term catheterization, a most common nosocomial infection in our medical care facilities is predominantly multi-microbial. Symptomatic UTIs are the common outcome of such bacteriuria and are associated with increased risk for blood-stream infections and increased mortality. , Achieving optimal management of polymicrobial UTIs among hospitalized and elderly populations is problematic since they contain high number of organisms with significant levels of antimicrobial resistance and high levels of invasiveness and are going untreated in clinical settings. ,, Infections caused by single organisms are usually treatable with an antibiotic regimen advised on the basis of antibiotic sensitivity tests (AST). Early species identification and AST of each urinary isolate may be of paramount benefit to the case of these patients.  Due to difficulties in identifying the organisms present in these cultures, clinical microbiology laboratories do not report organisms isolated from urine in mixed cultures unless there is significant count of a predominant organism.  This study was undertaken with the aim of reporting the major pathogens and to evaluate the clinical significance of mixed flora from urine samples of elderly and hospitalized (catheterized) patients. Also, it was aimed to study the drug resistance mechanisms revealed by the isolates from mixed flora and to understand the potential threat posed by microbes present in polymicrobial urine samples of the elderly and hospitalized (catheterized) patients and possibly reassessing the current diagnostic standard procedure.
| Materials and Methods|| |
The present prospective study was performed from July 2013 to December 2013 in the Microbiology Department of a teaching tertiary care hospital located in central India. The study protocol was approved by the ethical committee of our institute. Non-repetitive urine samples (midstream/catheterized/nephrostomy) from a total of 1471 patients were collected in sterile culture containers. Semi-quantitative culture of urine was done by a calibrated loop method  on blood agar, Mac Conkey agar, and UTI chromogenic media.  The culture plates were incubated at 37 o C for 18-24 hours under aerobic conditions. Identification of the bacterial growth was done by standard microbiological and biochemical techniques which were further confirmed by automated Vitek-2 compact (Biomeriux, France) bacterial identification system. , According to the definition, significant bacteriuria may range from > = 10 3 cfu/ml of urine in case of acute, uncomplicated UTI in women to > = 10 5 cfu/ml of urine in case of asymptomatic bacteriuria or complicated UTI in men. The mixed flora was considered significant only after the complete evaluation of clinical symptoms, history of the patient, age, gender, hospitalization status, days of hospital stay, catheterization/instrumentation status, pus cells, and bacterial count.
AST was performed by the Kirby-Bauer disc diffusion method on Muller-Hinton agar.  Dehydrated media and antibiotic discs were procured from Hi-media, Mumbai, India. The control strains used during the study were Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 25923). The selection of antibiotics and interpretation of inhibition zone sizes were done according to Clinical Laboratory Standards Institute (CLSI-2013) guidelines.  The antibiotic resistance pattern of individual isolates was evaluated. These results are not mentioned in the present manuscript but all the possible drug resistance mechanisms of each and every isolate were evaluated.
Phenotypic extended spectrum β-lactamase (ESBL) and Amp C confirmatory tests were also included in the routine susceptibility test.  While performing antibiotic sensitivity testing, ceftazidime plus clavulanic acid (30/10 μg) and cefotaxime plus clavulanic acid (30/10 μg) discs were also included along with ceftazidime (30 μg) and cefotaxime (30 μg) discs on Muller-Hinton agar. Organism was considered as ESBL producer if there was a > = 5 mm. increase in the zone diameter of ceftazidime/clavulanic acid disc and that of ceftazidime disc alone and/or > = 5 mm. increase in the zone diameter of cefotaxime/clavulanic acid disc and that of cefotaxime disc alone. Escherichia coli 25922 and a known in-house ESBL producer were used as negative and positive controls respectively. ,
Phenotypic Amp C screening and confirmatory tests were carried out simultaneously. Since there are no published criteria for phenotypic screening or confirmatory test for Amp C β-lactamases, isolates showing blunting of ceftazidime or cefotaxime zone of inhibition adjacent to cefoxitin disc or showing reduced susceptibility to either of the above test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as "screen positive" for Amp C detection,  whereas cefoxitin-cloxacillin double-disc synergy test was used to confirm Amp C production by the uropathogenic isolates. These isolates were simultaneously confirmed by placing the cefoxitin-cloxacillin (30 μg/200 μg) and cefoxitin (30 μg) at 15 mm apart on Muller-Hinton agar plate pre-inoculated with the test strain and incubated at 37 o C for 18-24 hours. A difference in the cefoxitin-cloxacillin inhibition zones minus the cefoxitin zones alone, of > = 4 mm. was considered indicative for Amp C production. 
Modified Hodge Test was performed simultaneously to detect carbapenemase producing uropathogens. A lawn culture was prepared on Muller Hinton agar by using an overnight culture of Escherichia coli ATCC 25922 adjusted to 0.5 McFarland's standard. The plate was left for drying for at least 15 minutes and then a disc of 10 μg meropenem was applied at the center of the plate. The test isolate was streaked from the edge of the disc to the periphery of the plate. Four isolates were tested per plate. After an overnight incubation at 37 o C under aerobic conditions, the clover leaf like indentation between the test streaks near the disc was considered as positive for carbapenemase production. 
Methicillin Resistant Staphylococcus Aureus (MRSA) detection and Vancomycin Resistant Enterococcus (VRE) detection was done by disc diffusion methods and by Vitek-2 compact automated system. 
| Results|| |
In the present prospective study, a total of 1471 urine samples aseptically collected from male and female, Inpatient department (IPD) and Outpatient patient department (OPD) were studied. Out of these, 608 (41.33%) samples were found to be positive and among them 59 (9.7%) samples had mixed flora (two pathogens in our case). The sex-wise and ward-wise distribution of samples have been mentioned in [Table 1], [Table 2], [Table 3]. The list of uropathogens isolated from male and female samples from IPD and OPD, and the various mechanisms of drug resistance exhibited by those uropathogens have been mentioned in [Table 4] and [Table 5].
|Table 1: Urine samples from male and female patients (IPD & OPD) showing total number of positive|
cultures with single and mixed flora
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|Table 2: Table showing ward-wise distribution of male IPD patients having mixed flora in their urine|
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|Table 3: Table showing ward-wise distribution of female IPD patients having mixed flora in their urine|
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|Table 4: Types of uropathogens isolated from IPD and OPD male patients and the various drug resistance mechanisms shown by them|
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|Table 5: Types of uropathogens isolated from IPD and OPD female patients and the various drug resistance mechanisms shown by them|
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Out of 59 samples with mixed flora, 50 (84.7%) were from IPD and nine (15.2%) were from OPD. Out of 59 samples, 34 (57.6%) were from male patients and 25 (42.3%) from female patients. In case of both male and female patients, the positive samples with mixed flora were recovered in patients above 21-years-of-age (with maximum in the age groups 41-60 years followed by 61-80 years in case of males and in the age groups 61-80 years in case of females followed by 21-40 years).
Out of the 28 IPD male patients, 27 (96.4%) were catheterized and one (3.57%) was non-catheterized. Ten patients were grouped as an elderly and catheterized both. The one who was not catheterized was an elderly patient of more than 80-years-of-age. All the six OPD male patients were non-catheterized but had urological problems. Out of 22 IPD female patients, 17 (77.2%) were catheterized and five (22.7%) were non-catheterized but had gynecological problems. Ten patients were grouped as elderly while eight were grouped as both elderly and catheterized. All the three OPD female patients were non-catheterized but two had urological and one had gynecological problem.
A total of 118 isolates were identified from 59 samples with mixed flora (two pathogens in each). Out of these, 100 (84.7%) isolates were obtained from IPD (56; 56%) males and (44; 44%) from IPD females; 18 isolates (15.7%) were obtained from OPD (12; (66.6%) males and six (33.3%) from OPD female patients. Out of 118 isolates, 88 (74.5%) out of which 48 were from males (38 IPD, 10 OPD) and 40 from females (34 IPD, six OPD) were Gram negative bacilli (GNB), 18 (15.2%) in which 13 were from males (12 IPD, one OPD) and five from females (five IPD, none from OPD) were Gram positive cocci (GPC) and 12 (10.1%) in whichseven from males (six IPD, one OPD) and five (five IPD, none OPD) were Candida species.
Among the 88 (74.5%) GNB isolates, Escherichia coli predominated in female samples (20; 22.7% females) followed by Klebsiella pneumonie (11; 12.5%) and Pseudomonas aeruginosa (8; 9%). Pseudomonas aeruginosa was the most dominating uropathogen among males (17; 19.3%) followed by Escherichia coli and (15; 17%) Klebsiella pneumonie (11; 12.5%). Five (5.5%) unusual Gram negative isolates were also detected in male patients as compared to only one (1.13%) isolate in females. Among the 18 (15.25%) Gram positive isolates, one (5.2%) was Coagulase negative Staphylococcus (CoNS) isolated from IPD male patient while 17 (94.4%) were Enterococcus fecalis (12; 66.6% from males and five; 27.7% from females).
Among the 34 urine sample from males, 10 (29.4%) samples had at least one isolate with one of the mechanisms of drug resistance (ESBL, AmpC, Methicillin resistance, VRE or carbapenemase production) while in six (17.6%) samples, both the isolates exhibited at least one mechanism of drug resistance. Among the 25 female urine samples, seven (28%) samples had at least one drug resistance mechanism in one of the two isolates while six (24%) samples had both the isolates exhibiting one or more mechanisms of drug resistance.
| Discussion|| |
In the present study, more male patients were suspected of UTI as compared to female patients but there was no remarkable difference in the number of positive cultures in both the sexes. In the present study, 59 (9.7%) samples were found to bear a mixed flora (two pathogens in our case). Our results were consistent with those of Nicolle et al., (10-25%) and Akkoyun et al., (11.8%) but was different from that of Mc Cue (16%). ,,,
Among the 59 samples revealing mixed flora, comparatively larger number which is 50 (84.7%) were from IPD as compared to nine (15.2%) OPD samples. Also, 34 (57.6%) samples were from male as compared to 25 (42.3%) female patients. These results can be attributed to the fact that there are more chances of getting mixed flora in hospitalized male patients. In our case, the group of patients that were detected to bear mixed culture can be either categorized as elderly (49.1%), catheterized (74.5%), elderly and catheterized both (30.5%), patients with urological (13.5%) and gynecological problems (10.1%). Several studies have confirmed that mixed flora are more pronounced in elderly and hospitalized patients often in association with long-term catheterization. ,,,,
Complicated UTIs are defined as those occurring in a functionally or anatomically abnormal urinary tract or caused by bacteria resistant to antibiotics. Basically, complicated UTIs occur in anyone who is not young, healthy, and non-gravid.  Multiple factors contribute to the high frequency of UTI in elderly patients. In both men and women, the post voiding residual volume increases with age. This is partly due to changes in pelvic musculature with age, as well as bladder function in women and prostatic hypertrophy in men. It is exacerbated in some cases by dementia, neuro-muscular disorders such as Alzheimer's disease, Parkinson's disease, and cardiovascular disease that are usually accompanied by a neurogenic bladder. Elderly men and women with history of diabetes, sickle cell anemia, HIV or other disorder that weakens the immune system, abuse of analgesics, constant use of anti-cholinergic drugs, those who are bed ridden and have incontinence of urine and bowel, more debilitated state with perineal soiling, less complete bladder emptying, bladder catheterization, and more often antibiotic prescription. It is well known fact that hospitalization may lead to dissemination of bacteria by means of instruments, bed pans and personnel are again at risk of complicated UTI. ,,, Diabetes mellitus, a common medical condition among the elderly patients is associated with mechanical and metabolic disturbances such as poor control of blood glucose, diabetic neuropathy with neurogenic bladder and chronic urine retention, more frequent catheterization and instrumentation of the urinary tract as well as diabetic micro- and macro-angiopathy. Conversely, UTIs can more often lead to the diabetic complication such as ketoacidosis or it is also suggested that in presence of bladder wall ischemia that is due to obstruction or atonia, bacteria can penetrate the bladder-lining that is usually resistant to microbial entry. 
Hormonally associated changes in the vaginal flora during and especially after menopause are the important factors in the pathogenesis of UTI in older women. In post menopausal women, the attenuated ovarian function leads to estrogen deficiency, thereby causing the disappearance of Lactobacilli, increased vaginal pH and augmented colonization of the vagina by more pathogenic flora from gastrointestinal tract. The existence of such colonization by enteric bacteria can be partially responsible for UTIs in older women. ,,, The high prevalence of UTI in postmenopausal women can also be attributed to risk factors such as women with parity, lower education, poor health, vaginal itching, vaginal dryness, diabetes requiring treatment, recurrent vulvo-vaginitis in women with diabetes, urge incontinence and history of UTI at younger ages. Recurrent and symptomatic UTI in such women are associated with incontinence, elevated post voiding residual urine, history of prior gynecological surgery and presence of cystocele. ,,,
Loss of prostatic bactericidal secretion during senescence or after prostectomy play an important role in increased incidence of UTIs among older men. Benign prostatic hyperplasia in older man causes compression of urethra and leads to bladder outlet obstruction. As a result, there is an incomplete emptying of the bladder, leading to urinary stasis thereby predisposing man to bladder infection. Bacterial prostatitis, once established is difficult to eradicate and can serve as a source for recurrent UTI over many years. There is a two-fold increase in incidence of UTI if incontinence is managed by means of external condom catheters in such cases. Even twisting and kinking of the condom or tubing and catheter manipulation are associated with an increased chance of infection. ,,
Nosocomial UTI account for 40% hospital acquired infections and are the most common nosocomial infections. Approximately, 80% of these infections are catheter-associated or occurs after other type of urologic instrumentation.  Catheter-related UTI occurs because catheters inoculate organism into the bladder and promote colonization by providing a surface for bacterial adhesions and causing mucosal irritation.  Comparison between monomicrobial and polymicrobial infection episodes shows that later are more frequently associated with urinary catheters.  Risk factors for bacteriuria in patients who are catheterized include longer duration of catheterization, colonization of the drainage bag, diarrhea, diabetes, absence of antibiotics, female gender, renal insufficiency, errors in catheter care, catheterization late in the hospital course and immune-compromised or debilitated states. 
Most elderly patients are categorized into the complicated UTI group. However, if the patient is older than 65 years it presents the only risk factor, the microbial flora differ only slightly from those in young women but in case of complicated UTI (as in hospitalized/hospitalized elderly in our case), the pathogen changes more markedly and the predominant Escherichia coli value falls from 70% in uncomplicated to 40% in complicated cases. , Our results, in case of male urine samples, clearly show that Escherichia coli have been replaced by highly resistant Pseudomonas. However, the predominant organism in case of female polymicrobial sample was Escherichia coli in our case which concurs with general consensus among published data concerning monomicrobial culture of UTI (Farajinia et al., Tal et al., and Brzuszkiewicz et al.). In our study more male patients were catheterized and hence were found to be infected with pathogenic isolates inhabiting hospital environment. Also, many more bacteria other than Escherichia coli inhabit hospital setting, they are frequently replaced by those nosocomial pathogens. Indwelling urinary catheter is used frequently in older population. Infection due to Escherichia coli predominates again in this case, but a wide variety of other organisms including yeast species are recovered. ,,,,, In our case, Candida was recovered in 9.3% cases with slightly higher percentage among male patients. This was again much higher as compared to study by Naber (0.25%) and much lesser than those of Akkoyun et al., (23%). , Pseudomonas is the most common hospital pathogens associated with polymicrobial than monomicrobial infections. They are also associated with biofilm growth in catheters. It was found to be higher in our case in males (17; 14.4%) as compared to females (8; 6.7%) attributing to more male catheterized patients as studied in our case. The percentage prevalence was much higher than the study by Akkoyun et al., (7.6%) The percentage prevalence of Klebsiella was also high in our case (22; 18.6%) that was much higher than the study by Akkoyun (6.8%).  A list of Gram negative isolate in our setting includes unusual pathogens like Morganella (2; 1.69%), Providencia (1; 0.84%) and Acinetobacter (1; 0.84%) prevalence of which were comparable with the studies by Naber which were 0.25%, 0.25%, and 1.5% respectively.  However, a study by Akkoun detected Acinetobacter on the higher side (4.2%).  Providencia has a unique hospital predilection and studies say that Providencia and Morganella are frequently isolated uropathogens in long term catheterized patients with polymicrobial bacteriuria, and in our case it was detected in an elderly and catheterized male patient. Though, Proteus is isolated in several clinical settings, it was not detected in our study.  Among the various Gram positive isolates, Enterococci predominated with a greater percentage (17 of 118 isolates; 14%) in the male patients and the results resembled almost those of Akkoyun et al., (11%) and Naber (16%). ,, Enterococcus, the third most commonly isolated organism (17; 14.4%) (almost equal to Pseudomonas) is known to be significantly more prevalent in polymicrobial culture than monomicrobial culture. Enterococcus was the second most prevalent in the study made by Croxall et al.,  Enterococcus were found in 17 (28.8%) samples out of which Escherichia coli and Enterococcus coinfection was found in 7 (11.8%) out of 59 samples. Coinfection of Enterococcus with other pathogens pose an important question for the antibiotic treatment of polymicrobial UTI as Enterococcus is known to be intrinsically resistant to many antibiotics including several first choice antibiotics to treat UTI. One of the Enterococcus isolate in our setting was also detected as VRE. Infection with VRE is significant issues for nosocomial infection control because they are associated with high patient-care cost and an increased mortality rate.  At the same time, coinfection of Enterococcus fecalis/Enterococcus faecium and uropathogenic Escherichia coli not only create difficulties in devising an antibiotic treatment regimen but also may exacerbate the pathogenicity of Escherichia coli (Lavigne et al., Montrantus et al.) Prescription of ineffective antibacterial agents can increase the selection pressure for antibiotic resistant agents within an infection. Due to diversity of pathogens present in pairs which may include coinfection by heterogenous group of pathogens such as GNB-GNB, GNB-GPC, GPC-GPC, GNB-Candida or GPC-Candida, the cases would remain untreated or incompletely treated if left undiagnosed under the current guidelines for UTI diagnosis.  Studies also highlight that Escherichia coli present in polymicrobial UTI samples are more invasive than those isolated from monomicrobial culture samples, thereby posing an increased risk to human health. Studies also prove that significant differences exist in susceptibility of bacteria in polymicrobial v/s monomicrobial cultures. ,, As it was evident from our data regarding various types of drug resistance mechanisms exhibited by the various urinary isolates of the mixed flora. Mortality is usually high in polymicrobial infection and is further increased if multiple organisms grow from blood rather than from urine.
Based on our study, we believe that, in properly collected urine samples, multiple growths can represent true mixed infection and should therefore be completely evaluated. Our study stresses over the proper evaluation of mixed flora from the urine samples of risk group patients before reporting them as contaminated samples. The pathogens present in the urine samples with mixed flora that are frequently dismissed as contaminants may constitute important drug resistant mechanisms. As a result, the patients with the mixed flora in their urine may be left untreated or inadequately treated. Therefore, there is an urgent need to modify the guidelines for UTI diagnosis with respect to high risk patients. Infections caused by variety of polyresistant microbiological agents are a challenge for medical community. Most of these are either acquired in hospital settings during instrumental procedures. The most important factor in the reduction of UTIs and sepsis is a reduction in use of the catheter and to be more careful in case of elderly male patients. The clinicians must be certain that the catheter inserted is absolutely necessary and should attempt to have the catheter removed as soon as possible. ,,
| Acknowledgement|| |
The authors wish to thank the Chairperson and Dean of the institute for providing laboratory facilities and healthy working atmosphere during the study period. The authors are also thankful to the technical staff of the institute for providing necessary helping hand during the endeavour.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]