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Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 02 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.702.415 Prevalence of Urinary Tract Infection Causing Microorganism and Determination of Susceptibility to Antibiotic among Slum Women of District Varanasi, India Bharti Singh1, Deepmala Katiyar2*, Ragini Tilak1, Ratan Kumar Srivastava3 and Rajveer Singh Chauhan4 1 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India 2 Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India 3 Department of Community Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India 4 VitaeGen Biotech- Educational and Research Institute (Run by RED Society), Varanasi, India *Corresponding author ABSTRACT Keywords Urinary Tract Infection (UTI), E. coli, Slums, Varanasi Article Info Accepted: 28 January 2018 Available Online: 10 February 2018 Urinary tract infections (UTIs) are among the most common bacterial infections. Enteric bacteria predominantly Escherichia coli) remain the most frequent cause of UTIs, although the distribution of pathogens that cause UTIs is changing. The study was based on primary survey to five different slums (Ravindrapuri extension, Nagwan, Bajardeehan, Batuapura and Ghasiyari Tola) of Varanasi city. This study is an attempt to determine the UTI status in slums of Varanasi city. During Questionnaire session we found that only 6% women were aware of UTI. 398 samples from slums were cultured on CLED, HI chrome, MSA, and preceded for biochemical identification followed by antimicrobial susceptibility tests using first line drug. 183 episodes of UTI were observed out of 398.We found 48%, 45%, 49%, 42%, 35.4% women suffered from UTI in Ravindrapuri Extension, Nagwan, Bajardeehan, Batuapura and Ghasiyari Tola slums respectively. Highest number of gram negative E.coli and gram positive S. aureus found in all slums. More important is the increase in resistance to some antimicrobial agents, mainly the resistance to ampicillin, cefozolin, gentamicin, ofloxacin seen in E. coli. Introduction Urinary tract infection (UTI) is the second most common infectious presentation in community medical practice. Worldwide, about 150 million people are diagnosed with UTI each year, and UTI are classified as uncomplicated or complicated (Stamm and Norrby, 2001). UTI is one of the most frequently encountered bacterial infections in infants and children, and the most common bacterial infection in infants under three months of age (Stanley, 2005). Approximately 50% of women will suffer from at least once urinary tract infection (UTI) during their adult life. According Standing Medical Advisory 3483 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Sub-group on Antimicrobial Resistance (1998), UTIs account for around 5% of consultations in General Practice and are the second commonest infection after respiratory infections. 15% of community use of antibiotics is for UTIs. A few women will suffer significant morbidity from the effects of pyelonephritis, an infection of the kidney substance, usually caused by infection ascending from the bladder. Most of those suffering morbidity will have underlying causator abnormalities. 25% of those who have had one infection will have at least one further infection, and sometimes, multiple recurrences (Stapleton, 1999). More than 95% of urinary tract infections are caused by a single bacterial species. E. coli is the most frequent infecting organism in acute infection (Ronald, 2002). Though urbanisation is an indicator of development, but it is also bitter fact that poverty is growing faster in urban areas than in rural areas. Being a complex socioeconomic process closely connected with scientific technological revolution, urbanisation exercises a growing influence on all aspects of society, reflecting the nature of economic and regional development (Rao and Padmanabha, 1999). Around the world, one billion people live in urban slums, which are typically overcrowded, polluted and lack basic services. One of the targets set by world leaders in 2000 was to improve significantly the lives of at least 100 million slum dwellers by 2020. The situation is worst in developing countries like India. Varanasi, one of the oldest living city of the world, also faces the challenges of rapid urbanisation. The slums of Varanasi portray a pathetic picture of life in slums (Tripathi, 2011). Total wards in Varanasi Metropolitan Corporation are 106, amongst these wards the slum dwells only in 61 wards. The population of urban poor and slum dwellers in Varanasi accounts for about 37.69% of the city population. Varanasi has 227 slums spread all over the city, either on government or private lands. About 50% of slums are located in central city, 13% in old city while 37% in peripheral areas. The average density in slum areas is approximately 330 persons per hectare in the city area (Jha and Tripathi, 2014). The study was attempted to explore UTI status and occurrence of bacterial strain of slum dwellers and suggest some measures of inclusive development and provide vision to improve quality of life in slums by reducing incidence of UTI. Materials and Methods The study area Varanasi (Kashi or Banaras) is one of the oldest living city in the world, with a continuous history dating back 3,000 to 5,000 years. It is known as cultural and spiritual capital of India. Varanasi (82°56’E – 83°03’E and 25°14’N) is located on the left crescent-shaped bank of the Ganga river. Slums in Varanasi city has been continuously increasing from 1941 to 2017. With the increase in population of the city, housing needs of the city also grew, which could not be met out by the formal housing market. Migrating population, which could not avail the facilities of formal housing market, satisfied their needs by occupying vacant land and this has resulted in formation of slums. Another factor, which contributes in the formation of slums, is proximity to work place and low level of income and un-affordability of houses People living in the slums of Varanasi city are poor working as labour and rickshaw pullers. Selected slums Ravindrapuri extension, Nagwan, Bajardeehan, Batuapura and Ghasiyari Tola Survey The following tool was used for survey. Questionnaire prepared in the local language provided to women. The questions were 3484 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 mainly dichotomous items with Yes OR No choices and avoided personal, complicated and misleading questions. The questions were direct and brief. It included: personal data, age, marital status, occupation, previous history of UTI, risk factors, hygiene habits, the antibiotics use. Characteristics manifestation developing colonies Diagnosis Microscopic characteristics developing colonies of the Observed qualities manifestation of the colonies developing on culture media form color, the surface of the colony, strength, transparency, smell, on HiChrome, MSA. of the Collection of Samples 867samples of urine were collected from 4 villages (Neguran, Mustafapur, Bindpurvan & Gauri) and 5 Slums Ravindrapuri extension, Nagwan, Bajardeehan, Batuapura, and Ghasiyari Tola. Brief history of patients was obtained about frequency of micturation, retention of urine, burning micturation, fever and chills. The samples were collected in pre-sterile, dry, wide-mouthed and leak proof universal plastic containers. Samples were immediately transported to the laboratory where they were processed promptly. Isolation of bacteria 1 ul (.001 ml) wire loop was sterilized and dipped into a urine sample. The loop was then streaked on the plate of cysteine lactose electrolyte deficient (CLED) Agar. The plates were incubated overnight at 37°C ċ for 24 hours. Following the appearance of growth, well-isolated bacterial colonies were processed biochemical and for antibiotic sensitivity tests. The number of colonies on CLED plate was counted and viable count of bacteria was reported for each specimen Identification of bacterial isolates Culture media were prepare for the purpose making biochemical test of the bacterial isolates and studied following the completion of diagnosis: Swabs were the work of the pure colonies segments of slides the colored character gram stain observed forms of cells, arranged, responding to gram stain Biochemical Tests Tests conducted a production of the enzyme Catalase, Oxidase, Coagulase as stated in Motility test, Urease, Indol, H2S Production, Citrate utilization, Sugar fermentation. Sensitivity to antibiotics From the use of first line drug of antibiotics are: determining sensitive and resistant bacteria to antibiotics by measuring the diameter of inhibition zone by mm and then compared with the standard diameters that installed in the standard scales. Results and Discussion A close ended questionnaire for the symptomatic women were designed and translated into local language to prevent misunderstanding. The questions were mainly dichotomous items with Yes OR No choices and avoided personal, complicated and misleading questions. The questions were direct and brief. It included: personal data, age, marital status, occupation, previous history of UTI, risk factors, hygiene habits, the antibiotics use. 3485 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Ravindrapuri extension slum On the basis of questionnaire, 110 samples of highly symptomatic women were then collected from Ravindrapuri extension slum. Samples were cultured in laboratory and then organized in different age groups. The result showed that the overall prevalence of UTIs in both age groups was 48%. In which, occurrence of UTI in 15-30 age group was found upto 64% while 31-45 age group showed 35.8 %. Thus, 15-30 age group show increased risk of infection than 31-45 age group females. Women of 15-30 years age group were reported to have two folds increase UTI as compared to the 31-45 age groups (Table 1). Almost all of the cases of UTIs were due to the gram negative bacteria with no prominent age group difference. The percentage of gram negative bacteria was 52.8%. The percentage of Lactose fermenting bacteria was 78.5%. Lactose fermenting (78.5%) bacteria indicated predominant frequency in both age groups. Non lactose fermenting bacteria was observed 21.4%. The age group 15-30 years females demonstrated about two folds increase of NLF than 31-45 age group females (Table 1). The most frequent causative agents of UTIs in the study of 15-30 age group reported as E. coli (20.58%) and Klebsiella (14.7%) followed by Pseudomonas (11.7%) and Acinetobacter (2.9%). Similar pattern also was observed in 31-45 age group E. coli (31.5%) and Klebsiella (15.7%) followed by Pseudomonas (10.5%). Gram positive were found 24.5% in total samples. In which age group 15-30 years of women showed 23.5% gram positive isolates while 26.3% recorded prominent prevalence in age group 31-45 years women. The most frequent gram positive causative agents in the study were found to be Coagulase negative staphylococci and Staphylococcus aureus followed by and Enterococcus faecalis. In the study of 15-30 age group was found to be Coagulase negative staphylococci (8.8%) and Staphylococcus aureus (8.8 %) followed by Enterococcus faecalis (5.8%). Similar pattern also was observed in 31-45 age group Coagulase negative staphylococci (15.7%) and Staphylococcus aureus (10.5%). In this study, fungal infection of Candida was also found to be as a causative agent of UTI. The total percentage of fungal infection incidence was 22.6 %. In age group 15-30 years women showed higher (26.4%) incidence of UTI than age group 31-45 which was found to be 15.7% (Table 1). Figure 1 shows the sensitivity of gram negative isolates to antibiotics used showed the highest sensitivity to Norfloxacin with percentage (92.86%) followed by Nitrofurantoin with percentage (89.29%), Levofloxacin (82.14%) while the proportion was less sensitive to Ampicillin with percentage (39.29%). It was the proportion of resistance to Ampicillin Cefazolin (32.14%, 32.1 %) followed by Gentamicin (21.43%) Ofloxacin (10.71%) Nitrofurantoin (10.71%). In gram positive isolates, the sensitivity to antibiotics indicated the highest sensitivity to Norfloxacin (100%) followed by Gentamicin (92.31%), Ofloxacin (84.62%). The less sensitive antibiotic was Ampicillin with percentage (38.46). It was reported that in all antibiotics Ampicillin was showed highest resistant pattern followed by Cefoxitin (23.08%), Ofloxacin (15.38%) and Gentamicin (7.69%). Norfloxacin was not reported any resistance pattern against isolates (Fig. 2). Figure 3 showed antifungal susceptibility test against antifungal drug Amphotericin B, Voriconazole, Fluconazole and Itraconazole. 3486 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Amphotericin B indicated highest sensitivity pattern with percentage 91.67% followed by Voriconazole (83.3%), Fluconazole (83.3%) and Itraconazole (66.7%). The highest resistant pattern reported in Itraconazole (16.67%) followed by Fluconazole (8.3%) and Voriconazole (8.3%). Amphotericin B was demonstrated no resistant pattern against isolates. Nagwan slum The 51 samples of highly symptomatic women were collected from Nagwan slum. The result showed that the overall prevalence of UTIs in both age groups was 45%. In which, occurrence of UTI in 15-30 age group was found upto 52.2% while 31-45 age group showed 47.8%. Thus, 15-30 age group show increased risk of infection than 31-45 age group females (Table 2). Almost all of the cases of UTIs were due to the gram negative bacteria with no prominent age group difference (Table 2). The percentage of gram negative bacteria was 52.2%. The percentage of Lactose fermenting bacteria was 91.6%. Lactose fermenting (91.6 %) bacteria indicated predominant frequency in both age groups. Non lactose fermenting bacteria was observed 14.2%. The most frequent causative agents of UTIs in the study of 15-30 age groups reported as E. coli (33.3%) and Klebsiella (8.3%) followed by Pseudomonas (8.3%) and Acinetobacter (8.3%) but in 31-45 age group Klebsiella (27.27%) was commonest UTI pathogens then E. coli (18.2%) in Nagawan slum. Gram positive were found 26.1% in total samples. In which age group 15-30 years of women showed 25% gram positive isolates while 27.3% recorded prominent prevalence in age group 31-45 years women. The most frequent gram positive causative agents in the study were found to be Coagulase negative staphylococci and Staphylococcus aureus. In the study of 15-30 age group was found to be Staphylococcus aureus (16.6 %) and Coagulase negative staphylococci (8.3%) but in 31-45 age group Coagulase negative staphylococci (27.3%) was more common pathogen for UTI. The study was also noted that fungal infection of Candida was also found to be as a causative agent of UTI. The total percentage of fungal infection incidence was 21.7 %. In age group 31-45 years women showed higher (27.3%) incidence of UTI than age group 15-30 which was found to be 16.66% Figure 4 shows Norfloxacin Nitrofurantoin and Levofloxacin antibiotics were used that showed the highest sensitivity of gram negative isolates while the proportion was less sensitive to Cefazolin and Ampicillin. In gram positive isolates, the sensitivity to antibiotics indicated the highest sensitivity to Norfloxacin (100%) followed by Ampicillin, Cefoxitin, Gentamicin and Ofloxacin but data also revealed that Ampicillin was resistant with percentage of 16.67. It was reported that in all antibiotics Ampicillin and Cefoxitin was showed highest resistant pattern. Norfloxacin, Gentamicin and Ofloxacin were not reported any resistance pattern against isolates (Fig. 5). Figure 6 showed antifungal susceptibility test against antifungal drug Amphotericin B, Voriconazole, Fluconazole and Itraconazole. Amphotericin B indicated highest sensitivity pattern with percentage 100% followed by Voriconazole (80%), Itraconazole (80%) and Fluconazole (60%). The highest resistant pattern reported in Itraconazole and Fluconazole. Amphotericin B and Voriconazole were demonstrated no resistant pattern against isolates. Bajardeehan slum The 115 samples of highly symptomatic women were then collected from Bajardeehan 3487 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 slum. Samples were cultured in laboratory and then organized in different age groups. The result showed that the overall prevalence of UTIs in both age groups was 49.5%. In which, occurrence of UTI in 15-30 age group was found upto 57.8% while 31-45 age group showed 42.1 %. Thus, 15-30 age group show increased risk of infection than 31-45 age group females (Table 3). Almost all of the cases of UTIs were due to the gram negative bacteria with no prominent age group difference (Table 3). The percentage of gram negative bacteria was 57.9%. The percentage of Lactose fermenting bacteria was 81.2%. Lactose fermenting (81.2%) bacteria indicated predominant frequency in both age groups. Non lactose fermenting bacteria was observed 18.2%. The age group 15-30 years females demonstrated about two folds increase of NLF than 31-45 age group females. The most frequent causative agents of UTIs in the study of 15-30 age group reported as E. coli (24.2%) and Klebsiella (15.2%) followed by Pseudomonas (12.1%) and Enterobacter (6.1%). Similar pattern also was observed in 31-45 age group E. coli (29.2%) and Klebsiella (16.6%) followed by Pseudomonas (8.3%) Enterobacter (4.1%). Gram positive were found 22.8% in total samples. In which age group 15-30 years of women showed 21.2% gram positive isolates while 25% recorded prominent prevalence in age group 31-45 years women. The most frequent gram positive causative agents in the study were found to be Staphylococcus aureus (12.1%) and Coagulase negative staphylococci (9.1) in 1530 age group but Coagulase negative staphylococci (16.6%) was observed higher than Staphylococcus aureus (8.3%). In this study, fungal infection of Candida was also found to be as a causative agent of UTI. The total percentage of fungal infection incidence was 19.2 %. In age group 15-30 years women showed higher (12.1%) incidence of UTI than age group 31-45 which was found to be 4.1%. The sensitivity of gram negative isolates to antibiotics used that showed the highest sensitivity to Norfloxacin and Levofloxacin with percentage (90.91%) followed by Nitrofurantoin with percentage (87.9%), Gentamicin (75.8%) and Ofloxacin (72.7%) while the proportion was less sensitive to Ampicillin with percentage (63.6%) (Fig. 7). It was the proportion of resistance to Gentamicin (32.1 %) followed by Ampicillin, Ofloxacin, Cefazolin and Levofloxacin. Ampicillin was indicated highest intermediate sensitivity (27.3%). In gram positive isolates, the sensitivity to antibiotics indicated the highest sensitivity to Norfloxacin (92.3%) followed by Ofloxacin, Cefoxitin. The less sensitive antibiotic was Ampicillin with percentage (61.5%). It was reported that in all antibiotics Ampicillin (23.1%) was showed highest resistant pattern. Gentamicin (23.1%) was indicated intermediate sensitivity against isolates (Fig. 8). Figure 9 showed antifungal susceptibility test against antifungal drug Amphotericin B, Voriconazole, Fluconazole and Itraconazole. Amphotericin B indicated highest sensitivity pattern with percentage 90.9% followed by Voriconazole (81.8%), Fluconazole (63.3%) and Itraconazole (54.7%). The highest resistant pattern reported in Itraconazole (36.4%) followed by Fluconazole (27.3%) and Voriconazole (9.1%). Amphotericin B was demonstrated no resistant pattern against isolates. 3488 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Table.1 Prevalence of different isolates of bacteria and fungi in both groups of Ravidrapuri slum Types of Microorganisms Gram Negative Gram positive Fungi 15-30 No. of isolates Escherichia coli 7 Klebsiella pneumoniae 5 P. aeruginosa 4 Acinetobacter 1 Staphylococcus aureus 3 Coagulase negative 3 Staphylococci Enterococcus faecalis 2 Candida 9 20.58 14.7 11.7 2.9 8.8 8.8 31-45 No. isolates 6 3 2 0 2 3 5.8 26.4 0 3 Percentage of Percentage 31.5 15.7 10.5 0 10.5 15.7 0 15.7 Table.2 Prevalence of different isolates of bacteria and fungi in both groups of Nagwan slum Types of Microorganisms Gram Negative Gram positive Fungi 15-30 No. of isolates Escherichia coli 4 K.pneumoniae 1 P. aeruginosa 1 Acinetobacter 1 S.aureus 2 Coagulase negative 1 Staphylococci E.faecalis 0 Candida 2 33.3 8.3 8.3 8.3 16.6 8.3 31-45 No. of Percentage isolates 2 18.18 3 27.27 0 0 0 0 0 0 3 27.27 0 16.6 0 3 Percentage 0 27.27 Table.3 Prevalence of different isolates of bacteria and fungi in both groups of Bajardeehan slum Types of Microorganisms Gram Negative Gram positive Fungi 15-30 No. of isolates Escherichia coli 8 K.pneumoniae 5 P. aeruginosa 4 Acinetobacter 0 E.aerogenes 2 S.aureus 4 Coagulase negative 3 Staphylococci E.faecalis 0 3489 24.24 15.15 12.12 0 6.06 12.12 9.09 31-45 No. of Percentage isolates 7 29.16 4 16.6 2 8.3 0 0 1 4.1 2 8.3 4 16.6 0 0 Percentage 0 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Table.4 Prevalence of different isolates of bacteria and fungi in both groups of Batuapura slum Types of Microorganisms 15-30 No. of isolates 3 1 1 1 2 2 2 2 Escherichia coli K.pneumoniae P. aeruginosa Acinetobacter E.aerogenes Staphylococcus aureus CONS E. faecalis Gram Negative Gram positive Fungi 31-45 Percenta No. of Percent ge isolates age 15.17 5 25 5.2 4 20 5.2 1 5 5.2 0 0 10.52 1 5 10.52 4 20 10.52 0 0 10.52 2 10 Table.5 Prevalence of different isolates of bacteria and fungi in both groups of Ghasiyari Tola slum Types of Microorganisms Gram Escherichia coli Negative Klebsiella pneumoniae P. aeruginosa Acinetobacter Gram E.aerogenes positive S.aureus Coagulase negative Staphylococci E.faecalis Fungi 15-30 No. of isolates 3 1 0 0 0 0 0 0 31-45 Percentage No. of isolates 75 3 25 0 0 0 0 0 0 1 0 2 0 0 0 0 Percentage 42 0 0 0 14.2 28.5 0 0 100 90 80 70 60 50 40 30 20 10 0 Sensitive Intermediate (1 0) Ce fa zo lin (3 G 0) en ta m ic O in f lo (1 xa 0) cin /C ip ro No (5 ) rfl ox ac Ni in tro (1 fu 0) ra nt oi n (3 00 Le ) vo f lo xa cin (5 ) Resistant lin pi ci Am Percentage of Isolates Fig.1 Antibiotic susceptibility pattern for gram negative bacteria of Ravindrapuri extension slum Antibiotic concetration (µg) 3490 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Fig.2 Antibiotic susceptibility pattern for gram positive bacteria of Ravindrapuri extension slum Percentage of Isolates 120 100 80 Sensitive 60 40 Intermediate Resistant 20 in (1 O 0) f lo xa cin /C ip ro (5 ) N or flo xa ci n (1 0) G en ta m ic A m pi ci lin C ef ox itin (1 0) (1 0) 0 Antibiotic Concentration (µg) Fig.3 Effect of antifungal drugs against isolated fungal Candida of Ravindrapuri extension slum women 100 90 Percentage of Isolates 80 70 60 Sensitive 50 Intermediate 40 30 Resistant 20 10 0 Amphotericin B Voriconazole Fluconazole Itraconazole Antifungal drug concetration Fig.4 Antibiotic susceptibility pattern for gram negative bacteria of Nagwan slum 100 80 Sensitive 60 40 Intermediate Resistant 20 in (1 O 0) f lo xa cin /C ip ro (5 ) No rfl ox ac in (1 Ni 0) tro fu ra nt oi n (3 00 ) Le vo f lo xa cin (5 ) G en ta m ic (3 0) Ce fa zo lin pi ci lin (1 0) 0 Am Percentage of Isolates 120 Antibiotic Concentration (µg) 3491 Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 3483-3497 Fig.5 Antibiotic susceptibility pattern for gram positive bacteria of Nagwan slum 100 80 Sensitive 60 40 Intermediate Resistant 20 in (1 0) O f lo xa ci n/ C ip ro (5 ) N or flo xa ci n (1 0) G en ta m ic C ef ox it i n n pi ci li m A (1 0) 0 (1 0) Percentage of Isolates 120 Antibiotic Concentration (µg) Fig.6 Effect of antifungal drugs against isolated fungal Candida of Nagwan slum women 120 Percentage of Isolates 100 80 Sensitive Intermediate 60 Resistant 40 20 0 Amphotericin B Voriconazole Fluconazole Itraconazole Antifungal drug Concentration Fig.7 Antibiotic susceptibility pattern for gram negative bacteria of Bajardeehan slum 3492