High Frequency of Multidrug Resistant Urinary Isolates in Pregnant Women in a Tertiary Care Hospital of Nepal

High Frequency of Multidrug Resistant Urinary Isolates in Pregnant Women in a Tertiary Care Hospital of Nepal Bhandari Pashupati1*, Dipak Raj Joshi2, Sharma Khem Raj2, Khanal Santosh1, Acharya Ganesh3, Adhikari Nabaraj2 1Department of Microbiology, National College (NIST), Khusibu, Kathmandu, Nepal 2Department of Microbiology, Kantipur College of Medical Sciences, Sitapaila, Kathmandu, Nepal 3Department of Pathology Laboratory, Paropakar Maternity and Women’s Hospital, Kathmandu, Nepal SOJ Microbiology & Infectious Diseases Open Access Research Article


Introduction
Urinary Tract Infection (UTI) is one of the most common bacterial infections accounting for about 25% of all types of infection in human and approximately 10% of the humans acquire UTI at some time during their lifetime [1].The Incidence of UTI is age and sex dependent and it occurs with higher frequency in female than in men because of shorter urethra and close proximity of urinary tract to anus and most prevalent age group experiencing UTI in females is 21-30 years age [2].Anatomical changes, for example ureteral dialation, increased bladder volume along with decreased bladder and ureteral tone will contribute to urinary stasis and ureterovesical reflux, in addition with physiological changes like glycosuria, during pregnancy which encourages bacterial growth in the urine thus leading to development of UTI [3,4].Among the pregnant women approximately 4 to 10% will have asymptomatic bacteriuria, and 1 to 4% will develop acute cystitis and 1 to 2% may develop severe acute pyelonephritis during the second half of pregnancy 5 .Major bacteria associated with UTI includes, E. coli, Klebsiella spp, Citrobactor spp, Enterobacter spp, Proteus spp, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase negative Staphylococci where E. coli alone accounts for 80 -90% infections [5], about 85% of community acquired UTIs, 50% of nosocomial UTIs and more than 80% of uncomplicated pyelonephritis [6].
Untreated asymptomatic bacteriuria increases the frequency of premature delivery, neonates with low birth weight and it is also likely to cause acute pyelonephritis at a rate of 20 to 30% [7].Studying antibiotic sensitivity profile of urinary isolates helps us to select drugs for individual patient treatment so that recurrence could be prevented.Additionally, resistance pattern of these organisms against drugs in clinical practice can be determined which will aid in prevention of emerging multidrug resistant strains.

Culture
Urine specimens were cultured by semi-quantitative culture technique.A loop full of well-mixed and uncentrifuged samples was inoculated using standard calibrated loop onto Blood Agar (BA) and Macconkey Agar (MA) plates and aerobically incubated at 37ºC for 24 h.After overnight incubation, colony count yielding bacterial growth of ≥ 10 5 CFU/ml was taken as being significant [11].If the culture indicates presence of two uropathogens both showing significant growth, definitive identification and antimicrobial susceptibility testing of both were performed whereas in cases of ≥ 3 pathogens, it was reported as multiple bacterial morphotypes and asked for appropriate recollection with timely delivery to laboratory [12].

Identification of Isolates
At first colony characteristics of isolated bacteria was observed on agar plates and Gram staining was done.Gram positive isolates were identified by performing catalase, oxidase, coagulase, and optochin sensitivity tests while for Gram negative bacteria, different biochemical tests, catalase, oxiadse, motility, hydrogen sulphide and indole production, citrate utilization, MR/ VP, urea hydrolysis, and triple sugar iron utilization were done and then identified based on their results [13].

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility test was performed by disc diffusion method as recommended by Clinical Laboratory Standard Institute (CLSI), 2012.Four to five different colonies of test organisms were touched with sterile loop and mixed with 2 ml of sterile saline and vortexed to create a smooth suspension.Turbidity of this solution was adjusted to a 0.5 McFarland standard which has corresponding bacterial concentration of approximately 150 million/ml.A sterile swab was dipped into the suspension, firmly pressed to remove excess fluid, and plated on Muller Hinton Agar (MHA).Discs were applied on MHA plates and incubated at 37°C for 24 hours.Zone of inhibition was measured and interpreted using the standard chart and organisms reported as susceptible, intermediate or resistant accordingly [14].Following antibiotic discs were used Ampicillin (10 µg), Cephalexin (30 µg), Ciprofloxacin (5 µg), Cotrimoxazole (25 µg), Gentamycin (10 µg), Nalidixic acid (30 µg), Nitrofurantoin (300µg), Norfloxacin (10 µg), Olfoxacin (10 µg).

Statistical Analyses
Data analyses were performed using Statistical Package for Social Science (SPSS) version 16.0 (SPSS Inc., Chicago, IL, USA) and Microsoft excel 2007 and correlation analysis was performed to determine relation between two or more variables.

Socio-demographic Characteristics
A total of 653 pregnant women were enrolled in this study with the age ranges between 17-30 years and all of them were married.Of considered variables, increasing trimester was significantly associated with UTI while showing no association with age (Table 1).

Antimicrobial Susceptibility Pattern of Gram Positive Bacteria
Nitrofurantoin, Norfloxin and Ofloxacin were found to be the drug of choice with a susceptibility of 94.73%, 78.94% and 78.94% respectively (Table 6).

Multiple Drug Resistance Patterns of the Isolates
Among the total 112 isolates, multi drug resistance (MDR = resistance in ≥ 3 drugs) was recorded in 82 (73.2 %) bacteria.MDR was observed in E coli, Klebsiella spp, Pseudomonas and Proteus spp only (Table 7).

Discussion
Different physiological changes like increased glucose, amino acids, vitamins content of the urinary tract along with other favorable anatomical changes during pregnancy fosters the bacterial growth during pregnancy [9].Prevalence of urinary tract infection in this study was 17.2% which is higher than prevalence of UTI reported in another research (9.8%) [15], but lower than study in Valley Maternity hospital, Kathmandu (44.6%) [16].This variation could be due to difference in the environment, social habit of community and standard of personal hygiene [17].All patients in our study were in age group 17-30 which corresponds to another study [18] and illustrates active pregnancy age group in Nepalese woman is in between these years.
There was significant positive association between increasing trimester of pregnancy with increasing UTI frequency while patients having at least higher secondary level of education experienced lower episodes of frequency was statistically significant.Similar to our findings, other studies have also shown significant statistical relationship between among these variables [19,20].Maintaining hygienic standards, sanitation practices and keeping the tract clean greatly reduce the risk to acquire urinary tract infections during pregnancy.

Microscopic observation Frequency (n) Percentage (%)
Pyuria (significant > 5/ hpf) 123 24.6 Haematuria (significant > 3/ hpf) 6 5.77 tract infection during pregnancy.E. coli is the most common microorganism in the vaginal and rectal area and due to anatomical and functional changes and difficulty of maintaining personal hygiene during pregnancy may increase the risk of acquiring UTI from E. coli [2,17].
Antibiotic resistant caused multi drug resistant organisms have severely impaired available treatment options which might lead to the situation to rely only on certain antibiotics like fosfomycin, colistin and carbapenems [23,24].We have found that Gram negative bacteria were most resistant to Cotrimoxazole (47.32%), quinolone [Ciprofloxacin and Ofloxacin (31.32% and 22.59% respectively)], and Nitrofurantoin (6.46%).In contrary, another study showed high resistance to different classes of antibiotics used: Co-trimoxazole (86.8%), quinolone (Ciprofloxacin and Ofloxacin 92.6%) in each and Nitrofurantoin (17.6%) [23].Local resistance pattern, nature of patients (ICU or OPD), easy availability and indiscriminate use of common drugs might account for this difference.
Our study showed that, resistant rate of Gram positive bacteria against Nitrofurantoin (5.27%), and Ofloxacin and Norfloxacin was 21.06% each.Generally, Gram positive bacteria have less resistance mechanism than gram negative bacteria as they are more easily inhibited by body's defense system than later, and also by routine drugs [24].
Multidrug resistant (resistance to ≥ 3 different structural drugs) was reported in 73.2% of isolates while in another study carried out at Kathmandu Model Hospital from Kathmandu in 2012 showed 41% MDR isolates [23].This clearly indicates rise in MDR isolates in uropathogens and could be consequence of indiscriminate antibiotic use and abuse [25].High frequency of MDR is alarming issue as this leaves us with potentially very few antibiotics to treat such MDR infected patients and risk patient lives.Drugs like ceftriaxone or carbapenems for example meropenem or imipenem could be the drug of choice in treating such multi drug resistant urinary bacterial isolates [26].Gram negative bacteria have several host adhesion factors that facilitates unique adhesion and adherence to host epithelial cells thereby preventing removal during urination and ultimately results invasion and pyelonephritis [22].

Conclusion
We have found E. coli as the most common isolates (63.4%) followed by Proteus spp (9%).In concurrent to our study, study from other Maternity hospitals of Kathmandu [3] and Pokhara [10] shown E. coli as dominant pathogen causing urinary

Figure 1 :
Figure 1: Growth pattern of bacterial isolates

Table 1 :
Prevalence of UTI and demographic characteristic of study population (N= 653).

Table 3 :
Frequency of different bacterial isolates

Table 2 :
Frequency of pus and red blood cells on microscopic observation.

Table 5 :
Individual antibiotic susceptibility pattern of E. coli, K. pneumoniae and P. vulgaris

Table 7 :
Frequency of multi drug resistant isolates