INTRODUCTION:

In any primary care centre setting, antibiotics are most commonly prescribed for lower urinary tract infections (UTI)¹. The second predominant bacteria associated infection in such a set-up is asymptomatic bacteriuria (ASB)². Asymptomatic bacteriuria, also called as, asymptomatic urinary infection, is referred to the isolation of bacteria, beyond the normal count, from urine samples. ASB has been observed to be prevalent women with urologic dysfunctions along with impaired voiding. The presence of active bacteria in the urine of women without apparent signs or symptoms of UTI is ASB which is an important predictor of UTI. However, even healthy women are diagnosed with ASB³. In developed countries ASB affects millions of people⁴.

Sexual activity, child bearing, short urethra, and lack of personal hygiene are the main reasons for ASB in women⁵. One of every three women are diagnosed with urinary tract infection before they reach the age of 24 and the others experience UTI at least once in their lifetime⁶. The chances of occurrence of ASB in pregnant women are much higher than non-pregnant women. The higher rate of infection in pregnant women is because of the urinary stasis caused by the activity of progesterone in pregnancy along with certain other morphological and physiological changes that occur during pregnancy⁷. UTI can be diagnosed by identifying certain urinary symptoms as well as isolation of an uropathogen in urine culture tests. There is 88% sensitivity and 92% specificity when bacteria are detected in urinalysis⁸. Urine culture detection for specific bacteria is the best way for diagnosis of UTI. A colony count of more than 100,000 colony forming units (CFU)/ml of bacteria indicate significant clinical infection of which E. coli accounts for 70-90% of all urinary tract infections⁹.

Treatment of UTIs using antimicrobial drugs plays an essential role in reducing the morbidity and mortality caused by such infections. Antibiotic treatment, unfortunately poses a challenge due to the resistance caused by the pathogens¹⁰. Hence screening for ASB and the treatment using antimicrobials with less resistance will certainly reduce the risk of pyelonephritis, an unwarranted complication of ASB among women⁵.

The main objective of this study is to isolate and identify the predominant uropathogen in women with ASB undergoing elective procedures like ovarian hyper-stimulation, embryo transfers and cervical encerclage as part of infertility treatment in our hospital. Further, the antibiotic sensitivity to these women with ASB were determined.

METHODS:

Study design and setting:

This is a cross sectional study that included 350 women attending the outpatient clinic at Sri Narayani Hospital and Research Centre – Vellore between July 2017 and October 2018 and who were planned for elective procedures like controlled ovarian hyper-stimulation, embryo transfer, and cervical cerclage as part of treatment for infertility. Women with more than 6-8 pus cells on urine routine examination were also included in this study. Mid stream urine sample collected early in the morning was received from the participants and the samples were subjected to urine analysis, urine culture and antimicrobial susceptibility testing. The study was done after being approved by the ethics committee of the institution.

Urine sampling and culture methods:

The women were given a clean leak proof, wide open, sterile sample container and asked to collect freshly voided clean-catch early morning midstream urine samples of about 15 ml. These samples were processed in the microbiology lab within 30 minutes of collection to ensure the maximum recovery of bacteria.

The urine sample was spread plate on MacConkey agar and incubated at 37ºC for 48 hours. Macroscopic examination of these agar plates was done for observation of bacterial growth. The colonies of the isolates on the plates were counted and calculated to evaluate the number of organisms per millilitre specimen.

Antibiotic susceptibility testing:

Antibiotic sensitivity testing was carried out using nutrient agar medium. The bacterial cultures were swabbed on nutrient agar plates. Antibiotic discs were then placed on the plates and incubated for 24-48 h to observe the zone of clearance¹¹. The zone of clearance was recorded to determine the sensitivity or resistance of the bacterial strains towards a particular antibiotic.

Data Analysis:

Continuous variables were presented as mean and standard deviation. Positive result was expressed as percentage with 95% confidence interval. Continuous measures between two groups were compared using Independent T test or Mann Whiteny test based on distribution of data. Categorical variables between the two organisms were compared using Chi square test. A P value<0.05 was considered statistically significant. Analysis was done in SPSS version 18.0.

RESULTS:

Demographic profile:

This cross-sectional study was conducted to analyse the microbiological profile and their sensitivity and resistance to antibiotics on urine culture tests in patients who were a part of infertility treatment, before cervical cerclage after conception by ART. Women, who had more than 6-8 pus cells in their urine routine examination were also checked during the study. A total of 350 women were chosen for urine culture analysis during the study and the antibiotic sensitivity pattern was studied. Out of the 66 women who had urine culture positive with the prevalence of colonisation 18.9 % (95% CI =14.9% - 23.4%), 43.94%²⁹ were planned for embryo transfer, 25.76%¹⁷ planned for cervical cerclage, 19.70%¹³ had more than 6-8 pus cells on urine routine examination and 10.61%⁷ were posted for oocyte retrieval procedure.

Age:

The mean age of patients of the urine culture positive women included in this study was 31 years with a minimum of 18 years and maximum of 46 years.

Isolated microorganism:

Only two organisms were isolated in the 66 samples collected, in which the most common pathogen found was E. coli 62.12%⁴¹ and the other organism was Klebsiella sp. 37.88%²⁵. The morphological and biochemical analysis of the isolated organisms are tabulated in table I and II respectively.

Table II: Biochemical Identification of Isolates

Sl. No.	Identification Test	Name of Organism
Sl. No.	Identification Test	*Escherichia coli*	*Klebsiella* sp.
1.	Motility	+ve	-ve
2.	Catalase	+ve	+ve
3.	Oxidase	-ve	-ve
4.	Methyl Red	+ve	-ve
5.	Voges Proskauer	-ve	+ve
6.	Oxidation/Fermentation 1. Arabinose 2. Fructose 3. Glucose 4. Lactose 5. Maltose 6. Mannose 7. Raphinose 8. Ribulose 9. Sorbitol 10. Galactose 11. Xylose	Fermentative + + + + + + - - + + +	Fermentative + + + + + + + + + +
7.	Indole	+ve	-ve
8.	Citrate	-ve	+ve
9.	Urease	-ve	+ve
10.	Nitrate Reduction Test	+ve	+ve
11.	Triple Sugar Iron Agar Test	+ve	+ve

Key: -ve: Negative, +ve: Positive

Table III: Susceptibility of antibiotics for E. coli

Antibiotics	S (%)	I (%)	R (%)
Ciprofloxacin	60.98(25)	14.63(6)	24.3
Norfloxacin	41.4(17)	12.2(5)	46.3
Ofloxacin	39.02(16)	31(13)	29.2
Cefdinir	41.4(17)	41.4(17)	17(7)
Cefixime	21.9(9)	24.3(10)	53.66
Cefotaxime	63.4(26)	14.6(6)	21.9(9)
Ceftriaxone	58.54(24)	24.3(10)	7(17.0)
Cefuroxime	46.34(19)	29.27(12)	24(10)
Nalidixic acid	14.6(6)	14.6(6)	70.7
Amikacin	60.68(25)	31.7(13)	7.3(3)
Aztreonam	31.71(13)	7.3(3)	25(60.9)
Ceftazidime	31.71(13)	31.71(13)	15(36.59)
Gentamicin	26.8(11)	53.66(22)	19.5(8)
Nitrofurantoin	34.15(14)	34.1(14)	31.7(13)

S (%): shows the percentage of sensitivity towards the antibiotics, I %: percentage of strains which have intermediate activity, R%: percentage of bacterial strains that are resistant towards the antibiotics

Table IV: Susceptibility of antibiotics for Klebsiella sp.

Antibiotics	S (%)	I (%)	R (%)
Ciprofloxacin	72%(18)	16(4)	3(12)
Norfloxacin	56%(14)	24(6)	20(5)
Ofloxacin	48%(12)	24(6)	28(7)
Cefdinir	48%(12)	40(10)	12(3)
Cefixime	24%(6)	24(6)	52(13)
Cefotaxime	68(17)	16(4)	16(4)
Ceftriaxone	60(15)	24(6)	16(4)
Cefuroxime	48(12)	36(9)	16(4)
Nalidixic acid	24(6)	12(3)	64(16)
Amikacin	64(16)	20(5)	16(4)
Aztreonam	36(9)	16(4)	48(12)
Ceftazidime	60(15)	24(6)	16(4)
Gentamicin	44(11)	40(10)	16(4)
Nitrofurantoin	16(4)	52(13)	32(8)

Table V: Antagonistic activity of the isolated strains

Antibiotics	*E. coli*	*Klebsiella* sp.	Total
Ciprofloxacin	24.39(10)	16(4)	21.2(14)
Norfloxacin	46.34(14)	24(6)	37.8(25)
Ofloxacin	29.2(12)	28.0(7)	28.7(19)
Cefdinir	17.0(7)	12.0(3)	15.1(10)
Cefixime	53.6(22)	52(13)	53(35)
Cefotaxime	21.9(9)	16.4(4)	16(4)
Ceftriaxone	17.0(7)	16(4)	16.6(11)
Cefuroxime	24.3(10)	16.4(4)	21.2(14)
Nalidixic acid	70.73(29)	64.0(16)	68(45)
Amikacin	7.3(3)	16(4)	10.6(7)
Aztreonam	60.9(25)	48.0(12)	56.06(37)
Ceftazidime	36.5(15)	16(4)	28.7(19)
Gentamicin	19.5(8)	16.0(4)	18.1(12)
Nitrofurantoin	31.7(13)	32(8)	31.8(21)

Antibiotic sensitivity pattern:

The antibiotic sensitivity and resistance of the isolated organisms were determined against 15 antibiotics which are tabulated in table III and IV. A total of 26 women (63.4%) with E. coli were sensitive to cefotaxime, followed by amikacin, ciprofloxacin 60.9%²⁵ and ceftriaxone 58.4%²⁴. Klebsiella sp., the other isolated strain, showed 72% sensitivity towards ciprofloxacin followed by cefotaxime 68%¹⁷, and amikacin 64%¹⁶. In both the groups, the number of women resistant towards nalidixic acid was much higher followed by cefixime. The E. coli isolated samples had a higher number of pus cells in the urine routine sample when compared to the group of samples infected with Klebsiella sp. A total of 13 patients had more than 15 pus cells in their urine culture sample out of which 61.54% (8) had significant bacteriuria (>10⁵). Figure 1 shows the zone of inhibition obtained during analyzing antibiotic sensitivity analysis of E. coli and Klebsiella sp.

(a) (b)

Figure 1: Antibiotic Susceptibility Test of Antibiotics against (a) E. coli and (b) Klebsiella sp.

DISCUSSION:

Treatment of urinary tract infection is made easier and without delay by studying the antimicrobial patterns and the traits of the bacteria involved in the infections. This study confirmed the prevalence of urinary colonisation along with the antibiotic susceptibility patterns among the women attending the clinic at Sri Narayani Hospital and Research Centre. The overall prevalence in our study was 18.9%. The type of population and the difference in sample size has influenced the prevalence rate in many cross-sectional studies. It has been noted that pregnant women are 20 to 30 times more at risk for urinary infection compared to the non-pregnant women of the same age group⁵. Our finding was much the same with 25% of women presenting with urine culture positive results after achieving successful pregnancy by assisted reproductive technology.

It is well known that E. coli is the most prevalent Gram-negative organism responsible for UTI. About 12% of men and 10-20% of women have been observed to have experienced an acute symptomatic UTI and many more develop asymptomatic bacteriuria¹². E. coli was the predominant pathogen isolated even in our study with the maximum number of pus cells in the sample when compared to the Klebsiella containing group of samples. The presence of E. coli and Klebsiella sp. were examined by culturing the urine samples on nutrient agar plates and MacConkey agar medium. Biochemical tests confirmed the predominant strains to be E. coli and Klebsiella sp. The data obtained after performing biochemical tests were compared to the standard biochemical chart for identification of the bacterial isolates. E. coli strains were indole, methyl red, urease and catalase positive. However, the Klebsiella sp. showed positive results to Voges Proskauer, citrate utilisation, urease, catalase and nitrate test. The reasons for abundance of E. coli and Klebsiella sp. in the samples might be due to fecal contamination, the high prevalence of this organism in toilets and also the shortness of the female urethra ^13,14,15.Klebsiella species is a Gram-negative opportunistic pathogen that is known to produce urinary tract infection, wound infection and blood infections ¹⁶. Earlier studies have reported that Klebsiella sp. is commonly found in the urine sample ¹⁷.

The mean age of women with culture positive results in this study was 31 years with a minimum of 18 years. The reason for this could be that most women in this age group are sexually active when compared to their very young and older counterparts. Sexual activity very clearly exposes women to uropathogens resulting in urine culture positive results¹⁷.

Susceptibility pattern was found to be similar in both the E. coli and Klebsiella sp. isolates samples with a higher proportion of sensitivity to ciprofloxacin. This type of susceptibility pattern with higher sensitivity to ciprofloxacin was also reported in a study conducted in India proving the efficacy of ciprofloxacin to be the most effective antibiotic against E. coli isolates¹⁸. Ciprofloxacin is a well-known fluoroquinolone drug which has potential to inhibit bacterial DNA gyrase. The presence of magnesium ions and the high acidic nature of un-infected urine reduces the effectiveness of ciprofloxacin. However, the presence of bacterial pathogens in the urine samples of infected women results in modification of the characteristics of urine samples and thus the effectiveness of the drug might not change and influence the inhibition of DNA gyrase¹⁹. This study revealed that both the organisms exhibited resistance to nalidixic acid followed by cefixime since these antibiotics have been used for a long term. Mutation in gyrA gene of bacterial isolates causes the strains to be resistant towards nalidixic acid²⁰. Easy access to commonly used drugs is one of the main reasons for self-medication and medicine abuse for high resistance²¹.

In this study, urine culture analysis was conducted at Sri Narayani Hospital and Research Centre anticipating any bacterial infection of the genital tract from the urinary tract may occur before embryo transfer. Bacterial contamination of the uterine cavity after a transcervical embryo transfer may affect the implantation rate and pregnancy outcome since the embryo transfer catheter tip gets contaminated²². Urinary tract infection is the most common infection during pregnancy. If asymptomatic bacteriuria is non treated properly, it may lead to acute cystitis, pyelonephritis and other maternal and fetal complications^23,24. Urine culture, as part of routine antenatal screening and before cervical cerclage is conducted since urinary tract infection is treatable and prevents unwarranted maternal and neonatal complications.

CONCLUSION:

It is well known that asymptomatic bacterial colonisation of the urinary tract is prevalent among women in the reproductive age group and specifically more in pregnant women. This could have consequences on pregnancy and hence this study was accomplished to determine the predominant bacterial strains and their sensitivity towards the antibiotics used for treatment. The common uropathogens isolated were E. coli and Klebsiella sp. This study can be a help to create awareness on personal hygiene, cleanliness and systematic screening for uropathogens in all women.

ACKNOWLEDGEMENT:

The authors would like to express their gratitude to the management of VIT, Vellore.

CONFLICT OF INTEREST:

The authors have no conflict of interest to declare.

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Received on 04.09.2020 Modified on 28.03.2021

Research J. Pharm.and Tech 2022; 15(2):566-570.

DOI: 10.52711/0974-360X.2022.00092

Isolate	Gram nature	Motility	Shape	Pigment	Elevation	Margin	Opacity	Surface
Escherichia coli	Gram negative rods	Motile	Circular	Cream-white	Convex	Entire	Translucent	Smooth
Klebsiella sp.	Gram negative coccobacilli	Non Motile	Irregular	Cream	Raised	Entire	Opaque	Glistening