Phylogenetic background of E . coli isolated from asymptomatic pregnant women from Kolkata , India

Introduction: Asymptomatic bacteriuria (ABU) in pregnancy generates medical complications. E. coli is the common etiologic agent responsible for ABU-associated infections. This study aimed to identify the phylogenetic background and drug resistance in asymptomatic E. coli from a pregnant population. Methodology: E. coli was confirmed biochemically from culture-positive urine samples collected from asymptomatic pregnant women. Phylogenetic typing was done by polymerase chain reaction (PCR). The isolates were subjected to antibiotic susceptibility testing and extended-spectrum beta-lactamase (ESBL) production. Statistical significance was determined using SPSS 17.0 software. Results: Bacteriuria was observed in 113 (22.6%) of 500 asymptomatic pregnant females. E. coli was reported in 44 (38.9%) of 113 isolates. The mean age-wise distribution was 25.14 ± 4.63. Although pathogenic phylogroup B2 was predominant (54.5%), incidence of nonpathogenic phylogroup B1 (27.3%) was found to be statistically significant (p ≤ 0.001), and B1 and B2 were correlated with respect to total ABU population. Antibiotic sensitivity against ampicillin (34.1%), ceftazidime (50%), cefotaxime (47.7%), ciprofloxacin (47.7%), amikacin (86.4%), nitrofurantion (79.5%), and co-trimoxazole (36.4%) was observed. Multidrug resistance (MDR) and ESBL production was reported in 26 (59.1%) of 44 and 18 (69.2%) of the 26 MDR isolates, respectively. A significant distribution of phylogroup B1 (p = 0.03) with drug resistance was also observed. Conclusions: This is the first study that reported significant incidence of non-pathogenic phylogroup B1 in asymptomatic E. coli with high incidence of MDR isolated from pregnant women in Kolkata, India. These varied resistance patterns present major therapeutic and infection control challenges during pregnancy.


Introduction
Urinary tract infections (UTIs) represent the most common bacterial infection in pregnancy and are classified as either asymptomatic or symptomatic.Asymptomatic bacteriuria (ABU) is defined as the presence of significant bacteriuria without the symptoms of an acute urinary tract infection, and it can lead to symptomatic infection and subsequent adverse effects on pregnancy continuation [1].Recently, it has been reported that UTIs usually develop in the third trimester [2].The susceptibility to UTIs during this period may be due to urethral dilatation, which is maximal in the 22nd-24th weeks of gestation [3].Untreated bacteriuria in pregnancy can lead to serious obstetric complications and poor maternal and perinatal outcomes, such as intrauterine growth restriction, preeclampsia, caesarean delivery, and preterm delivery [1,4].Women with asymptomatic bacteriuria during pregnancywhich can lead to acute respiratory distress, transient renal failure, sepsis, and shock during pregnancyshow a 20-to 30-fold increased risk of developing pyelonephritis compared with women without bacteriuria [4].Urine culture is recognized as the golden standard for detecting ABU showing bacterial growth > 10 5 colony-forming units (CFU)/mL.Therefore, all pregnant females should be screened for asymptomatic bacteriuria during their pregnancy, especially in their third trimester, as most cases of ABU (40%) are reported during this period of pregnancy; screening must be considered as a standard of obstetrical care that will help to minimize UTIassociated complications [2,5].
Escherichia coli have been shown in previous studies to be the dominant bacterial agents of ABU isolated from the urine of pregnant women [6,7].ABU is commonly caused by strains of lower virulence than the symptomatic uropathogenic E. coli strains, with a reduced frequency of virulence factor expression [8].Only 22% of strains of E. coli isolated from women with asymptomatic bacteriuria had the capacity to adhere to uroepithelial cells, compared with 75% in the group of women who developed acute urinary tract infections [9].E. coli isolates belong to four main phylogenetic groups: A, B1, B2, and D. Pathogenic isolates mainly belong to group B2 and, to a lesser extent, to group D, and most of the commensal or nonpathogenic strains belong to group A or group B1 [9,10].ABU strains were phylogenetically related to strains that cause symptomatic UTIs, as previously reported in other ABU situations [1].
Emergence of MDR has also been reported [11], and poses risk in prescribing specific drugs to treat the infection.Moreover, there no clear consensus in the literature on antibiotic choice or duration of therapy with bacteriuria in pregnant women, as drug resistance varies according to geographic location and may be related to misuse of antibiotics.Therefore, with increasing antibiotic resistance, considering local resistance rates must be considered when choosing therapy.This study was designed with the aim to determine the prevalence of E. coli in asymptomatic bacteriuria among pregnant woman from Kolkata, in eastern India, and to determine their phylogenetic background, antimicrobial resistance, and extendedspectrum beta-lactamase (ESBL) production.

Isolation and identification of E. coli
A total of 500 urine samples collected from asymptomatic pregnant women were screened for significant bacteriuria.Of these, 113 urine samples yielded significant growth.Significant monomicrobic bacteriuria was defined as culture of a single bacterial species from the urine sample at a concentration of > 10 5 CFU/mL.Only a single positive culture per patient was included in the analysis.E. coliwere further confirmed by standard biochemical tests [12].The media used during the study included MacConkey agar, Mueller-Hinton broth, and Luria Bertaini Broth (HiMedia, Mumbai, India).The cultures were incubated at 37°C for 24 hours.The E. coli isolates were sub-cultured in Luria Bertaini broth and maintained on MacConkey agar plates at 4°C for further study.The study protocol was approved by the institutional ethical committee.

Isolation of bacterial DNA
DNA for amplification was released from whole cells by boiling.Single colonies were harvested from the Luria Bertaini broth agar plates, suspended in 100 µL of sterile water, incubated at 100°C for 10 minutes, and centrifuged [13].The supernatant was used in subsequent polymerase chain reaction (PCR).

Phylogenetic analysis
The distribution of phylogenetic groups among the isolates was determined by individual PCR using gene-specific primers against the three DNA markers (chuA, yjaA,and the DNA fragment TSPE4.C2).The size of amplicons obtained as PCR products allowed the E. coli isolates to be classified into one of the four major E. coli phylogenetic lineages: A, B1, B2, or D [14].

ESBL confirmatory test
All the E. coli isolates collected from the urine of hospitalized patients resistant to at least CAZ and/or CTX were subjected to ESBL confirmatory test using CAZ and ceftazidime-clavulanic acid (CAC), and CTX and cefotaxime-clavulanic acid (CEC) combination disks.A difference of 5 mm between the zone of inhibition of a single disk and in combination with clavulanic acid (inhibitor) was confirmed as an ESBL-positive isolate [15].

Statistical analysis
The data were analyzed using SPSS version 17.0.The Chi-square test was used to compare categorical variables.A p value of < 0.05 was considered to be statistically significant after Yates correction.Correlation was ascertained by calculating Pearson's correlation coefficient.

Discussion
In this study, bacterial growth was observed in 22.6% of the asymptomatic pregnant women.The prevalence of asymptomatic UTIs has been previously reported to be 2% to 13% in pregnant women [5].This variation may be explained by the fact that there were differences in the environments, social habits of the community, socioeconomic status, and standard of personal hygiene and education of the patients who were studied.Furthermore, E. coli was the predominant pathogen associated with asymptomatic UTIs in pregnant women globally [4,5,10,13,16] as well as in India [11,17,18] and its sub-continent [6,19], as also observed in our study (38.9%).Earlier reports by Turpin et al. [20] and Akinloyeet al. [21] revealed that advanced maternal age (≥ 35 years) was a risk factor for asymptomatic bacteriuria.However, other reports worldwide [4,16,19,22,23] and nationwide suggest a high incidence of ABU in pregnant women in the 20-30 year age group [7,11,17,18], consistent with our results, which also revealed 68.2% incidence of ABU in pregnant women in the 22-29 year age group, indicating that ABU is a risk factor for asymptomatic UTIs in pregnant women in this region of the country.
A better understanding of what differentiates ABU from symptomatic bacteriuria could be particularly helpful in monitoring patients who are unable to report symptoms, and it could help to avoid complications during pregnancy.This study indicated predominance of B2 phylotype in the E. coli isolates associated with ABU (Figure 1), as shown in a previous study [1].However, significant incidence of non-pathogenic phylogroup B1 in the isolates investigated (p < 0.001) indicated association of isolates belonging to B1 phylogenetic origin in asymptomatic infection from this region in India.
In the present study, varied antibiotic resistance patterns were observed in the drug-resistant isolates, with resistance against ampicillin, ceftazidime, cefotaxime, ciprofloxacin, and co-trimoxazole being predominant.The isolates were highly sensitive to amikacin (86.4%) and nitrofurantoin (79.5%) (Table 1).These results were mostly in concurrence with other studies [11,17].Emergence of resistant ABU strains has also been reported [16]; these observations may be due to indiscriminate use of the antibiotics.Incidence of ESBL production among the MDR asymptomatic E. coli isolates was highly alarming and justifies the importance of routine screening for significant bacteriuria in pregnant women.Therefore, urine culture followed by an antimicrobial sensitivity test should be the method of choice for screening for ABU to prevent maternal and perinatal morbidity.There is no clear consensus in the literature on either the duration of therapy or the choice of antibiotic for bacteriuria in pregnancy.Increasing antibiotic resistance complicates empirical regimens, and local resistance rates need to be taken into consideration when deciding on therapy.
While it is generally assumed that virulence gene decay in ABU and bacterial adaptation to different infected hosts reflect the host response to infection, reduction in virulence potential may be related to the phylogenetic origin of the microbes.Our results reveal significant prevalence of phylogroup B1 (p ≤ 0.001)) in the asymptomatic E. coli isolates.Moreover, distribution of phylogroup B1 and B2 in the MDR isolates was significant (p = 0.03 and p ≤ 0.001, respectively) but in the drug-sensitive isolates, only B2 was significant (p ≤ 0.001).Therefore, these results indicate an association of the non-pathogenic phylogroup and drug resistance in the asymptomatic E. coli isolated from pregnant women in Kolkata.

Conclusions
The prevalence of MDR and ESBL-producing E. coli isolates associated with ABU in pregnant women from Kolkata, India, indicates that drug resistance among ABU is on the rise, and selection of appropriate antibiotics (after antibiotic susceptibility testing) is important and has to be addressed by policymakers in order to formulate a strict and safe antibiotic prescription policy for pregnant women to treat ABUassociated infection of the urinary tract.This report also, for the first time, shows a significant distribution of E. coli belonging to the non-pathogenic phylogenetic group B1 associated with ABU in pregnant women in Kolkata, India.