Escherichia coli bacteriuria in pregnant women in Ghana: antibiotic resistance patterns and virulence factors

Objectives The relevance of Escherichia coli associated bacteriuria infection in pregnant women is poorly understood, despite these strains sharing a similar virulence profile with other pathogenic E. coli causing severe obstetric and neonatal infections. We characterized and determined the antimicrobial susceptibility, resistance genes and virulence profiles of 82 E. coli isolates associated with asymptomatic bacteriuria in some pregnant in Ghana from February to August 2016 using Kirby–Bauer disc diffusion and polymerase chain reaction. Results High levels of antimicrobial resistance were observed to ampicillin (79.3%), tetracycline (70.7%) and cotrimoxazole (59.8%), except for cefuroxime (32.9%). Resistance genes analyses revealed 58.5% were positive for BlaTEM and 7.3% for aph(3)-Ia(aphA2). Virulence factors (VFs) was more widespread in pregnant women in the 2nd and 3rd trimesters than 1st trimester. VFs relating to adhesion (papC and iha), Protectins (traT), aerobactin acquisition (iutA) and iron acquisition systems (fyuA and irp2) were more prevalent in the resistant E. coli isolates. This study provides evidence for a link in bacteriuria and transmission of extra-intestinal E. coli in pregnant women to cause multi-resistant obstetric or neonatal infections. Considering the involvement of extra-intestinal E. coli in infections, results are helpful to develop strategies to prevent maternal and/ neonatal infections. Electronic supplementary material The online version of this article (10.1186/s13104-018-3989-y) contains supplementary material, which is available to authorized users.


Introduction
In pregnant women, the odds of acquiring urinary tract infections (UTI) from untreated bacteriuria is high, with consequent risk for preterm labour [1]. Pregnant woman diagnosed with bacteriuria are thus offered antibiotics to prevent complications [2,3]. The extraintestinal pathogenic E. coli (ExPEC) are a major cause of UTI in pregnancy [4][5][6]. The ExPEC harbour diverse but specific virulence factors (VFs) with the potential to colonize highly specialized ecological niches, such as the urogenital tract [7][8][9][10][11]. Furthermore, multidrug-resistance traits in many ExPEC strains involved in bacteriuria is increasing accounting for considerable amount of morbidity, and can lead to significant mortality in pregnant women with UTI [12][13][14][15]. There is however paucity of data in sub-Sahara Africa where such infections are likely to be common and devastating due to high exposure to infectious organisms and limited access to health care services [1,[13][14][15][16][17][18][19][20]. In Ghana, the aetiology of E. coli in bacteriuria are well documented [21][22][23][24][25] but often limited to phenotypic tests with little knowledge on virulence factors. This study aimed to characterize ExPEC strains, and determine their virulence and antimicrobial resistance potential in urine samples of pregnant women.
reported prevalence of UTI among pregnant women in Ghana (56.5%) [26], and D = allowable margin error of 0.05. The 400 pregnant women were recruited into this study after the provision of informed consent. Pregnant women on antibiotics were excluded. A self-administered questionnaire was used to obtain information on demographic and socio-economic characteristics (Additional file 1: S1 file). Mid-stream-clean-catch urine from participants were inoculated onto cysteine lactose electrolyte deficient (CLED) agar and incubated at 37 °C for 24 h [27]. Bacteria isolates were speciated with API 20E identification system (bioMerieux, France). For purposes of this study, only E. coli cultures were further analyzed [28].

Molecular characterization
Colonies of fresh bacterial culture were suspended in 200 ml of sterile water. The suspension was heated at 98 °C for 10 min and centrifuged at 17,900g for 5 min.

Statistical analysis
Data was analysed using GraphPad Prism software, version 6. Bacteriuria was defined as bacterial growth > 10 5 colony forming units/mL per urine sample on CLED. Associations between socio-demographic characteristics and development of UTI, phenotypic resistance, and virulence factors were done using Chi square test. p-values < 0.05 were considered significant.
In total, 49 (59.7%) ampicillin resistant isolates contained Bla TEM (Table 2). Pregnant women in the 2nd (24 isolates) and 3rd (18 isolates) trimesters had E. coli isolates with more Bla TEM gene compared to women in their 1st trimesters (5 isolates). The aminoglycoside genes aph(3)-Ia(aphA2) for gentamicin resistance was in 6 isolates from pregnant women in their 2nd and 3rd trimesters ( Table 2). All the E. coli isolates were screened for the presence of intI and intII, however only 10 of isolates were positive for intI, whilst two E. coli isolates contained intII, 58 of the isolates did not possess either intI or intII.
The distribution of the 82 E. coli isolates in relation to virulence genes from the various groups of pregnant women revealed 75.6% (62 isolates) E. coli contained two or more virulence genes (VFs) ( Table 2). The virulence score used to classify the ExPEC isolates was calculated using the total number of VFs genes. Isolates were classified as ExPEC if they were positive for two or more of the tested virulence genes [5]. The iutA (aerobactin acquisition), papC and iha (adhesins), fyuA and irp2 (iron capture systems), traT (protectins) were the common detected genes, whereas usp (uropathogenic-specific proteins) and some of the adhesin genes (hra, ibeA, and papG1) were the least.
VFs was widespread in pregnant women in the 2nd (30 isolates) and 3rd (25 isolates) trimesters than 1st trimester (12 isolates) ( Table 2). In this study, all the E. coli isolates in women in their 1st trimester were ExPEC. Whilst 19 pregnant women in their 2nd trimesters (20-29 years) were positive for ExPEC, 13 women in the 3rd trimester (20-29 years) were positive. In addition, in the age group 40-49 years, only 2 women in the 3rd trimester were ExPEC positive, whilst one woman in the 2nd trimester was positive (Table 2).

Discussion
There are few studies on the antimicrobial susceptibility and/or virulence of E. coli isolates colonizing the genital tract of pregnant women [39][40][41]. However, no studies have been carried out to compare virulence factors and antimicrobial resistance in E. coli from pregnant in Ghana. This study revealed the 42.75% of the pregnant women with UTI was slightly lower than the 56.5% previously reported in Ghana by Boye et al. [26]. Although findings are similar to 47.5% from Nigeria [42], it is lower than the 85% reported by Turay et al. [43]. However, the 42.75% in this study is higher than reports from Thailand (5.1%) and Ethiopia (18.8%) [44,45]. The difference in prevalences may be attributed to varied genital hygiene and socioeconomic conditions [46]. E. coli accounted for 47.95% of the UTI cases in the pregnant women. This is in conformity with   (1) previous studies from Sudan, Bangladesh, and Nigeria [47][48][49]. The high incidence of E. coli associated with UTI among the pregnant women may be attributed to poor genital hygiene practices [50]. Multiparity, gestational age, history of UTI and anatomic urinary tract abnormalities are reported to affect the frequency of bacteriuria during pregnancy [51,52]. Pregnant women in their 3rd trimester recorded the highest incidence of UTI (49.13%), followed by those in 2nd trimester (43.25%). Finding are conformity with studies from Bangladesh, Iran Ethiopia, Yemen, and India [48,[53][54][55]. Although Chi square exact test revealed a statistical association of UTI and gestational age (p = 0.002), it is in contrast to studies from Nigeria [20,43]. Furthermore, the prevalence of UTI was found to increase with parity in this study. This findings however are in contrast to Emiru et al. [51] and Nandy et al. [56] studies.
Escherichia coli isolates were highly resistant to ampicillin, and tetracycline. Findings are similar to earlier studies in Ghana [21,57,58]. The high levels of resistance can be attributed to abuse of these drugs over the years because the drugs are relatively cheap and easily accessible [21,58]. A considerable number of the bacteria harboured the iutA (aerobactin acquisition), papC and iha (adhesins), fyuA and irp2 (iron capture systems), and traT genes [59,60]. In contrast to Sáez-López et al. [41] study with pregnant women in Barcelona, the ExPEC isolates in this study showed high antimicrobial resistance as previously reported in some African countries [61,62]. In addition, the ampicillin resistant ExPEC isolates containing Bla TEM gene showed a greater number of VFs in comparison with tetracycline or gentamicin resistant isolates. Our findings however, are dissimilar to Ramos et al. [63] study with pregnant women in Sweden, Uganda, and Vietnam [63]. The differences in the studies may be due to varying geographical area, host physiological changes or susceptibility to E. coli isolates with pathogenic islands containing VFs [64].

Conclusion
In conclusion, our results demonstrates that antibiotic resistant ExPEC associated with UTI in some Ghanaian pregnant women have virulence properties which enables them to adhere, invade and utilize the iron acquisition systems. Information from this study is useful to develop appropriate interventions to avoid maternal and/neonatal infections with asymptomatic pathogens during obstetric care.

Limitations
The study focused on asymptomatic infection rather than symptomatic infection and included only few hospitals in Ghana, thereby not allowing extrapolation of our results to other regions.

Additional file
Additional file 1: S1 File. Questionnaire for demographic data collection.