Prevalence and molecular analyses of extended spectrum β-lactamase producing uropathogens among pregnant women, Jigawa state, Nigeria

Citation: Chinyere EL, Nura SM, Ahmad GM, Kemi AF, Sani MN (2020) Prevalence and molecular analyses of extended spectrum β-lactamase producing uropathogens among pregnant women, Jigawa state, Nigeria. J Clin Microbiol Biochem Technol 6(1): 033-038. DOI: https://dx.doi.org/10.17352/jcmbt.000041 increase in antibiotic resistance among these uropathogens are due to their ability to hydrolyse virtually all the beta-lactam antibiotics including the 3rd generation cephalosporins and carbapenems by producing ESBLs such as CTX-M enzymes (CTX-M-14 and CTX-M-15) or AmpC -lactamases and OXAtype beta-lactamases [2,3].


Introduction
Beta-lactamases are enzymes produced by bacteria that breakdown a particular class of antibiotics called the beta-lactams. As the bacteria developed resistance to one type of beta-lactam antibiotic, new antibiotic derivatives (cephalosporins, carbapenems and monobactams), which inturn made the bacteria to continually evolved and developed new set of enzymes described as extended-spectrum betalactamases (ESBLs) that can break down the newer derivatives . ESBLs are plasmid mediated enzymes that confer resistance to penicillin, third generation cephalosporins and aztreonam but are said to be inhibited by clavulanic acid [1]. The global Citation: Chinyere  ESBL producing bacteria such as E. coli, Klebsiella sp, Proteus sp and P. aeruginosa, have been found to continuously develop resistance to several antibiotics, which has resulted to wide spread cases of chronic urinary tract infections (UTI) in pregnant women. It has been widely reported that the bacteria harbour antibiotic resistant genes which can be horizontally transferred to other bacteria thus call for urgent research on their prevalence and management [5].
This study, intend to report on the prevalence and molecular typing of ESBL uropathogens among pregnant women, thereby providing an avenue for policy makers and researchers to build on effective management policies and public health strategies.

Study site
This study was carried out at the Rasheed Shekoni Teaching Hospital and Dutse General Hospital, Jigawa State, Nigeria.
According to National Population Commission, Jigawa state is one of the developing cities in the North-west of Nigeria, and is one of the less populated state in the Northern region.

Study population
The studied populations include samples from 200 pregnant women randomly collected attending Rasheed Shekoni Teaching Hospital and Dutse General Hospital Jigawa State, Nigeria. About 100 samples were collected from each hospital individually.

Samples collection
Structured questionnaires were used to collect demographic and clinical information from all patients. Clean catch midstream urine was aseptically collected and processed immediately into the Microbiology laboratory.

Specimen processing and isolation of the uropathogens
Laboratory analysis of the urine samples including macroscopic examination was conducted, to check out the color, turbidity and presence of blood. The uncentrifuged urine samples were mixed by rotating the plastic container before inoculating the surface of MacConkey agar and nutrient agar, followed by incubation at 37˚C for 24 h [6].

Identifi cation and characterization of the bacterial isolates
Further studies and identifi cation of the bacterial isolates was based on colony morphology, Gram staining, and biochemical characterization using standard method [6].

The antibiotic susceptibility tests
The antibiotic susceptibility test was carried out using the Kirby-Bauer disc diffusion method; isolated colonies was inoculated in Muller Hinton Agar plate and spread evenly over the surface, then the disc of commercially available discs (Oxoid Uk)) with standard antibiotic concentrations of Ceftazidime (30μg), Cefotaxime (30μg), Tetracycline (30μg), Augmentin (30μg), Ofl oxacin (5μg) and Gentamicin (10μg) were placed on the culture plate and incubate at 37˚C. After 24h the zone of inhibition around the discs were observed following recommendations of the Clinical and Laboratory Standard Institute [7].

Determination of β-lactamase production by the bacterial isolates
The double-disc synergy test was used for confi rming the identifi ed as potential ESBL producers [7].

Molecular analysis
These isolates were subjected to molecular characterization using 16S rRNA amplifi cation and sequencing. The product of PCR amplicons was separated using gel electrophoresis. The sequence was parsed for genomic analogy using EMBL Blastn database, in reference to Sun [8]. The MEGA-X software was used to construct the phylogenetic neighbor joining tree for species relationship. In addition, the evolutionary distance was established using bootstrapping.

Statistical analysis
The data obtained were analyzed using Statistical Package for Social Sciences (SPSS 20), all observed data was recorded as ±SEM (Standard Error of Means). Statistical signifi cant established at 95% confi dence interval P  0.05.

Results
The prevalence of UTI among the pregnant women was found to be 37.0%.

Demographic date of patient in relation to UTI
The prevalence of UTI is highest in DGH 54(27.0 %) than in RSTH 20(10 %), as clear in (Table 1). The age group UTI distribution within the studied population showed that the prevalence is lowest (4.5 %) in age group of 31-40, and highest (27.5 %) in age group of 21-30. Results showed that there is no statistical signifi cance difference in the prevalence of UTI(P<0.05) within the age group (X 2 = 5.759; P =0.056).
However, highest incidence of UTI is recorded in second trimester (19.0 %) followed by fi rst trimester (10.0 %), and is least in the third trimester (8.0%).

Prevalence of ESBL production among the bacterial uro pathogens
The recorded prevalence of UTI among the pregnant women is 74(37.0%) presented in Table 2

Susceptibility pattern of the isolated uropathogens to the selected antibiotics
The antibiotic susceptibility assay revealed the increased resistance of the uropatogens against Ofl oxacin, Gentamicin and Tetracycline (Table 3). The mo st effective antibiotic is ceftazidime (31.1%) that caused inhibition to almost all of the isolated bacterial uropathogens.

Comparative distribution of the ESBL-producing isolates between the two hospitals
Comparative distribution of the ESBL-producing bacterial isolates between the two hospitals is shown in Figure 1

Gene extraction and amplifi cation
The product of PCR amplicons were separated using gel electrophoresis are presented in Figure 2 and DNA base pair of 750pb

Post PCR phylogenetic analysis
The results for phylogenetic analaysis and molecular systematics of the isolates were presented in The highest incidence was observed among age group of 21-30(27.5%) and the lowest seen in 31-40(4.5%). This could probably be due to the fact that greater number of the subject fall within ages 21-30 which represents sexually and reproductively active age group. The analysis showed that there was no statistical signifi cant difference (X 2 =5.759 P=0.056) in the prevalence of UTI between the age groups. This study observed that multigravida had an increased risk factor of developing bacteriuria among pregnant women, which could be as a result of their reproductive system becoming more vulnerable to infections after successive pregnancies. Although this differs with that of Ranjan [12] who reported highest incidence in primigravida and lower incidence in multigravida (Table 1). More so, in this study, highest incidence of UTI is seen in second trimester (19.0%) followed by fi rst trimester (10.0%) and least is seen in third trimester (8.0%), the result was in contrast with Ranjan [12] whose highest incidence of UTI    [20], Onwuezobe [4], and Motayo [21] respectively. The antibiotic susceptibility testing revealed increasing resistance with Ofl oxacin, Gentamicin and Tetracycline that resisted strains of all isolated uropathogens (Table 3). This low sensitivity of these drugs may   be due to development of resistance genes by pathogens as a result of antibiotic abuse or self-medication [21][22][23][24][25][26].
In this study, the most effective antibiotic is ceftazidime that was sensitive to strains of all isolated uropathogens. The possible reason for the clinical effectiveness of ceftazidime compared to other antibiotics may be due to its limited use and abuse by patients. The molecular enumeration of ESBLproducing uropathogens was observed using 16S rRNA partial sequencing analyses [26][27][28][29][30][31][32][33].

Conclusion
The observed prevalence of UTI among pregnant women in this study was 37.0%. Past history of UTI, contraceptive use and cleaning method after urination or defecation contributed to constitute risk factors for acquiring UTI and the predominant uropathogens are Alcaligenes, Klebsiella species, S. aureus and Myroides. The prevalence of ESBL-producing Gram-negative uropathogens was 17.6%.
The ESBL-producing bacteria among UTI pregnant was found to be associated with risk factors such as past histories of exposure to a specifi c antibiotic, UTI and recent antibiotic use.