MOLECULAR CHARACTERIZATION OF MULTIDRUG RESISTANT BACTERIAL UROPATHOGENS FROM PREGNANT WOMEN

(A CASE STUDY OF IBADAN METROPOLIS, SOUTH-WEST NIGERIA)

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Department of Pharmaceutical Sciences

ABSTRACT
Urinary tract infection (UTI) is one of the commonest infectious diseases of women worldwide, and pregnant women are more predisposed. This study aimed to determine the prevalence of bacterial infecting agents in UTI during pregnancy, to establish their antimicrobial susceptibility profiles, identify the socio-economic factors that promote UTI. Virulence factor genes (VFGs) of the multi-drug resistant bacterial uropathogens were determined in order to establish their pathotypes. Clean catch mid-stream urine samples were collected, processed and analyzed from a total of 650 women on their first visit to antenatal clinics of three major health facilities in Ibadan metropolis. Demographic information was collected using structured questionnaires. Dip-stick leucocyte esterase test was performed and urinary isolates were identified using standard conventional biochemical tests and further confirmed to specie level with Microbact® identification kits (Oxoid). Modified Kirby-Bauer disc diffusion method was used to determine the antibiotic susceptibility profile and interpreted as specified by the Clinical Laboratory Standard Institute (CLSI).Genomic DNA extraction and amplification of specific virulence genes associated with UTI by polymerase chain reaction (PCR) was carried out. Extended spectrum beta-lactamase production by the most prevalent multi-drug resistant Gram negative isolate was determined phenotypically and genotypically using universal primers.Molecular phylogenetic analysis by maximum likelihood method (MLM) was carried out for some sequenced isolates, and an evolutionary analysis was conducted using MEGA 7®. Significant bacteriuria was observed in 246(37.8%) antenatal patients, with 149(60.6%) being asymptomatic. The major bacterial species recovered included Staphylococcus epidermidis 51(21.9%), Escherichia coli 48(19.5%), Staphylococcus aureus 27(10.9%), Streptococcusspp 17(6.0%), Providencia stuartii 12(4.8%), Proteus mirabilis 13(5.2%),Staphyloccocus saprophyticus 12(4.9%),Enterobacter agglomerans 8(3.2%). Others including Staphylococcus sppranged from 0.4% to 2.3%. The highest prevalence of bacteriuria with respect to gestational age, women?s age group, occupational status and educational level were 53.2% (n=131) third trimester, 44.2% (n=101) 25-30 years, 35.3% (n=87) housewives, and women with no formal education 32.1% (n=79) respectively and were statistically significant at P<0.05. Majority (69.5%) of the isolates were multidrug resistant. The isolates were most resistant to Cotrimoxazole (66.6%), and most susceptible to Nitrofurantoin (11.9% resistance). Phenotypic ESBL production was seen in 35(72.9%) of E. coli isolates; the ESBL genes amplified were bla CTX-M 10(100%), blaTEM 10(100%), bla OXA 10(100%), and bla SHV 7(70%). Virulence genes amplified in the analyzed MDR ESBL E. coli isolates were fim H 10(100%), pap 10(100%), cnf 5(50%), upa Bc 10(100%), qep 5(50%), Int 1 10(100%), IS 256 8(80%), amp C 8(80%), aac 1(10%), tsp 10(100%), yjaA 9(90%), but chuA gene was absent. Virulent extraintestinal phylogenetic group B2 strains of uropathogenic E. coli (UPEC) 7(70.0%) were identified. In the analyzed MDR S. aureus isolates, using simplex PCR, the following were amplified: mec A 2(20%), amp C 10(100%), ica D 8(80%), ica A 5(50%), Int 1 10(100%), IS 256 7(70%), upa Bc 10(100%), while pvl, hly, aer, qep and upa Cc genes were absent. The nucleotide sequence of 16SrRNA gene of all the MDR isolates used in the molecular analysis in comparison with those from GenBank database revealed 100-99% identity.