About 0.001 ml of samples where inoculated into Nutrient agar (Oxoid, United Kingdom) and MacConkey agar (Oxoid, United Kingdom) using micropipette. The media were prepared according to the manufacturer’s instructions. After inoculating sample on culture plates, urine microscopy was also done. At 2000 g for 5 min, each urine samples (5 - 10 ml) were centrifuged (using Teco diagnostic centrifuge), followed by microscopic examination (×40 objective) of the wet preparation of the sediment. The presence of pyuria (more than 5 white blood cells per high power field (HPF)) or any bacteria per HPF was noted as significant and suggestive of UTI [2]. Under aerobic condition at 37˚C for 24 hours, the plates were incubated and colony forming units (CFU) was determined and those that had ≥10⁵ CFU/mL were considered to be significant indication of UTI and were marked for further investigation. The isolates were confirmed with Chromogenic media (Uriselect^tm 4 Agar Bio-Rad Laboratory).

Using sterile wire loop, colonies were streaked on prepared molten CHROM agar plates and were incubated aerobically for 24 hours for colour change. Identification of each organism was done following the manufacturer’s colour guide; further identification was carried out using biochemical tests following standard techniques [13].

The antibiotics susceptibility test was performed using the Kirby Bauer disk diffusion technique [14] with commercially available disks (ABTEK laboratories). A 0.1 mL of 0.5 MacFarland standardized isolates (approximately 10⁷ CFU/mL) was used and the plates were incubated at 37˚C for 16 - 18 h. And according to Clinical Laboratory Scientific Institute (CLSI) approved standard guidelines, the inhibition zones diameter of the antibiotics were measured, recorded and interpreted. The antibiotics tested against Gram-negative bacterial isolate were Augmentine Ofloxacine Gentamicin, Nalixidic Acid, Nitrofurantoin, Cotrimoxazole, Amoxicilin and Tetracycline.

Following the initial antimicrobial screening tests, the minimum inhibitory concentrations of each antibiotic were determined by using broth tube microdilution method as described by Andrew [15], in accordance with CLSI approved standard for bacteria. The antibiotics were dissolved in their different appropriate diluent. Ofloxaxine 500 mg was dissolved in 500 mls of sterile distil water, gentamycin was dissolved in distil sterile water, while nitrofurantoin 100 mg was dissolved in 100 mL of Dimethylsulphuramide (DMF). 744 µl of sterile Mueller Hinton broth was added to 256 µl of antibiotics i.e. ofloxacine, gentamycin and nitrofurantoin given a total of 1000 µl. another 1000 µl of sterile Mueller Hinton broth is added to the tube containing the antibiotics and the broth to reduce the concentration to 128, then 1000 µl of is pipette into another sterile test tube given a concentration of 128. Another 1000 µl of sterile Mueller Hinton broth is added to the tube containing 128 concentrations to reduce the concentration to 64 and then serially transferring 1000 µl from it to the next tube and so on for 16, 8, 4, 2, 1, 0.5 and 0.25 µg/ml is achieved for the different antibiotics, about 1000 µl was removed from the last tube and discarded. 700 µl of the serially diluted different concentrations is pipette into the sterile eppendorf tubes then 10 µl of the standardized test organism were dispensed into the eppendorf tubes containing the different concentrations. The negative control tubes were different concentrations of antibiotics in MHB without the test isolates, while the positive control tubes contained 1000 µl broth medium and each of the test organisms. All the test tubes were incubated for 18 to 24 hours at 37˚C. The MIC was determined visually by inspecting the tubes for turbidity post-incubation (matching the test tubes with test organisms with the corresponding negative control tubes). The MIC was reported as the lowest concentration of the test antibiotics which resulted in 100% inhibition of the test organism.

The minimum bactericidal concentration of the antibiotics was determined by further sub-culturing from the tubes which showed no visible growth in the MIC assay onto freshly prepared Mueller Hinton agar (MHA) plates. The culture plate containing bacteria were incubated at 37˚C for 24 hours. The MBC was therefore taken as the lowest concentration that did not show any visible growth on the sub-cultured MHA plate [15].

Multiple Antibiotic Resistance Index (MARI), has been shown to be a cost effective and valid method of bacteria source tracking. Multiple antibiotic resistance indexes are calculated as the ratio of number of antibiotics to which an organism is resistant to total number of antibiotics to which the organism has been exposed [16]. The MAR indices of the isolates were calculated and noted.

Results were analysed using SPSS software version 25.0 for Windows (SPSS Inc. 2017 Chicago, Illinois, USA). Categorical variables were tested for association using Pearson Chi-square and Fisher exact test as appropriate. Significant level was set at P value of 0.05.

Of the 489 urine samples examined, the overall prevalence of urinary tract infection was 26.4%. The prevalence rate of UTI in the investigated areas such as Nsukka, Otukpo, Gboko and Kastina Ala was 31.8%, 17.5%, 34.3% and 17.1%, respectively (Table 1). Out of 130 positive samples, the prevalence of UTI was

NE: Number examined, NI: Number infected, P: Prevalence, Statistically significant at p < 0.05.

higher in males 81 (30.9%) than in females 49 (21.6%), but there was no statistically significant association between gender and UTI (p = 0.636) (Table 1). The age categorizations of the study subjects were 2 - 5, 6 - 9 and 10 - 12. This study revealed that the prevalence of UTI was greater among the age 2 - 5 (28.2%), followed by the age group 6 - 9 (26.9%), while the age group 10 - 12 had the least prevalence of 21.5% (Table 1); although there was no statistically significant association between UTI and the age groups (p = 0.870).

The result of dipstick urinalysis showed that out of 489 sample examined, 36 (7.4%) was positive for nitrite; 33 (6.8%) for bilirubin, 8 (1.6%) for blood, 10 (2.0%) for urobilinogen, 111 (22.7%) for protein, 31 (6.4%) for ketone, 86 (17.6%) for ascorbic acid and 22 (4.5%) for glucose. The pH categorisation shows that 292 (59.8%) had acidic pH urine followed by 142 (29.1) for neutral and 53 (10.9%) alkaline pH (Table 2).

The prevalence of uropathogens among children attending hospitals in all the study areas according to urine samples culture, showed that four Gram-negative organisms were isolated and they are Klebsiella pneumonae, E. coli, Pseudomonas spp. and Proteus mirabilis (Figure 1(a)). Klebsiella pneumonae, was the most predominant organism (88.8%) and it was more isolated from male children, followed by E. coli. It was observed that E. coli was the most predominant organism isolated from the female children (40.6%), and also Pseudomonas spp was seen more in male children (45.0%) and Proteus mirabilis was the least (13.8%) (Figure 1(a)). The bacteria with highest prevalence in Nsukka, Otukpo, Gboko, and Kastina Ala were Klebsiella pneumonae (46.4%) (Figure 1(b)), Klebsiella spp (58.3%) (Figure 1(c)), E. coli (38.5%) (Figure 1(d)), and Klebsiella spp (42.0%) (Figure 1(e)), respectively.

Evaluation of antibiotic susceptibility patterns of isolates to antibiotics used in the treatment of UTI in children presenting at some hospitals in the study areas.

The in-vitro antibiotic susceptibility tests were made on a total of 52 randomly selected isolates, which includes 16 from Nsukka, 12 from Otukpo, 16 from Gboko and 8 from Kastina Ala. This study showed that most of the isolate were highly resistant to Augmentin, Cotrimoxazoel, Amoxicilin and Tetracycline, while some of the isolate shows intermediate resistant to Nitrofurantoin and Nalixidic acid. Ofloxacine and Gentamicin were the most effective antibiotics against the isolates from all the study areas (Supplementary materials: Tables 1-4). K. pneumoniae isolates from Nsukka (56.3%), Otukpo (55.6%) and K. Ala

KEY: R = Resistant (+Intermediate), S = Sensitive. AUG = Augmentin, OFL = Ofloxacin, GEN = Gentamicin, NAL = Nalixidic Acid, NIT = Nitrofurantoin, COT = Cotrimoxazole, AMO = Amoxicilin, TET, Tetracycline.

(53.1%) were the most resistant to the test antibiotics, while E. coli isolates from Gboko had highest percentage of resistance (Table 3). More than 50% of each of the bacteria isolates were resistant to NAL, COT and TET (Table 4).

Overall average MAR index of all the isolates from the different region revealed that Pseudomonassp had the highest MARI of 0.80, followed by K. pneumoniae (0.73), and P. mirabilis (0.72) and E. coli (0.64) (Figure 2(a)). The average MAR index of isolates from the different study areas shows that Pseudomonassp isolates from Nsukka had the highest MARI at 0.9, followed by K. pneumoniae (all locations) and P. mirabilis (Nsukka) (0.8). E. coli had the least MAR index of 0.6 (Figure 2(a)).

The MICs of nitrofurantoin against Pseudomonas sp, K. pneumoniae, P. mirabilis, and E. coli isolates from all the study areas ranged from 8.0 - 128.0, 16.0 - 128.0, 16.0 - 64.0, and 8.0 - 64.0 µg/ml, respectively, while the MBCs were 64.0- > 128.0, 32.0- > 128.0, 128.0- > 128.0, and 32.0- > 128.0 µg/ml, respectively (Tables 5-8). The MICs of ofloxacine against the sameisolates ranged from 4.0 - 16.0, 4.0 - 32.0, 8.0 - 16.0, and 0.5 - 16.0 µg/ml, respectively, while the MBCs were 8.0 - 128.0, 16.0 - 128.0, 16.0 - 64.0, and 8.0 - 32.0 µg/ml, respectively (Tables 5-8). The MICs of gentamicin against the sameisolates ranged from 4.0 - 16.0, 8.0 - 64.0, 8.0 - 64.0, and 8.0 - 32.0 µg/ml, respectively, while the MBCs were 32.0- > 128.0, 32.0- > 128.0, 32.0 - 128.0, and 16.0 - 64.0 µg/ml, respectively (Tables 5-8).