Patients admitted to the Protestant Hospital of Ngaoundere with UTIs and antibiogram were included. Before inclusion in the study, an informed consent was signed by all participants. This cross-sectional study was conducted from April 2023 to August 2023. Sociodemographic data for these patients were recorded based on the documented of clinical diagnostics in their medical files. Sampling was performed during the study periods from patient who did not receive antimicrobial drugs at least seven days. Urine samples were collected at morning before first urination or 4 hours after a last miction. The urine sample were immediately transferred to the microbiology laboratory in the Protestant Hospital of Ngaoundere.

The samples were processed according to the standard procedures and germs were identified according to the European Committee on Antimicrobial Susceptibility Testing (CA-SFM/EUCAST) [12]. In brief, specimens were inoculated to medium (Cled) plate and incubated at 37˚C for 18 H to 24 H. Microscopical examination of Gram-stained smears confirmed. Bacterial colonies were subjected to biochemical reaction (oxidase, catalase, API 20E). Susceptibility of isolated bacteria to various classes of antibiotics was determined on Mueller Hinton agar (MHA) by Kirby-Bauer disc diffusion method, using commercial disks according to Clinical and Laboratory Standards Institute (CLSI) [13]. The isolates germs were determined against Amoxicillin/clavulanic acid (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), cefuroxime, cefixime, imipenem (10 µg), gentamicin (10 µg), Tobamycin (10 µg), Erythromycin, azithromycin, ciprofloxacin (5 µg), Norfloxacin (30 µg), Nitrofurantoin (50 µg).

The antimicrobial susceptibility testing of all isolates was done by the standard Kirby-Bauer disk diffusion method, using commercial disks according to Clinical and Laboratory Standards Institute (CLSI). When pure culture was obtained, a loopful of bacteria was then taken from a colony and transferred to a tube containing 5 mL of sterile distilled water and mixed gently until it formed a homogenous suspension. Then, the turbidity of the suspension was adjusted to the density of a McFarland 0.5 (Mary-l’Etoil, year) in order to standardize the inoculum size [14][15].

Antibiotic susceptibility testing was performed by Kirby-Bauer’s disk diffusion method on Muller-Hinton agar whereby 50 µl of bacterium suspension prepared above was poured on to the surface of the agar. A swab was then used to distribute the bacteria evenly over the entire surface of Mueller-Hinton agar and the surface was allowed to dry for about 5 minutes before placing antibiotic disc. Individual antibiotic dispensers were placed onto the surface of the agar using a pair of sterile forceps and the latter was then incubated upside down for 24 hours at 37˚C.

Antibiotic disks used are selected in relation to the frequent treatment used by the clinicians and pharmaceutical presentation depends on the age of the patients. The antibiogram was interpreted based on CLSI guidelines as follows. The results were reported as sensitive and resistant.

2.4.1. Screening of β-Lactamases Enzymes

1)ScreeningofExtended-Spectrumβ-Lactamase(ESBL)enzyme

Double-disk synergy test was applied to determine the presence of extended-spectrum β-lactamase (ESBL) production. Chances of ESBL enzyme screening were carried out if we observed any resistance in ceftazidime (caz ≤ 22 mm), or cefotaxime (ctx ≤ 27 mm) and cefodoxime ≤ 17 mm, ceftriazone (all third generation cephalosporins). A susceptibility disk containing amoxicillin-clavulanic acid was placed at the center of the plate, and disks containing ceftazidime (30 µg) was placed 30 mm from the amoxicillin-clavulanic acid (30 µg) disk. ESBL producing isolate was indicated by an extension of the inhibition zone of amoxicillin-clavulanic acid disk that is ≥5 mm than that of ceftazidime after 48 hours incubation at 37˚C aerobically and the result was noted [16].

2)ImipenemEDTACombineddisctest(CDT)forMetallo-betalactamase(MBL)

If any isolates from the antibiogram yielded a resistance in Imipenem or Ceftazidine or Meropenem, we proceeded to test for MBL production. An exactly 0.5 McFarland suspension of the pure bacteria culture was inoculated on the surface of Mueller-Hinton agar plate and then two imipenem (10 µg) and imipenem (10 µg) plus EDTA (20 µl of 0.1 M) disks was placed at distance of 25 mm apart and incubate the plate overnight at 37˚C. If the zone of inhibition (ZOI) of imipenem (10 µg) plus EDTA (20 µl) disks was greater than or equal to 5 mm than that of imipenem (10 µg), then the organism is an MBL producer [17].

2.4.2. Meticillin Resistant Staphylococcusaureus

An exact 0.5 McFarland suspension of the pure bacteria culture was inoculated on the surface of Mueller-Hinton agar plate and cefoxitin (10 µg) disks was placed and the plate incubate overnight at 37˚C. If zone of inhibition (ZOI) of Cefoxitin was less than or equal to 21 mm, the isolate was a Meticillin resistant Staphylococcusaureus (MRSA) strain (https://universe84a.com/collection/beta-lactamases/).

2.4.3. D-Zone Test (Erythromycin-Resistant Staphylococcusaureus (cMLS_B, iMLS_B and MS Strains)

For detecting erythromycin and clindamycin resistance, 15 μg erythromycin disks and 2 μg clindamycin disks were used. Interpretation of the diameters of zones of inhibition was as follows: for erythromycin = 23 mm; S, 14 - 22 mm; I, = 13 mm; R, and for clindamycin = 21 mm; S, 15 - 20 mm; I, = 14 mm; R. Intermediate resistant strains were considered resistant. Erythromycin-resistant S.aureus strains were selected for further studies [5]. D-testing was performed for erythromycin-resistant S.aureus strains according to the guidelines of the CLSI. Suspension equivalent to 0.5 McFarland of each freshly cultured isolate in sterile distilled water was prepared and used for inoculation of Mueller-Hinton agar plates. Erythromycin and clindamycin disks were placed on inoculated plates 15 mm apart (edge-to-edge). Plates were read after 18 h - 24 h incubation at 35˚C and the shape of the clindamycin zone was verified. Strains resistant to both antibiotics were considered to have cMLS_B resistance. Strains with flattening of the susceptible zone of inhibition to clindamycin adjacent to the erythromycin disk (D-shape) were considered to contain iMLS_B-resistance, while strains with circular zones were considered to contain MS resistance [18].

Overall, 108 patient’s urine samples were included in the current study. It shows that out of the 108, 30.56% (33) consisted of males while 69.44% (75) were females, a sex ratio of 2:27 in favour of the females (Table1). The overall prevalence of UTI infection is 37.03%. Of them 23 (57.5%) females and 17 (42.5%) males were infected.

Table1. Age and gender distribution in acquired UTI patients.

In our study, E.coli was the most encountered Enterobacteriae with 35% (14/40) followed by K.pneumoniae, 10% (4/40), while S.aureus on the other hand was the most encountered Gram-positive cocci, 25% (10) as shown in Table2 below.

Table2. Prevalence of each isolate obtained.

Most bacterial isolates were found resistant to imipenem (72.5%), amoxicillin/clavulanic acid 57.5%, ceftazidime (45%) but maximum sensitive to tobramycin (82.5%), ciprofloxacin (7.5%), cefixime (12.5%), cefuroxime (12.5%). Table3 shows the antibiotic susceptibility pattern of the all-bacterial isolates in this study. E.coli yielded a higher resistance rate for Amoxicillin/clavulanic acid (57.1%), ceftazidime (50.7%), and imipenem (71.4%) among K.pneumoniae more than 50% resistance was seen to gentamycin (75%), imipenem (100%), Amoxicillin/clavulanic acid (75%), ceftazidime (50%). S.aureus yielded a higher resistant rate to imipenem (100%) followed by erythromycin (70%) and azithromycin (50%).

Table3. Antimicrobial resistant rates of Enterobacteriaceae (n = 30) and Staphylococcus (n = 10) collected from clinical urine specimens of patients.

Out of 27 Enterobacteriaceae screened for ESBL production, 25.92% (7) were positive indicated by an extended of the zone of inhibition ≥ 5 mm of amoxicillin/clavulanic acid than ceftazidine. The highest frequency of ESBL enzymes was found among K.pneumoniae isolates (50%) (Table4).

Table4. Frequency of ESBL, MBL among Enterobacteriaceae members detected by DDST and imipenem EDTA combined disc test (CDT) phenotypic laboratory methods. E.coli presented the most ESBL phenotypes (57%), followed by K.pneumoniae (29%) and (14%) then other gram-negative rods.

ESBL: Extended spectrum beta lactamase MBL: Metallo beta lactamase.

Among the 10 Staphylococcusaureus isolate tested, 23.07% (3/10 strains) expressed the MRSA strain, with a ZOI ≤ 21 mm of Cefoxitin. For the D-test, 38.46% (5/10 strains) were found to exhibit the constitutive MLS_B resistance, and 15.38% (2/10 strains) the inducible MLS_B resistance, while 23.076% (3/10) expressed the MS_B resistance (Table5).

Table5. Frequency of iMLS_B, cMLS_B, MS_B and MRSA in Staphylococcusaureus detected by different phenotypic laboratory methods.

D-test: disk-test; iMLS_B: Induicible Macrolide Lincosamide Streptogramin B; cMLS_B: Constitutive Macrolide Lincosamide Streptogramin B; MS_B: Macrolide Streptogramin B; MRSA: Meticillin resistant Staphylococcusaureus.