A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina
ISSN: 2955 – 1145 (print); 2955 – 1153 (online)
ORIGINAL RESEARCH ARTICLE
1*Bello, B.Y., 1Manga, S.B., 1A.S Shehu., 2Maigandi, A., 3Salisu, H. And 4Shamsudeen, S.
1Faculty of Chemical and Life Sciences, Department of Microbiology, Usmanu DanFodiyo University, Sokoto, Nigeria
2Faculty of Agriculture, Department of Agric and Extension, Usman Dan Fodio University, Sokoto, Nigeria
3Faculty of Life Sciences, Department of Microbiology, Federal University Gusau, Zamfara, Nigeria
4Faculty of Life Sciences, Department of Microbiology, Bayero University, Kano, Nigeria
Corresponding author: Baheera Bello Yabo
baheerabello2010@gmail.com
Urinary tract infections (UTIs) during pregnancy pose significant health risks, and the rising prevalence of extended-spectrum β-lactamase (ESBL)-producing organisms further complicates treatment. This study determined the prevalence of ESBL-producing Gram-negative uropathogens and identified associated β-lactamase genes among pregnant women in Sokoto Metropolis. Midstream urine samples were processed using standard microbiological methods. ESBL detection was performed using the Double Disk Synergy Test, and PCR was used to identify blaTEM, blaSHV, and blaCTX-M genes. Of 14 multidrug-resistant isolates, 6 (42.9%) were ESBL producers. Escherichia coli (40%) and Enterobacter sp. (100%) showed the highest ESBL prevalence. Molecular analysis revealed blaSHV in 3 (60%) of the ESBL-positive isolates, while blaTEM and blaCTX-M were not detected. The predominance of SHV-type ESBLs highlights localized resistance patterns and underscores the need for continuous surveillance and antibiotic stewardship in antenatal care.
Keywords: UTI, ESBL, SHV, multidrug resistance
Urinary tract infections (UTIs) remain one of the most common bacterial infections affecting pregnant women worldwide, and they pose significant risks to both maternal and neonatal health. Infections during pregnancy may progress to pyelonephritis, preterm labor, low birth weight, and increased neonatal morbidity (Addis et al., 2021). Over the last decade, the rising trend of antimicrobial resistance among uropathogens has become a major concern in both developed and developing countries. Among these resistant organisms, extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria have emerged as some of the most problematic multidrug-resistant (MDR) pathogens (Shrestha et al., 2016).
ESBLs are plasmid-mediated enzymes that hydrolyze a wide range of β-lactam antibiotics, including penicillins, third-generation cephalosporins, and monobactams, although they are inhibited by β-lactamase inhibitors such as clavulanic acid (Mehmood et al., 2025). The global increase in ESBL-producing bacteria is largely due to their ability to produce β-lactamases such as TEM, SHV, and CTX-M variants, as well as AmpC and OXA-type enzymes, which confer resistance to virtually all β-lactam antibiotics, including the carbapenems (Igweonu, 2024; Uyanga et al., 2020). Importantly, these resistance genes are often carried on plasmids that can be horizontally transferred among bacteria, facilitating the rapid spread of resistance (Zhang et al., 2025).
ESBL-producing uropathogens, particularly Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa, have been implicated in recurrent and chronic UTIs (Chinyere et al., 2020). Their presence has complicated management options, especially in vulnerable populations such as pregnant women. The situation is further exacerbated by the reliance of many healthcare facilities in Nigeria on dipstick urinalysis rather than urine culture and sensitivity testing, which often results in empirical treatment failure (Onwuezobe & Orok, 2015; Belete, 2020). This study therefore aimed to detect and characterize ESBL-producing Gram-negative uropathogens and to identify the β-lactamase genes (blaTEM, blaSHV, blaCTX-M) responsible for resistance among isolates obtained from pregnant women attending antenatal clinics in Sokoto Metropolis, Nigeria.
Midstream urine samples were aseptically collected from pregnant women attending selected antenatal clinics in Sokoto Metropolis. Samples were cultured on CLED and MacConkey agar and incubated at 37°C for 24 hours. Significant bacteriuria was defined as ≥10⁵ CFU/mL. Isolates were identified using standard biochemical tests and confirmed by Gram staining.
All Gram-negative isolates were subjected to antibiotic susceptibility testing using the Kirby–Bauer disk diffusion method on Mueller–Hinton agar. Isolates showing resistance to third-generation cephalosporins (cefotaxime, ceftazidime, ceftriaxone) were further screened for ESBL production using the Double Disk Synergy Test (DDST) with amoxicillin-clavulanate (20/10 µg) placed between ceftazidime (30 µg) and cefotaxime (30 µg) disks. Enhancement of the inhibition zone toward the clavulanate disk was interpreted as positive for ESBL production (CLSI, 2022).
PCR amplification of ESBL resistance genes (blaTEM, blaCTX-M, blaSHV) was performed in a 27 µL reaction mixture containing 1 µL DNA template, 1 µL each of forward and reverse primers, and 12 µL FirePol Master Mix with PCR-grade water. Positive control strains were included for each gene, while sterile water served as a negative control. PCR cycling involved: initial denaturation at 95°C for 7 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing (gene-specific) for 1 min, and extension at 65°C for 8 min, followed by a final extension at 65°C for 16 min (Uyanga et al., 2020).
Agarose gel (1%) was prepared by weighing 0.5 gm Agarose in 50 ml 1XTris- Acetate EDTA (TAE) buffer and subjected to heat until the Agarose was completely dissolved and appeared as a clear transparent solution. The Agarose solution was allowed to cool to 50°C and then 1µL of ethidium bromide (0.5µg/ml) dye was added to it. Thereafter, the gel was poured into the gel casting tray with sealed edges and a gel comb was placed into the slots on the tray. The gel was allowed to solidify 30 min and then the comb was gently removed, and the gel slab along with the running tray was submerged carefully into the electrophoresis tank containing 1X TAEbuffer. A total volume of 8 µl amplicon was transferred on a clean Para film and mixed with 2 µl of 6X gel loading dye (Biolabs, UK) and loaded carefully into the wells of Agarose gel. To determine the size of the amplified PCR product, 1Kb DNA ladder (Biolabs, UK) were loaded in the first well. Electrophoresis was performed at 70 V for 1 hour and the mobility were monitored by the migration of the dye in the gel. After appropriate migration, the Agarose gel was visualized under UV trans-illuminator in a Bio-Rad gel documentation device and the results documented (Uyanga et al., 2020).
Out of 14 multidrug-resistant Gram-negative bacterial isolates obtained from pregnant women with urinary tract infections in Sokoto metropolis, 6 (42.9%) were confirmed as extended-spectrum β-lactamase (ESBL) producers, while 8 (57.1%) were non-ESBL producers.
Escherichia coli accounted for the highest number of isolates (n = 5), with 2 (40%) identified as ESBL producers. Klebsiella sp. contributed 3 isolates, of which 1 (33.3%) was ESBL-positive. Notably, all Enterobacter sp. (n = 2) with 2 (100%) exhibited ESBL production. One out of the 2 Proteus sp isolates (50%) was ESBL-positive, while none of the Pseudomonas aeruginosa isolates showed ESBL activity (Table 1).
These findings highlight a considerable prevalence of ESBL-producing uropathogens in the study population, underscoring the need for careful antibiotic selection and infection control in antenatal care settings.
PCR amplification was conducted on the 5 phenotypically confirmed ESBL-producing isolates to detect blaCTX-M, blaSHV and blaTEM genes. Amplicons of the expected product sizes were targeted: ~550 bp for blaCTX-M, ~450 bp for blaSHV, and ~850 bp for blaTEM. (Plates 1, 2 and 3). blaSHV was the only gene detected, with bands observed in 3 of the 5 isolates tested (60.0%). No amplification was observed for blaCTX-M (0/5; 0.0%) or blaTEM (0/5; 0.0%) (Table 2).
Table 1: Prevalence of ESBL production among the bacterial uropathogens.
| Isolates | Number | ESBL producers | Non-ESBL producers |
|---|---|---|---|
| Escherichia coli | 5 | 2 (40.0%) | 3 (60.0%) |
| Klebsiella sp | 3 | 1 (33.3%) | 2 (66.7%) |
| Enterobacter sp | 2 | 2 (100%) | 0 (0.0%) |
| Proteus sp | 2 | 1 (50%) | 1 (50%) |
| P. aeruginosa | 2 | 0 (0.0%) | 2 (100%) |
| Total | 14 | 6 (42.9%) | 8 (57.1%) |
Plate 1: Agarose Gel Electrophoresis of blaCTX-M Gene Amplification [Lane M: 100 bp DNA ladder; Lane NC: Negative control; Lanes 1–5: bacterial isolates. Absence of distinct band at~550 bp in all isolates confirm blaCTX-M negative.]
Plate 2: Agarose Gel Electrophoresis of blaSHV Gene Amplification [Lane M: 100 bp DNA ladder; Lane NC: Negative control; Lanes 1–5: bacterial isolates. Bands at ~450 bp in Lanes 1, 3, and 5 confirm blaSHV positivity.]
Plate 3: Agarose Gel Electrophoresis of blaTEM Gene Amplification [Lane M: 100 bp DNA ladder; Lane NC: Negative control; Lanes 1–5: bacterial isolates, at ~850 bp no bands observed, indicating absence of the blaTEM gene]
Table 2: Molecular detection of ESBL genes among phenotypically confirmed ESBL-producing isolates
| Gene detected | Amplicon size (bp) | No. of positive isolates | (%) |
|---|---|---|---|
| blaCTX-M | ~550 bp | 0 | 0.0 |
| blaSHV | ~450 bp | 3 | 60.0 |
| blaTEM | ~850 bp | 0 | 0.0 |
In this study, 42.9% of multidrug-resistant Gram-negative bacterial isolates from pregnant women with urinary tract infections were identified as extended-spectrum β-lactamase (ESBL) producers. This prevalence is notable and indicates a significant threat to empirical therapy in the study population.
The proportion observed aligns with reports from other regions of Nigeria and sub-Saharan Africa, where ESBL rates among Enterobacteriaceae range from 30–50% (Chinyere et al., 2020; Igweonu, 2024). For instance, a study in Enugu reported 40% ESBL production among E. coli and Klebsiella isolates (Onwuezobe and Orok, 2015), comparable to our findings of 40% in E. coli and 33.3% in Klebsiella sp. Similarly, 100% ESBL positivity among Enterobacter sp in this study is consistent with reports that this genus frequently harbors plasmid-mediated resistance determinants (Zhang et al., 2025).
The presence of ESBL-producing Proteus spp (50%) further highlights the widespread dissemination of resistance mechanisms across diverse Gram-negative pathogens. Conversely, the absence of ESBL production in Pseudomonas aeruginosa may reflect its reliance on other resistance mechanisms, such as efflux pumps and AmpC β-lactamases, rather than ESBL enzymes (Belete, 2020).
The 42.9% prevalence of ESBLs observed in this study is higher than reports from some regions of Nigeria (25–35%) but comparable to findings from Ethiopia (41%) and Cameroon (45%) (Tanko et al., 2020; Addis et al., 2021; Nzalie et al., 2016). The differences may be attributed to variations in antibiotic use, infection control practices, and availability of diagnostic facilities.
The molecular results of this study showed that blaSHV was the only ESBL gene detected, present in 60.0% of isolates tested, while neither blaCTX-M nor blaTEM was identified. This finding indicates that SHV-type β-lactamases are the predominant ESBL determinant among Gram-negative uropathogens from pregnant women in Sokoto metropolis.
This observation contrasts with global patterns, where blaCTX-M is reported as the most frequently detected ESBL gene (Bevan et al., 2017; Castanheira et al., 2021). In Nigeria, Akinyemi et al. (2021) documented CTX-M as the most common ESBL type in Lagos, while Uyanga et al. (2020) reported widespread co-existence of CTX-M, SHV, and TEM among E. coli, K. pneumoniae, E. cloacae isolates in South-south Nigeria. The absence of CTX-M in the present study may reflect geographical variation in gene distribution, differences in antibiotic usage practices, or limitations related to the relatively small number of isolates tested.
The predominance of blaSHV in this study aligns with earlier reports linking SHV enzymes to Klebsiella pneumoniae and other Enterobacteriaceae. The plasmid-mediated nature of SHV facilitates horizontal gene transfer, ensuring persistence in bacterial populations and enhancing the spread of resistance (Castanheira et al., 2021). Similar findings were reported in southeastern Nigeria, where Chioma et al. (2019) also documented the presence of SHV genes in uropathogenic E. coli and K. pneumoniae from pregnant women, emphasizing the continued clinical importance of SHV enzymes in maternal UTIs.
The absence of blaTEM in this study further supports the global trend of declining TEM prevalence as CTX-M and SHV have become more dominant in clinical isolates (Onyango et al., 2018; Askari et al., 2024). However, studies from other regions have reported different patterns. For example, Maina et al. (2012) in Kenya identified CTX-M as the most predominant gene in urinary isolates, while Muriuki et al. (2022) also documented high prevalence of CTX-M (95.6%) and TEM (95.6%) in comparison to SHV (21.7%). Similarly, Ghaddar et al. (2020) in Lebanon reported CTX-M (90.7%) as the most predominant ESBL gene, followed by TEM (88.4%) and SHV (44.2%). In Somalia, Hussein et al. (2021) found TEM (71.4%) and CTX-M (66.7%) to be the most frequent, with SHV detected in only 3.2% of isolates. In Iran, Amiri et al. (2015) reported TEM (66.7%) as the most common ESBL gene, followed by CTX-M (33.3%).
Among the 14 multidrug-resistant Gram-negative isolates, 6 (42.9%) were confirmed as extended-spectrum β-lactamase (ESBL) producers, with E. coli and Enterobacter spp. showing the highest ESBL prevalence. PCR amplification of the ESBL genes revealed the presence of the blaSHV gene in 3 (60%) of 5 tested isolates; blaTEM and blaCTX-M were not detected.
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