Prokaryotes (bacteria, yeast, etc) store their DNA only in the cytoplasm as cytoplasmic DNA (Ikwuka, 2023a). The discovery of antibiotics in the early twentieth century was a huge milestone achievement, especially for therapeutic and prophylactic purposes against a variety of bacterial infections. However, with time, some of these microorganisms continued to become resistant to these antibiotics. This resistance poses a huge threat to antibiotic therapy and puts a substantial clinical and financial burden on the healthcare system. The major cause of this phenomenon as observed by (Arlet, 2006) is the spread of plasmid-encoded Extended-Spectrum Beta-Lactamase (ESBL) genes, conferring resistance to third-generation cephalosporins introduced into the clinical practice in the early 1980s as a major breakthrough in the fight against beta-lactamase-mediated bacterial resistance. 

β-lactamases possess the ability to breakdown third-generation cephalosporins and aztreonam; and yet are inhibited by clavulanate. Moreover, ESBL-producing organisms also manifest co-resistance to many other classes of antibiotics, resulting in limited treatment option (Rawat, 2010). It has been established that chronic metabolic disorders have the ability to compromise immunity due to the activity of different systemic, immune inflammatory processes. Metabolic disorders e.g. Hypertension, Adiposity, Diabetes mellitus and Dyslipidemia collectively known as Metabolic Syndrome Diseases (MSDs) are diseases related to one another and have very high morbidity and mortality rates (Ikwuka, 2015; Ikwuka, 2017a; Ikwuka, 2017c; Ikwuka, 2023c; Ikwuka, 2023f; Virstyuk, 2016). Results obtained from different researches have shown that hypertension, diabetes mellitus, adiposity and dyslipidemia, asymptomatic hyperuricemia, systemic immune inflammation activation and fibrogenesis, can lead to kidney damage (Ikwuka, 2017d; Ikwuka, 2017e; Ikwuka, 2018a; Ikwuka, 2018c; Ikwuka, 2018d; Ikwuka, 2019a; Ikwuka, 2019c; Ikwuka, 2022; Ikwuka, 2023d; Virstyuk, 2017a; Virstyuk, 2018a; Virstyuk, 2019; Virstyuk, 2021a; Virstyuk, 2021b). Published research on ESBLs has now originated from more than 30 different countries, reflecting the truly worldwide distribution of ESBL-producing organisms. Extended-spectrum β-lacta-mases (ESBLs) are a group of plasmid-mediated, diverse, complex and rapidly evolving enzymes that are posing a major therapeutic challenge today in the treatment of hospitalized and community-based patients. Infections due to ESBL producers range from uncomplicated urinary tract infections to life-threatening sepsis (Rawat, 2010). Linked with the induction of oxidative stress are major free radicals. Among these major free radicals, superoxide anion, hydroxyl radical, and hydroperoxyl radical are of physiological significance. Non-radical of physiological significance is hydrogen peroxide (Ikwuka, 2023b, Ekechi, 2023a; Ama, 2023).

It has been noted that the treatment of choice for serious infections due to ESBL-producing organisms are Carbapenems, yet isolates resistant to Carbapenems have been reported recently (Rawat, 2010). ESBLs show an excellent example of the ability of gram-negative bacteria to develop novel resistance mechanisms to antibiotics mechanisms especially in the start of new antibiotic (Rawat, 2010). The rapid spread of ESBL-producing bacteria has gradually become of global importance, necessitating prompt actions such as continuous monitoring systems and thus, there is need for effective control of infections and public health practices which prevent the spread of infections. The change of anti-biotics in order to reduce the spread of antimicrobial resistance is also a considerable intervention strategy (Rawat, 2010). Use of antibiotics that kill bacteria in the colon which produce Vitamin K can lead to Vitamin K deficiency, which in turns results in decreased production of clotting factors II, VII, IX and X. Implicated antibiotics are broad-spectrum antibiotics e.g. fluoroquinolones, cephalosporins and other penicillin derivatives (Ikwuka, 2023e). Patients with Vitamin K deficiency are prone to different degrees of bleeding which can lead to anemia (Musa, 2023; Inya, 2023a; Inya, 2023b). Nevertheless, there is also need for new and effective treatment options in patients with Metabolic Syndrome Diseases. Sodium-Glucose Linked Transporter 2 (SGLT-2) inhibitors e.g. Dapagliflozin and Glucagon-like Peptide 1 Receptor Agonists (GLP-1 RAs) e.g. Liraglutide have been found to improve the efficacy of treatment and clinical course of type 2 diabetes mellitus and hypertension in patients with such comorbidities (Ikwuka, 2017b; Ikwuka, 2018b; Ikwuka, 2019b; Ikwuka, 2021; Virstyuk, 2017b; Virstyuk, 2018b; Virstyuk, 2018c). It has also been documented that coconut water has hepatorenal protective functions in alloxan-induced type 1 diabetes mellitus (Ekechi, 2023b). Not so much information is available for molecular detection of ESBL-producing S. typhi isolates in Nigeria. Therefore, this research was aimed at molecular detection of extended-spectrum beta-lactamase (ESBL) producing S. typhi from stools of patients attending Bingham University Teaching Hospital, Jos, Plateau State, Nigeria.

The study was carried out at Bingham University Teaching Hospital (BhUTH), formerly ECWA Hospital. BhUTH is located around Zaria bypass in Jos, Plateau State, North-Central Nigeria. BhUTH is a missionary-owned hospital, established in 1959 with a 250 bed capacity. Jos is the capital city of Plateau State with current metro area population of 1,001,000 in 2024, a 3.2% increase from 2023. Jos is situated at about 1,238 meters or 4,062 feet above sea level. 

Sample Collection 

A total of 353 stool samples were collected from suspected typhoid fever patients attending the Bing-ham University Teaching Hospital, Jos, Plateau State into labeled, sterile universal containers. The samples were collected between March to May 2020. The samples were carried to the Microbiology Laboratory of the same hospital and analyzed accordingly. Table 1 shows the primers, primer sequences with amplicon sizes for extended-spectrum beta-lactamase genes.

Table 1: Primers, primer sequences with amplicon sizes for extended-spectrum beta-lactamase genes.

Isolation of Salmonella typhi

S. typhi were isolated from stool samples of the patients using the following procedure described by (Cheesbrough, 2011; Ahmed et al., 2021). Samples from the patients were collected in sterile universal bottles. A thick suspension of the sample was placed in about 1ml of sterile Selenite F broth and incubated. A loopful of 24 hours old growth from the Selenite F broth was emulsified on freshly prepared XLD agar and SS agar plates; and were incubated aerobically for 24 hours at 370C. 

Identification of Salmonella typhi

Salmonella typhi isolates were identified using cultural, morphological and biochemical methods (see Table 2) and confirmed by PCR.

Table 2: Cultural, morphological and biochemical authentication of S. typhi isolated from stool of patients.

Antimicrobial Susceptibility Testing

The antimicrobial susceptibility testing of the bacterial isolates were carried out as described by Clinical and Laboratory Standards Institute (CLSI, 2017). Three (3) pure colonies of the isolates were inoculated into 5ml sterile 0.85% (w/v) NaCl (normal saline). Turbidity of the bacteria suspension was adjusted to the turbidity equivalent to 0.5 McFarlands standard. The McFarlands standard was prepared as follows: 0.5ml of 1.172% (w/v) BaCl2. H2O was added into 99.5ml of 1% (w/v) H2SO4. A sterile swab stick was soaked in standard-sized bacteria suspension and streaked on Mueller-Hinton agar plates and the antibiotic discs were placed aseptically at the center of the plates and allowed to stand for 1 hour for pre-diffusion. The plates were incubated at 37ºC for 24 hours. Results were read using Kirby Bauer interpretation chart, with zone of inhibition size ≤ 14mm indicating resistance. 

Determination of Multiple Antibiotics Resistance (MAR) Index

The MAR Index was determined according to the method of (Krumperman, 1983). From the result of the antibiotics susceptibility test, MAR Index was mathematically calculated as follows:

Phenotype Confirmatory Test for Extended-Spectrum Beta-Lactamase Production

The phenotypic confirmatory test for ESBL production by isolates jointly resistant to both third generation cephalosporins (Ceftazidime and Ceftria-xone) and Amoxicillin-Clavulanic acid were carried out using Double-Disc Synergy Test (DDST) method earlier described by (Jarlier, 1988). On sterile Mueller-Hinton agar plates and Amoxicillin-Clavulanate (30μg) disc placed at the center of the plate, 105cfu/ml bacterial suspension was streaked. Ceftriaxone (30μg) and Ceftazidime (30μg) discs were then placed 15mm (edge-to-edge) from the center disc. Enhancement of inhibitory zone in the area between the disc and any one of the β-lactam discs were compared with inhibitory zone on the far side of the drug disc and interpreted as indicative of the presence of an ESBL in the tested strain. For ESBLs production, Klebsiella pneumoniae was used as positive control while Escherichia coli were used as negative control.

DNA Extraction

Boiling method was used for DNA extraction as described by (Abimiku, 2016). Bacterial DNA was isolated from a 24-hour culture in Lysogeny broth (Luria-Bertani broth/LB broth) and was prepared according to the manufacturers protocol, after purification on MacConkey agar. Centrifugation at 3,200 rpm in a micro-centrifuge for 2 minutes at room temperature yielded the bacterial cells and the supernatant was thrown away. The bacterial cells were re-suspended in 1ml of sterile normal saline and the micro-centrifuge tubes were placed in the vortex for 5 seconds. Centrifugation was repeated at 3,200 rpm for 1 minute and the supernatant was then thrown away again. 0.5ml of sterile normal saline was added to the pellets and the tubes were vertexed for 5 seconds after which they were heated in the block heater at 90°C for 10 minutes. Following heating, rapid cooling was done by transferring the tubes into the freezer for 10 minutes. After centrifugation was done at 3,200 rpm for 1 minute, cell debris were removed. 300µl of the supernatant were transferred into a sterile 2ml appended tube as DNA and stored at -10°C until use.

DNA Amplification of Extended-Spectrum β-Lactamase Genes

In order to amplify the ESBL genes present in the isolates, Multiplex Polymerase Chain Reaction (PCR) was done. Using previously published primer sets and conditions, the presence or absence of blaCTX-M, blaSHV and blaTEM genes were tested for. Table 1 contains the list of the primer sequences and expected amplicon size for each gene. The reactions were carried out in 20µl reaction volume made up of 10µl of Mastermix (Qiagen), 0.32µl of primers (0.16µl each of forward and reverse primers), 3µl of DNA and 6.68µl of nuclease free water. The primer concentration reached at 0.2M. The reaction tubes were placed in the holes of the thermal cycler and the door of the machine closed. Conditions during the reactions were set as: 3 minutes of initial denaturation at 95°C, followed by 35 amplification cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 40 seconds, initial extension at 72°C for 50 seconds, final extension at 72°C for 3 minutes and a hold at 4°C indefinitely. The amplified bands were visualized under ultra-violet light and photographed. Reaction mixtures without a DNA template served as negative controls (Fontana, 2003; Rahman et al., 2019).

Statistical Analysis

All statistical analyses were carried out using PAST and Microsoft EXCEL for Windows version 21.0. 

Ethical Approval

Ethical approval for this study was received from the Health Research and Ethical Committee of Bingham University Teaching Hospital, Jos with Reference No.: NHREC/21/05/2005/00706. Voluntary verbal consent was gotten from all study participants after informed decision. Guardians served the purpose of consent for patients below 16 years of age.

This study recorded the occurrence of 37(10.5%) Salmonella typhi isolates out of three hundred and fifty-three (353) stool samples collected from patients suspected to have typhoid fever and attending Bingham University Teaching Hospital, Jos. Cultured phenotypic identification of the isolates showed milkfish single colonies with black center on SSA and black metallic sheen on XLD which were Gram Negative, rod shape. Biochemical tests reviewed motile, hydrogen sulphate production and indole negative, citrate negative, triple sugar iron test, with H2SO4 production. Some isolates which produced gas were identified as S. typhi (see Table 2). The antibiotic resistance patterns of the isolated 37(10.5%) S. typhi are presented in Table 3. 

All the 37 isolates showed resistance (i.e. 100% resistance) to Augmentin and Ampicillin while Nitrofurantoin was observed to be effective on the 37 S. typhi isolates (i.e. 100% susceptibility). The antibiotic resistance patterns of the other antibiotics include Ceftriaxone (78.3%), Amoxicillin (94.5%), Erythromycin (62.1%), Ceftazidime (67.5%), Streptomycin (63.2%), Ofloxacin (54.0%), Chloramphenicol (48.6%), Ciprofloxacin (59.4%), Cefuroxime (24.3%), and Gentamicin (54.0%). Table 4 depicts the phenotypic resistance profile of S. typhi isolates from stool samples of the patients. 

Table 4: Phenotypic resistance profile of S. typhi isolates from stool samples of patients, n=37.

The Multiple Antibiotics Resistance Index (MAR Index) of the 37 S. typhi isolated from the study was determined which showed the number of isolates and occurrence of 3(8.10%) with the highest MAR Index of 0.90 while number of isolates and occurrence of 2(5.40) with the lowest MAR Index of 0.20 (see Table 5).

Table 5: Multiple Antibiotics Resistance Index (MAR Index) of S. typhi isolates from stool samples of patients.

Genotypic frequency of ESBL resistance genes in the phenotypic ESBL producing isolates showed the occurrence of blaCTX-M 2(40.0%), blaSHV 3 (60.0%), and blaTEM 5(100.0%) and were detected on base pairs 857, 615, and 972 respectively. The occurrence of co-carriage blaTEM/blaSHV (60.0%) was high while the occurrence of blaSHV/blaCTX-M, blaTEM/blaCTX-M, and blaSHV/ blaTEM/ blaCTX-M were 40.0% as shown in Table 8. 

Table 8: Genotypic occurrence of Extended-spectrum beta-lactamase genes in phenotypic ESBL producing S. typhi from stool samples of patients.

The DNA bands of amplified ESBLs genes in phenotypic ESBL production is shown in Fig. 3 whereas the distribution patterns of ESBL genes on agarose gel electrophoresis is shown in Table 10.

Third generations cephalosporins remain the most commonly prescribed class of antibiotics for case management of typhoidal and non-typhoidal salm- onellosis in many countries of the world, including Nigeria (Akinyemi, 2014; Arlet, 2006). However in this study, the focus was placed on only typhoidal salmonellosis caused particularly by S. typhi. An increasing incidence of antibiotic resistance among Salmonella strains worldwide has been attributed to the unapproved and indiscriminate use of anti-biotics particularly in the developing countries (Mathew, 2007). Of recent, ESBLs have become a notable driver of drug resistance. Despite extensive distribution of ESBLs, the prevalence and pheno-typic characteristic among clinical isolates may differ in different geographical zones (Bradford, 2001). In addition, the prevalence of ESBLs between E. coli and Klebsiella has been frequently shown in many countries, but the emergence of ESBLs in Salmonella, which now confers serious clinical problem is noteworthy. These isolates were subjected to ESBLs testing because they were multi-drug resistant which only 5(1.4%) isolates showed positive while 32(9.0%) isolates were negative. Not many studies have been done on extended-spectrum beta-lactamase (ESBLs) producing S. typhi in Jos, Plateau State. Hence, there is limited information and data on previous studies. Finding from this present research shows the importance of S. typhi as a potential public health burden in Jos and the necessity of continuous surveillance. This study revealed a prevalence of 10.4% S. typhi isolates from stool samples of patients presenting with typhoid-like symptoms in BhUTH, Jos, Plateau State, Nigeria, which is low compared with the results of other studies e.g. 24.56% noted in West Bengal, India (Das, 2012), 32.1% observed in North Karnataka, India (Metri, 2011), and 53% seen in Mumbai (Rudresh, 2011). Nonetheless, a recent research in Bangladesh noted that the distribution of S. typhi shows remarkable seasonality, with higher prevalence seen during the raining season (May to October) (Ahmed, 2014). Thus, the low prevalence of S. typhi noted in this present study could be explained by the fact that the period of samples were collected during the dry season (March 2020 to May 2020). Phenotypic confirmatory test method also showed a 1.4% prevalence rate of ESBLs producing S. typhi in this study. A limited number of studies have been carried out to ascertain the prevalence of ESBLs producing Salmonella strains with zero to very low prevalence rates. In addition, resistance to antibiotics was remarkably higher among ESBL producers than some non-ESBL producers (see Table 3). Ceftazidime and Ceftriaxone resistance in this study were found to be significantly linked to ESBLs production in the isolates. This agrees with the recent finding which showed that most detected ESBLs have special affinity to degrade Ceftazidime (Bradford, 2001). Another study has also reported Ceftazidime to be efficient in screening isolates as potential ESBL producers (Vahaboglu, 2001). 

Of interest in this study is the fact that some of the bacteremic strains of S. typhi that produced these ESBL genes can be used to explain why continuous fever occurs in affected patients despite them receiving treatment. The resultant consequence of this is the potential for spread of emerging blaTEM, blaSHV, and blaCTX-M producing S. typhi in Jos and its environs which will increase the prevailing public health burden. Continuous surveillance of blaTEM-producing pathogens is requires to guide prophylactic strategies in Nigeria generally. blaTEM gene cluster carriage has been linked with increased resistance to Ceftriaxone. Thus, the clinical practice of switching Salmonella bacteremic and febrile patients who failed treatment with Ceftriaxone and Ceftazidime empirically to Cefuroxime also possess the risk of treatment failure in this environment. For these patients, this present study recommends the use of either Levofloxacin, Imipenem, Azithromycin or Nitrofurantoin for their treatment. In treating multidrug-resistant infections, the clinical effective-ness of these antibiotics has been reported in Tanzania (Mshana, 2011), in Nepal (Pokharel, 2006), and in India (Pathak, 2012). These antibiotics are also included in the drug formulary of many hospitals in Lagos State and other states in Nigeria, where they are used for treatment of in-patients and out-patients affected by other bacterial infections (Aibinu, 2003; Akinyemi, 2014). In addition to the results of the conjugation experiment revealing that the ESBL genes carried by S. typhi was plasmid-mediated, the results also showed the potential for fast spread of this genetic marker to other members of Enterobacteriaceae such as E. coli, which are often seen in polymicrobial infections in Plateau State and other states of Nigeria. Similar plasmid-mediated transfer of blaTEM, blaSHV, and blaCTX-M resistance to antibiotics such as Cefuro-xime, Aminoglycosides, and beta-lactamase inhi-bitor-containing antibiotics such as Augmentin, as demonstrated in this present study, have been docu-mented by previous researchers from other countries of the world (Mshana, 2011; Jin, 2006; Bado, 2012; Fischer, 2014). Moreover, additional antibiotic resistance conferred by this genetic marker may also lead to the emergence of pan-resistant S. typhi in Nigeria, as was recently reported in some Asian countries (Tadesse, 2014). The spread of ESBL-producing S. typhi to other states in Nigeria and other neighboring countries is also a possibility.

The incidence of infections with S. typhi is growing worldwide especially in poor developing countries where clean water supply is scarce and unhygienic sanitary conditions prevail. The prevalence of S. typhi from stool samples of patients in this study is relatively low. However, subsequent detection of extended-spectrum beta-lactamase (ESBL) genes in the isolates has proven to be a potential serious public health issue and almost all the antibiotics tested were not effective against the isolates except Cefuroxime and Nitofurantoin. Hence, these two drugs should be considered for typhoid infection treatment. Continuous, vigorous and collaborative measures such as public health education and mass surveillance will help to further deepen the know-ledge on the mode of transmission, prevention, treatment and evolution of these resistant isolates.

The authors wish to thank in a special way, all the patients and their guardians who unanimously parti-cipated in this study following informed decision.

All the authors of this manuscript agreed that they have no confliction to make the manuscript publishable.