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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                                    Research Article

Fecal carriage of extended spectrum beta-lactamase and fluoroquinolone resistant gene in non-typhoidal Salmonella enterica isolates from food-producing animals and humans

Uchenna Ogbu Nwosu ¹ , Francis Amadi Ibiam² , Christiana Onyemaechi Amadi-Ibiam³ , Chidinma Stacy Iroha⁴ , Christiana Inuaesiet Edemekong⁵ , Ikemesit Udeme Peter⁶* ,  Ifeanyichukwu Romanus Iroha ¹   

¹ Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, P.M.B. 53, Nigeria 

² Department of Otorhinlaryngology (ENT), Alex Ekwueme Federal University Ndufu-Alike, P.M.B. 1010, Ikwo, Ebonyi State, Nigeria

³ Department of Fisheries and Aquaculture, Faculty of Agriculture, Ebonyi State University, Abakaliki, P.M.B. 53, Nigeria 

⁴ Department of Pharmacy, Alex Ekwueme Federal University Teaching hospital Abakaliki, Ebonyi State, P. M. B. 102, Nigeria

⁵ Department of Biotechnology, Faculty of Pure and Applied Science, Federal College of Dental Technology and Therapy, Trans-Ekulu, P.M.B. 01473, Enugu, Nigeria.

⁶ Department of Public Health, Faculty of Health Technology and Engineering, Federal College of Dental Technology and Therapy, Trans-Ekulu, P.M.B. 01473, Enugu, Nigeria.

Non-typhoidal salmonellosis refers to illnesses caused by all serotypes of Salmonella except for Typhi, Paratyphi A, Paratyphi B (tartrate negative), and Paratyphi C ^{1, 2}. (Gal-Mor et al., 2014; Majowicz et al., 2020). The disease Salmonellosis is a major public health problem worldwide. NTS usually causes self-limiting gastroenteritis associated with nausea, abdominal pain, vomiting and inflammatory diarrhoea. In some cases, specific strains among the serovars can cause bacteremia majorly in young children and immune compromised patients. Incubation of NTS after ingestion of the pathogen is between 6 and 12 h ¹. (Gal-Mor et al., 2014). It is estimated that, each year in the United States, about 1.4 million persons are infected with Non-typhoidal Salmonella, which results in 15,000 hospitalizations and 580 deaths ^{2, 3}.  (WHO, 2017; Majowicz et al., 2020). In many regions such as South East Asia, there is an absence of official Salmonella surveillance data but it is estimated that up to 22.8 million cases occur annually with 37,600 deaths ².(Majowicz et al., 2020). There are more than 2,500 serovars of Salmonella enterica that have been identified. However, the majority of human cases of Non-typhoidal salmonellosis are caused by a limited number of serovars, which may vary from country to country and over time ⁴. (Hendriksen et al., 2011). Invasive Salmonella spp. can spread beyond the gastrointestinal mucosa to infect other sites such as the bloodstream, the meninges, bone or joint spaces ^5. (Crump et al., 2011). Serovars such as S. choloraesuis and S. dublin cause more invasive disease than other serovars ⁶. (Jones et al., 2008). S. enteriditis and S. Typhimurium have traditionally been the most frequently isolated serovars from humans worldwide ^{4, 7}. (Hendriksen et al., 2011; Fashae et al., 2010). Some other serovars have been reported to be more prevalent in specific regions or within countries, such as the prevalence of S. weltevreden and S. stanley in some South East Asian countries ^{4, 8}. (Hendriksen et al., 2011; Lee et al., 2009). However, Non-typhoidal Salmonella spp. are zoonotic agents and foods of animal origin are the main sources for Non-typhoidal Salmonella spp. transmission ⁷. (Fashae et al., 2010).

Human Non-typhoidal (NT) salmonellosis manifests clinically as self-limiting gastroenteritis in healthy individuals but may be severe in populations with compromised or low immunity (the young, the elderly and people with debilitating disease conditions) especially in developing nations ⁹. (Hohmann, 2001). Transmission of NT Salmonella to humans is mostly linked to the consumption of contaminated poultry and poultry products ^{10, 11}. (Braden, 2006; Heredia and Garcia, 2018). While in the developed world, the incidence of Salmonella contamination along the food chain is treated seriously with proactive measures incorporated in the food chain to prevent outbreaks ¹². (Álvarez-Fernández et al., 2012); the reverse is the case in the developing nations where food-borne infections are given less attention. The lack of focused surveillance systems and data collection on circulating Salmonella serovars in most developing countries make it difficult to define the severity of the problem. One major contributing factor may be due to the burden of other debilitating infections such as human immunodeficiency virus (HIV) that relegate foodborne infections like NT salmonellosis to the priority list in most developing countries. 

Previous studies on NT salmonellosis in Nigeria have revealed diverse Salmonella serovars in both animals and man ^{7, 13, 14}. (Fashae et al., 2010; Smith et al., 2016; Agbaje et al., 2019). However, the risk of infection with Salmonella has been worsened by the acquisition and spread of resistance traits to antimicrobials, a possible consequence of excessive and widespread use of antimicrobials in animal productions ^{15, 16}. (Ojo et al., 2012; Omoshaba et al., 2017). Studies have been undertaken largely on human clinical isolates exhibiting resistance to particular antimicrobials, such as fluoroquinolones and beta-lactams ¹⁷. (Onyenwe et al., 2020). AMR to beta-lactams antibiotics mostly occurs due to the carriage of extended-spectrum beta-lactamase (ESBL) resistance plasmids in NTS. ESBLs are often plasmid-mediated enzymes and have various genotypes ^{17, 18} (Onyenwe et al., 2020; Joseph et al., 2023). Through mutational events of amino acids surrounding the active site, ESBL genes evolved from the most predominant ESBL genes such as TEM-1, TEM-2, and SHV-1 genes ^{17, 18}. (Onyenwe et al., 2020; Joseph et al., 2023). This led to the emergence of ESBLs with expanded substrate profiles that empowers them with the ability to hydrolyze all penicillins, cephalosporins, and monobactams. Interestingly, in the past decade, the massive spread of CTX-M-type ESBL gene which has become the main epidemic genotype worldwide has been described ^{17, 19, 20}. (Onyenwe et al., 2020; Yhiler et al., 2019; Sharma et al., 2010).  Study has shown that the majority of ESBLs in Salmonella are derivatives of the TEM, SHV, and CTX-M (cefotaximase) β-lactamase families ²¹. (Eguale et al., 2017) including the report of OXA in Salmonella species ¹⁹. (Yhiler et al., 2019) commonly found in Pseudomonas aeruginosa. Additionally, fluoroquinolone resistance target sites in E. coli are the topoisomerases, such as DNA gyrase (topoisomerase II) - the primary site, and topoisomerase IV - the secondary target which are both vital enzymes in bacterial DNA replication ^{21, 22}. (Eguale et al., 2017; Dupouy et al., 2019). Plasmid-mediated quinolone resistance (PMQR) gene, the first identified PMQR gene, also termed qnrA1, was discovered in 1998 ^{21, 23}. (Eguale et al., 2017; Martínez-Martínez et al., 1998). Plasmids which harbor PMQR genes may also carry genes exhibiting resistance to beta-lactams ²⁴. (Jeong, 2011). Although AMR genes can spread clonally; the transmission of mobile genetic elements harboring AMR genes between bacteria, including from commensal to pathogenic Enterobacteriaceae is evident  ²⁵. (Newire et al., 2013). In Nigeria, beta-lactams and fluoroquinolones are commonly used in the treatment of Gram-negative bacterial infections in both human and veterinary medicine. However, the misuse of these antibiotics has ostensibly led to an increase in bacterial resistance; thus, resulting in "difficult-to-treat’’ bacterial infections.

Despite the arrays of studies highlighting enteric bacteria harboring blaTEM, blaSHV, blaOXA-1, blaCTX-M, qnr A, qnrB, qnrS genes ^{19, 22, 26, 27}. (Yhiler et al., 2019; Dupouy  et al., 2019; Aasm¨ae et al., 2019; Ugbo et al., 2020), there is paucity of information regarding their prevalence in human and food-producing animal.  Thus, this study was designed to ascertain the fecal carriage of extended spectrum beta lactamase and quinolone resistant non-typhoidal Salmonella enterica isolates from food-producing animals and humans. 

2. METHODS

2.1 Sample collection

Three hundred (300) fecal samples were collected using sterile universal containers from food-producing animals namely (Chicken [100], Pig [100] and humans (100) in Onicha Local Government Area of Ebonyi State. Human stool samples were aseptically collected from different wards which includes A&E, Male surgical ward, Female surgical ward, theatre ward, Labour ward, Orthopaedic ward, Pediatric ward, Female medical ward, Male medical ward, GOPD ward. Ten samples were collected randomly from each ward with their age discrepancies from General Hospital Onicha Igboeze, Isu Health Centre, Enyibuchiri Health Center Abaomege, Oshiri Health Centre and Ukawu Health Centre. All human and animal fecal samples collected were labeled and analyzed within 2 hrs of sample collection for bacteriological identification.

2.2 Isolation and identification of Salmonella species

The collected samples were analyzed for the presence of Salmonella specie by inoculating a loopful of each sample into a separate tube of sterile nutrient broth and incubate at 37°C for 24 hrs. After overnight incubation, a loopful of the turbid broth culture was aseptically seeded by streaking on sterile solidified Salmonella/Shigella agar (SSA) and was incubated at 37° C for 24 h. Salmonella specie from positive cultures were identified by their characteristic appearance (color, consistency, shape) on the differential media, motility, and biochemical tests as previously described as reference in the microbiology practical handbook ²⁸. (Iroha et al., 2019).

2.3 Detection of Extended‑spectrum beta‑lactamase (ESBL)-producing Non typhoidal Salmonella species

The production of ESBL was phenotypically confirmed by the Double Disk Synergy Test (DDST) method using Non-typhoidal Salmonella isolates which exhibited reduced susceptibility to 2^nd and 3rd generation cephalosporins as previously described (Joseph et al., 2023). All non-typhoidal Salmonella inoculum were adjusted to 0.5 McFarland turbidity standards and aseptically swabbed on the Mueller-Hinton agar (MHA) plates. Thereafter, a disc of amoxicillin/clavulanic acid (20/10 μg) was placed at the centre of the plate while ceftazidime (30 μg) and cefotaxime (30 μg) discs were each adjacently placed 15 mm away from the centre disc of amoxicillin- clavulanic acid. Plates were then incubated for 18–24 hrs at 37 ◦C. ESBL production was phenotypically confirmed by an expansion of the zone of inhibition for either ceftazidime or cefotaxime in the presence of amoxicillin-clavulanic acid than in its absence giving a dumb-bell shape ¹⁸. (Joseph et al., 2023).

2.4 Molecular typing of ESBL and fluoroquinolone encoding genes 

Extraction of DNA was done with the ZR fungal/bacterial DNA kit (Cat number: D6005). PCR mix contained up of 12.5 μL of Taq 2X Master Mix (New England Biolabs, M0270); 1 μL each of 10 μM forward and reverse primers; 2 μL of DNA template and 8.5 μL Nuclease free water. Oligonucleotide nucleotide primers used are shown in Table 1. The following PCR condition was used: An initial denaturation for 5 mins at 94 ◦C, followed by 36 cycles of denaturation for 30 secs at 94 ◦C, annealing for 30 secs at 55 ◦C, and elongation for 45 s at 72 ◦C; followed by a final elongation step for 7 min at 72 ◦C, and hold temperature at 4 ◦C. Electrophoresis was run at 80–150 V for about 1–1.5 h. Amplified PCR products were then visualized under UV transilluminator. Positive controls used for PCR assays were previously sequenced isolates that harbored the tested genes.

Table 1: Primer Sequence

Statistical analysis

The raw data obtained in the course of this study were presented as mean ± standard deviation in tables and bar charts while relevant data were interpreted using simple descriptive statistics such as minimum, maximum, and one-way analysis of variance (ANOVA) with the aid of IBM Statistical Package for Social Sciences (SPSS) version 22 and Microsoft Excel 2013 software. P < 0.05 was considered to be statistically significant.

The distribution of NTS accounted for 25 % and 17 % in poultry and pig fecal sample respectively while 60 % and 40% were phenotypic ESBL producers respectively and were statistically significantly (P˂ 0.05). Also, none of the 16 (58 %) NTS isolated from humans were ESBL producers (Table 2). Samples gotten from the Laboratory and theatres had the highest frequencies of isolated Salmonella species with 17 (34 %) and 9 (16 %). They were also the only isolates that were significantly (P˂ 0.05) ESBL-producers with a frequency of 6 (86 %) and 1 (14 %) respectively (Table 3).

PCR analysis with β-lactam specific primer detected the presence of blaOXA 50 % and 50 %, blaSHV 36 %, and 64 %, blaTEM 43 % and 57 %, blaCTX-M 36 % and 64 % in poultry and pig respectively Combination of β-lactam gene blaOXA + blaSHV + blaCTM was observed to be 0.0% isolate of human, poultry and pig origin. Fluorquinolone resistant gene QnrA was Present in 0 and 100 % of poultry and pig, then QnrB was 40 % and 60 % present in poultry and pig isolate. QnrS was Present in 64 % isolate of poultry and 13 % of pig. There was no Co-expression of fluoroquinolone and β-lactam gene- Qnr +blaOXA +blaSHV+blaCTM seen in ESBL-producing Salmonella (Table 4).

Table 2: Frequency of ESBL-producing Salmonella and Non-typhoidal Salmonella species from fecal samples of food-producing animals and humans in Onicha Local Government Area

 (Mean ± SD = 2.1 ± 0.521, ∑fv = 5.00)

KEY: NTS = Non-typhoidal Salmonella species, SD = Standard Deviation, ∑V = Summation of variables. Values represent means of data ± Standard Deviation (SD). Data was statistically analyzed at 95% level of confidence (P < 0.05).

Table 3: Frequency of ESBL-producing Salmonella species from fecal samples of humans in a hospital in Onicha Local Government area with respect to wards of admission

Key: SD = Standard Deviation, ∑V = Summation of variables. Values represent means of data ± Standard Deviation (SD). Data was statistically analyzed at 95% level of confidence (P < 0.05), A & E = Accident and emergency ward, GOPD = General outpatient department.

Table 4: Molecular Detection of Extended Spectrum β-Lactamase and Fluoroquinolone Resistant Genes in non-typhoidal Salmonella Isolates

Key: F-β: Fluoroquinolone and β-lactam

ESBL and fluoroquinolone gene were not detected from human sample. Also, resistance due to ESBLs was demonstrated by using the phenotypic confirmatory disc diffusion method, which is relatively cheaper and easy to carry out. However, the sensitivity of this method substantiate with molecular detection techniques, which are more sensitive and reproducible for confirmation of false positive results.

In contrast with our findings, molecular analysis of Salmonella strains demonstrated high dissemination of blaTEM (71.4%; 40) followed by blaCTX-M-1 (48.2%; 27), and blaSHV (19.6%; 11) ³². (Al- Mayahi and Jaber, 2020). These findings are close to other results in Iraq ³³. (Aljanaby and Medhat, 2017), Pakistan ³⁴. (Saeed et al., 2020), and Bangladesh ³⁵. (Ahmed et al., 2014). TEM, SHV and CTX-M has been reported in most studies in Nigeria ^{17, 19, 36, 37, 38}. (Onyenwe et al., 2020; Yilher et al., 2019; Ugwu et al., 2020; Akinyemi et al., 2015; Iroha et al 2012) while in Abakaliki and Afikpo, Qnr gene was not reported ²⁹.(Egwu et al., 2023). 

 The absence of ESBL and florouquinolone gene can be used to explain the need for continuous use of this drug class for treatment of persistent salmonelloisis infection in patients. 

blaOXA, blaTEM, blaSHV and blaCTX-M was common among food-producing animals. The emergence of ESBL-producing Salmonella reported, could be due to selective pressure imposed by the inappropriate use of broad-spectrum antibiotics such as the third generation cephalosporin as growth promoter and the treatment of bacterial infection in animal husbandry. Interestingly, in this study, some of the strains from food producing animal that produced ESBL gene can be used to explain why persistent salmonelloisis could occurs among the populace despite them receiving treatment with 3GCs. The implication of this is the potential for spread of emerging ESBL producing S. typhi in Onicha, which will add to the prevailing public health burdens in the state.

PCR analysis with β-lactam specific prime detected the presence of blaOXA 50 % and 50 %, blaSHV 36 %, and 64 %, blaTEM 43 % and 57 %, blaCTX-M 36 % and 64 % in poultry and pig respectively. Combination of β-lactam gene blaOXA + blaSHV + blaCTM was observed to be 0.0% isolate in human, poultry and pig samples. Salmonella spp. co-producing CTX-M- and TEM-type β -lactamases have been documented in a few case reports from Nigeria ^{17, 19, 36}. (Onyenwe et al., 2020; Yilher et al.,2019; Ugwu et al., 2020) Bangladesh ³⁹. (Ahmed et al., 2012) and India ⁴⁰. (Karthikeyan et al., 2011). Plasmid analysis of the resistant isolate revealed that the blaCTX-M and blaTEM genes were located in the same plasmid, which carried the ISEcp1 element upstream of the blaCTX-M gene to facilitate mobilization and expression ⁴⁰. (Karthikeyan et al., 2011). The present study is important in understanding the mechanism of resistance operating in these common pathogens, which are also endemic in most area in Nigeria.  

Fluorquinolone resistant gene QnrA was present in 0 % and 100 % of poultry and pig respectively, while QnrB was 40 % and 60 % present in poultry and pig isolates respectively. QnrS was present in 64 % isolates from poultry and 13 % from pigs. Studies have shown quinolone resistance in salmonellae to be as a result of mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV encoding (parC and parE) genes ^{17, 19, 41}. (Onyenwe et al., 2020; Yhiler et al., 2019; Ye et al., 2018). Other studies have also reported the presence of plasmid mediated quinolones resistant (PMQR) genes carried by the ESBL-producing plasmid, which facilitates the selection of higher-level resistance to quinolone drugs ^{17, 42, 43, 44}. (Onyenwe et al., 2020; Riyaaz et al., 2018; Carfora et al., 2018; Jacoby et al., 2014; Kongsoi et al., 2015).

There was no Co-expression of fluoroquinolone and β-lactam gene- Qnr +blaOXA +blaSHV+blaCTM seen in ESBL-producing non-Typhoidal Salmonella. This study has shown the presence of resistant genes encoding floroqunonlone and ESBL producing NTS predominantly in poultry and pigs. This could be associated with the extensive use of this antibiotic during chicken and pig rearing/production. There is an indication that the route of acquiring these genes are zoonotic and continually eating under cooked pork and chicken, poor hygienic handling of meat and careless playing with domestic animals can transfer these genes to humans and if not controlled can lead to outbreak of Salmonellosis among human population.

There is greater prevalence of genes encoding beta-lactamases and fluoroquinolone resistant (blaTEM, blaSHV, blaCTX-M, blaOXA and blaCMY, (qnrA, qnrB and qnrS) present in poultry and pig than in humans. This demonstrated a significant threat in spread of genes across human and can cause an outbreak if control approach is not put in place. ESBL producing NTS are more prevalent in animal than human and the danger associated to this is that continually eating under cooked chicken and pork plus other poor hygienic handling of this animal can actually be a route to human infection. The wide spread of fluoroquinolone and ESBL producing NTS could be associated with the extensive use of antibiotic during chicken and pig rearing/production and Salmonella isolates from human may be due to unhygienic handling and consumption of under cooked chicken and pork. It is of public health importance because consumers are exposed to the risk of infection by fluoroquinolone and ESBL-NTS producing strain from the chicken and pork. This further highlights the need for rational use of antibiotics in livestock, poultry and pig farming, proper meat handling/cooking practices and enforcement of standard food safety by governmental regulatory agencies so as to prevent the risk of ESBL and fluoroquinolone resistant bacteria mediated foodborne diseases.

Acknowledgement: We acknowledge the support of the staff of Ebonyi State Ministry of Health, Abakaliki, Nigeria and also thank Professor. I. R. Iroha for his unflinching support 

Author’s Contributions: All authors investigated the study, did literature searches and did data Validation and Visualization. All the authors reviewed and approved the final draft, and are responsible for all aspects of the work

Funding Source: None

Conflict of Interest: None

Ethical consideration: Ethical approval with reference No:  SMOH/ERC/042/21 was obtained from the Research and Ethics Committee of Ebonyi State Ministry of Health, Abakaliki, Nigeria. All experiment in this study was executed following relevant national and international guidelines

REFERENCES

1. Gal-Mor O, Boyle EC, Grassl GA, Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ, Frontier Microbiology, 2014; 5:391-392. https://doi.org/10.3389/fmicb.2014.00391 PMid:25136336 PMCid:PMC4120697

2. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'Brien SJ, Jones TF, Fazil A, Hoekstra RM, Studies ICoEDBoI: The global burden of nontyphoidal Salmonella gastroenteritis, Clinical Infectious Disease, 2020; 50(6):882-889. https://doi.org/10.1086/650733 PMid:20158401

3. World Health Organisation. Integrated Surveillance of Antimicrobial Resistance in Foodborne Bacteria: Application of a One Health Approach. Geneva: World Health Organization; 2017.

4. Hendriksen RS, Vieira AR, Karlsmose S, Lo Fo Wong DM, Jensen AB, Wegener HC, Aarestrup FM. Global monitoring of Salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007, Foodborne Pathogens and Disease, 2011; 8:887-900. https://doi.org/10.1089/fpd.2010.0787 PMid:21492021

5. Crump JA, Medalla FM, Joyce KW, Krueger AL, Hoekstra RM, Whichard JM, Barzilay EJ, Emerging Infections Program NARMS Working Group, 2011, Antimicrobial resistance among invasive nontyphoidal Salmonellaenterica isolates in the United States: National Antimicrobial Resistance Monitoring System, 1996 to 2007, Antimicrobial Agents and Chemotherapy, 2011; 55:1148-1154. https://doi.org/10.1128/AAC.01333-10 PMid:21199924 PMCid:PMC3067073

6. Jones TF, Ingram LA, Cieslak PR, Vugia DJ, Tobin-D'Angelo M, Hurd S, Medus C, Cronquist A, Angulo FJ, Salmonellosis outcomes differ substantially by serotype, Journal of Infectious Diseases, 2008; 198:109-114. https://doi.org/10.1086/588823 PMid:18462137

7. Fashae K, Ogunsola F, Aarestrup FM, Hendriksen RS, Antimicrobial susceptibility and serovars of Salmonella from chickens and humans in Ibadan, Nigeria, Journal of Infection in Developing Countries, 2010; 4:484-494 https://doi.org/10.3855/jidc.909 PMid:20818100

8. Lee HY, Su LH, Tsai MH, Kim SW, Chang HH, Jung SI, Park KH, Perera J, Carlos C, Tan BH, Kumarasinghe G, So T, Chongthaleong A, Hsueh PR, Liu JW, Song JH, Chiu CH, High rate of reduced susceptibility to ciprofloxacin and ceftriaxone among nontyphoid Salmonella clinical isolates in Asia, Antimicrobial Agents and Chemotherapy, 2009; 53:2696-2699. https://doi.org/10.1128/AAC.01297-08 PMid:19332677 PMCid:PMC2687261

9. Hohmann E L, Nontyphoidal Salmonellosis, Clinical Infectious Diseases, 2001; 32(2):263-9 https://doi.org/10.1086/318457 PMid:11170916

10. Braden CR, Salmonella enterica serotype Enteritidis and eggs: a national epidemic in the United States, Clinical Infectious Diseases, 2006; 43(4):512-517. https://doi.org/10.1086/505973 PMid:16838242

11. Heredia N, García S, Animals as sources of food-borne pathogens: A review. Animal Nutrition, 2018; 4(3):250-255. https://doi.org/10.1016/j.aninu.2018.04.006 PMid:30175252 PMCid:PMC6116329

12. Álvarez-Fernández E, Alonso-Calleja C, Garcia-Fernández C, Capita R, Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry in Spain: comparison between 1993 and 2006, International Journal of Food Microbiology, 2012; 153(3):281-7 https://doi.org/10.1016/j.ijfoodmicro.2011.11.011 PMid:22208955

13. Smith, S, Braun S, Akintimehin F, Fesobi T, Bamidele M, Coker A, Monecke S, Ehricht R, Serogenotyping and antimicrobial susceptibility testing of Salmonella species isolated from retail meat samples in Lagos, Nigeria. Molecular and Cellular Probes, 2016; 30(4):189-194 https://doi.org/10.1016/j.mcp.2016.04.001 PMid:27133921

14. Agbaje M, Lettini AA, Ojo OE, Longo A, Marafin E, Antonello K, Dipeolu MA. Antimicrobial resistance profiles of Salmonella serovars isolated from dressed chicken meat at slaughter in Kaduna, Nigeria, Revue d'elevage et de medecine veterinaire des pays tropicaux, 2019; 72(4):1-8 https://doi.org/10.19182/remvt.31484

15. Ojo OE, Awosile B, Agbaje M, Sonibare AO, Oyekunle MA, Kasali OB, Quinolone resistance in bacterial isolates from chicken carcasses in Abeokuta, Nigeria: A retrospective study from 2005-2011, Nigerian Veterinary Journal, 2012; 33(2):483- 491.

16. Omoshaba EO, Olufemi FO, Ojo OE, Sonibare AO, Agbaje M, Multidrug-resistant Salmonellae isolated in Japanese quails reared in Abeokuta, Nigeria, Tropical Animal Health and Production, 2017; 49(7):1455-1460. https://doi.org/10.1007/s11250-017-1347-z PMid:28717851

17. Onyenwe NE, Nnamani ND, Okoro JC, Nwofor CN, Jesumirhewe C, Prevalence and gene sequencing of extended spectrum β-lactamases producing Salmonella enterica serovar. Typhi from South-East Nigeria, African Journal of Pharmacy and Pharmacology, 2020; 14(7):192-202 https://doi.org/10.5897/AJPP2020.5115

18. Joseph I S, Okolo I O, Udenweze E C, Nwankwo C E, Peter I U, Ogbonna I P, Iroha I R, Comparison of Antibiotic-Resistant Pattern of Extended Spectrum Beta-Lactamase and Carbapenem- Resistant Escherichia coli Isolates from Clinical and Non-Clinical Sources, Journal of Drug Delivery and Therapeutics, 2023; 13(7):107-118 https://doi.org/10.22270/jddt.v13i7.5918

19. Yhiler NY, Bassey BE, Paul I, Francis U M, Anne A, Okocha-Ejeko A, Antimicrobial resistance pattern in Salmonella enterica from clinical and poultry sources in Calabar, Nigeria, Journal of Microbiology and Antimicrobials, 2019; 11(2):5-10. https://doi.org/10.5897/JMA2019.0413

20. Sharma J, Sharma M, Ray P, Detection of TEM and amp; SHV genes in Escherichia coli and amp; Klebsiella pneumoniae Isolates in a Tertiary Care Hospital from India, Indian Journal of Medical Research, 2010; 132:332-336.

21. Eguale T, Birungi J, Asrat D, Njahira M N, Njuguna J, Gebreyes, G, Wondwossen, A, Gunn JS, Appolinaire-Djikeng, A, Engidawork E, Genetic markers associated with resistance to beta-lactam and quinolone antimicrobials in non-typhoidal Salmonella isolates from humans and animals in central Ethiopia, Antimicrobial Resistance and Infection Control, 2017; 6:13-14. https://doi.org/10.1186/s13756-017-0171-6 PMid:28105330 PMCid:PMC5240271

22. Dupouy V, Abdelli M, Moyano G, Arpaillange N, Bibbal D, Cadiergues, M. C, Prevalence of beta-lactam and quinolone/fluoroquinolone resistance in enterobacteriaceae from dogs in France and Spain: Characterization of ESBL/pAmpC isolates, genes and conjugative plasmids, Frontier in Veterinary Science, 2019; 6:279-281 https://doi.org/10.3389/fvets.2019.00279 PMid:31544108 PMCid:PMC6730528

23. Martínez-Martínez L, Pascual A, Jacoby GA, Quinolone resistance from a transferable plasmid, Lancet of Infectious Disease, 1998; 351:797-799, https://doi.org/10.1016/S0140-6736(97)07322-4 PMid:9519952

24. Jeong HS, Prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase and AmpC beta-lactamase in Enterobacteriaceae, Korean Journal of Laboratory Medicine, 2011; 31:257-264 https://doi.org/10.3343/kjlm.2011.31.4.257 PMid:22016679 PMCid:PMC3190004

25. Newire E A, Ahmed S F, House B, Valiente E, Pimentel G, Detection of new SHV-12, SHV-5 and SHV-2a variants of extended spectrum beta-lactamase in Klebsiella pneumoniae in Egypt, Annal of Clinical Microbiology and Antimicrobial Agent, 2013; 12:16-340, https://doi.org/10.1186/1476-0711-12-16 PMid:23866018 PMCid:PMC3723734

26. Aasm¨ae B, H¨akkinen L, Kaart T, Kalmus P, Antimicrobial resistance of Escherichia coli and Enterococcus species isolated from Estonian cattle and swine from 2010 to 2015, Acta Veterinary Scandinavian, 2019; 61:5-34, https://doi.org/10.1186/s13028-019-0441-9 PMid:30665443 PMCid:PMC6341677

27. Ugbo EN, Anyamene CO, Moses IB, Iroha IR, Babalola OO, Ukpai EG, Chukwunwejim CR, Egbule CU, Emioye AA, Okata-Nwali OD, Igwe OF, Ugadu, IO, Prevalence of blaTEM, blaSHV, and blaCTX-M genes among extended spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae of clinical origin, Gene Report, 2020; 21:34-45. https://doi.org/10.1016/j.genrep.2020.100909

28. Iroha IR, Orji JO, Onwa NC, Nwuzo AC, Okonkwo EC, Ibiam EO, Nwachi AC, Afuikwa FN, Agah VM, Ejikeugwu EPC, Agumah NB, Moses IB, Ugbo E, Ukpai EG, Nwakaeze E A, Oke B, Ogbu L and Nwunna E, Microbiology practical handbook. (Editor; Ogbu. O), 1st Edition. Charlieteximage Africa (CiAfrica Press), 2019; 344.

29. Egwu, E, Ibiam FA, Moses IB, Iroha CS, Orji I, Okafor-Alu F N, Eze CO, Iroha IR, Antimicrobial susceptibility and molecular characteristics of beta-lactam- and fluoroquinolone-resistant E. coli from human clinical samples in Nigeria, Scientific African, 2023; 21:18-63. https://doi.org/10.1016/j.sciaf.2023.e01863

30. Ahmed AM, Motoi Y, Sato M, Maruyama A, Watanabe H, Fukumoto Y, Shimamoto T, Zoo animals as reservoirs of Gram-negative bacteria harboring integrons and antimicrobial resistance genes, Applied Environmental Microbiology, 2007; 73:6686-6690. https://doi.org/10.1128/AEM.01054-07 PMid:17720829 PMCid:PMC2075039

31. Robicsek A, Jacoby GA, Hooper DC, The Worldwide Emergence of Plasmid-mediated Quinolone Resistance, Lancet Infectious Disease, 2006; 6:629-640. https://doi.org/10.1016/S1473-3099(06)70599-0 PMid:17008172

32. Al-mayahi F S, Jaber SM, A preliminary study of multiple antibiotic resistance (MAR) and extensively drug-resistant (XDR) of bacterial causing typhoid fever isolated from stool specimens in Al-Diwaniya, Iraq, Eurasia Journal of Bioscience, 2020; 14:2369-2378.

33. Aljanaby A A I, Medhat A R, Prevalence of Some Antimicrobials Resistance Associated-Genes in Salmonella typhi isolated from Patients Infected with Typhoid Fever, Journal of Infection Developing Countries, 2017; 14(2):169-176.

34. Saeed M, Rasool MH, Rasheed F, Saqalein M, Extended-spectrum beta-lactamases producing extensively drug-resistant Salmonella Typhi in Punjab, Pakistan, Journal of Infection Developing Countries, 2020; 14(2):169-176. https://doi.org/10.3855/jidc.12049 PMid:32146451

35. Ahmed D, Ud-Din AI, Med S, Wahid S U H, Mazumder R, Nahar K, Hossain A, Emergence of blaTEM Type Extended-Spectrum β- Lactamase Producing Salmonella species in the Urban Area of Bangladesh, Journal of Antimicrobial Agents and Chemotherapy, 2014; 7(15):310-3. https://doi.org/10.1155/2014/715310 PMid:25101188 PMCid:PMC4003836

36. Ugwu MC, Shariff M, Nnajide C, Beri M, K, Okezie UM, Iroha IR, Esimone CO, Phenotypic and Molecular Characterization of β-Lactamases among Enterobacterial Uropathogens in Southeastern Nigeria, Canadian Journal of Infectious Diseases and Medical Microbiology, 2020; 12:1975-1978 https://doi.org/10.1155/2020/5843904 PMid:32184910 PMCid:PMC7060859

37. Akinyemi KO, AIwalokun B, Alafe OO, Mudashiru S A, Fakorede S, blaCTMgroup extended spectrum beta lactamase-producing Salmonella typhi from hospitalized patients in Lagos, Nigeria, Infection and Drug Resistance, 2015; 8:99-106. https://doi.org/10.2147/IDR.S78876 PMid:25999745 PMCid:PMC4437039

38. Iroha I.R, Chika E, Ogonna A, Chidinma I, Monique A, Ikechukwu M, Stanley E, Emmanuel N, Ngozi A, Agabus N, Prevalence and Antibiogram of Salmonella species isolated from poultry products, Journal of Advanced Veterinary and Animal Research, 2016; 3(4):353-359. https://doi.org/10.5455/javar.2016.c-172

39. Ahmed AM, Shimamoto T, Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt. Microbiology and Immunology, 2012; 56:254-261. https://doi.org/10.1111/j.1348-0421.2012.00429.x PMid:22500933

40. Karthikeyan K, Thirunarayan M, Krishnan P, CTX-M15 type ESBL producing Salmonella from a Paediatric Patient in Chennai, India, Indian Journal of Medical Research, 2011; 134:487-489.

41. Ye Q, Wu Q, Zhang S, Zhang J, Yang G, Wang J, Xue L, Chen M, Characterization of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae From Retail Food in China, Frontiers in Microbiology, 2018; 9:1709-1710. https://doi.org/10.3389/fmicb.2018.01709 PMid:30135680 PMCid:PMC6092486

42. Riyaaz A A A, Perera V, Sivakumaran S, de Silva N, Typhoid Fever due to Extended Spectrum β-Lactamase-Producing Salmonella enterica Serovar Typhi: A Case Report and Literature Review, Case Reports in Infectious Diseases, 2018; 12(23):34-66. https://doi.org/10.1155/2018/4610246 PMid:29666727 PMCid:PMC5832104

43. Carfora V, Alba P, Leekitcharoenphon P, Ballarò D, Cordaro G, Di Matteo P, Donati V, Ianzano A, Iurescia M, Stravino F, Tagliaferri T, Battisti A, Franco A, Colistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016-2017), Frontiers in Microbiology, 2018; 9:18-80. https://doi.org/10.3389/fmicb.2018.02395 PMid:30344517 PMCid:PMC6186965

44. Jacoby GA, Strahilevitz J, Hooper DC, Plasmid-mediated Quinolone Resistance, Microbiology Spectrum, 2014; 2(6):20-13. https://doi.org/10.1128/microbiolspec.PLAS-0006-2013 PMid:25584197 PMCid:PMC4288778

45. Kongsoi S, Yokoyama K, Suprasert A, Utrarachkij F, Nakajima C, Suthienkul O, Suzuki Y, Characterization of Salmonella Typhimurium DNA gyrase as a Target of Quinolones, Drug Testing and Analysis, 2015; 7:714-720. https://doi.org/10.1002/dta.1744 PMid:25381884