Occurrence of Extended-Spectrum Beta-Lactamase and Quinolone Resistance Genes among Escherichia coli and Klebsiella pneumoniae Isolated from Poultry, Domestic Pigs and Environ in Msimbazi River Basin in Tanzania

: We investigated the occurrence and distribution of genes encoding for extended-spectrum beta-lactamase (ESBL) production and quinolone resistance among multi-drug resistant (MDR) Escherichia coli and Klebsiella pneumoniae isolated from poultry, domestic pigs, and environment samples in the Msimbazi basin in Tanzania. A total of 130 non-duplicated isolates obtained from the poultry (n = 40), domestic pigs (n = 52) and environment (n = 38) were screened for ESBL genes ( bla CTX-M , bla TEM , and bla

The high level of antimicrobial use (AMU) and antimicrobial resistance (AMR) poses a threat to public and animal health given the fact that they are associated with the development and spread of antimicrobial resistance genes, which can be transferred across animal, human, and environment compartments as shown in a previous study (Minja et al., 2021;Subbiah et al., 2020).Indeed, conjugative plasmids have been responsible for disseminating blaCTX-M-15 among humans, animals, and the environment (Minja et al., 2021).Additionally, the resistant genes blaCTX-M, qnrS, and aac(6)-lb-cr were found to circulate between humans, domestic and companion animals, fish and environment (Subbiah et al., 2020).
In East Africa, a range of genetic diversity of ESBL and carbapenem-resistant genes from Escherichia coli and Klebsiella pneumoniae isolates including blaTEM, blaOXA-1, blaCMY-2, blaCTX-M, blaSHV, NDM, VIM, IMP, KPC, and blaOXA-48 from human, animals and the environment have been reported which were attributed to a range of sources including mutations, selection pressure and genes transfer (Katale et al., 2020).In Tunisia, a study by (Grami et al., 2016) identified IncH12-type plasmid spreading the blaCTX-M-1 and mcr-1 in farm chick, which were reported in cattle in France and food samples in Portugal (Valiakos and Kapna, 2021).In Nigeria, the plasmid-mediated quinolone resistance (PMQR) from Escherichia coli strains was recovered from healthy pigs and poultry that resembled those of human origin (Fortini et al., 2011).Furthermore, the spread of similar antimicrobial resistance genes from the community, human, animals, and environment have been reported in Tanzania, Cameroon, Nigeria, Angola, and Netherlands (Adesoji et al., 2015;Dohmen et al., 2015;Founou et al., 2018;Moremi et al., 2017;Ribeiro et al., 2016).A review study reported that humans exposed to poultry farms carried carbapenem-resistant Enterobacteriaceae (CRE) with close relation to the environmental isolates (Köck et al., 2018).
The Msimbazi river basin in Tanzania has a myriad of human activities making it a hot spot for the spread of AMR bacteria and resistant genes between humananimal-environmental compartments (Kayombo and Mayo, 2018;Kahangwa, 2021;Mrutu et al., 2013).Phenotypic studies conducted in the basin showed very high levels of ESBL-producing and quinolone resistant strains among bacteria isolated from poultry, domestic pigs, and the environment (Kimera, et al., 2021a,b).However, all these studies were limited to phenotypic screening of antibiotic resistant pathogens and none focused on the occurrence of resistant genes.Hence, the objective of this paper was to investigate the occurrence of extended-spectrum beta-lactamase (ESBL) and quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from poultry, domestic pigs and environ in Msimbazi River Basin in Tanzania.

MATERIAL AND METHODS
Bacteria isolates: A total of 130 non-duplicate Escherichia coli (n=95) and Klebsiella pneumoniae (n=35) isolates from poultry (n=40), domestic pigs (n=52), and environment (n=38) were selected from stored isolates obtained between July 2020 and April 2021 (Kimera, et al., 2021a,b) These isolates were randomly selected from a total of 504 isolates from domestic pigs (n=193), poultry (n=151) and environment (n=160) and depicted non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012).The environmental samples included effluents from domestic animals, abattoirs, commercial factory effluents, crop soil, river water, and sediments.For the river water, samples were collected from a distance of at least 0.5 m from the shore and at a depth of 20 to 50 cm.The community and industrial effluents were collected at up to five different points per site using a sterile trowel and placed in a 50mL sterile falcon tube (BD, Nairobi, Kenya).
The faecal materials were obtained from the cloaca and rectum of the poultry and domestic pigs and were collected aseptically using sterile cotton swabs (Himedia, Mumbai, India).Detailed descriptions of the sampling site, sampling protocol, and sample processing have previously been described (Kimera, et al., 2021 a,b).The Escherichia coli and Klebsiella pneumoniae isolates were inoculated on nutrient agar and incubated aerobically at 37 o C for 24 hours prior to further processing.
DNA extraction: Briefly, a half loop of a freshly bacterial culture was suspended in 100µL of sterile DNAse free water for DNA extraction.DNA was extracted by boiling in a water bath at 100 o C for 10 minutes, followed by centrifugation at 1500 rpm for 3 minutes as described previously (Valat et al., 2016).The supernatant containing DNA was transferred into a sterile Eppendorf PCR tube (Eppendorf AG, Hamburg, Germany) and centrifugation and separation of supernatant were repeated three times.The concentration of DNA was determined by a Nanodrop spectrophotometer (Biochrom LTD, Cambridge, England) at 260/280 wavelength (ranging from 1.5 to 1.8).The DNA was stored at -20 o C, before being used for detection of resistant genes.
Screened genes: ESBL genes (blaCTX-M, blaTEM, and blaSHV) and PMQR genes (qnrA, qnrB, qnrS, qnrC, qnrD, qepA, and aac(6)-Ib-cr were the resistant genes screened.The selection of resistant genes to be screened was based on the isolates that demonstrated to be positive for ESBL and PMQR from the previous studies published in the links https://doi.org/10.3390/antibiotics10040406and https://doi.org/10.3390/antibiotics10040343. For quality check, internal quality control was performed by running each DNA sample in duplicates and for external quality control, known control organisms harboring blaCTX-M, blaTEM, and blaSHV and quinoloneresistant genes (qnrA, qnrB, qnrS, qnrC, qnrD, qepA, and aac(6)-Ib-cr were included in each run from DNA extraction to detection of ESBL and quinolone resistant genes.The negative control was a DNA-free template (nuclease-free water).

PCR reaction mixture for the detection of ESBL and PMQR genes:
The One Tag Master Mix Hot Start DNA polymerase kit (New England Biolabs, Ipswich, MA, USA) was used in the detection of resistance genes.Total PCR reaction volume was 25µL, consisting of One Tag Master Mix 2X Standard buffer 12.5µL, 10µM forward primer 0.5µL, 10µM reverse primer 0.5µL, nuclease-free water 9.5µL.The mixture was then aliquoted to the PCR tubes and thereafter 2µL of each DNA extract was added to the PCR tubes containing the Master Mix.The primers used in amplification of the respective resistance genes are listed in Table 1 below.The working buffer was prepared by measuring 980mL of the distilled water and mixed with 20mL of Tris-acetate -EDTA (TAE) buffer stock solution in a conical flask.Thereafter, 1.5% of the Agarose gel was prepared by measuring 1.5 gram of agarose (Merck, SA), which was then dissolved in 100mL of the working TAE buffer solution and mixed by heating in a microwave until completely melted.
To facilitate visualization of DNA 5µL gel red stain (Sigma-Aldrich, USA) was added to the agarose gel.After cooling the solution to about 60 o C it was then poured into the casting tray containing sample comb and allowed to solidify at room temperature.The amplicons of each sample was mixed with 2µL loading dye and a 100-base pair marker (Thermo Scientific, (EU) Lithuania) was included for DNA band size estimation.All gels were run in 0.5xTBE buffer at 100V for 45 minutes, and visualized by UV transillumination.Those that produced bands were termed positive, whereas those that did not show bands were termed negative.

Statistical analysis:
The data generated in this study was entered into an Excel spread sheet version Office 2010 and then transferred to Statistical package for social sciences (SPSS) version 20.0 for Windows (IBM Corp, Armonk, NY, USA) software for statistical analysis.Descriptive analysis was conducted to compare the frequencies of ESBL and PMQR genes between poultry, domestic pigs, and the environmental isolates.The Chi-square test was used to determine the difference in occurrence of resistant genes in Escherichia coli and Klebsiella pneumoniae isolates and a p-value of less than 0.05 was considered significant.

RESULTS AND DISCUSSION
Distribution of ESBL and quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolates: Table 2 and Figure 1 show isolation frequencies of different ESBL and quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolates.As shown in Fig 1, Klebsiella pneumoniae isolates had a lower frequency of occurrence for blaCTX-M but higher level of blaTEM, compared to Escherichia coli, which had higher level of blaCTX-M but less blaTEM genes.Additionally, Klebsiella pneumoniae isolates had higher occurrence of quinolone resistant genes qnrB, qnrS and aac ( 6)lb-cr, compared to qnrB, qnrS and aac ( 6)-lb-cr among Esherichia coli isolates.However, the only statistically significant difference was the higher occurrence of blaTEM among Klebsiella pneumoniae isolates (Table 1).Figure 2 and 3 shows the gel electrophoresis bands of some of the isolates that were positive for quinolone-resistant genes (qnrS) and the ESBL genes (blaTEM).

E. coli K. pneumoniae Total
Distribution of ESBL and quinolone resistance genes in isolates from domestic pigs, poultry and environmental samples: As shown in Table 3, occurrence of resistance genes were highest in isolates from the environmental samples (86%, 33/38), followed by poultry (72.5%, 29/40), and domestic pigs (21.4%, 9/52).The most frequent resistance gene was qnrS, followed by blaCTX-M and blaTEM.The occurrence of blaTEM was significantly higher in environmental (18.4%, 7/38) and poultry (15.8%, 6/40) but not found in domestic pigs.Significant differences were also observed for qnrS, in poultry, environment and domestic pigs, respectively.This study aimed to determine the occurrence of ESBL and PMQR genes among Escherichia coli and Klebsiella pneumoniae isolates obtained from poultry, domestic pigs and the environment in the Msimbazi river basin the most densely populated area in Tanzania (Kayombo and Mayo, 2018;Sawe et al., 2019).The area has very high anthropogenic activities including high concentration of pharmaceutical and commercial industries, hospital, veterinary and human drug sellers, crops and animal farming with intense use of pesticides and antimicrobials and environmental contamination that have been linked with antimicrobial use and resistance (Kayombo and Mayo, 2018;Sawe et al., 2019).We hypothesized that these anthropogenic activities may lead to the occurrence and spread of antimicrobial resistance genes including those encoding for ESBL production and PMQR as previously reported (Alonso et al., 2017;Salah et al., 2019).The most commonly detected ESBL genes were blaCTX-M, while quinolone resistance genes were qnrS, qnrB and aac (6)-lb-cr.This finding is in line with those of other studies (Athanasakopoulou et al., 2021;Kammili et al., 2020;Moremi et al., 2021;Seni et al., 2017) which reported predominance of blaCTX-M and qnrS in isolates from human, animals and environment in Escherichia coli and Klebsiella pneumoniae species.This finding suggests serious concern given the fact that these beta-lactams and quinolone classes of antimicrobials are the affordable and most commonly used in treatment, prophylaxis and metaphylaxis, with limited alternative options (Mdegela et al., 2021;Rasmussen et al., 2015) The observed level and pattern of resistance imply that most of currently used antimicrobials in animal infections might be ineffective for their intended use and may contribute to the emergence and spread of resistant bacteria causing infections that are difficult to treat both in human and animal.We did not find the ESBL genes blaSHV and quinolone resistant genes qnrA, qnrC and qnrB, which have been reported by other studies (Alouache et al., 2014;Fortini et al., 2011;Salah et al., 2019).These variations between and within countries could be due to differences in the extent and types of antimicrobial usage in animal production, variations in policies and regulations regarding usage and disposal of antimicrobial agents as well as differences in public awareness, warrant geographic specific surveillance of AMR (Ejaz et al., 2021;Ohene Larbi et al., 2021).We found out that Escherichia coli had a slightly higher level of ESBL genes blaCTX-M compared to Klebsiella pneumoniae isolates, the variations that are supported by the previous studies (Athanasakopoulou et al., 2021;Salah et al., 2019).Higher level of blaCTX-M in Escherichia coli than Klebsiella pneumoniae isolates found in the present study is consistent with that done in Ghana (Georgina Solano-Gálvez et al., 2021).On the contrary, Klebsiella pneumoniae isolates had higher occurrence of quinolone resistance genes qnrB, qnrS and aac ( 6)-lb-cr, compared to Escherichia coli isolates, a finding which is consistent with that of isolates from chicken and domestic pigs in Nigeria (Fortini et al., 2011).These variations could be associated with intrinsic differences in antimicrobial resistant genes (ARGs) expression (Wyres and Holt, 2018).We observed co-existence of ESBL and quinolone resistance genes in some of the isolates from poultry, domestic pigs and the environmental samples, with the most frequent combination of blaCTX-M + qnrS in Klebsiella pneumoniae and blaTEM + qnrS both in Escherichia coli and Klebsiella pneumoniae, and blaCTX-M + aac(6)lb-cr + qnrS in Escherichia coli, as has been reported by others studies (Alouache et al., 2014;Ben Sallem et al., 2012).Similar observations have been reported in Togo and Algeria where coexistence of ESBL and quinolone resistant genes among Escherichia coli and Klebsiella pneumoniae isolates collected from human specimens, cattle, and environment (Alouache et al., 2014;Salah et al., 2019) which has been attributed to shared resistance mechanisms (Alouache et al., 2014;Samreen et al., 2021).The co-existence of resistant genes plays a potential role in the transmission of resistant genes that calls for across human and animal compartments thus highlighting the importance of collaborative One Health efforts to address the threat.Regarding sample sources, isolates from environmental samples had higher level of resistant genes, followed by poultry and domestic pigs indicating the role of environment as being a reservoir of ARGs originating from human anthropogenic activities (Ben Said et al., 2016;Fletcher, 2015;Ram and Kumar, 2020).
This study which has provided useful insights on the occurrence of ESBL and quinolone resistance genes in poultry, domestic pigs and environment has some limitations.The use of conventional PCR, instead of whole genome sequencing (WGS) limited information on genetic relatedness and unveiling of potential novel resistance mechanisms of the isolates.Secondly, screening of all resistant genes encoding for ESBL production and PMQR such as blaAmpC, VEB, OXA, PER, oqxA, and oqxB was not done and this might have underestimated level of ESBL and PMQR genes across poultry, domestic pigs and the environment compartments.Nonetheless, results of this investigation provides a foundation for more detailed molecular studies, given the paucity of such studies, especially those involving environmental surveillance in the region.

Conclusions:
The wide-spread presence of several genes encoding for quinolone resistance and ESBL production isolates recovered from this study certainly contributes to the growing burden of AMR in the study area, with adverse clinical outcomes, economic and societal impact.The interactions between animals, humans and the environment are likely to be responsible for occurrence of resistant bacteria.We therefore recommend interventions that should focus on the implementation of comprehensive and wellcoordinated One Health approach.
The ethical clearance for the study was issued by the National Institute for Medical Research (NIMR) of Tanzania (Reference No. NIMR/HQ/R.8a/Vol.IX/3133), and the Muhimbili University of Health and Allied Sciences permit no.DA.282/298/01.C.The permission to work in the study area was sought from the Municipal Directors Office (Dar es Salaam).

Fig 1 .
Fig 1. Distribution of ESBL and quinolone resistance genes in Escherichia coli (n=95) and Klebsiella pneumoniae (n=35) isolated from domestic pigs, poultry and environmental samples.

Table 1 .
Primers and PCR conditions used for screening ESBL and PMQR resistant genes.

Table 2 .
Frequency of ESBL and quinolone resistant genes from Escherichia coli and Klebsiella pneumoniae isolates

Table 3 .
Distribution of ESBL and quinolone resistance genes isolated from domestic pigs, poultry, and environmental samples Combination of blaCTX-M + qnrS in Klebsiella pneumoniae and blaTEM + qnrS both in Escherichia coli and Klebsiella pneumoniae had highest occurrence.

Table 4 .
Co-existence of ESBL and quinolone resistance genes in Escherichia coli and Klebsiella pneumoniae isolates.