ANTIBIOTIC RESISTANCE IN BACTERIAL PATHOGENS ISOLATED FROM CATTLE DUNG AND ITS CONTAMINATED SOIL

attle dung is used as organic fertilizer and alternative source of fuel or biogas but could also be a source of antibiotic resistance genes in the environment. This study isolated, identified and assessed antibiotic susceptibility pattern of bacteria from cattle dung and its contaminated soil. Bacteria isolation and identification were based on standard techniques while hemolytic activity was used to confirm potential pathogenic bacteria. Antibiotic susceptibility pattern of isolated pathogens were assayed by disk diffusion method. Among isolated bacteria, Staphylococcus spp had highest occurrence of 23.8 % while Micrococcus spp was the least at 1.3 %. Hemolytic bacteria isolates were Staphylococcus aureus (16.5 %), Bacillus spp (17.4 %), Nocardia spp (4.6 %), Escherichia coli (29.4 %), Pseudomonas spp (13.8 %), Serratia marcersens (2.8 %) and Salmonella spp (15.6 %). High resistance (100 %) against Ampiclox (30 μg) was observed in all Staphylococcus aureus and Bacillus spp isolates while Pseudomonas aeruginosa isolates showed 100 % resistance to Ofloxacin (30 μg). Most Gram-positive bacterial isolates were majorly resistant to Beta lactams while Gram negative bacteria were resistant to Fluoroquinolones antibiotics. Multiple antibiotics resistant index (MARI) was measured at greater than 0.2, and was observed in 71.5 % of the hemolytic pathogens. Antibiotics resistance in hemolytic bacterial pathogens from this study is indicative of environmental sources of antibiotic resistance and possible adverse effects on human health.


INTRODUCTION
Antibiotics have been used as panacea for the treatment of infections in both humans and animals for decades (WHO, 2014). They are often included in feed additives at small doses for growth promotion of animals used for meat, which account for large proportions of the global use of veterinary antibiotics (You and Silbergeld, 2014). The development of large-scale concentrated animal feeding operations (CAFOs) has increased the extensive use of veterinary antibiotics in the treatment of infections, prevention of diseases and promotion of growth (Sarmah et al., 2006;Jechalke et al., 2014;Van Boeckel et al., 2015).
Substantial proportions of the veterinary antibiotics administered are excreted in un-metabolized forms or as active metabolites in the soil (Sarmah et al., 2006). Evidence has shown that antibiotic residues can adversely affect microbial processes in the environment such as nutrient cycling and pollutants degradation (Sarmah et al., 2006;Jechalke et al., 2014). Similarly, antibiotics administered to animals provide selective advantages for antibiotic resistant bacteria (ARBs) to develop in animal intestines, which end up in their dung and eventually in the soil (Muurinen et al., 2017).
Animal manure could be a reservoir of bacteria carrying Antibiotic Resistant Genes (ARGs) and Mobile Genetic Elements (MGEs) such as plasmids (Feng-Hua et al., 2015;Yijun et al., 2018). When manure is used as fertilizer, residual antibiotics and ARGs could disperse into agricultural soils, which may exert selection pressure on antibiotic resistance (Martinez, 2009;Obuekwe and Osariemen, 2020). Antibiotic-resistance genes from the soil resistome can enter the food chain via contaminated crops or groundwater, and have potential consequences for human health if transferred to human pathogens (Marti et al., 2013;Nikolina et al., 2014;Adebisi et al., 2016). There is need to curb this problem by laying emphasis on improving productivity with less dependence on antibiotics especially for developing economics like Nigeria (Chollom et al., 2018). Again, strict control measures should be placed on antibiotics use through government policies and regulations, and where antibiotics are needed, there should be veterinarian prescription before use (Piddock et al., 2016;Chollom et al., 2018). This study assessed antibiotic susceptibility pattern of pathogens from cattle dung and its contaminated soil.

SAMPLES COLLECTION
Fresh cattle dung, cattle dung manure and dung contaminated soils were collected in five (5) replicates from four (4) cattle settlement/market (Federal Staff road, Dumez junction, Upper Mission and Eyaen) within Benin City. Sterile plastic bags were used for fresh cattle dung and manure collection while cattle dung contaminated soils were collected using soil augers at a depth of 6 inches and then placed in a sterile plastic bag. Fifteen (15) samples of each category were collected for analysis, and the samples were collected during the rainy season in 2018. Methodology flowchart is shown in Figure 1.

ISOLATION AND IDENTIFICATION OF BACTERIA
The spread plate method was used for bacterial isolation. Ten-fold serial dilution of each sample was prepared aseptically in physiological saline up to10 -6 dilutions. Thereafter, l ml aliquots of 10 -5 and 10 -6 dilutions were plated on nutrient agar (Himedia) and incubated at 37 °C for 24 -48 h under aerobic conductions (APHA, 1999). Further identification was based on Gram's stain and biochemical tests (Cheesebrough, 2006) (Table 1).

HEMOLYSIN PRODUCTION
All bacterial isolates from samples were also cultured on 5 % sheep blood agar (SBA) media as described by Pavlov et al. (2004) and Ryan et al. (2014). Plates were incubated at 37 °C for 24 h and growth was then observed for hemolytic activity of the bacteria. Greenish zone around the culture would indicate α-hemolysis while clear transparent zone indicated β-hemolysis.

INDEX OF BACTERIAL PATHOGENS
Hemolysin producing isolates were sub-cultured on nutrient agar, and pure colonies of pathogenic bacteria were obtained. These were picked to make suspensions in 1ml sterile normal saline that was adjusted to an equivalence of a 0.5 McFarland standard. Subsequently, sterile Mueller-Hinton agar (Oxoid, UK; Bauer et al., 1966) plates were inoculated with them by spreading 0.1ml of each pathogenic bacterial suspension on the entire surface of the plate and then antibiotic discs were inserted. The following antibiotic sensitivity discs and concentrations were used for The multiple antibiotic resistance MAR index was determined for each bacterial pathogen by dividing the number of antibiotics the pathogenic bacteria was resistant to by the total number of antibiotics tested (Adenaike et al., 2016).

RESULTS
A total of sixty (60) samples of fresh cattle dung, manure and dung contaminated soils collected from four settlements within Benin City were microbiologically assessed. Out of these samples, one hundred and fifty-one (151) bacteria were recovered and were identified as Staphylococcus spp., Staphylococcus aureus, Micrococcus spp, Bacillus spp., Nocardia spp, Escherichia coli, Neisseria spp., Pseudomonas spp, Serretia marcescens and Salmonella spp (Table 1).

Salmonella spp
However, Micrococcus spp, Nocardia spp, Neisseria spp and Serretia marcescens were absent in both fresh cattle dung and cattle dung manure samples but present in dung contaminated soil (Table 2).

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Obuekwe and Offodile., 2020 | 70  (Table 2). These isolates were further tested for virulence factor using hemolysin production and it was observed that a total number of 109 isolates from each sample were pathogenic. Bacillus spp, Pseudomonas spp, Staphylococcus aureus, Salmonella spp, Nocardia spp, E. coli and Serretia marcescens were α and β hemolysin positive. However, no hemolysin production was observed with Staphylococcus spp, Micrococcus spp and Neisseria spp (Table 3).

Total number of isolates from each sample that are pathogenic = 109
Legend: N/%: Total number of bacteria/ percentages of occurrence; hemolysin +ve: percentage number of bacterial positive for hemolysin production.
Isolated hemolytic bacterial pathogens were subsequently subjected to common antibiotics to assess their resistance patterns as well as their percentage multiple antibiotic resistance indices (Table 4).
Staphylococcus aureus isolated from fresh cattle dung manure showed high resistance to Ampiclox and Amoxicillin (100 %) and least resistance to Erythromycin and Cotrimoxazole (14.3 %). Similar pattern was also observed with Bacillus spp which were resistant to Ampiclox, Amoxicillin and Ceftriaxone but not to Gentamycin and Streptomycin (14.3 %) ( Figure 3A). The highest antibiotic resistance in E. coli isolates from cattle dung manure was observed with Ofloxacin (88.9 %) however, they were least resistant to Streptomycin and Chloramphenicol (11.1 %). Pseudomonas spp and Salmonella spp were highly resistant to Ofloxacin (100 %) but Pseudomonas spp was least resistant to Augmentin (0 %) while Salmonella spp was least resistant to Gentamycin (0 %) ( Figure 3B).
Amongst the three (3) categories of samples analyzed, fresh cattle dung contained the highest population of antibiotic resistant pathogens, followed by cattle dung manure, then cattle dung contaminated soil.
Multiple antibiotics resistance indices (MARI) of the hemolytic bacterial pathogens from all samples showed that highest percentage multi drug resistant population was observed in Salmonella spp (94.1 %). This was followed  (Mbah et al., 2016).
In this study Staphylococcus spp had the highest percentage occurrence of 23.8 % which may be associated with mastitis (Roberts et al., 2018). This was followed by E. coli (21.2 %) and Bacillus spp (12.6 %) while Micrococcus spp and Serretia marcescens were the least bacterial isolates with percentage occurrence of 1.3 % and  (Adebisi et al., 2016;Obuekwe and Osariemen, 2020).
Virulence factor based on hemolysin production by these isolates (Bacillus spp, Pseudomonas spp, Staphylococcus aureus, Salmonella spp, Nocardia spp, E. coli and Serretia marcescens) grouped them as pathogenic.
Hemolysin is a bacterial protein that breaks down the membrane of red blood cells causing the release of hemoglobin.
It acts by integrating into the membrane of the red blood cell and either punching a hole through the membrane or disrupting the structure of the membrane in some other ways (Verdon et al, 2009). One hundred and nine (109) out of these bacterial pathogens were positive for α-hemolytic and β-hemolytic production which is an important virulence determinant of a disease outcome, and pathogenicity of commonly recognized pathogens such as Shiga-toxin producing E. coli, Salmonella, and Staphylococcus aureus (Spiehs and Goyal, 2007;Divyakolu et al., 2019;Obuekwe and Osariemen, 2020). E.coli is naturally found in the intestinal tract of animals, and shed with faeces however, Pathogenic hemolytic E. coli is known to cause many enteric diseases such as traveler's diarrhea and other forms of diarrhea (Carbone et al 2002;Adebisi et al., 2016).
Presence of high level of resistance to the tested antibiotics was observed in this study.  (Qingxiang et al., 2013). Resistance gene profiles are believed to play a very important role in mediating and transferring resistance to antibacterial drugs in the bacteria population. They can be localized in discrete transposable elements of DNA called transposons, which are mobile, and can move from one DNA molecule to another (Yijun et al., 2018). This can lead to the rapid spread of antibiotic resistance in a bacteria

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Obuekwe and Offodile., 2020 | 78 population, and explains the emergence of multi-resistant strains (Nain et al., 2015). Bacteria use three main strategies to get protected against β-lactams: alteration in Penicillin Binding Proteins (PBPs) which reduces the affinity of βlactams, efflux pumps which remove the antibiotic from the bacterial periplasmic space, and production of βlactamases which hydrolyze the ring of β-lactams (Zapun et al., 2008;Bush, 2013). However, resistance to quinolones has been a problem ever since Nalidixic acid was introduced into clinical medicine more than 40 years ago (Vranakis et al., 2013).
Generally, three mechanisms of resistance to quinolones are currently recognized: mutations that alter the drug targets, mutations that reduce drug accumulation and plasmids that protect cells from the lethal effects of quinolones (Hooper and Jacoby, 2015 Luis Martinez., 2009). Nonetheless, low resistances to tested antibiotics were seen in Norcadia spp and Serratia marcersens which were exclusively isolated from cattle dung contaminated soil as well as Some E. coli isolates that were also susceptible to chloramphenicol. This is in agreement with Barbara et al. (2006) who reported antibiotics susceptibility of Nocardia spp. Comparably, Abu and Wondikom, (2018) reported the sensitivity of bacterial isolates to similar antibiotics such as Ciprofloxacin and Ofloxacin and opined their value as empiric antibiotic therapy for enteric infections. It has been shown that resistance to ciprofloxacin is usually associated with resistance to other macrolides, lincosamides, and type B streptogramin, and is referred to as MLS resistance (Ndirika et al., 2016).
Multiple drug resistance was seen in pathogenic Staphylococcus aureus, Bacillus spp, Escherichia coli, Pseudomonas aeruginosa and Salmonella spp in this study. This may be attributed to the presence of resistance determinants on plasmids with similar selective markers or as a result of independent, simultaneous development of resistance to different agents (Abu and Wondikom, 2018). These suggest that bacteria have the unique characteristics of being able to transfer resistance genes from one bacterium to another in different population and habitats (Mandal et al., 2011). Multiple antibiotic drug resistance profiles have also been reported in enteric bacteria from both human and animal sources (Ikpeme et al., 2011). Persistent multiple drug resistance of most isolates to appropriate drugs of choice are of great public health concern and calls for periodic monitoring of antibiograms to detect possible changing patterns (Davies, 2014).