Molecular Detection of Beta Lactamase Genes from Salmonella Typhimurium Isolated from Poultry Droppings in Nyanya, Abuja

Molecular Detection of Beta Lactamase Genes from Salmonella Typhimurium Isolated from Poultry Droppings in Nyanya, Abuja

Kanu N.V., Orji S.L., Okafor H.A., Adama R. I., Mustapha, F.A., Oluwafemi M. P., Offiong A. B., Tsebam B.C., Donli, S.C and Ekwe E.O

National Biotechnology and Research Development Agency, Lugbe Abuja, Nigeria

DOI: https://doi.org/10.51584/IJRIAS.2024.908020

Received: 28 June 2024; Revised: 07 July 2024; Accepted: 12 July 2024; Published: 02 September 2024

ABSTRACT

Salmonella infections are one of the major global public health problems. During the last decade, antibiotic resistance and multiresistance of S. typhimurium. This study aimed at molecular detection of Beta Lactamase genes from S. typhimurium Isolated from poultry droppings in Nyanya, Abuja. Fecal droppings samples were collected from two different Poultry farms using sterile screw tubes. S. Typhimurium was isolated and identified using standard microbiology methods. Antimicrobial susceptibility was carried out using Clinical and Laboratory Standards Institute method. Out of 180 samples collected the overall occurrence of S. typhimurium was 20 (11.1%). S. typhimurium isolated were higher in Farm A 9(10.0%) than in farm B 11(12.2%). The S. typhimurium isolates from Farm A were highly resistant to Ampicillin with 77.7% but less resistant to gentamicin with 33.3%, ofloxacin, ciprofloxacin and Sulphamethoxazole/trimethoprim with 44.4%. Isolates from Farm B were highly resistant to Ampicillin and Sulphamethoxazole/trimethoprim 81.8%, but less resistant to Cefuroxime 36.3%. the most common phenotypic resistant observed were C-CIP-CAZ-CRO-AUG- OFL -SXT- CEF (22.2%) from Farm A. CIP-CRO-CAZ- AMP -AUG- OFL -SXT- CEF (18.1%) from Farm B, The commonest MAR index observed in this study was 0.8(33.3%) in farm A and (36.3%) in farm B. The order of occurrences of the 4 Beta Lactamase genes detected was: blaCTX-M-9 and blaSHV (75.0%) > bla CMY2 and Bla CTX-M (50.0%). The resistance observed in this study indicates it will be difficult to use commonly use antibiotics to treat infection caused by S. typhimurium isolated in the study location

Keywords; Antibiotic, Resistance, S. typhimurium, Fecal droppings, Beta Lactamase

INTRODUCTION

Poultry and poultry meat products are considered as some of the main carriers of Salmonella Species and represent a significant share of the attributed sources of salmonellosis in Humans. The widespread occurrence of Salmonella in natural environment and the Intensive husbandry practice used in the meat, fish and shellfish industries have been a significant problem in public health [1].

Beta-lactamases are ancient bacterial enzymes that attacks the beta-lactam ring. The class B beta-lactamases (the metallo- beta-lactamases) are divided into three subclasses: B1, B2 and B3. Subclasses B1 and B2 are theorized to have evolved about one billion years ago and subclass B3s is theorized to have evolved before the divergence of the Gram-positive and Gram-negative eubacteria about two billion years ago [2].

Penicillinase was the first β-lactamase to be identified. It was first isolated by Abraham and Chain in 1940, Among Gram-negative bacteria, the emergence of resistance to expanded-spectrum cephalosporins has been a major concern. Salmonella infections are one of the major global public health problems [3].

During the last decade, antibiotic resistance and multi-resistance of Salmonella spp. have increased a great deal, especially in developing countries with an increased and indiscriminate use of antibiotics in the treatment of humans and animals. Multidrug-resistant (MDR) strains of Salmonella are now encountered frequently worldwide and the rates of multidrug-resistance have increased considerably in recent years [4,5]. Even worse, some variants of Salmonella have developed Salmonella metabolism and its effect on host colonization of food-producing animals by Salmonella generally results in asymptomatic colonization of the intestines. Recently, they have begun to recognize that nutritional requirements of Salmonella play an important role in colonization. Salmonella typhimurium possesses three mannose-family sugar phosphotransferase (PTS) [6] permeases which are absent in most closely related bacteria and whose expression is controlled by the transcription factor, Consistent with the hypothesis that Salmonella’s nutritional requirements play a role in colonization, and are important for Salmonella survival within the chicken gastrointestinal tract [7, 8]. This study focus on molecular detection of Beta Lactamase genes from Salmonella Typhimurium Isolated from poultry droppings in Nyanya, Abuja

METHODS

Study Area

The study was carried out in the satellite town in Abuja called Nyanya which is under Abuja Municipal `Area Council, it is bordered on the north and northeast by a range of hills, to the east and southeast by Maraba district of Nasarawa state to the south by Federal housing karu site and to the west by Abuja city

Media

Bacteriological media that was used, Methyl Red Voges Proskauer Medium, xylose-lysine deoxycholate (XLD) agar, Bismute susphate agar (BSA) and Salmonella-Shigella agar (SSA), Nutrient Agar, Simmon’s Citrate Agar, Urea broth, and Peptone Water, Mueller-Hinton Agar (MHA)

Primers

The primers used in this study and their characteristics are as given in table 3.1.

Table 1: Primers sequence, target genes and their amplicon sizes

Sample Size Determination

The sample size was calculated manually using the formula below as earlier described by [10]

N=Z²P∑/d²

Where: N= desired sample size (when the population >10,000); Z= standard normal deviate, usually set at 1.96, which usually correspond to 95% confidence level; P= proportion in the target population, set at 50% (0.5) d= tolerated margin of error.

The proportion was estimated as p< (0.5) and q< (0.5) for non-infection confidence estimated used was 95% (≥1.5) confidence interval with degree of accuracy of d (0.05). The designed effect of 1 was used. The sample size was obtained

N= (1.16)2× 0.5× 0.5÷ (0.05)2

=0.9404÷0.0025 = 180

Sample collection

A total of one hundred and eight (180) poultry fecal droppings samples were collected using sterile screw tubes. The samples were then transported to microbiology laboratory department of Nasarawa State University Keffi for the analysis.

Isolation of Salmonella species

A loopful of the poultry fecal droppings sample was transferred into 5ml enrichment nutrient agar broth and incubated at 37⁰C for 24 h. After incubation, all the tubes showing turbidity after incubation were bacteriologically cultured on xylose-lysine deoxycholate (XLD) agar and Bismute susphate agar (BSA) (Oxoid, UK) for the selective isolation of Salmonella species. The plates were then incubated at 37^oC for 24 hours for bacteria. After incubation, the plates were observed for growth and subsequently sub-cultured unto freshly prepared media to obtain pure isolates while other conditions for incubation remain the same according to the methods describe by Langkabel et al. [11].

Identification of the Isolates

Representative colonies were chosen from each of the cultured plate on the bases of their colonial and morphological characteristics. Pure bacterial colonies were identify using Gram staining reaction and biochemical tests according to the methods of Cheesbrough [12]

Gram Staining Examination

The Gram staining technique was carried out as described by Cheesbrough [12] with some modifications; A small portion of cultural organism was transferred onto a clean grease-free glass slide, and emulsified in a drop of distilled water until a thin homogeneous film is obtained, then the wire loop was ` re-sterilized and the thin homogeneous film was allowed to air-dry, and heat-fixed by passing through the flame. The slide was then flooded with crystal violet for 1 minute, and then rinsed with distilled water. The smear was again flooded with Lugol’s iodine for 1 minute, and rinsed with distilled water and then decolorized, rapidly with acetone alcohol until no more colour appeared to flow from the preparation and rinsed appropriately with distilled water. The smear was then counter stained with neutral red for 1 minute and rinsed with distilled water and allowed to air dry and viewed microscopically using x100 oil immersion objective. Gram positive organism retains the dark blue colour inferred by the iodine/crystal violet complex, while Gram negative organisms appear red, maintaining the colour of the secondary dye. Salmonella species is a gram negative cocco-rod.

Biochemical tests

The conventional and commercial (Microgen Bioproducts) biochemical identification tests were used. Conventional biochemical tests were carried out according to the methods of Cheesbrough [12] and observed for reactions typical of Salmonella such as Triple Sugar Iron Agar (TSI); Indole Test; Lysine Decarboxylase Test:  Methyl Red Test; Citrate Utilization test; Urease test; Motility Test

Salmonella serotyping

Salmonella serotyping was carried out by standard slide agglutination test (CEN ISO/TR 6579-3:2014) with polyvalent and monovalent somatic (O) and flagella (H) antisera (Statens Serum Institute Denmark and Sifin, Berlin, Germany). Firstly, suspected colonies were picked up and tested with somatic O polyvalent and O polyvalent group antisera. In the case of a positive reaction, testing according to the Kaufman White scheme would be applied. If the suspect colony did not show any reaction with O polyvalent antisera, Salmonella species and subspecies was identified by biochemical properties. Serotyping results was evaluated according to the Kaufmann White Salmonella serotyping scheme as described by Ibrahim et al. [13]

Determination of Antibiotic Susceptibility of the S. Typhi isolates

Antimicrobial Susceptibility Testing of the isolates were carried out using Kirby-Bauer disc diffusion method as described by the National Committee for Laboratory Standards (NCCLS) now the Clinical and Laboratory Standards Institute (CLSI) [14].

Preparation and Standardization of Inoculum: At least three well isolated and identical colonies were selected and transferred aseptically with a wire loop into 5 ml sterile Mueller-Hinton broth. The tubes were incubated at 37ºC for 18h to match the turbidity of the 0.5 McFarland barium sulphate standard prepared by mixing 9.95 ml of 1% H₂SO₄ with 0.05 ml of 1.175%  BaCl₂.2H₂O. Culture whose turbidity exceeded that of 0.5 McFarland was re-adjusted to equivalence using sterile broth. The turbidity of the standard and the test inocula was compared by holding both tubes in front of a stripped white background.

Inoculation of agar plates and application of antibiotics discs: Within 15 min of adjusting the turbidity of the inocula, sterile cotton swabs were immersed into the properly diluted inocula, then rotated firmly several times against the upper inside wall of the tubes to express excess fluid and used to inoculate (by streaking) the entire agar surface of the agar plates. Antimicrobial agent impregnated discs were aseptically placed on the inoculated agar using antimicrobial disc forceps. The discs were deposited such that the centers are 24 mm apart and left to stand for 1 h for pre-diffusion.  The plates were subsequently incubated at 37ºC for 24 h.

Results and Interpretation: The diameters (in millimeters) of the various zones of inhibition of growth around each disc was measured with the aid of a ruler on the under surface of the plates without opening the lid. The results would be interpreted as susceptible, intermediate or resistant depending on the diameter of the zone of inhibition measured in millimeter as described in the CLSI [14] interpretation criteria.

Determination of Multiple Antibiotic Resistance (MAR) Index

The MAR Index was determined according to the method of Ibrahim et al. [13] as described by Ngwai et al. (2014). From the results of the antimicrobial susceptibility testing, MARI was calculated using the following formula:

MARI=a/b

Where a = number of antibiotics to which an isolate is resistant to

b = number of antibiotics against which the isolate was tested.

An isolate with MARI ≥ 0.2 is regarded to have originated from an environment where antibiotics are frequently used [13].

Phenotypic Detection of Extended Spectrum Beta-Lactamase

Extended Spectrum Beta-Lactamase production was detected by the conventional Double Disc Synergy Test (DDST) using ceftazidime (30 μg) and cefotaxime (30 μg) discs (placed 15 mm apart from the centre of the discs) with amoxicillin/clavulanic acid (20/10 μg) disc as recommended by the CLSI [14]. Positive result is indicated when the inhibition zones around any of the cephalosporin discs are augmented in the direction of the disc containing clavulanic acid. An increase of ≥ 5 mm in the inhibition zones of either cephalosporin in combination with clavulanic acid compared to the cephalosporin alone was interpreted as ESBL positive [14].

Molecular Detection of Extended-Spectrum Beta-Lactamase Resistance

1. DNA Extraction

The DNA was extracted from ESBL producing isolates using boiling method as described by Ibrahim et al. [13]. Briefly following, 1 pure colony of ESBL producing isolate, was inoculated into 2 ml of LB broth and incubated at 37ºC for 8 h and 200 μl of LB culture was transfer into Eppendorf tube and centrifuge in micro centrifuge as 3200 rpm for 2 min at room temperature and the supernatant was discard living the cells and the cells were wash twice with washing buffer. About 0.5ml of sterile phosphate buffer was added to the pellet and vortex for 5 sec after which it was heated at 90ºC for 10 min and rapid coiling was done by transferring the tubes into freeze for 10 min and thereafter it was centrifuge at 3200 rpm for 1 min to separate the DNA and the cell containing the DNA debris and 300 μl of supernatant, was transferred into 2 ml Eppendorf tube and stored at -10ºC until use.

2. DNA Amplification of Extended Spectrum β-Lactamase Genes

multiplex Polymerase Chain Reaction (PCR) was performed in order to amplify the ESBL genes present in the isolates. The presence of blaCTX-M, blaSHV and blaTEM genes were tested for using previously published primer sets and conditions. The primer sequences and expected amplicon size for each gene are listed in Table 1. The reactions were carried out in 20 μl reaction volume made up of 10 μl of Mastermix (Qiagen), 0.32 μl of primers (0.16 μl each of forward and reverse primers), 3 μl of DNA and 6.68 μl of nuclease free water. The primer concentration stood at 0.2 M. The reaction tubes were placed in the holes of the thermal cycler and the door of the machine was 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 sec, annealing at 56°C for 40 sec, initial extension at 72°C for 50 sec, final extension at 72°C for 3 min and a hold at 4°C infinitely

RESULTS

Isolation and Identification of Salmonella Typhimurium

The cultural, Morphological and Biochemical characterization of Salmonella typhimurium from poultry droppings in some selected poultry farm in Nyanya- Abuja as shown in Table 2. shows that Salmonella typhimurium is Gram negative staining, the morphology of the isolated bacterium was small, Gram negative rod shape bacteria, on xylose lysine Deoxycholate (XLD) plates, the presumptive Salmonella typhimurium colonies appeared red with black centre appearing shows the presumptive and the biochemical characteristics of the Salmonella isolates.

Occurrence of Salmonella typhimurium

Out of the 180 samples collected twenty 20 (11.1 %) S. typhimurium were isolated. Farm A 9(10.0%) were isolated and from farm B 11(12.2%) were isolated as shown in Table 3.

Table 2: Cultural, Morphological and Biochemical Characteristics of Salmonella typhimurium

OXD=Oxidase MOT= Motility test; UR: Urease test= TDA; LYS= Lysine utilization; H₂S= hyudrogen sulphide ; ONPG= Onpg-Galactosidase test; NIT= Nitrate test; LAC= Lactose fermentation test; MAL=Maltonate test; IN= Indole fermentation test; MR : Methyl red MCA: MacConkey agar; SSA: Salmonella-Shigella agar; BSA: Bismuth Sulfite agar

Table 3: Percentage occurrence of Salmonella typhimurium in relation to Selected Poultry Farms

Antibiotics resistance of Salmonella typhimurium isolates

The S. typhimurium isolates from Farm A were highly resistant to Ampicillin with 77.7% followed by Ceftazidime and Chloramphenicol with 66.6%, Cefuroxime, Cefotaxime and Amoxicillin-Clavulanic acid with 55.5% but less resistant to gentamicin with 33.3%, ofloxacin, ciprofloxacin and Sulphamethoxazole/trimethoprim with 44.4% each. From Farm B the isolates were highly resistant to Ampicillin and Sulphamethoxazole/trimethoprim with 81.8%, Ceftazidime and Chloramphenicol with 72.7%, Cefotaxime with 63.6% but less resistant to Cefuroxime with 36.3%. as shown in Table 4.

Antibiotics Resistance Phenotypes

The antimicrobial resistance Phenotypes of isolates from Selected Poultry Farms is as shown in Table 5. The isolates were distributed into different resistant phenotypes and the commonest were C-CIP-CAZ-CRO-AUG- OFL -SXT- CEF (22.2%) from Farm A, CIP-CRO-CAZ- AMP -AUG- OFL -SXT- CEF (18.1%) from Farm B, C-CIP-CRO-AUG-AMP-SXT- CEF (12.5%) respectively.

Table 4. Antibiotics resistance of Salmonella typhimurium from the poultry Farms in part of Nyanya-Abuja.

 Table 5. Antibiotics Resistance Pattern against Salmonella typhimurium Isolates

Key: OFX = Ofloxacin; CXM = Cefuroxime, AMC = Amoxicillin-Clavulanic acid; CIP = Ciprofloxacin; TE = Tetracycline, NOR = Norfloxacin; CN = Gentamicin; CTX = Cefotaxime; C = Chloramphenicol; STX = Trimethroprim-sulfamethoxazole.

Multiple Antimicrobial Resistance (MAR) index        

The MAR index of the isolates from Selected Poultry Farms is as shown in Table 6. The isolates were distributed into different MAR index and the commonest MAR index were 0.8(33.3%) from farm A and (36.3%) from farm B,

Occurrence of ESBL resistance genes

The occurrences of the ESBL resistance genes in the 4 cefuroxime, cefotaxime and amoxicillin-clavulanic acid resistant isolates are as given in Table 7. The order of occurrences of the 4 genes detected was: blaCTX-M-9 and blaSHV (75.0%) > bla CMY2 and Bla CTX-M (50.0%).

Table 6. Multiple Antibiotic Resistance (MAR) Index

Table 7 Occurrence of ESBL resistance genes

Plate 4.1: Agarose gel electrophoresis showing the amplified ESBL genes in antibiotic-resistant Salmonella typhimurium.  Lane 1and 4 represent  esbl gene; lane 2-4 represent blaCTX-M-9 (876bp) gene, lane 2 and 3 represent bla CMY2 (720bp) gene;  lane 2 and 4 represent blaCTX-M (651bp) gene and lane 2,3 and represent blaSHV  (615bp) gene while lane M represent 1000 bp molecular ladder

DISCUSSION OF FINDINGS

Salmonella Typhimurium were isolated from 180 faecal droppings across the two (2) Local poultries in Nyanya-Abuja. The isolation of Salmonella serotype such as S. typimurim in our study indicate the public health significance of these Salmonella typhimurium [15] as poultry product contaminated with Salmonella may pose serious health hazards [16,17]. S. typimurium has been implicated in bacteremia, septicemia and gastroenteritis.

The overall prevalence of Salmonella typhimurium isolated from faecal droppings in the poultry farms in Nyanya-Abuja was 11.1 %. The high prevalence of Salmonella typhimurium recorded in this study may be attributed to the improved hygienic condition in the poultry farms and the administration of antibiotics into the feeds and water in the farm [18]. This may account for the higher prevalence obtained in our study than Salihu et al., [19] in Nassarawa State, Nigeria, reported a prevalence of 2.5% which was lower than 11.1% reported in this study. In addition, Obi and Ike [4] reported a prevalence of 1.3% in Intensively Reared and backyard chickens in Nsukka Area, Nigeria. However, relatively lower but similar prevalence of 0.85 % was reported by Aiyelu [20] who isolated Salmonella Arizonae from chicken in Zaria, Kaduna State. The difference in prevalence observed may be due to the fact that the presence and distribution of Salmonella serotypes vary from region to region as reported by Uyttendaele et al.[21], Dominquez et al.[22], Agada et al., [23]. Similarly, the isolation rates might depend on the sampling plan as well as the detection limit of the methods of isolation adopted (15, 22].

The result of the antibiotic resistance in is present study showed that the four (4) Salmonella isolates were resistant to the various antibiotics used in the study. These organisms harbour multiple resistance genes. These findings agree with that of Kristiansen et al. [24] who reported multiple resistance characteristics to chloramphenicol, amoxicillin, sulfonamides and ampicillin. This indicates that fewer drugs would be available to treat infectious diseases caused by Salmonella typhimurium. Resistance to sulphamethoxazole – Trimethoprim and ampicillin among Salmonella isolates from poultry has been previously reported in Jos, Nigeria [23], in Ibadan, [25].  The observed resistance to sulphamethoxazole – Trimethoprim is likely due to its high usage in animal production [26].

The Salmonella showed resistance (66.6%) to the cephalosporins, this finding agrees with the 59.5% found in Salmonella species from poultry reported by Agada et al. [23] in Jos, Nigeria. Also, Arlet et al. [27] documented that Salmonella species is the most common serovars associated with extended spectrum cephalosporins resistance in poultry. The resistance to cefotaxime (100%) to Salmonella Arizonae observed in this finding is higher than 94.73% observed by Sivakumar et al. [28]. Results of multi drug resistant Salmonella recorded in this study was similar to the findings of other researchers (Davis et al., 2005; Akinyemi et al., 2007; Mohammed et al., 2010). In many of these studies, non-typhoidal Salmonella species were found to be resistant to Amoxicillin, co-trimoxazole, chloramphenicol, ciprofloxacin, sulphamethoxazole–Trimethroprim, Tetracycline, Cefotaxime. Similarly, Okeke et al. (2005) also reported that multiple antimicrobial resistances are common in Salmonella species in developing countries.

Multidrug resistant Salmonella reported in this study is in agrees with the findings of Hemen et al. [29] in Benue State Nigeria who reported the isolation of multi drug resistant Salmonella in poultry environment that was resistant to chloramphenicol, Augmentin and ampicillin. Fasure et al. [30] also revealed the emergence of multi – drug resistant Salmonella serotypes in Ogun state, Nigeria. In his study, resistant to ampicillin (100%), Tetracycline (90.6%), resistant strains were observed. Similarly, Mohammed et al. [31], also reported that Salmonella isolate from a poultry farm in Jos was resistant to Gentamicin and Ciprofloxacin. It was observed that 100% of the Salmonella species were resistant to the antibiotics in the study. The routine use of antibiotics for agricultural purposes, and disease prevention could be responsible for the emergence of multi-drug antibiotic resistance [30].

Polymerase Chain Reaction detection of antibiotic resistance genes analysis revealed that 75.0% and 50.0% of the isolates had bla-CMY, blaCTX-M-9, Bla CTX-M and blaSHV genes of the AmpC family. Salmonella resistance to cefuroxime, cefotaxime and amoxicillin-clavulanic acid could be due to the secretion of beta-lactamases into the periplasmic fluid. This enzyme hydrolyses the beta-lactam ring in beta-lactams into beta – amino acids which have no anti-microbial activity. The genes encoding for beta lactamases are typically carried on plasmids [32]. The presence of beta-lactamases in this study is in line with the findings of Abdel-Maksoud et al. [33, 34] who reported the presence of these genes in Salmonella isolates from poultry samples. The detection of these genes in this study may be due to the widespread use of antibiotics in animals for disease prevention and animal production. Thus, the spread of resistance genes in poultry flock [35] that can infect human directly or transfer antibiotic resistance genes to other human pathogen [36].

CONCLUSION

In this study Salmonella typhimurium were isolated from two poultry farms sampled. The occurrence of the isolates was high from farm A. the isolates were highly resistance to most of commonly used antibiotics to treat infection caused by Salmonella species. Most of the isolates were multidrug resistant and beta-lactam producing isolates. The beta-lactam resistant genes detected were blaCTX-M-9 and blaSHV, bla CMY2 and Bla CTX-M.

COMPLIANCE WITH ETHICAL STANDARDS

Disclosure of conflict of interest

Authors declare no conflict of interest

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