Antimicrobial Resistance Profiling and Virulence Factors of Gram-Negative Bacteria Recovered from Poultry

Abstract

Poultry farming is a significant sector of the global agriculture industry that has been identified as a major reservoir for pathogenic organisms. The widespread and indiscriminate utilization of antibiotics in poultry production, for both therapeutic and growth-encouraging purposes, has contributed to the emergence and spread of antibiotic-resistant strains. Poultry litter harbors antimicrobial remnants and antibiotic-resistant bacteria, when used as fertilizer brings potential environmental risks posed by pharmaceutical residues and antimicrobial-resistant bacteria. The focus of this investigation was to determine the susceptibility patterns of poultry isolates to different antibiotic classes. Sum of 19 Gram negative bacterial isolates were detected from poultry litter and feed samples. The isolates were classified into three different bacterial genera namely Escherichia spp., Proteus spp., and Pseudomonas spp.. The isolates showed a moderate degree of resistance to nitrofurantoin, doxycycline hydrochloride, amoxiclav, and ampicillin, where 35.7% were resistant to ampicillin, 28.5% to cefotaxime and 7.14% to chloramphenicol. Variation in virulence factors expression among the isolates and their association with multidrug resistance (MDR) were also included in the study. The occurrence of moderate-level of resistance in poultry strains is an alarming situation posses great economic and major public health concern. These observations point to poultry farms as potential reservoirs of bacteria resistant to antibiotics that pose a risk of transmission to humans via the food chain. Hence, evaluating and ensuring the utilization of antibiotics in synergistic combinations is crucial to achieve effective outcomes and minimize the risk of emerging resistance development.

Keywords

Introduction

Table 1: Source distribution of identified bacterial genera from poultry

Table 2: Antibiogram of bacterial isolates (n=19) showing resistance (Red) and sensitive (Yellow) and distribution of MDR & MDS isolates among sample source.

Table 3: Expression of virulence factors among Multidrug resistant (MDR) and Multidrug sensitive (MDS) bacterial phenotypes.

Determination of virulence attributes

Poultry isolates were further evaluated for the presence of selected virulence traits using phenotypic tests. Overall 89.74% isolates were biofilm producers and 78.94% were found motile. Lipase and siderophore production was observed among 73.68% isolates. The expression of protease as a virulence factor was observed for 52.63% isolates. A total of 6 MDR (multidrug resistant) and 13 MDS (multidrug sensitive) phenotypes were observed in the present study. All studied virulence factors were equally expressed among MDR isolates from both the sources. However, a notable difference was identified in protease production among MDS isolates from poultry litter and feed samples (Table 3).

DISCUSSION

This study aimed to evaluate the emergence of MDR isolates among bacteria recovered from poultry. A tremendous increase in the accumulation of resistance determinants in clinically important bacteria is a worldwide problem. In the present study, 19 Gram negative bacterial isolates were isolated and identified. Out of these, 13 were from poultry litter and 6 were from poultry feed samples. The most predominant isolate was Proteus spp., followed by Escherichia spp., and Pseudomonas spp. The isolates expressed a moderate degree of resistance to nitrofurantoin, doxycycline hydrochloride, amoxyclav, and ampicillin in the present study. However, a low resistance to cefotaxime, cefepime, and chloramphenicol was observed.  A total of 57.1% isolates of Proteus spp. showed resistance to doxycycline hydrochloride and nitrofurantoin, 35.7% were resistant to amoxyclav, 28.5% were resistant to ampicillin, 14.2% were resistant to cefotaxime, and 7.14% were resistant to cefepime and chloramphenicol. However, 100% isolates of Escherichia spp. were resistant to amoxyclav and 66.6% of isolates were resistant to ampicillin followed by 33.3% who were resistant to nitrofurantoin and doxycycline hydrochloride each. 50% isolates of Pseudomonas spp. were resistant to amoxyclav and chloramphenicol each.  Most of the isolates were sensitive to other antibiotics (gentamycin, tobramycin, ceftriaxone, norfloxacin, and amikacin) used in the study. A study executed by Ezekiel et al., 2011^[14] observed the high resistance among E. coli isolates recovered from poultry feed samples against nitrofurantoin (100%) and amoxicillin (93.3%). Eyasu et al., 2017^[15] observed high level of antimicrobial resistance among E. coli poultry isolates towards ampicillin (94.2%), followed by penicillin (92%), tetracycline (73%), erythromycin (66%) and the lowest resistance was recorded for kanamycin (2%). A study carried out by Shrestha et al., 2017^[16] reported that the E. coli isolates were resistant to ampicillin whereas the isolates were sensitive to doxycycline hydrochloride and gentamicin. Sebastian et al., 2021^[17] reported that the E. coli isolates were resistant to ampicillin, amoxicillin, amikacin and cefotaxime in their study. In our study, the rate of resistance among Escherichia spp. against amoxicillin (100%) and ampicillin (66.6%) was higher than in the study reported by Bushen et al., 2021^[18]. In their study, E. coli demonstrated a 91.7% resistance rate against ampicillin, 66.7% against amoxicillin-clavulanic acid, and 54.2% against chloramphenicol. P. mirabilis isolates also exhibited resistance to ampicillin, amoxicillin-clavulanic acid, and chloramphenicol in their study which was higher as compared to our study. Overall 31.5% of isolates were identified as multiple drug-resistant bacteria in this study. Out of 6 MDR isolates, 1 (33.3%) was of Escherichia spp., and 5 (35.7%) isolates belonged to Proteus spp. A study conducted by Ibrahim et al., 2021^[19] reported a high level, i.e., 90% of the multidrug resistant isolates from poultry litter. However, Okonko et al., 2010^[20] reported a moderate percentage of MDR isolates among E. coli (27.3%), and  Proteus mirabilis (45.5%) isolates recovered from poultry feed. In the present study, almost 50% of Proteus spp. from poultry litter were reported as MDR resembling the findings of the study conducted by Bushen et al., 2021^[18] who observed 50% of P. mirabilis poultry isolates as MDR. Despite high concentrations of incorporated antibiotics, multidrug-resistant organisms are still frequently observed. This points to the possibility that low-potency antibiotics used during feed production have compromised their intended effectiveness. Consequently, evaluating and adopting synergistic antibiotic combinations are necessary to enhance effectiveness and minimize the risk of emergence of resistance. This might challenge the effectiveness of modern antimicrobial agents. The presence of clinically important virulence factors such as lipase, protease, siderophore production, biofilm formation, and motility among Multidrug resistant (MDR) & Multidrug sensitive (MDS) bacterial phenotypes has also been conducted in the present study.  The occurrence of virulence factors among poultry isolates might result in increased morbidity and mortality rate in poultry farms. A total of 73.6% isolates were lipase and siderophore producers, and 52.6% of isolates were protease producers. Proteus spp. is the most significant producer of lipase, protease and siderophore especially among the isolates recovered from the poultry litter, while Pseudomonas spp. and Escherichia spp. showed minimal to no production.  A total of 79% were found to be motile in our study which comprise of Proteus species (n=10), Escherichia (n=03) and Pseudomonas species (n=02). Channa et al., 2019^[21] observed that all the E. coli isolates from poultry litter were non motile in their study. The similar results as our study were reported by Danbappa et al., 2018^[22]. They observed that all poultry strains of E. coli and Proteus spp. were motile. In our study, 10.5% isolates were non biofilm producers, 36.8% were moderate producers, 42.1% were weak producers, and 10.5% were strong biofilm producers. Similar study carried out by Suresh et al., 2016^[23] found that out of 6 isolates, 3 (50%) were strong biofilm producers and Marek et al., 2021^[24] reported that out of 87 isolates of Coagulase Negative Staphylococcus, 69 isolates were biofilm producers, out of these, 24 (27.6%) produced weak biofilms, 11 (25.3%) produced moderate biofilms, and 13 (26.4%) produced strong biofilms. Conclusively, biofilm formation potentially enhances their survivability in any environmental condition. Such biofilm-producing strains are also implicated in chronic infections and are known to intensify disease severity. In the current study, significant numbers of bacterial isolates recovered from poultry litter and feed were resistant against one or more antibiotics regularly prescribed to treat human infections. The multidrug resistance was observed majorly among poultry litter isolates in the present study which may a consequence of irrational or indiscriminate antibiotics usage. These observations imply that poultry farms could act as potential reservoirs for antibiotic-resistant bacteria capable of spreading to humans via the food chain. The occurrence of moderate-level of resistance in poultry isolates is alarming situation possess great economic and major public health concern. The proper initiatives should be taken to control the emergence of such drug resistance pathogens. Although molecular methods could have been used to identify the isolates, financial constraints prevented access to commercial services. However, molecular techniques will be employed in the near future to accurately identify the bacterial species. The studied isolates will also be examined to confirm the presence and expression of virulence genes in both resistant and susceptible strains.

CONCLUSION

This study highlights the presence of multidrug-resistant gram-negative bacteria including Escherichia spp., Proteus spp., and Pseudomonas spp. in poultry litter and feed samples, with notable resistance to antibiotics such as ampicillin, cefotaxime, and doxycycline. The detection of virulence factors among these isolates further underscores the potential threat they pose to public health, particularly through transmission along the food chain. The findings highlight poultry farms as significant reservoirs for antimicrobial-resistant pathogens and underscore the need for prudent antibiotic use in animal agriculture. Implementing surveillance programs, promoting antibiotic stewardship, and exploring synergistic antibiotic combinations are essential strategies to restrict the emergence and dissemination of resistance. These measures are critical for safeguarding both human and animal health in the face of rising antimicrobial resistance.

REFERENCE

1. Roy CR, Ahmed T and Uddin MA: Microbiological analysis of poultry feeds along with the demonstration of the antibiotic susceptibility of the isolates and the antibacterial activity of the feeds, Bangladesh Journal of Microbiology (2019), 34(2):103-7.
2. Bolan NS, Szogi AA, Chuasavathi T, Seshadri B, Rothrock MJ and Panneerselvam P: Uses and management of poultry litter, World’s Poultry Science Journal (2010), 66(4):673-98.
3. Davies J and Davies D: Origins and evolution of antibiotic resistance, Microbiology and Molecular Biology Review (2010), 74(3):417-33.
4. Vaarst M, Steenfeldt S and Horsted K: Sustainable development perspectives of poultry production, World’s Poultry Science Journal (2015), 71(4):609-20.
5. Talukder M, Islam M, Ievy S, Sobur M, Ballah F, Najibullah M, Sarker S, Rahman M, Haque A: Detection of multidrug resistant Salmonella spp. from healthy and diseased broilers having potential public health significance, Journal of Advanced Biotechnology and Experimental Therapeutics (2021), 4(2):248.
6. Ali J, Sohail A, Wang L, Rizwan Haider M, Mulk S and Pan G: Electro-microbiology as a promising approach towards renewable energy and environmental sustainability, Energies (2018), 11(7):1822.
7. Kempf I, Jouy E, Granier SA, Chauvin C, Sanders P, Salvat G and Madec JY: Comment on “Impact of antibiotic use in the swine industry”, by Mary D. Barton [Current Opinion in Microbiology (2014), 19:9-15], Current Opinion in Microbiology (2015), 26:137-8.
8. Founou LL, Founou RC and Essack SY: Antibiotic resistance in the food chain: a developing country-perspective, Frontiers in Microbiology (2016), 7:1881.
9. Exner M, Bhattacharya S, Christiansen B, Gebel J, Goroncy-Bermes P, Hartemann P, Heeg P, Ilschner C, Kramer A, Larson E, Löffler H, Mielke M, Mühlig A, Oltmanns D, Pauli G, Ryll S, Schmithausen RM, Werner G, Widmer AF, Wischnewski N and Wilke F: Antibiotic resistance: What is so special about multidrug-resistant Gram-negative bacteria?, GMS Hygiene and Infection Control (2017), 12:Doc05.
10. Bettiol E and Harbarth S: Development of new antibiotics: taking off finally?, Swiss Medical Weekly (2015), 145:w2059.
11. Marshall BM and Levy SB: Food animals and antimicrobials: Impacts on human health, Clinical Microbiology Reviews (2011), 24(4):718-33.
12. Clinical and Laboratory Standards Institute (CLSI): Performance standards for antimicrobial susceptibility testing. 32nd ed. CLSI supplement M100. Wayne, PA: CLSI, 2022.
13. Minhas B, Chandel V, Minhas N, Attri S, Singha A and Thakur V: Identification, antimicrobial resistance profiling and virulence factors of bacterial isolates recovered from human clinical cases, Journal of Pure and Applied Microbiology (2024), 18(4):2850-61.
14. Ezekiel CN, Olarinmoye AO, Oyinloye JMA, Olaoye OB and Edun AO: Distribution, antibiogram and multidrug resistance in Enterobacteriaceae from commercial poultry feeds in Nigeria, African Journal of Microbiology Research (2011), 5(3):294-301.
15. Eyasu A, Moges F and Alemu A: Bacterial isolates from poultry litters and their antimicrobial susceptibility patterns in Gondar, Northwest Ethiopia, International Journal of Microbiology Research and Reviews (2017), 6(2):197-204.
16. Shrestha A, Bajracharya AM, Subedi H, Turha RS, Kafle S, Sharma S, Kattel HP and Raut S: Multi-drug resistance and extended spectrum beta lactamase producing Gram negative bacteria from chicken meat in Bharatpur Metropolitan, Nepal, BMC Research Notes (2017), 10(1):574.
17. Sebastian S, Tom AA, Babu JA and Joshy M: Antibiotic resistance in Escherichia coli isolates from poultry environment and UTI patients in Kerala, India: A comparison study, Comparitive Immunology Microbiology & Infectious Diseases (2021), 75:101614.
18. Bushen A, Tekalign E and Abayneh M: Drug- and multidrug-resistance pattern of Enterobacteriaceae isolated from droppings of healthy chickens on a poultry farm in Southwest Ethiopia, Infection and Drug Resistance (2021), 14:2051-8.
19. Ibrahim A and Habibu UA: Isolation and characterization of multidrug-resistant Escherichia coli from poultry litter samples from selected farms in Kano metropolis, Nigeria, Nigerian Journal of Microbiology (2021), 35(1):5623-9.
20. Okonko IO, Nkang AO, Fajobi EA, Mejeha OK, Udeze AO, Motayo BO, Babalola ET, Ejembi J, Onoja BA and Eze VC: Incidence of multi-drug resistant (MDR) organisms in some poultry feeds sold in Calabar metropolis, Nigeria, Journal of Agriculture and Food Chemistry (2010), 9(3):514-32.
21. Channa IA, Kalhoro NH, Channa AA, Korejo NA, Sethar A, Soomro AH, Munir U, Soomro H and Yaseen G: Prevalence, biochemical characteristics and antibiotic susceptibility of pathogenic bacteria in poultry litter, Sindh University Research Journal (2019), 51(3):533-40.
22. Danbappa AAR, Alhassan KA and Shah MM: Isolation and identification of microbial contaminants associated with commercial poultry feeds, Journal of Applied Advanced Scientific Research (2018), 3(5):142-7.
23. Suresh RM, Raja SS and Vijayakumar R: Comparative evaluation of methods to detect biofilm formation by bacterial isolates of poultry environments, Scientific Transactions in Environment and Technovation (2021), 10(1):1-5.
24. Marek A, Pyzik E, Stepien-Pysniak D, Dec M, Jarosz LS, Nowaczek A and Sulikowska M: Biofilm-formation ability and the presence of adhesion genes in coagulase-negative Staphylococci isolates from chicken broilers, Animals (2021), 11(3):728.