Prevalence of Antibiotic Resistance Non-Typhoidal Salmonella in Controlled Layer Poultry Sheds

Muhammad Rashid1, Adil Shahzad1,2,3, Hafiz Qadeer Ahmed2,4*, Muhammad Hamza5, Muhammad

Fayyaz Akhtar1, Syed Faizan ul Hassan1, Rabia Zahid1, Sultan Ali1

1Institute of Microbiology, University of Agriculture, Faisalabad-38000, Pakistan

2Livestock and Dairy Development Department, Government of Punjab, Pakistan

3College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA.

4College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China.

5Faculty of Biological Sciences, Department of Microbiology, Quaid-I-Azam University, Islamabad, Pakistan.

Abstract | Salmonella is a major zoonotic pathogen causing salmonellosis in poultry and gastroenteritis in humans. In poultry, Salmonella is the leading cause of bacterial infections causing watery diarrhea, inadequate food intake, poor growth, and low egg production. This research sought to identify Salmonella in controlled layer poultry sheds from poultry droppings and detect its pattern of antibiotic resistance. A total of 100 samples (25 from each shed) were collected and enriched in selective tetrathionate broth and grown on selective media salmonella-shigella agar. Salmonella was confirmed by examining the colony morphology, microscopic features, and results of biochemical tests such as the indole test, Voges-Proskauer test, methyl red test, oxidase test, catalase test, triple sugar iron test, and citrate test. Out of 100 samples, 22 samples were confirmed for having Salmonella. The Kirby-Bauer disk diffusion method was used to check the susceptibility of different antibiotics and the zone of inhibition was measured and compared to Clinical and Laboratory Standards Institute (CLSI 2023). When susceptibility was measured, all Salmonella isolates were resistant to ampicillin, 86.36% of isolates were resistant to Nalidixic acid and 77.27% of the isolated bacteria were resistant to tetracycline. Ceftriaxone and Tazobactam were the most sensitive drugs against the Salmonella isolates. PCR results showed that the blaTEM gene was the most detected resistant gene in the Salmonella isolates. These results confirm the prevalence of antibiotic resistance Salmonella species in the poultry environment. Therefore, to prevent the emergence and spread of drug-resistant Salmonella isolates, reasonable utilization and stringent control techniques for antimicrobials in control and treatment are required.

Novelty Statement | This research belongs to importance of zoonosis of Salmonella spp., antibiotic resistance which may be caused by the egg consumption from layer sheds used to treat the layer bird. The studies highlight the importance of this aspect and investigated the prevalence in selected area to highlight the potential issue.

Article History

Received: September 21, 2024

Revised: March 10, 2025

Accepted: March 24, 2025

Published: May 13, 2025

Authors’ Contributions

MR, AS did the material preparation,

lab testing and experiments. MH and

RZ analyzed the data. MFA, SFUH

wrote the first draft of the manuscript. SA supervised the research. HQA reviewed and edited the manuscript.

All authors read and approved

the final manuscript.

Keywords

Salmonella, Occurrence, Antibiogram, blaTEM gene, Antimicrobial resistance

Copyright 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Corresponding Author: Hafiz Qadeer Ahmed

[email protected]

To cite this article: Rashid, M., Shahzad, A., Ahmed, H.Q., Hamza, M., Akhtar, M.F., Hassan, S.F. and Zahid, Z., 2025. Prevalence of antibiotic resistance non-typhoidal Salmonella in controlled layer poultry sheds. Punjab Univ. J. Zool., 40(1): 37-42. https://dx.doi.org/10.17582/journal.pujz/2025/40.1.37.42

Introduction

Increased in Antibiotic resistance is a substantial concern in the poultry industry, specifically about bacteria like Salmonella. In Pakistan, the poultry sector is vital for the economy, contributing around 1.3% to the GDP. Commercial production was started in the 1960s, now provides a considerable portion of the daily protein intake for the population (Hussain et al., 2015). However, it faces key challenges, including Salmonella contamination and antibiotic resistance. Salmonella is a bacterium that can cause foodborne illnesses. Two primary species include: Salmonella enterica and Salmonella bongori (Hassena et al., 2022). With nearly 2600 identified serovars, some such as S. typhi and S. paratyphi cause typhoid and paratyphoid fever, while others like S. typhimurium and S. enteritidis are commonly found in poultry and can infect both birds and humans (Hendriksen et al., 2011; Cui et al., 2021). Typhoid toxin is important in the pathogenicity of these bacteria (Andesfha et al., 2019). Salmonella can form biofilms, enabling them to persist for extended periods in animals and humans (Afshari et al., 2018).

Salmonella infections are often occur through consuming contaminated food, eggs, or poultry meat. Approximately 20% of the world’s poultry products are contaminated with Salmonella. This bacterium not only causes illness in birds and reduces egg and meat production but also potential health risk to humans (Hassena et al., 2022). Young chicks are particularly vulnerable due to their immature immune system and weak intestinal microflora (Pan and Yu, 2013).

Salmonella infections are a common cause of illness in poultry and potential threat to foodborne diseases in humans (Ahmad et al., 2022). Most salmonellosis outbreaks have been linked to chicken products contaminated with S. typhimurium and S. enteritidis (Cui et al., 2021). Poultry diseases, especially those caused by Salmonella, result in significant economic losses for the industry (Rosengren et al., 2010).

Antibiotics are normally used to promote growth and treat infections in birds (Beier et al., 2013). However, the use of key antibiotics has been banned in many regions. For instance, antibiotics used as growth promoters were banned in the EU in 2006 and in the US in 2017, though they remain permitted in some countries like China and Brazil (Peruzy et al., 2020).

In Pakistan, while antibiotics for growth promotion are banned, their use for treating sick birds continues. This raises concerns about the emergence of antibiotic-resistant Salmonella strains in poultry sheds. However, there is limited data on the prevalence and antibiotic resistance of Salmonella in controlled layer sheds in Pakistan. This lack of information hinders the development of effective strategies to control Salmonella and protect public health. This study aims to address this gap by examining the prevalence and antibiotic resistance profiles of non-typhoidal Salmonella isolated from controlled-layer poultry sheds in Jhang, Punjab, Pakistan.

Materials and Methods

Salmonella isolation and biochemical identification

A total of 100 samples were collected from the feces of egg-laying poultry birds from the four control layer sheds of the Jhang region of Pakistan, following approval from the Institutional Ethical Committee. 1 gram of each sample was measured through weighing balance and added to the 9 ml of buffered peptone water for non-selective bacterial enrichment. Selective pre-enrichment was done for 24 h at 37 °C temperature, followed by streaking a loopful of the enrichment broth on the selective media salmonella -shigella agar and incubated for 24-48 h at 37 °C, Figure 1. Suspected Salmonella black centered or colorless colonies on SS agar were streaked to xylose-lysine-desoxycholate agar and also observed under a microscope, Figure 2. Colony characteristics showed black dot-centered pinkish colonies on XLD agar, which were further identified by biochemical tests including, the Tripple sugar iron test, IMViC test, and oxidase test (Ashrafudoulla et al., 2021). The entire procedure for isolating and detecting Salmonella was carefully followed, as per ISO-6579-2017.

 

 

Table 1: Primer sequence used to target blaTEM gene.

Gene name	Sequence	AT (°C)	Amplification size (bp)	References
blaTEM	F. 5′-ATCAGCAATAAACCAGC-3′ R. 5′-CCCCGAAGAACGTTTTC-3′	54°c	516	(Yukawa et al., 2019)

 

Antimicrobial susceptibility test

Antimicrobial susceptibility test is used to check the ability of pathogenic microorganisms to develop resistance to different antibiotics. The resistance to antibiotics was checked by the Kirby-Bauer disk diffusion method. Muller Hinton agar was poured into the Petri plates and allowed to cool. A loopful of the bacterial culture was streaked on the agar using sterilized cotton swabs. Antibiotic disks were then placed on the agar using a sterilized tweezer. The plates were then incubated at 37 C for 24 h and inhibition zone was measured (Igbinosa et al., 2022). Susceptibility of various antibiotics was checked: Piperacillin/Tazobactam, Tetracycline, Amikacin, Ciprofloxacin, Nalidixic acid, ceftriaxone, Ampicillin, Chloramphenicol, and Gentamycin.

 

Molecular detection of antibiotic-resistant gene (blaTEM)

Polymerase chain reaction was used to detect the blaTEM gene in Salmonella. The conditions that were used for PCR were: initial denaturation was done at 94°C for 30 sec, annealing was done at 54 °C for 30 sec and extension was carried out at 72°c for 1 min. All isolates of Salmonella showed the presence of a blaTEM-resistant gene (Colom et al., 2003).

Results

Antimicrobial resistance profiling

Antimicrobial susceptibility test was performed with commonly used antibiotics as shown in Figure 3. All Salmonella isolates were resistant to ampicillin. 86.36% and 77.27% of Salmonella isolates showed resistance to Nalidixic acid and tetracycline respectively. Ciprofloxacin, chloramphenicol, gentamycin, and sulphamethoxazole were intermediate sensitive to salmonella. Ceftriaxone, tazobactam, and amikacin were the most sensitive drugs to salmonella 91%, 82%, and 75%, respectively Figure 4.

 

Molecular detection of antimicrobial-resistant gene

Polymerase chain reaction was used for the detection of blaTEM resistant gene in salmonella isolates. The primer sequence used to target the gene is given in Table 1. PCR showed most prevalent resistant gene was found to be blaTEM. Agarose gel electrophoresis results are shown in Figure 5.

 

Discussion

The poultry sector is facing significant risks due to the prevalence of Salmonella in poultry. Salmonella is an important zoonotic pathogen that causes various diseases in poultry (Kuźmińska-Bajor et al., 2023). It is transmitted through contaminated food to humans and causes gastroenteritis and septicemia. According to estimates, Salmonella is responsible for 155,000 fatalities and 93 million gastrointestinal infections per year (Yu et al., 2021).

In this study, Salmonella in poultry was examined for its prevalence, antibiotic resistance pattern, and the presence of antibiotic-resistant genes for β-lactam antibiotics. The samples were collected from Jhang, Punjab, Pakistan poultry sheds. Out of the 100 fecal samples, 22 found positive for Salmonella. Previous research conducted in Pakistan showed a 25.67% prevalence of Salmonella (Siddique et al., 2021). This is almost similar to our results. Our results are also comparable to another study on Salmonella prevalence in Karachi, Pakistan, which reported a 21.34% rate of Salmonella prevalence (Hussain et al., 2020). A previous study showed a higher prevalence of Salmonella than our results, finding a 38% prevalence in Hyderabad, Pakistan (Soomro et al., 2010). Another study reported a higher prevalence of Salmonella in Punjab, Pakistan, at 44.66% (Yasmin et al., 2020).

The disc diffusion method was used to assess Salmonella susceptibility to several antibiotics, revealing the bacterial resistance pattern to drugs. In the present study, all the isolates were found to be resistant to ampicillin, followed by 86.36% being resistant to nalidixic acid, and 72.27% being resistant to tetracycline. This is similar to a previous study in which ampicillin resistance was found in 76.66% of isolates, the highest resistance among other antibiotics (Rafiullah et al., 2018). Salmonella’s strong resistance to ampicillin may indicate its overuse in common illnesses. Another study showed that isolated Salmonella strains from poultry exhibited 100% resistance to nalidixic acid. In this study, Salmonella showed 86.36% resistance to nalidixic acid, which is highly concordant with previous research. In a previous study, Salmonella demonstrated 77% resistance to tetracycline (Kayalvizhi and Antony, 2011). The present research showed 72.27% resistance to tetracycline, indicating that resistance to tetracycline has increased to a dangerous level due to the unusual or overuse of antibiotics in poultry.

In our study, Ceftriaxone was the most sensitive drug against Salmonella isolates. The previous study also showed that ceftriaxone was the most sensitive drug against Salmonella isolates (Veeraraghavan et al., 2021). Another study found that Tazobactam was the most sensitive drug against Salmonella (Uddin, 2018). In our study, Tazobactam-Piperacillin was also among the most sensitive drugs against bacteria Salmonella.

Furthermore, the presence of the blaTEM gene was detected in all the Salmonella isolates. The bacteria displayed resistance to different antibiotics specifically β-lactam antibiotics. The analysis of the genes (in bacteria) causing resistance to β-lactam antibiotics was done by using a polymerase chain reaction. The blaTEM gene was found to be the most prevalent gene in the bacteria causing resistance. Previous studies also demonstrated that the blaTEM gene was the most detected resistant gene in Salmonella (Igbinosa et al., 2022). Other studies also confirmed the presence of blaTEM resistant gene is salmonella isolates (Siddiky et al., 2021).

This study has some limitations. The sample size was relatively small, with only 100 fecal samples, which might not fully represent the overall prevalence and resistance patterns of Salmonella in the region. Additionally, the study was geographically limited to Jhang, Punjab, Pakistan, which may not reflect the situation in other areas. Future research should also investigate the mechanisms behind antibiotic resistance in Salmonella and explore alternative strategies to combat this issue, such as the development of new antibiotics or the use of bacteriophage therapy.

Conclusions

This study indicates the high prevalence of antibiotic-resistant Salmonella in poultry, which poses a major health threat to the poultry sector. This study indicated that Tazobactam and Imipenem are the most effective drugs against Salmonella isolates. Salmonella isolates show maximum resistance to ampicillin, tetracycline, and Nalidixic acid. PCR showed positive results for the presence of blaTEM resistant gene. Antibiotics and other antimicrobial agents should only be used when absolutely essential and in the appropriate doses. Overuse or incorrect use of these medications can contribute to the emergence of drug-resistant Salmonella. The study emphasizes the concern over the increase and dissemination of Salmonella variants that have developed resistance to frequently used antibiotics. Drug-resistant Salmonella strains can be difficult to treat because they do not respond to routine antibiotic treatments. There should be procedures put in place to strictly regulate the usage of antimicrobial drugs. This can involve rules for administering antimicrobial agents, controlling their use, and putting in place strategies to limit the spread of antibiotic-resistant microbes.

Declarations

Funding

The study received no external funding.

Data availability

The data supporting the findings of this study is included in the article. Additional datasets generated or analyzed during the study are available from the corresponding author upon reasonable request.

IRB approval and ethical statements

Study was approved by University of Agriculture Faisalabad Pakistan Institutional Ethical Committee/research committee with strict observance of animal rights during study.

Declaration of generative AI and AI-assisted technologies in the writing process

No generative AI and AI-assisted technologies were used in the writing process.

Conflict of interest

The authors have declared no conflict of interest.

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