AMR is a complex, multifaceted problem that impacts human, environmental and animal health and is one of the most significant global public health threats (Moses et al., 2025). Bacterial AMR alone was associated with 4.95 million deaths globally in 2019 (Yusuff et al., 2023). The susceptibility or resistance of bacteria to antimicrobial drugs is not uniform. There is considerable variation in the degree of resistance within the bacterial groups, largely depending on the bacterial species and specific resistant gene present.  Resistance development in bacteria occurs over time as they gradually adapt to their environment to ensure their survival (Uddin et al., 2021). Antimicrobial drugs exert selection pressure by eliminating susceptible bacteria and allows resistant strains to dominate. This, in turn, facilitates the transfer of AMR genes to other bacteria, including both commensal and pathogenic organisms (Kumar et al., 2022; Abreu et al., 2023). The issue is further compounded by the potential for AMR to spread through the food chain, amplifying the risk of resistance transmission to humans through contaminated food and water sources (Fernandez-Trapote et al., 2024; Sunder et al., 2021). Such dynamics underscore the urgent need for a coordinated global response to mitigate the spread of AMR and to reduce its devastating public health impact.
       
To mitigate AMR, surveillance is an essential tool for determining the burden of AMR and providing the necessary information for effective control measures. Countries have implemented surveillance and monitoring programs to detect AMR over the past decade. Several tools are available for the detection of AMR, each with its own advantages and limitations. Most of the screening programs focus on isolating indicator bacteria and assessing their resistance patterns towards clinically useful antibiotics. This method allows detection of AMR genes within these indicator bacteria. However, such culture-based methods are time-consuming and are often selective, screening only specific AMR bacteria, which may limit their ability to detect a broader range of resistant strains.
       
It is increasingly recognized that detecting AMR genes, rather than identifying individual bacterial species, is critical in combating AMR. The Direct PCR method, which detects AMR genes in any organism within a sample, offers a broader perspective of resistance across entire microbiota of the sample. This approach provides a more comprehensive understanding of AMR, reflecting the resistance patterns of a wider range of microorganisms present in the sample.
       
Thus, in this study, we aimed to compare two methods of studying AMR. The first method involved culture-based screening of AMR genes is focused on isolation and detection of AMR genes in Escherchia coli (Gram-negative indicator bacteria) and Staphylococcus aureus (Gram-positive bacteria). In the second method, we employed culture-independent commercially available direct PCR master mix for the detection of AMR genes without the need for DNA extraction. The samples included broiler meat samples, fecal sample from broiler chicken and water samples collected from slaughter house.  The two methods were compared for their sensitivity, specificity and concordance of detection.

Study area and sample size
 
A total of 150 different samples were collected from the retail broiler outlets across 15 zones in Chennai district, Tamil Nadu, India between January 2024 to May 2024. The samples included broiler Chicken meat, cloacal swabs from live chicken and water from the slaughter house water cutting area. Around 25 g of chicken meat samples were collected in sterile polythene bags while cloacal samples were taken using sterile swabs and placed in 3 ml sterile Phosphate Buffered Saline (PBS). Around 100 ml of water samples were collected in sterile containers. All the samples were properly labeled and transported immediately to the Department of Veterinary Pharmacology and Toxicology, Madras Veterinary College, Chennai, in an ice box and stored at 4°C until further processing.
 
Isolation and identification of E. coli and S. aureus
 
The samples were inoculated in Nutrient Broth or brain heart infusion broth and grown aerobically at 37°C for 18-24 hours. A loopful of bacteria from the broth was streaked onto MacConkey Agar or Mannitol salt agar (MSA) and incubated at 37°C for 18-24 hours. Selective pink colonies from MacConkey agar were sub-streaked in Eosin Methylene Blue (EMB) agar while golden yellow colonies from MSA agar were sub streaked into MSA agar and incubated at 37°C for 18-24 hours. The presumptive colonies that produced a metallic sheen on EMB agar and golden yellow colonies on MSA were subjected to the Gram’s staining and biochemical tests (IMViC test, Catalase and Oxidase test) for tentative identification of E. coli and S. aureus.
 
Bacterial DNA extraction
 
Bacterial DNA was extracted by heat lysis following the procedure described by Arora et al. (2006) with slight modifications. Pure bacterial cultures grown in nutrient broth were centrifuged at 4°C for 5 min at 8000 rpm to pellet the cells. The pellet was washed with sterile nuclease free water (NFW) and then re-suspended in sterile NFW. The suspension was placed in a water bath at 95°C for 10 min and then immediately transferred to-20°C for snap chilling for 10 min. The suspension was then centrifuged for 5 min at 8000 rpm and the supernatant, containing the DNA was collected and used. The purity of the DNA was determined by calculating the ratio of absorbance at 260 nm and 280 nm using a spectrophotometer Nanodrop 2000 (Thermo-Scientific, Wilmington, DE, USA).
 
Molecular confirmation of E. coli and S. aureus
 
Molecular confirmation of E. coli and S. aureus was done by PCR using primers specific to E. coli 16S rRNA gene and S. aureus nuc gene (Table 1). The PCR reaction was carried out with a final volume of 25 µl, consisting of 12.5 µl Emerald AmpR GT PCR master mix (Takara), 1 µl of forward primer, 1 µl of reverse primer, 2 µl bacterial DNA and 8.5 µl of NFW in a thermal cycler (BIO-RAD T100^tm thermal cycler, Singapore). The cyclic conditions are given in Table 2. PCR products were loaded onto agarose gels for analysis. The gels were stained using ethidium bromide and electrophoresis was run at 100 V for 30 min. The gels were visualized using UV transilluminator and photographed using the Bio-Rad Gel Doc 1000 gel documentation system (Bio-Rad, USA).




 
Detection of Antimicrobial Resistant Genes in E. coli and S. aureus
 
The isolates were tested for the presence of resistance genes using primers specific to tetA, sul1, qnrA, blaOXA-1 and blaCTX-M-1 (Table 1). The PCR reaction was carried out in a final of 20 μl, consisting of  10 ìl of Emerald AmpR GT PCR master mix (Takara), 1 ìl of forward primer and 1 ìl of reverse primer of respective resistant genes, 2 μl of DNA template and 6 μl of NFW in a thermal cycler [BIO-RAD T100tm thermal cycler, Singapore]. The cyclic conditions were given in Table 2. PCR products were loaded onto agarose gels for analysis. The gels were stained using ethidium bromide and electrophoresis was run at 100 V for 30 min. The gels were visualized using UV transilluminator and photographed using the Bio-Rad Gel Doc 1000 gel documentation system (Bio-Rad, USA).

Identification of antibiotic resistance genes by direct PCR technique
 
For direct detection of AMR genes from the samples without nucleic acid extraction, Platinum Direct PCR Universal Master Mix (Invitrogen, Thermo Fisher Scientific, USA) was used according to the manufacturer’s instructions.
       
The lysis solution was prepared by adding 0.6 µl of Proteinase K to 20 µl of Lysis buffer and vortexing the mixture. 20 µl of Lysis Solution was taken in a micro centrifuge tube and sample (0.5 mm tissue/1 µl water/1 µl cloacal sample) was then added and placed in a PCR machine and incubated at 98°C for 5 min. After incubation, the lysis solution was centrifuged and the supernatant collected was used for the PCR reaction.
       
The samples were tested for the presence of the same five resistance genes using primers specific to tetA, sul1, qnrA, blaOXA-1 and blaCTX-M-1 (Table 1). 20 μl reaction mixture contains 10 µl of Platinum™ Direct PCR Universal Master Mix, 1 µl of Forward primer, 1 µl of Reverse primer, 4 µl of Platinum™ GC Enhancer, 3 µl of Nuclease-free Water and 1 µl of Sample supernatant. The contents are mixed and then centrifuged briefly. The cyclic conditions of PCR conditions are given in Table 2 and the reaction carried out in a thermal cycler [BIO-RAD T100tm thermal cycler, Singapore].  PCR products were loaded onto agarose gels for analysis. The gels were stained using ethidium bromide and electrophoresis was run at 100 V for 30 min. The gels were visualized using UV trans illuminator and photographed using Bio-rad Gel Doc 1000 gel documentation system (Bio-rad, USA).
 
Statistical analysis
 
The descriptive statistics for the data was given by MS Office 2016 Excel. The two methods for the detection of AMR genes were compared with culture dependent method by Cohen’s kappa statistics using SPSS (Statistical Package for the Social Sciences) software version 2.0.

Identification of AMR genes by culture-dependent method
 
The prevalence of E. coli in chicken meat, cloacal sample and water sample were 26%, 80% and 24%, respectively while that of S. aureus were 30%, 4% and 18%, respectively. The positivity of AMR genes in E. coli and S. aureus were given in Table 3. Among the E. coli isolates from chicken meat, sul1 (69.23%) most prevalent followed by tetA (53.85%) and blaOXA-1 (53.85%). In E. coli isolates from cloacal samples, tetA (100%) was most common followed by sul1 (85%) and blaOXA-1 (62.5%). In E. coli isolates from water samples, tetA and blaOXA-1 were present in all isolates, while sul1 is present in 75% of the isolates.


       
In S. aureus isolated from chicken meat, the most common resistance genes were tetA (86.67%) and sul1 (86.67%) followed by qnrA (40%). Similar to this, tetA (88.89%) was most prevalent in S. aureus isolates from water samples followed by sul1 (73.08%). None of the S. aureus isolates showed the presence of blaCTX-M-1.
 
Detection of AMR genes by rapid direct PCR method
 
A rapid direct PCR assay was used to detect antimicrobial resistance (AMR) genes in the collected samples and the results were given in Table 4 and Fig 1-3. In cloacal samples, tetA was most prevalent followed by sul1, blaOXA-1, blaCTX-M-1 and qnrA. The prevalence of tetA was highest in cloacal samples (70%) followed by chicken meat (36%) and water samples (28%). Similarly, sul1 and qnrA were more prevalent in cloacal samples (64% and 18%) compared to chicken meat (46% and 10%) and water samples (36% and 8%). blaOXA-1 was most commonly found in cloacal samples (44%) followed by water (14%) and chicken meat (6%) samples. blaCTX-M-1 was present in 26% of cloacal samples whereas it is present only in 2% of both chicken meat and water samples.








 
Performance of AMR gene detection by direct PCR method compared to culture-based DNA extraction method              
 
The performance of Direct PCR method in detecting AMR genes was compared with the culture-dependent method. The sensitivity, specificity, kappa value and concordant value were given in Table 5.


       
In chicken meat, the direct PCR method showed moderate to substantial agreement with the culture-dependent method, with concordant values in the range of 76.15% to 87.72%. However, the sensitivity and specificity varied depending on the AMR gene and sample type. In chicken meat, direct PCR method demonstrated 100% sensitivity in detecting blaOXA-1 with a specificity of 84.78%. For tetA, sul1 and qnrA, sensitivity ranged from 72.73% to 78.57% and the specificity ranged from 78.95% to 96.55%. For blaCTX-M-1, this method showed 50% sensitivity and 98% specificity.
       
In cloacal sample, the direct PCR method showed moderate to almost perfect agreement with the culture-dependent method, with concordant values exceeding 78% The method demonstrated very good sensitivity for tetA, sul1, blaOXA-1 and blaCTX-M-1 genes, ranging from 80-100% with specificity of 61.54% to 83.33%. However, the sensitivity for the qnrA gene (66.67%) was lower compared to other genes.
       
In water samples, the direct PCR method showed moderate to substantial agreement with the culture-dependent method, with the concordant values above 77%. Similar to meat sample, the direct PCR method was 100% sensitive in detecting blaCTX-M-1 in water samples, with a specificity of 97.96%. The sensitivity for other genes ranged from 50%  to 81.25% while specificity ranged from 82.22% to 96.77%.
       
In this study, we evaluated the reliability and utility of Direct PCR method for detecting AMR genes, comparing with that of conventional approach, wherein bacterial isolation followed by detection of AMR genes is required. In the direct PCR method, PCR was performed directly on the samples without the need for DNA extraction. We found strong concordance (>76%) and ‘moderate to almost perfect’ agreement between Direct PCR method and the conventional method of AMR detection.
       
Using the Direct PCR method, we were able to successfully detect all the five AMR genes of interest in chicken meat, cloacal sample and water sample. However, the performance indicators of the method, such as sensitivity, specificity, kappa ratings and concordant value, varied depending on gene and sample matrix. This variation in the method’s performance may be attributed to differences in the sample matrix, which can influence the method’s performance (Ahlstrom et al., 2023).
       
The method demonstrated good sensitivity and specificity in detecting all AMR genes. Though the sensitivity of the method in detecting blaCTX-M-1 in chicken meat and qnrA in water samples was 50%, the method’s specificity was excellent (97-98%), highlighting its reliability for accurately identifying negative results.
       
The major advantage of the direct PCR method is its rapid detection of AMR genes, making it suitable for screening programs. The results can be obtained within 4-5 h, while the culture-dependent method requires 4-5 days. However, it is to be borne in  mind that the direct PCR method will not be able to link the bacterial species from which the resistance gene originates. Unlike the culture-dependent method, the direct PCR method only detects the presence of AMR genes and it cannot link the genotypic presence of resistance gene with the phenotypic resistance.
       
The choice of molecular method of detecting AMR gene depends on various factors, such as sample type and the research objective. Bacterial isolation is necessary if the study aims to identify AMR genes associated with a bacterium or mobile genetic element. Similarly, for epidemiological studies which aimed to correlate AMR gene with the bacterial species, bacterial culture is necessary. In AMR monitoring or surveillance programs, which aim to estimate the burden of AMR genes across samples or locations, the Direct PCR method may be a viable option for detecting AMR genes present in the entire microbiota. It is suggested that the abundance and spread of antibiotic resistance should be investigated at the gene level, rather than at the bacterial level, for epidemiological purposes, to analyze the spread of resistance (Di Francesco et al., 2021).
       
The Direct PCR assay employed in this study was reliable and rapid in detecting AMR genes in chicken meat, broiler cloacal sample and environmental sample. The Direct PCR method offers a promising alternative to traditional culture-based techniques, providing faster, more efficient and broader detection of AMR across diverse sample types and could be highly valuable for large scale screening of AMR genes.

In conclusion, the direct PCR method optimized in this study demonstrated high efficiency and reliability for the detection of AMR marker genes in chicken meat, cloacal samples, and water samples. The assay exhibited excellent sensitivity and specificity, with strong concordance to conventional culture-based AMR detection methods. The elimination of the prior DNA extraction step streamlines the workflow, shortens processing time, simplifies procedures and reduces contamination risk. The standardized direct PCR protocol represents a rapid, practical, and highly effective alternative to traditional approaches for large-scale AMR gene surveillance and monitoring programs.

The authors acknowledge Tamil Nadu Veterinary and Animal Sciences University for providing financial support to carry out this research work.

The authors declare that they have no conflicts of interest.