Prevalence, phenotypic characterization and Antibiogram of Pseudomonas aeruginosa isolates recovered from oropharyngeal swabs of Pigs in and around Tirupati

Introduction

Pig farming is an important component of the livestock sector in India, contributing significantly to rural livelihoods and food security (DAHD, 2023). Respiratory diseases remain one of the major constraints affecting pig production by causing reduced growth, increased mortality, and economic losses (Manasa et al., 2025) [13] . Among bacterial pathogens, Pseudomonas aeruginosa is a Gram-negative, aerobic, opportunistic bacterium capable of causing respiratory infections, septicemia, wound infections, and otitis in animals. Although P. aeruginosa is not considered a primary respiratory pathogen in pigs, it frequently acts as a secondary invader in animals affected by stress, viral infections, or poor management practices (Wood et al., 2023) [20] . The bodies of animals and humans host a variety of ecological niches that support diverse populations of microorganisms. While most studies on microbiota have concentrated on the intestinal tract or faeces, other body areas, such as the nasal passages, also contain significant microbial populations. However, research on the nasal microbiota in pigs has been limited, with culture-based studies primarily targeting specific pathogens (Weese, 2013; Holman et al., 2017; Correa-Fiz et al., 2016) [5,9,19] . The emergence and spread of multiple drug-resistant pathogenic bacteria is a major global health concern. Moreover, P. aeruginosa exhibits intrinsic resistance to many antimicrobials and readily acquires additional resistance mechanisms, making infections difficult to treat. The emergence of multidrug-resistant (MDR) Pseudomonas strains in food-producing animals [15] . Despite its importance, limited information is available regarding the prevalence and antimicrobial resistance patterns of Pseudomonas spp. in pigs in Andhra Pradesh. Therefore, this study is proposed to investigate the occurrence of Pseudomonas aeruginosa in the nasal swabs of pigs collected from different farms in and around Tirupati, Andhra Pradesh and characterization of the isolates by phenotypic methods.

Materials and methods

Study location

A cross-sectional study was designed to study the antimicrobial resistance patterns of the Pseudomonas aeruginosa isolates recovered from pigs of various farms located in and around Tirupati. The present study was carried out in the Department of Veterinary Public Health and Epidemiology, College of Veterinary Science, Tirupati during the period January 2026 to June 2026.

Sampling details

A total of 100 oropharyngeal swabs were collected randomly and aseptically from pigs of various farms located in and around Tirupati, Andhra Pradesh. The samples were collected using sterile swabs and transported to the department under chilled conditions for further processing and for bacteriological analysis.

Isolation and Identification of Pseudomonas aeruginosa by conventional method

Initially, the oropharyngeal swabs were inoculated in the Tryptone Soya Broth (TSB) and incubated at 37 0 C for 24 hrs under aerobic conditions for pre-enrichment. Following incubation, the enriched cultures were streaked on to Cetrimide agar and incubated at 37 0 C for 24 hrs. Cetrimide agar is a selective and differential medium for the identification of Pseudomonas aeruginosa in which Cetrimide acts as detergent which inhibits most bacteria and enhances the production of two pigments pyocyanin and pyoverdin.

Biochemical characterization of presumptive P.aeruginosa isolates

After incubation, the plates were examined for the presence of typical P.aeruginosa colonies, characterized by flat, spreading colonies with irregular margins, blue-green or yellow-green pigmentation, characteristic fruity or grape like odour. Presumptive colonies were subjected to Grams staining, which revealed gram negative slender rods. These presumptive isolates were further characterized using biochemical tests, including the IMViC tests. The isolates showing negative for Indole, Methyl Red and Voges-Proskauer test, along with positive Citrate test, in combination with the characteristic colony morphology and gram staining reaction.

Phenotypic characterization of antimicrobial resistant P.aeruginosa isolates

All the confirmed P.aeruginosa isolates were tested for antibiotic susceptibility against commonly used antibiotics in human and veterinary therapy by the Kirby Bauer disc diffusion method on Mueller Hinton agar using commercial discs (Himedia Pvt. Ltd, India). Initially, each confirmed isolates were inoculated into Tryptone Soya Broth (TSB) and incubated at 37 0 C for 24 hrs. Further, the concentration of turbidity was adjusted to 0.5 McFarland Standard. A sterile swab was dipped in to the culture and pressed against the walls of the test tube to remove excess culture. Later, the swab was spreaded on to Mueller Hinton agar and the selected antibiotic discs (Ceftriaxone, Ciprofloxacin, Chloramphenicol, Gentamicin, Erythromycin, Tetracyline, Ampicillin and Amikacin) were gently pressed against the culture spreaded Mueller-Hinton agar plates and incubated at 37°C for 18-24 hours. The diameters of inhibition zones were measured to the nearest millimeter, and the results were interpreted as resistant (R), intermediate (I), or susceptible (S) according to CLSI guidelines (CLSI, 2014). Isolates showing resistance to three or more antibiotics were considered multidrug resistant (MDR), and the resistance rates were classified as highly resistant (>50%), moderately resistant (10-50%), and low resistance (<10%) (Schwarz et al., 2010) [15] .

Evaluation of Multiple Antibiotic Resistance Phenotypes (MARPS) and Multiple Antibiotic Resistance (MAR) Index for P.aeruginosa isolates

The multiple antibiotic-resistant phenotypes (MARPs) of P.aeruginosa were evaluated for isolates that exhibited resistance to three or more antibiotics according to the method adapted from Iwu and Okoh, 2020. MAR index was calculated for all isolates as per procedure given by Krumperman (1983) by the formula a/b, where 'a' represents the number of antibiotics to which the isolate is resistant and 'b' the total number of antibiotics to which the isolate is exposed [10,12] .

Results and discussion

Out of the 80 oropharyngeal samples screened, only 11 samples (13.75%) were confirmed as P. aeruginosa by conventional methods, viz., cultural and biochemical tests. These findings almost similar to Donnik et al., 2020 who recorded 16% prevalence of P.aeruginosa from various sample types viz., wash offs from pigs and surfaces, skin of pigs, feedstuff and premises, water samples of different pig farms. Although the sample types differed between the two studies, the prevalence observed in the present investigation was comparable to that reported by Donnik et al., 2020, suggesting the widespread occurrence of P.aeruginosa in pig farm environments. In contrast to this, Chakraborty et al., 2020 from India recorded zero and 6% prevalence from oropharyngeal swabs and faecal samples from pigs respectively [7] . Similarly, the present study prevalence is almost similar to the findings of Karthik et [2,3,11,17] . The information regarding the prevalence of P.aeruginosa in oropharyngeal swabs of pigs is published scarce. Most available studies have focused on experimental respiratory infection models, airway colonization, or isolation from nasal swabs, lungs and other anatomical sites rather than natural colonization of the oropharynx. Consequently, the data related to the prevalence of P.aeruginosa in oropharyngeal swabs of pigs under filed conditions remain limited, highlighting the need for epidemiological investigations in pig populations. The eleven P.aeruginosa isolates recovered from oropharyngeal swabs of pigs were studied for phenotypic characterization of antimicrobial resistance patterns by disc diffusion method. Out of these 11 isolates, ten isolates (91%) were resistance to ampicillin and erythromycin, while nine isolates (82%) were resistance to ceftriaxone and tetracycline, six isolates (55%) resistance to ciprofloxacin (Figure 1), four isolates (36%) to chloramphenicol, and three isolates (27%) were resistance against gentamicin and amikacin respectively (Table 1).

Many researchers have reported varying antimicrobial resistance patterns in Pseudomonas aeruginosa and have identified multidrug-resistant (MDR) isolates from different food animal sources, including chicken, fish, mutton, pork, and beef (Chakraborty et al., 2020; Abbas et al., 2022; Swetcha et al., 2023; Sowmya et al., 2024). The differences observed in antimicrobial resistance profiles among studies may be attributed to variations in antimicrobial usage practices, geographical locations, management systems, and the presence of diverse resistance mechanisms in P. aeruginosa (Zeng et al., 2014) [1,3,17,18] . 2).

Heat map of antibiotic resistance detected in P.aerugionsa isolates A Multiple Antibiotic Resistance (MAR) index greater than 0.2 indicates that the isolates may have originated from highrisk sources where antibiotics are frequently used, reflecting a greater risk of exposure to antimicrobial agents (Maurice et al., 2018). In contrast, a MAR index of less than 0.2 suggests that the isolates originated from sources where antibiotics are seldom or never used and therefore represent a lower risk of antimicrobial exposure [14] .

In the present study, all the eleven P.aeruginosa isolates recovered had the MAR index of greater than 0.2, ranged from 0.5-1.0, which indicates that these isolates falls under high risk category with high risk potential to favour the spread of antimicrobial resistance pathogens in both animals and humans. The antimicrobial resistance patterns observed in the present study highlight the need for the judicious prudent use of antibiotics at both veterinary and public health levels. Regular monitoring and continuous surveillance of the antimicrobial susceptibility profiles of P.aeruginosa are essential for the early detection of emerging resistance trends, guiding appropriate antimicrobial therapy, preventing treatment failures and minimizing the development and spread of resistance to commonly used antibiotics for the treatment of pseudomoniasis.

Acknowledgement

AcknowledgementThe authors are grateful to SVVU, Tirupati for providing facilities to carry out the research work.

References

1. Abbas R, Nawaz Z, Siddique AB, Aslam R, Rafique A, Zahoor MA, Qamar MU, Ahmad MZ, Jalees MM, Qasim M, Alsayeqh AF. Molecular detection of biofilm production among multidrug resistant isolates of Pseudomonas aeruginosa from meat samples. Pak Vet J. 2022;42(4):505-510.
2. Bantawa K, Rai K, Subba Limbu D, Khanal H. Foodborne bacterial pathogens in marketed raw meat of Dharan, eastern Nepal. BMC Res Notes. 2018;11(1):618.
3. Chakraborty S, Dutta TK, Roychoudhury P, Samanta I, Lalhruaipuii, Kalai S, Bandyopadhyay S. Molecular characterization of biofilm-producing Pseudomonas aeruginosa isolated from healthy pigs and chicken in India. Indian J Anim Res. 2020;54(11):1400-1407. DOI:10.18805/IJAR.B-3903.
4. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Twenty-fourth informational supplement. CLSI document M100-S24. Wayne (PA): Clinical and Laboratory Standards Institute; 2014.
5. Correa-Fiz F, Fraile L, Aragon V, et al. Piglet nasal microbiota at weaning may influence the development of Glässer's disease during the rearing period. BMC Genomics. 2016;17(1):404.
6. Department of Animal Husbandry and Dairying. Basic Animal Husbandry Statistics 2023. New Delhi: Ministry of Fisheries, Animal Husbandry and Dairying, Government of India; 2023.
7. Donnik IM, Krivonogova AS, Isaeva AG, Shkuratova IA, Moiseeva KV, Musikhina NB. Special features of Pseudomonas aeruginosa strains in animal and poultry farms in the regions with various levels of man-made pollution. Agron Res. 2020;18(4):2365-2373.
8. Food and Agriculture Organization of the United Nations. The FAO Action Plan on Antimicrobial Resistance 2021-2025. Rome: FAO; 2023.
9. Holman DB, Brunelle BW, Trachsel J, et al. Metaanalysis to define a core microbiota in the swine gut and respiratory tract. Sci Rep. 2017;7:12548.
10. Iwu CD, Okoh AI. Characterization of antibiogram fingerprints in Listeria monocytogenes recovered from irrigation water and agricultural soil samples. PLoS One. 2020;15(2):e0228956.
11. Karthik J, Poornasree M, Swetha CS, Ramya P, Suresh Y. Molecular characterization and antibiogram of Pseudomonas aeruginosa isolated from various meat samples in and around Tirupati, Andhra Pradesh. Indian J Vet Sci Biotechnol. 2026;22(3):15-19.
12. Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol. 1983;46(1):165-170.
13. Manasa NS, Shilpa VT, Girish BC, Karabasanavar N, Ravikumar P, Satheesha SP, Ravikumar BP. Respiratory infection of pigs in and around Hassan District, Karnataka. J Anim Res. 2025;15(5):177-184.
14. Maurice Bilung L, Sin Chai L, Tahar AS, Ted CK, Apun K. Prevalence, genetic heterogeneity, and antibiotic resistance profile of Listeria spp. and Listeria monocytogenes at farm level: a highlight of ERIC-and BOX-PCR to reveal genetic diversity. Biomed Res Int. 2018;2018:3067494. DOI:10.1155/2018/3067494.
15. Schwarz S, Silley P, Simjee S, Woodford N, Van Duijkeren E, Johnson AP, Gaastra W. Editorial: Assessing the antimicrobial susceptibility of bacteria obtained from animals. J Antimicrob Chemother. 2010;65(4):601-604.
16. Songer JG, Post KW. Veterinary Microbiology: Bacterial and Fungal Agents of Animal Disease. St. Louis: Elsevier Saunders; 2005.
17. Sowmya R, JagadeeshBabu A, Chinta SS, Sreedevi B, Suresh Y, Vinayaka Siddhartha P, MadhavaRao T. Molecular characterization and antibiogram of Pseudomonas aeruginosa isolated from fish sold in markets of Tirupati, India. Fish Technol. 2024;61(4):357-366.
18. Swetcha M, Sureshkhannan S, Porteen K, Ezhilvelan S. Isolation and molecular confirmation of Pseudomonas aeruginosa in chicken meat, mutton and pork. Int J Curr Microbiol Appl Sci. 2023;12(8):113-123.
19. Weese JS. The microbiome of the respiratory tract in animals. Vet Clin North Am Food Anim Pract. 2013;29(2):315-329.
20. Wood SJ, Kuzel TM, Shafikhani SH. Pseudomonas aeruginosa: Infections, animal modeling, and therapeutics. Cells. 2023;12(1):199. DOI:10.3390/cells12010199.
21. World Health Organization. Global Antimicrobial Resistance and Use Surveillance System (GLASS) Report 2023. Geneva: World Health Organization; 2023.