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Open Access

Peer-reviewed

Research Article

First report of MDR virulent Pseudomonas aeruginosa in apparently healthy Japanese quail (Coturnix japonica) in Bangladesh

  • Alamgir Hasan ,

    Contributed equally to this work with: Alamgir Hasan, Md. Tanjir Ahmmed

    Roles Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯
  • Md. Tanjir Ahmmed ,

    Contributed equally to this work with: Alamgir Hasan, Md. Tanjir Ahmmed

    Roles Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯
  • Bushra Benta Rahman Prapti,

    Roles Data curation, Methodology, Writing – review & editing

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯
  • Aminur Rahman,

    Roles Visualization, Writing – review & editing

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯
  • Tasnim Islam,

    Roles Visualization, Writing – review & editing

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯
  • Chandra Shaker Chouhan,

    Roles Visualization, Writing – review & editing

    Affiliation Department of Medicine, Bangladesh Agricultural University, Mymensingh, Bangladesh

    ⨯
  • A. K. M. Anisur Rahman,

    Roles Formal analysis, Validation, Writing – review & editing

    Affiliation Department of Medicine, Bangladesh Agricultural University, Mymensingh, Bangladesh

    ⨯
  • Mahbubul Pratik Siddique

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Validation, Writing – review & editing

    mpsiddique@bau.edu.bd

    Affiliation Department of Microbiology and Hygiene, Mymensingh, Bangladesh

    ⨯

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen associated conditions like septicaemia, respiratory disorders, and diarrhoea in poultry, particularly in Japanese quail (Coturnix japonica). The infection causes huge economical losses due to its high transmissibility, mortality and zoonotic potential. This study aimed to isolate, identify, detect virulence genes, and profile multidrug resistance (MDR) of P. aeruginosa from Japanese quail. Oral and rectal swabs were collected from 110 apparently healthy quail birds across various districts in Bangladesh. Bacterial isolation and identification were performed using cultural, morphological, biochemical, and polymerase chain reaction (PCR) methods. Antibiotic susceptibility was assessed using the disc diffusion method, and virulence genes were detected through PCR. Multivariable logistic regression was used to identify risk factors for P. aeruginosa infection. Both conventional and PCR methods revealed that 25 (22.73%) of the quail birds were positive for P. aeruginosa. The results showed that quail birds in Narsingdi were five times more likely to harbor Pseudomonas species (OR: 5.02; 95% CI: 1.34–18.84) compared to those in Mymensingh Sadar. Additionally, quail birds younger than eight weeks had nearly six times higher odds (OR: 5.93; 95% CI: 1.96–17.91) of carrying Pseudomonas compared to older birds. Female quail birds had almost four times higher odds (OR: 3.77; 95% CI: 1.30–10.93) of harboring Pseudomonas species than males. All 25 P. aeruginosa isolates exhibited multi drug-resistance (MDR) patterns. Virulence gene analysis revealed the consistent presence of exoA and rhlR in all isolates, while exoS, exoY, rhlI, and rhlAB showed variable distribution. The high prevalence of MDR and virulent P. aeruginosa in apparently healthy quail birds particularly in Mymensingh and Dhaka divisions, highlights the urgent need for a comprehensive ‘One Health’ approach to proactively address and mitigate the potential risk this organism poses to both quail and human populations.

Citation: Hasan A, Ahmmed MT, Prapti BBR, Rahman A, Islam T, Chouhan CS, et al. (2025) First report of MDR virulent Pseudomonas aeruginosa in apparently healthy Japanese quail (Coturnix japonica) in Bangladesh. PLoS ONE 20(1): e0316667. https://doi.org/10.1371/journal.pone.0316667

Editor: Morteza Saki, Ahvaz Jundishapur University of Medical Sciences: Ahvaz Jondishapour University of Medical Sciences, ISLAMIC REPUBLIC OF IRAN

Received: September 25, 2024; Accepted: December 14, 2024; Published: January 24, 2025

Copyright: © 2025 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data generated during this study is included in the paper and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: CONFLICT OF INTEREST 19th September 2024 Title: First Time Detail Virulence and antibiogram profiling of Pseudomonas aeruginosa isolated from apparently healthy Japanese quail (Coturnix japonica) in Bangladesh DECLARATION AND STATEMENT We know of no conflicts of interest associated with the publication. We declare that this manuscript is original and has not been submitted to anywhere else for publication. The manuscript nicely falls under the scope of your journal. Sincerely yours, Mahbubul Pratik Siddique, PhD Corresponding author and Professor Department of Microbiology and Hygiene Bangladesh Agricultural University Mymensingh-2202 Cell Phone: +880-1751-945123 Email: mpsiddique@bau.edu.bd https://www.researchgate.net/profile/Mahbubul-Siddique https://scholar.google.com/citations?user=YmxVEzEAAAAJ&hl=en

Introduction

Since early 1980s, poultry farming has been emerged as an industry in Bangladesh, and the majority of these poultry are indigenous chickens and ducks, which showed no satisfactory performance [1]. Apart from native poultry, people living in rural and semi-urban areas want a particular kind of bird that can be raised with minimal effort and yield greater financial returns in a short amount of time [2]. The Japanese quail (Coturnix japonica) has various characteristics for profitable farming, such as, higher growth rate, earlier sexual maturity, higher rate of egg production, close succession (3–4 generations per year) and less hatching period [3]. Moreover, due to small size and stout body configuration, it attributes to the less feed cost, less space requirements and most importantly, less vulnerability to common infectious diseases [3]. High potential exists for the development of Japanese quail eggs as a less expensive source of protein, particularly in developing nations [4].

The sustainability of quail farming, like that of other poultry, relies on effective management, breeding, feeding, and the implementation of disease prevention, control, and treatment measures. Japanese quail are susceptible to a variety of bacterial infections [5]. Colibacillosis, colisepticemia, fowl cholera, fowl typhoid, and salmonellosis are bacterial illnesses that have been observed in Japanese quail [6]. However, Pseudomonas infection in Japanese quail has been looked for unknown reason [7]. Martinez-Laorden et al. (2023) reported Pseudomonas spp. as the most prevalent bacterium in Japanese quail [8]. Again, Pseudomonas aeruginosa (P. aeruginosa) is the most prevalent species of the genus Pseudomonas that impact quails, leading to significant financial losses, elevated morbidity, and elevated mortality rates [9,10].

The bacterium P. aeruginosa, a Gram-negative, motile, opportunistic bacterium, is prevalent in the hospital and other environments and it is a normal component of the flora of both immune compromised hosts and healthy people [11]. It causes nosocomial and ventilator-associated infections with a high mortality [12]. P. aeruginosa possesses various virulence factors like Pili, exoenzyme S (encoding gene rhlI), LasB elastase, exotoxin A (encoding gene exoA), and Phospholipase C, Rhamnolipids (encoding genes rhlR, rhlAB), whereas the initiation of chronic infections involves alginate and siderophores [13]. Among the five protein secretion systems (type I, II, III (ExoS, ExoY), V, and the latest type VI), P. aeruginosa frequently employs these systems to release proteinaceous virulence factor [14,15]. Notably, the type III secretion system enables the direct injection of exotoxins into host cells upon contact, subverting host cell defences and signalling pathways [16]. Bifunctional proteins ExoT and ExoS, possessing ADP-ribosyl transferase and GTPase activating domains, impact the assembly of the actin cytoskeleton in response to extracellular signals by acting on Ras-related GTP-binding proteins like Rho, Rac, and Cdc42 [17]. Additionally, Exotoxin A inhibits protein synthesis by ADP-ribosylating eukaryotic elongation factor 2 [18]. ExoU exhibits phospholipase activity, rupturing eukaryotic membranes upon entering the cytoplasm, while ExoY possesses adenylate cyclase activity [19]. Moreover, P. aeruginosa utilizes quorum sensing (QS), a cell-density-dependent mechanism, to regulate the production of various virulence factors [20]. The mechanisms of disease occurrence also involve the bacterium’s ability to form biofilms, which contribute to the persistence of P. aeruginosa in various environments, making eradication challenging [21,22]. Infections caused by P. aeruginosa include pneumonia, urinary tract, wound and soft tissue infections.

Multidrug resistance (MDR) has been increasing globally, posing a significant public health threat due to irrational use of antibiotics, growth promoter and hormones in the livestock sector, particularly in poultry [23,24]. Often, farmers sell birds without considering the recommended withdrawal periods of these drugs, resulting in drug residues remaining in the food chain from animal products [25–27]. This can ultimately lead to the development of MDR in humans through horizontal gene transfer, selective pressure, gene transfer mechanisms, and ecosystem interactions [25,28]. Recent investigations have highlighted the emergence of multidrug-resistant bacterial pathogens from diverse sources, emphasizing the necessity for judicious antibiotic use. Furthermore, routine antimicrobial susceptibility testing is crucial for identifying appropriate antibiotics and screening for the emergence MDR strains [21,29–33], as the preventive measures for infectious diseases are largely lacking, and the predominant approach to control such diseases relies heavily on antimicrobial therapy [34]. However, it is crucial to recognize that AMR poses a significant public health emergency in both human and veterinary medicine [35]. The widespread overuse of antibiotics, used for growth-promotion, metaphylactic, and prophylactic purposes in both human medicine and animal husbandry, has led to the emergence and spread of antibiotic-resistant bacteria and resistance genes. This exacerbates the risks to public health and the environment [21,36]. The impact of antimicrobial resistance includes treatment failures, resulting in economic loses, and the potential to serve as a reservoir of resistant bacteria. Therefore, the selection of appropriate antimicrobial agents should be a top priority, emphasizing the need for judicious and responsible use to mitigate the adverse consequences associated with antimicrobial resistance. Identifying disease-causing agents in commercial quail farms is crucial [37] for developing sustainable farming practices [5]. While conventional methods (cultural, morphological and biochemical) are important for bacterial detection, confirmatory diagnosis necessitates the use of polymerase chain reaction (PCR) and virulence gene detection. Existing reports on quail diseases in Bangladesh primarily focus on clinical signs and histopathology [5], and antimicrobial resistance [18], with a notable gap in molecular-based studies for agent and virulence gene detection.

Therefore, this research aims to isolate and identify P. aeruginosa from Japanese quails, identify risk factors, detect virulence genes and antibiotic resistance in the bacterial isolates.

Materials and methods

Ethical statement

All the animal experimental procedures were approved by the Ethical Approval Committee, Bangladesh Agricultural University, Mymensingh-2202. All the animals were handled during sample collection in compliance with the local animal welfare regulations and maintained according to standard protocols. The approval number is “AWEEC/BAU/2023(73), Date: 24.12.23.

Sample collection

Samples were collected from apparently healthy live birds in two commercially recognized poultry divisions belonging to Dhaka (Narsingdi and Gazipur districts) and Mymensingh (Mymensingh and Jamalpur districts). A total of 220 samples (110 orals and 110 cloacal swabs) were collected in Luria Bertani (LB) broth from 110 apparently healthy quails from different quail shops (quail selling points) situated in Mymensingh Sadar [n = 60 (30 male + 30 female)], Jamalpur [n = 20 (10 male + 10 female)], Narsingdi [n = 20 (10 male + 10 female)], and Gazipur [n = 10 (5 male +5 female)].

Bacterial strains, media and growth

The primary enrichment was made in Luria-Bertani (LB) broth (Hi-Media, India), Nutrient broth (Hi-Media, India), and Brain heart infusion (BHI) broth. Subsequently, cultured onto LB agar (Hi-Media, India), BHI agar ((Hi-Media, India), Nutrient agar (Hi-Media, India), Tryptic soy (TSA) agar (Hi-Media, India), MacConkey (Hi-Media, India) agar and Cetrimide agar (Hi-Media, India) as selective media. Furthermore, suspected colonies were streaked on 10% Bovine Blood (BB) agar and incubation at 37°C for 24 hours. Bacterial growth was initially confirmed by visually observing turbidity and then by measuring optical density (OD) using UV-Vis spectrophotometry. After 24h of incubation, the cultured broth was used to measure the OD, while the fresh LB, Nutrient broth, and BHI broth served as negative control (zero). The morphological and staining properties of the isolated bacteria were documented by the Gram’s staining method through microscopic observations (100X), utilising a single pure colony obtained from a P. aeruginosa subculture. To confirm P. aeruginosa, indole synthesis, methyl red test, Voges-Proskauer test, hydrogen sulphide production, oxidase and catalase tests, and sugar fermentation tests were used. The Motility Indole Urease (MIU) test was also utilized to examine the organism’s motility.

Genomic DNA extraction and PCR amplification

Genomic DNA extraction from the isolates was performed through the boiling and thawing technique [38]. Subsequently, NanodropTM (Thermo Fisher Scientific, USA) was used for both quantifying and assessing the quality of the extracted DNA. To standardise the 16S rDNA oligonucleotides, bacterial DNA was mixed yielding an average concentration of 130 ng/μl and a purity of 2.10. The PCR reaction volume of 20 μl comprised 3 μl DNA template, 1 μl each of forward and reverse primers, 10 μl of PCR Master Mix, 2X (Promega, USA), and 5.5 μl of nuclease free water. Table 1 provides details on the primers and techniques used for the PCR reactions [39]. Analysis of the PCR end products was conducted using a 1.5% agarose gel. Post-electrophoresis, the gel was immersed in an ethidium bromide solution (concentration approximately 0.2–0.5 μg/mL) for 10 minutes and subsequently visualised using a UV transilluminator.

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Table 1. List of primers, with oligonucleotide sequences, amplification conditions, amplicon size and references, which were used to detect Pseudomonas at genus and species level, as well as to detect the virulence genes by PCR.

https://doi.org/10.1371/journal.pone.0316667.t001

Virulence profile of P. aeruginosa

The primer sets for PCR-based detection of the virulence-associated genes, along with the amplification protocols, were chosen from previously published papers with slight modifications, as summarized in Table 1. The visualization of PCR end products followed the procedure mentioned earlier.

Antibiotic susceptibility of P. aeruginosa

The antibiogram test utilized commonly used antibiotics at the field level through the disc diffusion method [41]. In brief, the turbidity of all isolates was adjusted to a 0.5% McFarland standard by culturing them in LB broth (incubated at 37°C for approximately 4 hours in a shaking incubator). Subsequently, the isolates were inoculated onto MHA (Liofilchem, Italy). Following inoculation, the antibiotic discs were placed and the entire setup was incubated at 37°C for 18 hours to measure the diameter of the inhibition zone. To determine sensitivity or resistance of the isolates the Clinical and Laboratory Standards Institute (CLSI, 2019) standard interpretation chart for the zone of inhibition was referenced [42]. The antibiotic discs used in this study included Beta lactam Ampicillin G (AMP, 25 μg/disc), Ceftriaxone (CTR, 30 μg/disc), Cephradine (CE, 30 μg/disc), Meropenem (MEM, 10 μg/disc), Aztreonam (ATM, 30 μg/disc) Non-beta lactam Streptomycin (S, 10 μg/disc), Gentamycin (GEN, 30 μg/disc), Neomycin (N, 30 μg/disc), Doxycycline (DO, 30 μg/disc), Oxytetracycline (O, 30 μg/disc), Cotrimoxazole (COT, 25 μg/disc), Cefoxitin (FOX, 30 μg/disc), Chloramphenicol (C, 30 μg/disc), Florfenicol (FFC, 25 μg/disc), Nalidixic acid (NA, 30 μg/disc), Ciprofloxacin (CIP, 5 μg/disc), and Levofloxacin (LEV 5 μg/disc). Among the discs, CTR and GEN were purchased from HiMedia Laboratories Private Limited (Maharashtra, India) and rest were from Oxoid Limited (Hampshire, UK).

Statistical analysis

The data on location, age, gender, sample site, and test results, were first entered into Excel 2010 and then transferred to R 4.3.2 [43] for analysis. A quail bird was considered as affected if it tested positive for Pseudomonas spp. either in rectal or oral swabs. Continuous variable age was converted into categorical variable (≤ 8 and > 8 weeks). The prevalence of Pseudomonas spp. across different variables was estimated using the ‘tabpct’ function of the R ‘epiDisplay’ package [44]. The association between Pseudomonas spp. infection and all explanatory variables was first investigated using univariable logistic regression model. Following that, multivariable logistic regression analysis was performed, incorporating only those explanatory variables with a univariable p-value of ≤ 0.2. Prior to conducting the multivariable logistic regression analysis, the variation inflation factor (VIF) was used to assess multicollinearity among the explanatory variables, with a cut-off level set at < 5. A backward model selection strategy was implemented for the final model selection, and the overall fit of the model was evaluated using the Hosmer-Lemeshow test. The dataset used to identify risk factors for P. aeruginosa infection in quail birds is available in Supplementary File 1. The correlation coefficient between phenotypic resistance and the virulence profile was calculated using the ’cor’ function in R version 4.3.2 [43].

Sequencing and phylogenetic analysis

For the sequencing of the 16S rRNA gene, two isolates of Pseudomonas spp. (PCR detection up to genus level; 618 bp) were selected, ensuring coverage from both divisions (Dhaka and Mymensingh). Additionally, two isolates of P. aeruginosa (PCR detection at species level; 956 bp) were chosen from each division. The PCR was conducted using the aforementioned primer sets, reaction mixtures and thermal profiles. Successful amplifications were confirmed through 1.5% agarose gel electrophoresis and staining with ethidium bromide (EtBr) (Research-Lab Fine Chem Industries, Mumbai, 400 002, India), followed by visualization under a UV trans-illuminator (Biometra, Germany).

The amplified PCR products were purified using the QIAamp PCR product purification kit (QIAgen GmbH, Hilden, Germany). Subsequently, the purified PCR products underwent commercial sequencing at the National Institute of Biotechnology, Savar, Dhaka, Bangladesh, using the Sanger Dideoxy sequencing method. For sequencing a single amplicon, both forward and reverse primers were used. Raw sequence data were aligned, considering the forward sequence and the reverse complement of reverse sequence, and then edited using CLC sequence viewer v8 (http://www.clcbio.com) and BioEdit v7.2 [45] sequence editing and analysis tools. To determine sequence similarities with sequences already deposited in the NCBI GenBank, the edited sequences were subjected to BLAST. The GenBank accession numbers assigned to the present isolates are OR617385 to OR617388.

To assess the evolutionary relationships, a total of 56 partials and complete 16S rRNA gene sequences of P. aeruginosa were retrieved from NCBI GenBank. These sequences represent data from every division and source in Bangladesh (at least one from each division and source), neighbouring countries (India, Pakistan, China, Japan, Thailand) and at least one sequence from each continent. The 56 downloaded sequences, along with four of our sequences, were aligned using MUSCLE within MEGA11: Molecular Evolutionary Genetics Analysis version 11 software [46] package and exported in MEGA format. Finally, the MEGA11 software package was used to construct a phylogenetic tree employing 1000 Bootstrap replications. The tree was further annotated using the Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation [47].

Results

Pseudomonas aeruginosa, a significant bacterial pathogen, poses potential threats to both animal and human health. In this study, our primary objectives were to investigate the prevalence, antibiotic resistance patterns, and virulence gene profiles of P. aeruginosa in apparently healthy quail birds in Bangladesh. We aimed to explore potential risk factors associated with the presence of this pathogen.

Phenotypic characteristics of the recovered isolates

The bacterial isolates showed robust growth in LB broth, characterized by dense turbidity and the formation of a pellicle. In nutrient broth at 37°C for 24 hours under aerophilic conditions, the culture displayed turbidity with a green pigment, while BHI broth resulted in turbidity only. On LB agar, the organism demonstrated an earthy odour and irregular swarming growth. Conversely, on BHI agar, the isolates produced slimy mucoid colonies. Nutrient agar revealed small, irregular bluish-green colonies with a sweet odour. Cetrimide agar exhibited circular, smooth, small to medium-sized, mucoid greenish-blue opaque colonies. MacConkey agar displayed colourless, circular smooth, mucoid transparent colonies, while blood agar showed, irregularly edged circular flat smooth greyish non-haemolytic white colonies. Gram staining identified the organism as Gram-negative, rod-shaped, and arranged singly, in pairs, or in short chains. Furthermore, the Motility Indole Urease/Urea (MIU) medium test confirmed the organism’s motility, but it tested negative for indole and urease. Suspected Pseudomonas spp. was found to be positive for Dextrose, Maltose, and Sucrose fermentation with acid and gas production, while testing negative for Lactose and Mannitol. Additionally, the organism exhibited a positive Methyl Red (MR) test but tested negative for the Indole and Voges-Proskauer tests.

Confirmation by PCR

Genus level identification was confirmed by the presence of a positive band at 618 base pairs during gel electrophoresis (Fig 1A). Subsequently, specific detection of P. aeruginosa was confirmed by the presence of a 956 bp rRNA gene product (Fig 1B).

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Fig 1. Agarose gel electrophoresis results of PCR amplification targeting 16S rRNA gene for the genus- and species-specific detection of Pseudomonas spp. and P. aeruginosa, respectively.

(a) genus-specific PCR amplification of Pseudomonas spp. and positive bands appeared at 618 bp; (b) species-specific detection of P. aeruginosa, with amplicon size of 956 bp. In both cases, Lane M: 100 bp DNA ladder; NC: Negative control; Lane 1-9/10: Positive isolates.

https://doi.org/10.1371/journal.pone.0316667.g001

Prevalence of P. aeruginosa

Of the 110 quail birds’ rectal and oral swabs, cultural, Gram staining, and biochemical tests showed the presence of Pseudomonas spp. In 25 (22.73%) samples (Table 2). Specifically, Pseudomonas spp. were detected in 19.09% of rectal swabs, 6.36% of oral swabs, and 2.73% of samples obtained from both rectal and oral swabs (Table 2). Moreover, all isolates conventionally characterized were also confirmed positive in PCR amplification using specific primer sets. Fig 2 shows the district-level prevalence of Pseudomonas spp. infections.

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Fig 2. District-level prevalence of Pseudomonas spp. infections across the study area.

The map was generated using Python with the Matplotlib and GeoPandas libraries. The district-level shapefile for Bangladesh was obtained from GADM (https://gadm.org/).

https://doi.org/10.1371/journal.pone.0316667.g002

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Table 2. Prevalence of the isolated bacteria (culture and PCR-based).

https://doi.org/10.1371/journal.pone.0316667.t002

Risk factors analysis for isolated P. aeruginosa

The multivariable analysis incorporated four variables that showed associations with P. aeruginosa infection in quail. at p-values ≤0.2 in the univariable analysis (Table 3). The final multivariable logistic regression model identified three specific variables as risk factors (Table 4) for P. aeruginosa infection in quail. In Narsingdi, quail birds were found to be five times more likely to harbor Pseudomonas spp. (OR: 5.02; 95% CI:1.34–18.84) compared to those in Mymensingh Sadar. Quail birds younger than eight weeks had nearly six times higher odds (OR: 5.93; 95% CI: 1.96–17.91) of carrying Pseudomonas spp. compared to their older counterparts. Similarly, female quail birds showed almost four times higher odds of harbouring Pseudomonas spp. (OR: 3.77; 95% CI: 1.30–10.93) than male birds (S1 Table).

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Table 3. Univariable association between Pseudomonas spp. infection and explanatory variables.

https://doi.org/10.1371/journal.pone.0316667.t003

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Table 4. Risk factors identified in the final multivariable model for Pseudomonas spp. infection in quail birds.

https://doi.org/10.1371/journal.pone.0316667.t004

Distribution of virulence genes of P. aeruginosa

The presence of virulence genes among the 25 isolates (S2 Table) was as follows: exoA (100%; amplicon size 396 bp), exoS (88%; amplicon size 118 bp), exoT (64%; amplicon size 152 bp), exoY (84%; amplicon size 289 bp), rhlAB (76%; amplicon size 151 bp), rhlR (100%; amplicon size 730 bp), rhlI (64%; amplicon size 625 bp). Notably, none of the isolates tested positive for exoU, lasI and lasA. The individual profile of virulence genes for each isolate is represented in Table 5, and the overall prevalence is visualized in Fig 3. Further, the PCR amplification results for each positive virulence gene is shown Fig 4.

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Fig 3. Frequency distribution of virulence genes in isolated P. aeruginosa.

https://doi.org/10.1371/journal.pone.0316667.g003

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Fig 4. Virulence genes detection in PCR confirmed P. aeruginosa isolates.

[A] exoA (396 bp), [B] exoS (118 bp), [C] exoT (152 bp), [D] exoY (289 bp), [E] rhlAB (151 bp), [F] rhlR (730 bp), and [G] rhlI (625 bp). However, there were no exoU, lasI and lasA found in any isolates. In all cases, the Lane M = 100 bp DNA ladder; NC = negative control; and clear and consistent band showing lanes indicated positive isolates.

https://doi.org/10.1371/journal.pone.0316667.g004

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Table 5. Frequency distribution of phenotypic resistance and virulence gene profiles of P. aeruginosa from rectal and oral swabs of quail birds with details antimicrobial categories and agents used to define MDR, XDR, PDR and MARI.

https://doi.org/10.1371/journal.pone.0316667.t005

Antimicrobial susceptibility testing of P. aeruginosa

The analysis of the results of antibiotic resistance profile of P. aeruginosa was illustrated in the Fig 4. The results revealed that 92% of the isolates showed resistance to ampicillin, all isolates showed 100% resistance to 1st generation cephalosporin (Cephradine). Furthermore, resistance to other β-lactams varied, with cefoxitin displaying 76% resistance, while ceftriaxone and meropenem exhibited 100% susceptibility. Within the aminoglycoside group, streptomycin showed 28% susceptibility and 72% intermediate resistance. Conversely, Neomycin, Gentamycin, Cotrimoxazole, Chloramphenicol, Florfenicol, Ciprofloxacin, Levofloxacin demonstrated 100% susceptibility. On the other hand, the tetracycline group and quinolone group including Oxytetracycline, Doxycycline, and Nalidixic acid, showed 92% and 100% resistance respectively. The antibiotic resistance patterns for each isolate were showed in Fig 5. Moreover, Fig 6 illustrated the isolate-wise distribution of virulence genes and the count of phenotypic antimicrobial resistance in P. aeruginosa isolates from quail birds in both the Mymensingh and Dhaka divisions.

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Fig 5. Isolate-wise antibiotic resistance patterns of the isolated P. aeruginosa from oral and rectal swabs of quail birds.

https://doi.org/10.1371/journal.pone.0316667.g005

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Fig 6. Isolate-wise with total number-based virulence genes occurrence and phenotypic antimicrobial resistance in the P. aeruginosa isolates.

https://doi.org/10.1371/journal.pone.0316667.g006

Phenotypic multi drug-resistance (MDR) nature of P. aeruginosa isolates

All P. aeruginosa isolates obtained from rectal and oral swabs of quail birds displayed a multidrug-resistant (MDR) phenotype. The 25 isolates from both rectal and oral swabs exhibited resistance to Aztreonam, Doxycycline, Chloramphenicol, Florfenicol, and Cefuroxime (ATM, DO, C, FFC, CXM), out of the 18 tested antibiotics. Among the 21 rectal swab isolates, nine exhibited resistances to a broader spectrum of nine antibiotics including Aztreonam, Doxycycline, Chloramphenicol, Florfenicol, Cefuroxime, Oxytetracycline, Ampicillin, Nalidixic acid, and Cefoxitin (ATM, DO, C, FFC, CXM, OT, AMP, NA, FOX,) (Table 5). In contrast, all 25 isolates were found to be susceptible to nine antibiotics, namely Meropenem, Ceftriaxone, Cephradine, Streptomycin, Neomycin, Gentamycin, Cotrimoxazole, Ciprofloxacin, and Levofloxacin. Though, all the 25 isolates were found as MDR, no isolate was detected as Extensively drug resistant (XDR) or pan drug resistant (PDR) (Table 5). A very weak positive linear relationship (r = 0.11) was observed between phenotypic resistance patterns and virulence profile (Fig 7).

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Fig 7. Correlation between phenotypic resistance patterns and virulence profiles.

https://doi.org/10.1371/journal.pone.0316667.g007

The multiple antibiotic resistance index (MARI) analysis showed that all the isolates showed MARI values of more than 0.3 values, where PM11 and PD21 isolates had 0.352 (lowest among others), however, 13 isolates (PM1-2, PM5, PM7, PM9-10, PM16, PD17-19, and PD23-25) showed highest MARI (0.471). The rest 10 isolates had MARI value of 0.412 (Table 5).

Sequencing and phylogenetic analysis

The phylogenetic analysis of the sequenced PCR amplification products revealed that, at the genus level, the Pseudomonas sp. isolates from quails in both Narsingdi (GenBank Accession No. OR617385) and Mymensingh (GenBank Accession No. OR617386) districts exhibited no evolutionary differences. Moreover, they displayed a close relationship with Pseudomonas isolates from milk (GenBank Accession No. MN464147; raw milk from Sylhet) and rhizosphere soil (GenBank Accession No. MG786551; source region not mentioned), respectively. Similarly, the analysed sequenced products of P. aeruginosa from quails in both Narsingdi (GenBank Accession No. OR617387) and Mymensingh (GenBank Accession No. OR617388) regions clustered very closely with the P. aeruginosa isolates from municipal waste in the Sylhet region (GenBank Accession No. MW040069) and hospital wastes from Khulna region (GenBank Accession No. MG735679), respectively (Fig 8).

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Fig 8. Phylogenetic relationship of the isolated P. aeruginosa from quail birds of Narsingdi and Mymensingh regions of Bangladesh.

The sequenced and analysed isolates were marked by various colour.

https://doi.org/10.1371/journal.pone.0316667.g008

Discussion

Our findings highlight the prevalence of multi- drug-resistant and virulent potential P. aeruginosa in apparently healthy quail birds reared in Mymensingh and Dhaka divisions of Bangladesh. Young female birds should be regularly screened for future surveillance. We suggested a ‘One Health’ approach to proactively address and mitigate any future disasters caused by this organism in quail and human populations.

P. aeruginosa infection in quail birds poses a potential public health threat due to the zoonotic nature of this pathogen. The antibiotic resistance, pathogenicity, and transmission behaviour of P. aeruginosa are growing concern in livestock and public health, and environmental health [48]. Therefore, studying Pseudomonas infections in quail birds offers valuable insights into pathogenicity, genetic variation, and resistance patterns, which can help inform effective treatment and prevention strategies. To address this issue, the present study was designed to provide a detailed analysis of the virulence and antibiogram profiling of P. aeruginosa isolated from apparently healthy Japanese quail (Coturnix japonica) in Bangladesh.

The susceptibility of young birds to P. aeruginosa infections is well-documented, with the potential for mechanical transmission through skin injuries or contaminated needles used in vaccinations. Factors such as the bird’s immunological status and concurrent illnesses can increase their vulnerability [49]. This study identified PCR positive isolates of P. aeruginosa, in quails at a proportion of 22.73%. Notably, there is a scarcity of studies on P. aeruginosa in poultry in Bangladesh. A recent study by Bakheet and Torra (2020) reported a 24% positivity in poultry [50], while another study found a 28.3% prevalence in broiler chickens from Ismailia Governorate, Egypt [51]. A higher prevalence (34%) of P. aeruginosa was reported in broiler chickens [52], while a 20% prevalence was observed in broiler chickens using 16S rDNA [53]. In this study, the prevalence of P. aeruginosa in quail birds from Narsingdi district was significantly higher than in Mymensingh. This variation may be due to differences in management practices, agro-climatic conditions such as rainfall, humidity, and temperature, as well as the local prevalence of P. aeruginosa in the Narsingdi region. The impact of regional factors as a risk factor aligns with findings from human infections, where regional variations also contribute to differences in prevalence [54,55].

In terms of age, the study found that the prevalence of P. aeruginosa in young birds (<8 weeks old) was six times higher than in older birds (>8 weeks old). This may occur because young birds have an underdeveloped immune system, which limits their ability to produce specific antibodies and T cells, as well as a lower number of phagocytic cells. As a result, their immune system struggles to effectively recognize and eliminate pathogens like P. aeruginosa [56,57]. Additionally, nutritional deficiencies and immature mucosal surfaces increase the permeability and susceptibility of young birds, facilitating the colonization of P. aeruginosa. This observation is consistent with existing literature [31,57,58]. These studies highlighted the heightened susceptibility of young chicks to P. aeruginosa infections. Additionally, mortality was noted to be higher in young birds [59], further emphasizing the vulnerability of this age group.

Furthermore, the study identified a higher prevalence of P. aeruginosa in female birds compared to male birds This finding aligns with studies on human infections, where female patients are more susceptible than males [60,61] The female reproductive hormones may contribute to increased vulnerability to P. aeruginosa infection [62].

Understanding the pathophysiology of opportunistic pathogens such as P. aeruginosa is important and characterizing the virulence factors that constitute its defensive mechanisms is instrumental in exploring novel antimicrobial strategies, especially for multidrug-resistant (MDR) strains. P. aeruginosa secretes various extracellular components to support its survival and enhance virulence [63,64]. The four effector proteins, ExoS, ExoT, ExoU, and ExoY, secreted by P. aeruginosa through the type III secretion system, play a pivotal role in its pathogenicity [65]. These proteins impact critical host cell activities, including signal transduction and cytoskeletal organization [66]. Wareham and Curti [67] described ExoS and ExoT as bifunctional toxins capable of activating GTPase and ADP-ribosyltransferase activities. In this study, we identified the virulence genes exoS (88%) and exoT (64%), consistent with their findings, which noted that ExoT exhibits lower ADP-ribosyltransferase activity than ExoS [67]. Additionally, ExoU, known for its phospholipase activity, causes eukaryotic membrane rupture upon entering the cytoplasm, while ExoY possesses adenylate cyclase activity. The presence of exoY (84%) in our study suggests that P. aeruginosa exhibited adenylate cyclase activity, whereas exoU was not detected. Furthermore, the cytotoxin protein synthesis gene, exoA, was found in 100% of the isolates, a result similar to Ghazaei [68], who reported exoA in 83.33% of animal samples. The quorum sensing (QS) systems of P. aeruginosa, particularly the conventional LuxI/R-type systems—LasI/R and RhlI/R—play a crucial role in regulating virulence factor synthesis and biofilm formation. According to Bakheet and Torra [50], these systems generate and detect 3OC12-homoserine lactone and C4-homoserine lactone, respectively. Interestingly, the lasI gene, responsible for secreting key virulence factors such as pyocyanin and elastase, was not detected in any isolates in this study, which contrasts with Bratu et al. [69], who reported the presence of lasI in 100% of P. aeruginosa strains. Similarly, lasR was present in 75% of strains in studies by Kebede [70] and Saleh et al. [71]. Despite the absence of the lasI gene, QS activity was confirmed in this study through the detection of rhlR (100%), rhlI (64%), and other markers. Furthermore, P. aeruginosa can develop antibiotic resistance through mutations in its DNA or via horizontal gene transfer. The presence of plasmids, integrons, and transposons carrying antibiotic-resistant genes in P. aeruginosa is well documented [55]. The multidrug-resistant characteristics of P. aeruginosa, coupled with its potential for transmission to humans and animals, make it a significant public health concern [57].

Notably, 92% of isolates displayed resistance to ampicillin, while all isolates exhibited 100% resistance to first-generation cephalosporin (Cephradine). These findings align with those of Shahat et al. [53] and are consistent with reports from other studies, which also identified high levels of resistance to Tetracycline and Ampicillin [72–74].

Interestingly, within the β-lactam category, cefoxitin exhibited 76% resistance, while ceftriaxone and meropenem showed complete susceptibility (100%). These results contrast with the general resistance trend observed in the β-lactam group, highlighting the nuanced response of P. aeruginosa to different antibiotics within the same class. In the aminoglycoside group, streptomycin displayed 28% susceptibility and 72% intermediate resistance. However, neomycin, gentamycin, cotrimoxazole, chloramphenicol, florfenicol, ciprofloxacin, and levofloxacin all demonstrated 100% susceptibility. These findings align with Shahat et al. [54] and support the effectiveness of ciprofloxacin and gentamycin against P. aeruginosa, as noted by Mohammad [75]. Conversely, the tetracycline and quinolone groups, including oxytetracycline, doxycycline, and nalidixic acid, showed 92% and 100% resistance, respectively. The high resistance to nalidixic acid (80%) is consistent with observations by [76]. Additionally, it was noted that 96% of isolates had Multiple Antibiotic Resistance Index (MARI) values greater than 0.2, indicating a high-risk source of antibiotic-resistant bacterial contamination [77]. These findings closely align with the report by Afunwa et al. [78].

Conclusion

This study highlights the concerning prevalence of Pseudomonas aeruginosa in quail birds. Younger and female quail from Narsingdi district should be prioritized for future surveillance and control measures. Infected birds should be treated with antibiotics to which P. aeruginosa is susceptible. The consistent presence of virulence genes exoA and rhlR, combined with the genetic diversity observed in other virulence markers, underscores the pathogenic potential of the isolates. These findings emphasize the need for further research into the epidemiology, virulence profiling, and genetic diversity of P. aeruginosa in quail, which is crucial for developing effective preventive strategies, including vaccines.

Supporting information

S1 Table. Gender wise positive distribution of P. aeruginosa at different areas.

https://doi.org/10.1371/journal.pone.0316667.s001

(DOCX)

S2 Table. Sample wise positive distribution of different virulence genes of P. aeruginosa.

https://doi.org/10.1371/journal.pone.0316667.s002

(DOCX)

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