---
title: "Genomic and antibiogram characterization of Pasteurella multocida type A from layer chickens"
id: "plos-one-21-isolation-antibiogram-and-whole-genome-sequence-analysis-of-pasteurella"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-21-isolation-antibiogram-and-whole-genome-sequence-analysis-of-pasteurella"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357005"
published_at: "2026-08-31T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Genomic and antibiogram characterization of Pasteurella multocida type A from layer chickens
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-21-isolation-antibiogram-and-whole-genome-sequence-analysis-of-pasteurella
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357005)
- **Published At:** 2026-08-31T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This study isolated and characterized **Pasteurella multocida** type A from commercial layer hens in Bangladesh using culture, biochemical tests, PCR, antimicrobial susceptibility testing, and whole-genome sequencing (WGS). - A total of 80 organ samples (heart, liver, spleen) were collected from layer farms in Trishal, Valuka, and Gazipur; 12 isolates (15%) were confirmed as P. multocida type A by molecular assays targeting **KMT1** and **capA**. - Antimicrobial susceptibility by disc diffusion against 16 drugs showed phenotypic resistance to **cefixime, ceftriaxone, ertapenem,** and **meropenem** and highest susceptibility to **ciprofloxacin, doxycycline, tetracycline,** and **levofloxacin**. - A representative isolate underwent Illumina MiSeq WGS. RAST annotation reported a genome size of 2,346,689 bp, 2,205 predicted coding sequences, 59 RNA genes, and a GC content of 40.2%. - Bioinformatic analyses identified multiple virulence-associated genes including **wecA, galU, manB, rfaD, rfaE, rfaF, lpxB, lpxC,** and **msbA**, supporting pathogenic potential and possible mechanisms of immune evasion. - PathogenFinder classified the isolate as a potential human pathogen with a probability score of 0.891. - Phylogenetic analysis (MEGA X, maximum likelihood) placed the isolate in a monophyletic clade with closest genetic relatedness to strains from Australia, China, and Malaysia. - The study provides the first WGS-based genomic data for P. multocida type A from Bangladeshi layer hens, offering actionable information for surveillance, outbreak investigation, and selection of targeted antimicrobials for fowl cholera control.
## Clinical Analysis & Structured Key Points
Isolation, antibiogram and whole genome sequence analysis of Pasteurella multocida type A from layer birds | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Peer Review Reader Comments Figures Figures Abstract Fowl cholera (FC), caused by Pasteurella multocida type A, represents a major threat to poultry production worldwide. Detailed characterization of the genotypic, phenotypic, and antimicrobial resistance profiles is essential for advancing epidemiological surveillance and guiding effective intervention strategies. A total of 80 suspected FC field samples were collected from which P. multocida was isolated and identified through conventional culture techniques, Gram staining, and PCR. Antimicrobial susceptibility testing was performed using the disc diffusion method with 16 antimicrobial drugs. Whole-genome sequencing of a representative isolate was conducted using the Illumina MiSeq platform, and genomic analysis was carried out with multiple bioinformatics tools. P. multocida type A was confirmed in 12 isolates (15%). RAST annotation revealed a genome size of 2,346,689 bp with 2,205 predicted coding sequences, 59 RNA genes, and a GC content of 40.2%. Phenotypic analyses showed resistance to cefixime, ceftriaxone, ertapenem, and meropenem, while the highest sensitivity was observed for ciprofloxacin, doxycycline, tetracycline, and levofloxacin. PathogenFinder predicted the isolate as a potential human pathogen with a probability score of 0.891. Virulence-associated genes identified included wecA, galU, manB, rfaD, rfaE, rfaF, lpxB, lpxC, and msbA , supporting the pathogenic potential of the isolates and suggesting a possible role in host immune evasion. Phylogenetic analysis using MEGA X with the maximum likelihood method revealed that the isolate was clustered within the monophyletic clade and that it showed closest genetic relatedness to strains from Australia, China, and Malaysia. These findings highlight the genomic basis of virulence and antimicrobial resistance in P. multocida type A from Bangladeshi layer for the first time. The isolate characterized in this study provides valuable genomic insights and the selection of targeted antimicrobials to improve fowl cholera control strategies. Citation: Shahid MAH, Kabir A, Das S, Rahman MT, Nazir KHMNH (2026) Isolation, antibiogram and whole genome sequence analysis of Pasteurella multocida type A from layer birds. PLoS One 21(8): e0357005. https://doi.org/10.1371/journal.pone.0357005 Editor: Faham Khamesipour, MOHME: Iran Ministry of Health and Medical Education, IRAN, ISLAMIC REPUBLIC OF Received: April 30, 2026; Accepted: August 11, 2026; Published: August 31, 2026 Copyright: © 2026 Shahid 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 whole genome shotgun sequencing data is present in NCBI and is publicly available on BioProject JAODLP000000000.1 ( https://www.ncbi.nlm.nih.gov/nuccore/JAODLP000000000.1/ ). If any other information is needed, the author will provide it. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1. Introduction Commercial poultry farming in Bangladesh began in the 1980s and has expanded rapidly in recent decades, driven by the intensive rearing of high-yielding chick strains. The sector plays a pivotal role in improving human nutrition through the supply of meat and eggs, while also contributing to income generation and poverty alleviation. Currently, more than 150,000 commercial farms, with an estimated investment of 350 thousand million Bangladeshi Taka, support the livelihoods of approximately six to eight million people in Bangladesh [ 1 ]. Despite favorable agro-climatic conditions, the growing poultry industry in Bangladesh faces recurrent outbreaks of infectious and noninfectious diseases, leading to weight loss, reduced egg production, and elevated mortality, and therefore considerable economic losses [ 1 ]. Among bacterial diseases, Fowl Cholera (FC) is a critical threat, significantly hampering poultry production [ 2 ]. FC is an acute septicemic disease with high morbidity and mortality in chickens and is prevalent in 25–35% of Bangladeshi poultry populations, with higher rates in backyard flocks (59–72%) compared to layers (12.5%) and broilers (4.25%) [ 3 – 5 ]. The disease is caused by P. multocida , a commensal bacteria species of the upper respiratory tract that can also induce acute septicemia in cattle, sheep, and goats, resulting in heavy economic losses [ 6 , 7 ]. Based on capsular antigens, P. multocida is divided into five capsular types (A, B, D, E, F) and 16 serovars based on lipopolysaccharide antigens [ 8 ]. In Asian countries, P. multocida types A:1, A:3, and D are commonly associated with FC, with serovar A:1 causing up to 80% mortality [ 9 ]. Layers are particularly susceptible compared to younger chickens, making the disease especially problematic in egg-producing flocks [ 10 ]. Traditional approaches for pathogen characterization, including phenotypic and molecular methods as well as antimicrobial susceptibility testing, are routinely used to manage outbreaks. However, these approaches have limitations such as low genomic resolution and the target-specific scope of molecular assays [ 11 , 12 ]. Conventional genotypic tools frequently detect antimicrobial resistance (AMR) genes but not virulence determinants, thereby limiting their utility for comprehensive outbreak investigations [ 13 ]. In contrast, whole-genome sequencing (WGS) provides high-resolution information on virulence, resistance, host adaptation, and evolutionary relationships. Advances in sequencing platforms and bioinformatics pipelines have reduced costs while increasing speed and accuracy [ 14 , 15 ]. Numerous studies now demonstrate that WGS offers a rapid, cost-effective, and superior method for outbreak analysis compared with traditional approaches [ 16 – 19 ]. In Bangladesh, although phenotypic methods are widely used to characterize P. multocida , genetic information is rarely integrated into outbreak management. Some locally produced vaccines are applied in the field, but vaccination failures remain common, likely due to inadequate genomic insights into circulating strains [ 10 , 20 , 21 ]. To date, no whole-genome sequence data are available for P. multocida type A isolates from layer hens in Bangladesh. Therefore, this study was undertaken to isolate and identify P. multocida from recent field cases of fowl cholera in layer hens using cultural, biochemical, and molecular approaches; to determine and screening its antimicrobial susceptibility profile by disc diffusion, and to elucidate its genomic features through next-generation sequencing. 2. Materials and methods 2.1. Study areas and collection of samples Samples were collected from commercial layer farms located in Trishal (24.5816°N, 90.3948°E, n = 5 farm), Valuka (24.4078°N, 90.3865°E, n = 5 farm), and Gazipur (25.6135°N, 83.5070°E, n = 5 farm). Each farm housed more than 1,000 hens, and at least five hens were sampled per farm. In total, 80 layer hens, each including heart, liver, and spleen tissues, were obtained from layer hens that had died with clinical suspicion of fowl cholera. Most hens were in mid- to late-laying age. According to farm owners, routine vaccination was practiced and flock management followed Department of Livestock Services (Bangladesh) guidelines for feeding and lighting. As these were private commercial farms, detailed information on flock size, production performance, or concurrent disease events was not disclosed. As the birds had either already died or were clinically ill and had been brought to the Veterinary Diagnostic Center for diagnosis, sampling was conducted by a registered veterinarian with the consent of the farm owners and transported to the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, in an icebox under cold-chain conditions for subsequent bacterial isolation. Therefore, no additional ethical approval was required for this study. 2.2. Isolation and Identification through cultural and biochemical examination Each tissue sample (5–10 g of infected organ) was inoculated into fresh nutrient broth (Himedia, India) and incubated aerobically at 37 °C for 24 h in a bacteriological incubator. Growth-positive broths were streaked onto nutrient agar (Himedia, India) and incubated at 37 °C for 24 h. Suspected Pasteurella colonies were subcultured on 5% bovine blood agar (Himedia, India) for enrichment and streaked onto MacConkey agar (Himedia, India) for further confirmation. Representative colonies were examined morphologically by Gram staining and microscopy under oil immersion (100X), and pure isolates were obtained for downstream analysis. Biochemical characterization of the isolates was performed using a panel of tests, including methyl red–Voges Proskauer (MR-VP), indole, catalase, oxidase, and carbohydrate fermentation (dextrose, maltose, lactose, mannitol, and sucrose) [ 5 ]. For preservation, pure isolates were maintained either in 70% glycerol stocks or on blood agar slants at refrigerated conditions to ensure retention of original characteristics. 2.3. DNA extraction and molecular detection by PCR A pure P. multocida colony was suspended in 200 μL of deionized water, subjected to heat lysis at 94 °C for 10 min, followed by cold shock on ice for 10 min. The lysate was centrifuged at 10,000 rpm for 10 min, and the supernatant was used as the DNA template [ 22 ]. PCR assays targeting the KMT1 and capA genes were performed to confirm P. multocida type A. The primers used in this study are listed in Table 1 . Each 25 μL PCR reaction consisted of 12.5 μL 2X PCR Master Mix (GoTaq® G2 Green Master Mix, Promega, USA), 1 μL of each forward and reverse primer (10 μM), 2 μL of DNA template, and 8.5 μL of nuclease-free water [ 23 ]. Download: PNG larger image TIFF original image Table 1. List of primers used for the detection of P. multocida type A. https://doi.org/10.1371/journal.pone.0357005.t001 The thermal cycling profile for the KMT1 gene included: initial denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 9 min, and a 4 °C hold. For the capA gene, the profile was: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 1 min 30 s; followed by a final extension at 72 °C for 5 min, and a 4 °C hold. PCR products were resolved by electrophoresis on a 1.5% agarose gel (Sigma) at 100 V for 30 min, stained with ethidium bromide (0.5 μg/mL) for 10 min, and destained in distilled water for 10 min. Gels were visualized and documented using a UV Solo transillumination (Biometra, Germany) [ 24 , 25 ]. 2.4. Antimicrobial sensitivity test The antimicrobial susceptibility profile of the isolates was determined using a panel of commercially available antimicrobial discs (Himedia, India) selected for antimicrobial resistance surveillance and comparison with previous studies. The antimicrobial tested were ampicillin (AMP, 10 μg); cefixime (CFM, 5 μg); ceftriaxone (CTR, 30 μg); chloramphenicol (C, 30 μg); ciprofloxacin (CIP, 5 μg); colistin (CL, 10 μg); doxycycline (DO, 30 μg); enrofloxacin (ENR, 5 μg); erythromycin (E, 15 μg); ertapenem (ETP, 10 μg); gentamycin (GEN, 10 μg); levofloxacin (LEV, 5 μg); meropenem (MEM, 10 µg); neomycin (N, 30 µg); norfloxacin (NOR, 10 µg); tetracycline (TE, 30 µg). Zone diameters were measured and interpreted according to Clinical and Laboratory Standards Institute [ 26 ] and European Committee on Antimicrobial Susceptibility Testing [ 27 ] guidelines breakpoints. Isolates were classified as sensitive, intermediate, or resistant. 2.5. Whole-genome sequence and data analysis Based on PCR and antimicrobial sensitivity pattern, one representative isolate (PM58 or BAUFCTA) which showed the most antimicrobial resistance was selected for whole-genome sequencing through Illumina MiSeq Sequencer (NSU, Dhaka) [ 28 ]. DNA was extracted by Wizard genomic DNA extraction kit (Promega, USA) according to the manufacturer’s instructions. The sequenced file was assembled by SPAdes 3.9 [ 29 ] and annotated with RAST server [ 30 ]. Annotated data were analyzed using different bioinformatic tools such as The Comprehensive Antibiotic Resistance Database (CARD) for identification of resistance genes [ 31 ] ( http://card.mcmaster.ca ), Virulence Finder Data Base ( https://www.mgc.ac.cn/VFs/ ) to identify virulence genes, SpeciesFinder 2.0 [ 32 ] ( https://cge.food.dtu.dk/services/SpeciesFinder/ ) for species identification, and Pathogenfinder (Version 2) to identify gene families that ( https://genepi.food.dtu.dk/pathogenfinder ) [ 33 ] correlate with pathogenicity. Core-genome alignment was generated using Roary v3.11.2, based on genome annotations produced with Prokka v1.14.5 ( https://github.com/tseemann/prokka ). The phylogenetic tree was constructed using the maximum likelihood method by CLC Genomics Workbench (Qiagen) with the bootstrap value of 1000 [ 34 ]. 3. Results 3.1. Isolation and molecular detection In the present study, 80 suspected FC cases from commercial layer farms were examined. On nutrient agar, the isolates produced spherical, smooth, opaque, white, and round colonies. On 5% bovine blood agar, colonies were small, round, opaque, and non-hemolytic, with a characteristic musty odor, while no growth was observed on MacConkey agar. Microscopically, the organisms appeared as Gram-negative coccobacilli occurring singly or in pairs. Biochemical characterization showed fermentation of dextrose, sucrose, and mannitol without gas production, but no fermentation of lactose or maltose. The isolates were positive for indole, catalase, and oxidase, and negative for methyl red and Voges-Proskauer tests. Samples passed the cultural and biochemical properties used for molecular detection by PCR. Overall P. multocida type A was confirmed in 15% (12/80) of cases based on cultural, biochemical, and PCR confirmation of KMT1 and capA genes ( Fig 1 and Fig 2 ). Download: PNG larger image TIFF original image Fig 1. Amplification of P. multocida species specific gene ( KMT1 ). Lane 1, 2: Negative and Positive Control; Lane 3: 100-bp DNA ladder; Lane 4-15: Representative P. multocida isolates. https://doi.org/10.1371/journal.pone.0357005.g001 Download: PNG larger image TIFF original image Fig 2. Amplification of P. multocida type A specific gene ( capA ). Lane 1, 2: Negative and Positive Control; Lane 3: 100-bp DNA ladder; Lane 4-15: Representative P. multocida isolates. https://doi.org/10.1371/journal.pone.0357005.g002 3.2. Antimicrobial sensitivity test The antimicrobial resistance profiles of the 12 P. multocida type A isolates are summarized in Fig 3 . All isolates (100%) were resistant to ampicillin, cefixime, ceftriaxone, ertapenem, and meropenem. Resistance was also observed for neomycin (75.0%, 9/12), tetracycline (66.7%, 8/12), colistin (58.3%, 7/12), and erythromycin (58.3%, 7/12). Lower resistance frequencies were detected for chloramphenicol (16.7%, 2/12) and enrofloxacin (16.7%, 2/12), whereas all isolates remained susceptible to ciprofloxacin, doxycycline, gentamicin, levofloxacin, and norfloxacin ( Fig 3A ). Download: PNG larger image TIFF original image Fig 3. A) The resistance profile of each P. multocida isolate against the tested antibiotics along with the number and percentage of isolates resistant to each antimicrobial, and B) the multidrug resistance (MDR) patterns observed among the isolates. https://doi.org/10.1371/journal.pone.0357005.g003 Analysis of multidrug resistance (MDR) patterns revealed five distinct resistance profiles among the isolates ( Fig 3B ). The predominant pattern (P3) was observed in 66.7% (8/12) of isolates, each showing resistance to eight antimicrobial agents. The remaining four MDR patterns (P1, P2, P4, and P5) were each detected in 8.3% (1/12) of the isolates, exhibiting resistance to 5, 7, 9, and 10 antimicrobial agents, respectively. These findings demonstrate the predominance of a common MDR phenotype among the P. multocida isolates examined. 3.3. Genomic features The isolate PM58, which exhibited the highest level of antimicrobial resistance, was selected for whole-genome sequencing and subsequent analyses. Genome annotation using the RAST server provided an overview of the genome’s taxonomy ID, domain, taxonomy, list of closest neighbors, genome size, GC content percentage, the genome’s N50 and L50 (statistics of a set of contigs or scaffold length), number of contigs (with PEGs), number of subsystems, number of coding sequences and number of RNAs which are listed in Table 2 . SpeciesFinder server finds out the strain based on 16s RNA. Here, we can see that the BAUFCTA was identified as P. multocida Strain HN06. Download: PNG larger image TIFF original image Table 2. Genome assembly and annotation statistics of BAUFCTA. https://doi.org/10.1371/journal.pone.0357005.t002 In the distance tree analysis ( S2 Fig ), the query isolate BAUFCTA (highlighted in yellow) clustered within a monophyletic clade containing closely related P. multocida reference genomes, supporting its taxonomic assignment as P. multocida . Core genome phylogenetic analysis showed that the Bangladeshi isolate BAUFCTA (highlighted in red) clusters with several international reference strains, forming a strongly supported lineage ( Fig 4 ). PathogenFinder server predicted the BAUFCTA genome as Human pathogenic, and its probability was 0.891. Download: PNG larger image TIFF original image Fig 4. Maximum-likelihood core-genome phylogenetic tree showing the evolutionary relationship of the Bangladeshi P. multocida isolate BAUFCTA (highlighted in red) with selected publicly available reference genomes. The tree was constructed using CLC Genomics Workbench v22 based on core-genome alignment with 1,000 bootstrap replicates. Bootstrap values are indicated at the corresponding nodes. https://doi.org/10.1371/journal.pone.0357005.g004 The SEED Viewer estimates the proportion of genes assigned to subsystems and categorizes them according to functional roles. We have given ( S3 Fig ) the subsystem feature counts for the BAUFCTA genome. Using Virulence Factor Data Base P. multocida BAUFCTA strain was found to harbor multiple virulence-associated genes involved in lipopolysaccharide (LPS) biosynthesis, modification, and export ( Table 3 ). Genomic analysis also identified multiple AMR determinants, including CRP (efflux regulation), tufA (elfamycin), uhpT (fosfomycin), and parC (fluoroquinolone) ( S1 Table ). Download: PNG larger image TIFF original image Table 3. Genome‑wide distribution of virulence factors‑associated genes, and their features in BAUFCTA strain. https://doi.org/10.1371/journal.pone.0357005.t003 4. Discussion The overall P. multocida type A was confirmed 15%. This prevalence is higher than that earlier reported by Hasan et al. [ 4 ], who found 12.05% in layer chickens, but lower than rates described in backyard flocks, where prevalence up to 59.72% has been reported [ 3 ]. Variations may be explained by differences in flock type, age, breed, season, biosecurity, farm hygiene, nutrition, and vaccine quality. In particular, FC is more frequent in layers at the onset of lay or at older ages [ 10 ]. It is well recognized that suboptimal nutrition can impair immune function (e.g., vitamin deficiencies) a
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