Developing a PmSLP3-based vaccine formulation that provides robust long-lasting protection against hemorrhagic septicemia-causing serogroup B and E strains of Pasteurella multocida in cattle.

Background: Pasteurella multocida is a bacterial pathogen that causes a variety of infections across diverse animal species, with one of the most devastating associated diseases being hemorrhagic septicemia. Outbreaks of hemorrhagic septicemia in cattle and buffaloes are marked by rapid progression and high mortality. These infections have particularly harmful socio-economic impacts on small holder farmers in Africa and Asia who are heavily reliant on a small number of animals kept as a means of subsistence for milk and draft power purposes. A novel vaccine target, PmSLP-3, has been identified on the surface of hemorrhagic septicemia-associated strains of P. multocida and was previously shown to elicit robust protection in cattle against lethal challenge with a serogroup B strain. Methods: Here, we further investigate the protective efficacy of this surface lipoprotein, including evaluating the immunogenicity and protection upon formulation with a variety of adjuvants in both mice and cattle. Results: PmSLP-3 formulated with Montanide ISA 61 elicited the highest level of serum and mucosal IgG, elicited long-lasting serum antibodies, and was fully protective against serogroup B challenge. Studies were then performed to identify the minimum number of doses required and the needed protein quantity to maintain protection. Duration studies were performed in cattle, demonstrating sustained serum IgG titres for 3 years after two doses of vaccine and full protection against lethal serogroup B challenge at 7 months after a single vaccine dose. Finally, a serogroup E challenge study was performed, demonstrating that PmSLP-3 vaccine can provide protection against challenge by the two serogroups responsible for hemorrhagic septicemia. Conclusion: Together, these data indicate that PmSLP-3 formulated with Montanide ISA 61 is an immunogenic and protective vaccine against hemorrhagic septicemia-causing P. multocida strains in cattle.

Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Puzzling encounter of idiopathic pasteurella bacteraemia.

Pasteurella multocida is a gram-negative coccobacillus that is commonly transmitted through animal bites including cats and dogs. The degree of infection can be worrisome in the immunosuppressed population with a stark correlation in patients with cirrhosis. However, taking that population into account, only 13 cases of P. multocida bacteraemia have been recorded with the majority of those cases having cirrhotic liver disease along with multiple comorbidities. Here, we present an elderly patient with only pertinent medical history of mixed hyperlipidaemia who presents after a mechanical fall with acute renal failure and septic shock secondary to P. multocida bacteraemia.

Dissecting aortitis in a goat associated with Pasteurella multocida and Staphylococcus spp infection.

Reports of primary cardiovascular disease in goats are rare and most commonly include ventricular septal defect, valvular endocarditis, traumatic pericarditis, ionophore poisoning and nutritional cardiomyopathies. We now report the pathological findings in a 67 kg, 6-year-old, adult female Boer goat that presented with neurological signs (ie, head pressing, unsteadiness and paddling) and hyperthermia 2 days prior to death. Lack of therapeutic response to meloxicam and penicillin‒streptomycin and poor prognosis led to euthanasia of the animal. At necropsy, the main findings included severe aortic dissection with luminal thrombosis and stenosis, and pulmonary congestion and oedema. Histological examination of the aorta revealed severe chronic granulomatous and fibrosing dissecting aortitis with mineralization. Bacterial culture of the affected aortic segment resulted in isolation of a profuse growth of Pasteurella multocida and a moderate growth of Staphylococcus spp. Histopathological findings in the central nervous system were consistent with neurolisteriosis.

CXCL8 Knockout: A Key to Resisting Pasteurella multocida Toxin-Induced Cytotoxicity.

Pasteurella multocida, a zoonotic pathogen that produces a 146-kDa modular toxin (PMT), causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. However, its mechanism of cytotoxicity remains unclear. In this study, we expressed PMT, purified it in a prokaryotic expression system, and found that it killed PK15 cells. The host factor CXCL8 was significantly upregulated among the differentially expressed genes in a transcriptome sequencing analysis and qPCR verification. We constructed a CXCL8-knockout cell line with a CRISPR/Cas9 system and found that CXCL8 knockout significantly increased resistance to PMT-induced cell apoptosis. CXCL8 knockout impaired the cleavage efficiency of apoptosis-related proteins, including Caspase3, Caspase8, and PARP1, as demonstrated with Western blot. In conclusion, these findings establish that CXCL8 facilitates PMT-induced PK15 cell death, which involves apoptotic pathways; this observation documents that CXCL8 plays a key role in PMT-induced PK15 cell death.

Integrative and conjugative elements of Pasteurella multocida: Prevalence and signatures in population evolution.

Pasteurella multocida (P. multocida) is a bacterial pathogen responsible for a range of infections in humans and various animal hosts, causing significant economic losses in farming. Integrative and conjugative elements (ICEs) are important horizontal gene transfer elements, potentially enabling host bacteria to enhance adaptability by acquiring multiple functional genes. However, the understanding of ICEs in P. multocida and their impact on the transmission of this pathogen remains limited. In this study, 42 poultry-sourced P. multocida genomes obtained by high-throughput sequencing together with 393 publicly available P. multocida genomes were used to analyse the horizontal transfer of ICEs. Eighty-two ICEs were identified in P. multocida, including SXT/R391 and Tn916 subtypes, as well as three subtypes of ICEHin1056 family, with the latter being widely prevalent in P. multocida and carrying multiple resistance genes. The correlations between insertion sequences and resistant genes in ICEs were also identified, and some ICEs introduced the carbapenem gene blaOXA-2 and the bleomycin gene bleO to P. multocida. Phylogenetic and collinearity analyses of these bioinformatics found that ICEs in P. multocida were transmitted vertically and horizontally and have evolved with host specialization. These findings provide insight into the transmission and evolution mode of ICEs in P. multocida and highlight the importance of understanding these elements for controlling the spread of antibiotic resistance.

Chicken gasdermins mediate pyroptosis after the cleavage by caspases.

Gasdermin (GSDM) proteins are executioners of pyroptosis in many species. Gasdermin proteins can be cleaved at their linker region between the amino domain (NT) and carboxyl domain (CT) by enzymes. The released GSDM-NTs bind cell membrane and form pores, thereby leading to the release of cellular components and lytic cell death. GSDM-mediated pyroptosis is considered to play important role in immune responses. However, little is known about the GSDM proteins and GSDM-mediated pyroptosis in birds. In the current study, genes encoding chicken gasdermin A (chGSDMA) and chGSDME were cloned. The cleavage of chGSDMA and chGSDME by chicken caspase-1 (chCASP1), chCASP3 and chCASP7 and the cleavage sites were determined. The chGSDMA-NT obtained form chCASP1-mediated cleavage and chGSDME-NT obtained from chCASP3/chCASP7-mediated cleavage could bind and damage cell membrane and lead to cell death of HEK293 cells. chGSDMA-NT also strongly localized to and formed puncta in nucleus. Besides, both chGSDMA-NT and chGSDME-NT showed growth inhibition and bactericidal activity to bacteria. In chickens challenged with Pasteurella multocida and Salmonella typhimurium, the expression of chGSDMA and chGSDME was upregulated and the activation of chCASP3 and the cleavage of chGSDME were observed. The work provides essential information for expanding our knowledge on pyroptosis in birds.

Pharmacokinetic/pharmacodynamic evaluation of gamithromycin against rabbit pasteurellosis.

BACKGROUND: Gamithromycin is an effective therapy for bovine and swine respiratory diseases but not utilized for rabbits. Given its potent activity against respiratory pathogens, we sought to determine the pharmacokinetic profiles, antimicrobial activity and target pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with therapeutic effect of gamithromycin against Pasteurella multocida in rabbits. RESULTS: Gamithromycin showed favorable PK properties in rabbits, including high subcutaneous bioavailability (86.7 ± 10.7%) and low plasma protein binding (18.5-31.9%). PK analysis identified a mean plasma peak concentration (Cmax) of 1.64 ± 0.86 mg/L and terminal half-life (T1/2) of 31.5 ± 5.74 h after subcutaneous injection. For P. multocida, short post-antibiotic effects (PAE) (1.1-5.3 h) and post-antibiotic sub-inhibitory concentration effects (PA-SME) (6.6-9.1 h) were observed after exposure to gamithromycin at 1 to 4× minimal inhibitory concentration (MIC). Gamithromycin demonstrated concentration-dependent bactericidal activity and the PK/PD index area under the concentration-time curve over 24 h (AUC24h)/MIC correlated well with efficacy (R2 > 0.99). The plasma AUC24h/MIC ratios of gamithromycin associated with the bacteriostatic, bactericidal and bacterial eradication against P. multocida were 15.4, 24.9 and 27.8 h in rabbits, respectively. CONCLUSIONS: Subcutaneous administration of 6 mg/kg gamithromycin reached therapeutic concentrations in rabbit plasma against P. multocida. The PK/PD ratios determined herein in combination with ex vivo activity and favorable rabbit PK indicate that gamithromycin may be used for the treatment of rabbit pasteurellosis.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Pasteurella multocida activates apoptosis via the FAK-AKT-FOXO1 axis to cause pulmonary integrity loss, bacteremia, and eventually a cytokine storm.

Pasteurella multocida is an important zoonotic respiratory pathogen capable of infecting a diverse range of hosts, including humans, farm animals, and wild animals. However, the precise mechanisms by which P. multocida compromises the pulmonary integrity of mammals and subsequently induces systemic infection remain largely unexplored. In this study, based on mouse and rabbit models, we found that P. multocida causes not only lung damage but also bacteremia due to the loss of lung integrity. Furthermore, we demonstrated that bacteremia is an important aspect of P. multocida pathogenesis, as evidenced by the observed multiorgan damage and systemic inflammation, and ultimately found that this systemic infection leads to a cytokine storm that can be mitigated by IL-6-neutralizing antibodies. As a result, we divided the pathogenesis of P. multocida into two phases: the pulmonary infection phase and the systemic infection phase. Based on unbiased RNA-seq data, we discovered that P. multocida-induced apoptosis leads to the loss of pulmonary epithelial integrity. These findings have been validated in both TC-1 murine lung epithelial cells and the lungs of model mice. Conversely, the administration of Ac-DEVD-CHO, an apoptosis inhibitor, effectively restored pulmonary epithelial integrity, significantly mitigated lung damage, inhibited bacteremia, attenuated the cytokine storm, and reduced mortality in mouse models. At the molecular level, we demonstrated that the FAK-AKT-FOXO1 axis is involved in P. multocida-induced lung epithelial cell apoptosis in both cells and animals. Thus, our research provides crucial information with regard to the pathogenesis of P. multocida as well as potential treatment options for this and other respiratory bacterial diseases.

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia.

Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.

Pathological analysis and etiological assessment of pulmonary lesions and its association with pleurisy in slaughtered pigs.

The intensification of pig farming has posed significant challenges in managing and preventing sanitary problems, particularly diseases of the respiratory complex. Monitoring at slaughter is an important control tool and cannot be overstated. Hence, this study aimed at characterizing both macroscopical and microscopical lesions and identifying the Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae (Mhyo), and Pasteurella multocida (PM) associated with pleurisy in swine. For this, a selected slaughterhouse in São Paulo State underwent a thorough examination of carcasses on the slaughter line, followed by lung sampling. The carcasses and lungs underwent macroscopical examination and were classified according to the score of pleurisy and lung samples were allocated into five groups, being: G0: score 0 - no lesions; G1: score 1; G2: score 2; G3: score 3; and G4: score 4. In total, 217 lung fragments were collected, for the histopathological evaluation and detection of the following respiratory pathogens: APP, Mhyo, and PM by qPCR. The results demonstrated that Mhyo and APP were the most prevalent etiological agents (single and co-identification) in lung samples, in different scores of pleurisies, while bronchopneumonia and bronchus-associated lymphoid tissue (BALT) hyperplasia lesions were the most frequent histopathological findings. Positive correlations were found between the quantification of APP DNA with 1) the score of pleurisy (R=0.254); 2) with the score of lung consolidation in all lung lobes (R=0.181 to R=0.329); and 3) with the score of lung consolidation in the entire lung (R=0.389). The study brings relevant information regarding the main bacterial pathogens associated with pleurisy in pigs and helps with understanding the relationship between the abovementioned pathogens and their impact on the respiratory health of pigs.

Chronic Pasteurella Multocida Bronchitis Diagnosed 3 Years After Onset of Symptoms.

We report a case of chronic infection with Pasteurella multocida in the lower respiratory tract in a man with a cat. A 77-year-old man presented with recurrent hemoptysis accompanied by bronchiectasis and an opacity in the left lung on chest computed tomography. Although the patient was seropositive for Mycobacterium avium complex, repeated sputum cultures were negative for any specific pathogen. Three years later, he was referred to our hospital for hemoptysis with enhanced opacity in the lower lobe of the left lung. Culture of bronchial lavage fluid obtained via bronchoscopy was positive for P. multocida. The patient was treated with amoxicillin-clavulanic acid for 14 days and was instructed to avoid close contact with his cat. His symptoms and chest imaging findings improved and have not recurred during more than 1 1/2 years of follow up. P. multocida can cause chronic lower respiratory infections.

Rare case of septic shock combined with meningitis caused by Pasteurella multocida without a history of cat and dog bites.

BACKGROUND: Pasteurella multocida is a zoonotic pathogen that mainly causes local skin and soft tissue infections in the human body through cat and dog bites. It rarely causes bacteraemia (or sepsis) and meningitis. We reported a case of septic shock and meningitis caused by P. multocida in a patient without a history of cat and dog bites. CASE PRESENTATION: An 84-year-old male patient was urgently sent to the emergency department after he was found with unclear consciousness for 8 h, accompanied by limb tremors and urinary incontinence. In the subsequent examination, P. multocida was detected in the blood culture and wound secretion samples of the patient. However, it was not detected in the cerebrospinal fluid culture, but its DNA sequence was detected. Therefore, the patient was clearly diagnosed with septic shock and meningitis caused by P. multocida. The patient had no history of cat or dog contact or bite. The patient was subsequently treated with a combination of penicillin G, doxycycline, and ceftriaxone, and he was discharged after 35 days of hospitalisation. CONCLUSION: This report presented a rare case of septic shock and meningitis caused by P. multocida, which was not related to a cat or dog bite. Clinical doctors should consider P. multocida as a possible cause of sepsis or meningitis and should be aware of its potential seriousness even in the absence of animal bites.

Pasteurella multocida activates Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis.

Pasteurella multocida is an opportunistic zoonotic pathogen that primarily causes fatal respiratory diseases, such as pneumonia and respiratory syndromes. However, the precise mechanistic understanding of how P. multocida disrupts the epithelial barrier in mammalian lung remains largely unknown. In this study, using unbiased RNA-seq analysis, we found that the evolutionarily conserved Hippo-Yap pathway was dysregulated after P. multocida infection. Given the complexity of P. multocida infection associated with lung injury and systemic inflammatory processes, we employed a combination of cell culture models, mouse models, and rabbit models to investigate the dynamics of the Hippo-Yap pathway during P. multocida infection. Our findings reveal that P. multocida infection activates the Hippo-Yap pathway both in vitro and in vivo, by upregulating the upstream factors p-Mst1/2, p-Lats1, and p-Yap, and downregulating the downstream effectors Birc5, Cyr61, and Slug. Conversely, pharmacological inhibition of the Hippo pathway by XMU-MP-1 significantly rescued pulmonary epithelial cell apoptosis in vitro and reduced lung injury, systemic inflammation, and mouse mortality in vivo. Mechanistic studies revealed that P. multocida induced up-regulation of Rassf1 expression, and Rassf1 enhanced Hippo-Yap pathway through phosphorylation. Accordingly, in vitro knockdown of Rassf1 significantly enhanced Yap activity and expression of Yap downstream factors and reduced apoptosis during P. multocida infection. P. multocida-infected rabbit samples also showed overexpression of Rassf1, p-Lats1, and p-Yap, suggesting that P. multocida activates the Rassf1-Hippo-Yap pathway. These results elucidate the pathogenic role of the Rassf1-Hippo-Yap pathway in P. multocida infection and suggest that this pathway has the potential to be a drug target for the treatment of pasteurellosis.

Pathogenomic analysis and characterization of Pasteurella multocida strains recovered from human infections.

Pasteurella multocida is an upper respiratory tract commensal in several mammal and bird species but can also cause severe disease in humans and in production animals such as poultry, cattle, and pigs. In this study, we performed whole-genome sequencing of P. multocida isolates recovered from a range of human infections, from the mouths of cats, and from wounds on dogs. Together with publicly available P. multocida genome sequences, we performed phylogenetic and comparative genomic analyses. While isolates from cats and dogs were spread across the phylogenetic tree, human infections were caused almost exclusively by subsp. septica strains. Most of the human isolates were capsule type A and LPS type L1 and L3; however, some strains lacked a capsule biosynthesis locus, and some strains contained a novel LPS outer-core locus, distinct from the eight LPS loci that can currently be identified using an LPS multiplex PCR. In addition, the P. multocida strains isolated from human infections contained novel mobile genetic elements. We compiled a curated database of known P. multocida virulence factor and antibiotic resistance genes (PastyVRDB) allowing for detailed characterization of isolates. The majority of human P. multocida isolates encoded a reduced range of iron receptors and contained only one filamentous hemagglutinin gene. Finally, gene-trait analysis identified a putative L-fucose uptake and utilization pathway that was over-represented in subsp. septica strains and may represent a novel host predilection mechanism in this subspecies. Together, these analyses have identified pathogenic mechanisms likely important for P. multocida zoonotic infections.IMPORTANCEPasteurella multocida can cause serious infections in humans, including skin and wound infections, pneumonia, peritonitis, meningitis, and bacteraemia. Cats and dogs are known vectors of human pasteurellosis, transmitting P. multocida via bite wounds or contact with animal saliva. The mechanisms that underpin P. multocida human predilection and pathogenesis are poorly understood. With increasing identification of antibiotic-resistant P. multocida strains, understanding these mechanisms is vital for developing novel treatments and control strategies to combat P. multocida human infection. Here, we show that a narrow range of P. multocida strains cause disease in humans, while cats and dogs, common vectors for zoonotic infections, can harbor a wide range of P. multocida strains. We also present a curated P. multocida-specific database, allowing quick and detailed characterization of newly sequenced P. multocida isolates.

Molecular characterization of Pasteurella multocida from cats and antibiotic sensitivity of the isolates.

BACKGROUND: Companion animals, including dogs and cats, are frequently identified as sources of Pasteurella multocida, a bacterium that can be transmitted to humans and cause infections. OBJECTIVES: This survey defines the prevalence, antibiotic sensitivity, capsular types, lipopolysaccharide (LPS) types and virulence factors of P. multocida isolated from cats. METHODS: A total of 100 specimens from various cat breeds were collected. P. multocida was characterized using both biochemical tests and PCR. Genotypes of isolates were determined using capsular and LPS typing methods. Additionally, virulotyping was performed by detecting the presence of 12 virulence-associated genes. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: The prevalence of P. multocida in cats was 29%. Among the isolates, the majority were capsular type A (96.5%) and type D (3.4%), with a predominant presence of type A. Twenty-six of the isolates (89.66%) belonged to LPS genotype L6, whereas three isolates (10.3%) belonged to genotype L3. Among the 12 virulence genes examined, sodC, oma87, ptfA, nanB and ompH showed remarkable prevalence (100%). The toxA gene was detected in four isolates (13.8%). Variations were observed in other virulence genes. The nanH gene was present in 93.1% of the isolates, whereas the pfhA gene was detected in 58.6% of the isolates. The exbD-tonB, hgbB, sodA and hgbA genes showed prevalence rates of 96.5%, 96.5%, 96.5% and 82.8%, respectively. Additionally, particular capsule and LPS types were associated with specific virulence genes. Specifically, the toxA and pfhA genes were found to be more prevalent in isolates with capsular type A and LPS genotype L6. Most isolates were resistant to ampicillin, clindamycin, lincomycin, streptomycin and penicillin. CONCLUSIONS: According to this epidemiological and molecular data, P. multocida from cats possess several virulence-associated genes and are resistant to antimicrobial medicines commonly used in humans and animals. Thus, it is crucial to consider the public health concerns of P. multocida in humans.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

Discovery of the tigecycline resistance gene cluster tmexCD3-toprJ1 in Pasteurella multocida strains isolated from pigs in China.

Pasteurella multocida is a leading cause of respiratory disorders in pigs. However, the genotypes and antimicrobial resistance characteristics of P. multocida from pigs in China have not been reported frequently. In this study, we investigated 381 porcine strains of P. multocida collected in China between 2013 and 2022. These strains were assigned to capsular genotypes A (69.55%, n = 265), D (27.82%, n =106), and F (2.62%, n = 10); or lipopolysaccharide genotypes L1 (1.31%, n = 5), L3 (24.41%, n = 93), and L6 (74.28%, n = 283). Overall, P. multocida genotype A:L6 (46.46%) was the most-commonly identified type, followed by D:L6 (27.82%), A:L3 (21.78%), F:L3 (2.62%), and A:L1 (1.31%). Antimicrobial susceptibility testing showed that a relatively high proportion of strains were resistant to tetracycline (66.67%, n = 254), and florfenicol (35.17%, n = 134), while a small proportion of strains showed resistance phenotypes to enrofloxacin (10.76%, n = 41), ampicillin (8.40%, n = 32), tilmicosin (7.09%, n = 27), and ceftiofur (2.89%, n = 11). Notably, Illumina short-read and Nanopore long-read sequencing identified a chromosome-borne tigecycline-resistance gene cluster tmexCD3-toprJ1 in P. multocida. The structure of this cluster was highly similar to the respective structures found in several members of Proteus or Pseudomonas. It is assumed that the current study identified the tmexCD3-toprJ1 cluster for the first time in P. multocida.