Emerging resistance to florfenicol in Actinobacillus pleuropneumoniae isolates on two Italian pig farms.

Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a highly contagious lung infection. The control of this respiratory disease remains heavily reliant on antibiotics, with phenicols being one of the primary classes of antibiotics used in pig farming. In the present study, we describe three isolates (B2278, B2176 and B2177) of A. pleuropneumoniae resistant to florfenicol attributed to the presence of the floR gene, which were obtained from two pig farms in Italy. Florfenicol susceptibility tests indicated that B2176 exhibited an intermediate susceptibility profile, while B2177 and B2278 were resistant. All three isolates belonged to serovar 6 and tested positive for the presence of the floR gene. Whole genome sequencing analysis revealed that isolates B2176, B2177 and B2278 harbored genes encoding the toxins ApxII and ApxIII, characteristic of strains with moderate virulence. Moreover, phylogenetic analysis demonstrated that these isolates were closely related, with single nucleotide polymorphisms (SNPs) ranging from 8 to 19. The floR gene was located on a novel 5588 bp plasmid, designated as pAp-floR. BLASTN analysis showed that the pAp-floR plasmid had high nucleotide identity (99 %) and coverage (60 %) with the pMVSCS1 plasmid (5621 bp) from Mannheimia varigena MVSCS1 of porcine origin. Additionally, at least under laboratory conditions, pAp-floR was stably maintained even in the absence of direct selective pressure, suggesting that it does not impose a fitness cost. Our study underscores the necessity of monitoring the spread of florfenicol-resistant A. pleuropneumoniae isolates in the coming years.

An investigation of the transmission of Actinobacillus pleuropneumoniae within vertically integrated systems using whole genome sequencing.

Actinobacillus pleuropneumoniae (APP) causes significant economic losses to the swine industry. Antibiotic treatment can be challenging due to its clinical urgency and the turnover of antimicrobial susceptibility results from the diagnostic laboratory. The aim of this study was to evaluate the vertical transmission of APP within integrated systems as a criterion for optimising antimicrobial treatment in the field, using whole genome sequencing (WGS). Additionally, the genetic variability of Spanish APP isolates has been assessed to decipher antimicrobial resistance (AMR) determinants, toxin presence, serotype, and phenotype/genotype concordance of AMR. A total of 169 isolates from clinical cases of porcine pleuropneumonia with known antimicrobial susceptibility profiles were sequenced. Additionally, 48 NCBI assemblies were included to perform a phylogenetic analysis. Phylogenetic analysis revealed high association between phylogenetic clusters, serotypes, and presence of toxins that are associated within vertically integrated systems by its epidemiological link. Concordance between presence of AMR determinants (genotype) vs in-vitro antimicrobial susceptibility pattern (phenotype) was acceptable for amoxicillin, florfenicol, oxytetracycline, and enrofloxacin using epidemiological cut-off values (ECOFFs), but low concordance was observed for doxycycline and trimethoprim-sulfamethoxazole (T/S). On the other hand, using CLSI clinical breakpoints (CBPs), concordance was acceptable for florfenicol and enrofloxacin and not evaluated for doxycycline, oxytetracycline, trimethoprim-sulfamethoxazole (T/S), and amoxicillin because no CBP are available for them. Finally, WGS has demonstrated the clonality between isolates that shared a common origin (grandmother's farm) and resistance phenotype, suggesting vertical transmission of this pathogen and supporting the use of the epidemiological approach as a good criterion to optimise the antimicrobial use.

Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

The cAMP receptor protein gene contributes to growth, stress resistance, and colonization of Actinobacillus pleuropneumoniae.

Porcine infectious pleuropneumonia (PCP) is a severe disease of porcine caused by Actinobacillus pleuropneumoniae (APP). The spread of PCP remains a threat to the porcine farms and has been known to cause severe economic losses. The cAMP receptor protein (CRP) serves as a pivotal player in helping bacteria adapt to shifts in their environment, particularly when facing the challenges posed by bacterial infections. In this study, we investigated the role of CRP in APP. Our results revealed that crp mutant (Δcrp) strains were more sensitive to acidic and osmotic stress resistance and had lower biofilm formation ability than wild-type (WT) strains. Furthermore, the Δcrp strains showed deficiencies in anti-phagocytosis, adhesion, and invasion upon interaction with host cells. Mice infected with the Δcrp strains demonstrated reduced bacterial loads in their lungs compared to those infected with the WT strains. This study reveals the pivotal role of crp gene expression in regulating pleuropneumonia growth, stress resistance, iron utilization, biofilm formation, phagocytosis, adhesion, invasion and colonization. Our discoveries offer novel perspectives on understanding the development and progression of APP infections.

De novo identification of bacterial antigens of a clinical isolate by combining use of proteosurfaceomics, secretomics, and BacScan technologies.

Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.

Aislamiento e identificación de actinobacillus pleuropneumoniae en pulmones de cerdos con pleuroneumonóa crónica sacrificados en el rastro municipal de mérida, Yucatán, México / Isolation and identification of actinobacillus pleuropneumoniae in pigs lungs with chronic pleuropneumonia at the slaugterhouse of Merida, Yucatan, Mexico

Introducción. El objetivo del presente estudio fue estandarizar la técnica de siembra directa e implementar la prueba de aglutinación rápida en placa para el aislamiento e identificación de Actinobacillus pleuropneumoniae en pulmones de cerdos con pleuroneumonía crónica provenientes del rastro municipal de Mérida, Yucatán, México. Materiales y Métodos. De julio de 1996 a febrero de 1997 se tomaron en el rastro municipal de la ciudad de Mérida, 250 muestras de pulmones de cerdos con lesiones de plueroneumonía en los lóbulos caudales. Las muestras se sembraron en agar sangre con cepa nodriza de Staphylococcus aureus, posteriormente fueron incubadas a 37§C por 24 horas en aerobiosis. Las colonias que presentaron satelitismo, beta hemólisis y brillantes, fueron consideradas colonias sospechosas a A pleuropneumoniae. Para la confirmación de A pleuropneumoniae, se realizaron las pruebas bioquímicas de urea, glucosa, lactosa, mannitol, dulcitol, CAMP y NAD por el método de micrométodos. La prueba utilizada para la serotipificación de A pleuropneumoniae fue aglutinación rápida en placa. Resultados. En 129 (51.6 por ciento) pulmones se aisló A pleuropneumoniae. De las 129 muestras positivas a A pleuropneumoniae, en 113 (87.6 por ciento) muestras se obtuvo cultivo puro (sin la presencia de otras bacterias) y en 16 (12.4 por ciento) muestras A pleuropneumoniae estuvo acompañado de otras bacterias (15 con P multocida y 1 con A pyogenes). Los serotipos identificados de A pleuropneumoniae fueron: serotipo 1 en 88 (68.2 por ciento) cepas, serotipo 7 en 29 (22.5 por ciento) cepas, serotipo 5 en una (0.8 por ciento) cepa y 11 (8.5 por ciento) cepas tuvieron reacción cruzada entre los serotipos 1 y 7. Discusión. La selección de la muestra es uno de los factores más importantes para lograr el aislamiento de A pleuropneumoniae por medio de la técnica de siembra directa. La técnica de siembra directa para el aislamiento de A pleuropneumoniae en pulmones con pleuroneumonía crónica en cerdos de rastro puede ser utilizada para el diagnóstico de la enfermedad.

Consumption of blood coagulation factors associated with Actinobacillus pleuropneumoniae infection

O efeito da infecçäo causada por Actinobacillus pleuropneumoniae no sistema da coagulaçäo do sangue de leitöes foi estudado. 25 leitöes desmamados isentos de organismos patogênicos específicos (IOPES) foram distribuídos de forma aleatória em 2 grupos. 10 leitöes firam infectados com 5x10(6) UFC de Actinobacillus pleuropneumoniae sorotipo 1, e 15 leitöes usados como controles negativos. Reduçöes significativas nas concentraçöes (Pó0.005) dos fatores de coagulaçäo do sangue IX, VIII, VII, X e V foram demonstradas. O tempo parcial ativado da tromboplastina aumentou enquanto que o tempo da protrombina (em porcentagem) diminuiu. A concentraçäo de antitrombina III diminui de forma significativa (Pó0.005). As alteraçöes observadas no tempo de trombina e na quantidade de fibrinogênio estäo relacionadas com a formaçäo de fibrina no processo de coagulaçäo sanguínea. Em consequência disso, a hemorragia pulmonar e a formaçäo de coágulos podem ser observados em pulmöes de leitöes infectados com Actinobacillus pleuropneumoniae