Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV preinfection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (,1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon c. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools

<i>AppΔapxIΔapxIIC</i> cell culture supernatant and PRRSV effects on mRNA quantification of type I (IFNα, IFNβ) and type II (IFNγ) interferons.

<p>qRT-PCR results expressed in relative expression (ΔΔCT) for IFNα (A), IFNβ (B) and IFNγ (C) in SJPL cells. The cells were mock infected or infected with 0.5 MOI of PRRSV for 4 hours then treated without or with <i>AppΔapxICΔapxIIC</i> cell culture supernatant for 48 hours. Poly (I:C) and LPS were used as positive controls. Data labeled with superscripts of different letters indicates that these sets of data are statistically different (P<0.05).</p

Antiviral activity of <i>AppΔapxIΔapxIIC</i> supernatant against several animal DNA and RNA viruses in SJPL infected cells.

<p>All experiences were performed at least 2 times.</p><p>Statistical <i>P</i> value compared to <i>AppΔapxIΔapxIIC</i> untreated cells: <i>P</i> = 0.15.</p><p>Statistically significative compared to <i>AppΔapxIΔapxIIC</i> untreated cells: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p><p>Statistically significative compared to other viruses: <i>^aP</i><0.01.</p

Bacterial adherence over time of <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> in PRRSV co-infected SJPL and MARC-145 cells.

<p>SJPL (A) and MARC-145 (B) cells were infected with or without PRRSV at an MOI of 0.5 during 72 hours, and then cells were co-infected with <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> at an MOI of 10. Bacterial adherence was measured in CFU per well after 1, 2 and 3 hours post bacterial infection as described in Auger <i>et al</i>., 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098434#pone.0098434-Auger1" target="_blank">[20]</a>. Values are presented as ± Standard Deviation (SD). No statistical significance was obtained following two-away ANOVA analysis. All experiments were repeated 3 times.</p

<i>AppΔapxIΔapxIIC</i> cell culture supernatant <1 kDa fraction antiviral activity against PRRSV.

<p>Detection of the N viral protein in PRRSV infected SJPL cells by immunofluorescence. SJPL cells were infected with 0.5 MOI of PRRSV for 4 hours then incubated with DMEM culture medium alone (DMEM) (A) or either a DMEM culture medium fraction of <1 kDa (DMEM <1 kDa) (B) or a <i>AppΔapxIΔapxIIC</i> cell culture supernatant <1 kDa fraction (<i>App</i><1 kDa) (C) added to complete SJPL culture medium for 48 hours. White scale bar represents 200 µm. Pictures were taken at 100X magnification.</p

Cytotoxicity over time of <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> in PRRSV co-infected SJPL and MARC-145 cells.

<p>SJPL (A and B) and MARC-145 cells (C and D) were infected with or without PRRSV at an MOI of 0.5 during 72 hours, and then cells were co-infected with <i>App</i> (for 1 or 2 hours) (A and C, respectively) or with <i>AppΔapxIΔapxIIC</i> (for 4, 5 and 6 hours) (B and D, respectively) at an MOI of 10. Cytotoxicity was measured in % using lactate dehydrogenase (LDH) CytoTox assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098434#pone.0098434-Auger1" target="_blank">[20]</a>. Values are presented as ± Standard Deviation (SD). Two-away ANOVA analysis was used to obtain statistical data. *<i>P</i><0.05. All experiments were performed 3 times.</p

Drug resistance phenotypes and genotypes in Mexico in representative gram-negative species: Results from the infivar network.

AimThis report presents phenotypic and genetic data on the prevalence and characteristics of extended-spectrum β-lactamases (ESBLs) and representative carbapenemases-producing Gram-negative species in Mexico.Material and methodsA total of 52 centers participated, 43 hospital-based laboratories and 9 external laboratories. The distribution of antimicrobial resistance data for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa in selected clinical specimens from January 1 to March 31, 2020 was analyzed using the WHONET 5.6 platform. The following clinical isolates recovered from selected specimens were included: carbapenem-resistant Enterobacteriaceae, ESBL or carbapenem-resistant E. coli, and K. pneumoniae, carbapenem-resistant A. baumannii complex, and P. aeruginosa. Strains were genotyped to detect ESBL and/or carbapenemase-encoding genes.ResultsAmong blood isolates, A. baumannii complex showed more than 68% resistance for all antibiotics tested, and among Enterobacteria, E. cloacae complex showed higher resistance to carbapenems. A. baumannii complex showed a higher resistance pattern for respiratory specimens, with only amikacin having a resistance lower than 70%. Among K. pneumoniae isolates, blaTEM, blaSHV, and blaCTX were detected in 68.79%, 72.3%, and 91.9% of isolates, respectively. Among E. coli isolates, blaTEM, blaSHV, and blaCTX were detected in 20.8%, 4.53%, and 85.7% isolates, respectively. For both species, the most frequent genotype was blaCTX-M-15. Among Enterobacteriaceae, the most frequently detected carbapenemase-encoding gene was blaNDM-1 (81.5%), followed by blaOXA-232 (14.8%) and blaoxa-181(7.4%), in A. baumannii was blaOXA-24 (76%) and in P. aeruginosa, was blaIMP (25.3%), followed by blaGES and blaVIM (13.1% each).ConclusionOur study reports that NDM-1 is the most frequent carbapenemase-encoding gene in Mexico in Enterobacteriaceae with the circulation of the oxacillinase genes 181 and 232. KPC, in contrast to other countries in Latin America and the USA, is a rare occurrence. Additionally, a high circulation of ESBL blaCTX-M-15 exists in both E. coli and K. pneumoniae