Global spread of the linezolid-resistant Enterococcus faecalis ST476 clonal lineage carrying optrA.

OBJECTIVES: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

Detection and molecular analysis of Pseudorabies virus from free-ranging Italian wolves (Canis lupus italicus) in Italy - a case report.

BACKGROUND: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In mammals, the infection is usually fatal and typically causes serious neurologic disease. This study describes four Aujeszky's disease cases in free-ranging Italian wolves (Canis lupus italicus). In Italy, the wolf is a strictly protected species and is in demographic expansion. CASE PRESENTATION: Three wolves (Wolf A, B, and C) were found in a regional park in Northern Italy, and one (Wolf D) was found in Central Italy. Wolf A and D were alive at the time of the finding and exhibited a fatal infection with epileptic seizures and dyspnoea, dying after a few hours. Wolf B presented scratching lesions under the chin and a detachment of the right earlobe, whilst Wolf C was partially eaten. The wolves showed hepatic congestion, diffuse enteritis, moderate pericardial effusion, severe bilateral pneumonia, and diffuse hyperaemia in the brain. The diagnostic examinations included virological analyses and detection of toxic molecules able to cause serious neurological signs. All four wolves tested positive for pseudorabies virus (PrV). The analysed sequences were placed in Italian clade 1, which is divided into two subclades, "a" and "b". The sequences of Wolf A, B, and C were closely related to other Italian sequences in the subclade b, originally obtained from wild boars and hunting dogs. The sequence from Wolf D was located within the same clade and was closely related to the French hunting dog sequences belonging to group 4. CONCLUSION: Results showed the presence of PrV strains currently circulating in wild boars and free-ranging Italian wolves. The genetic characterisation of the PrV UL44 sequences from the four wolves confirmed the close relationship with the sequences from wild boars and hunting dogs. This fact supports a possible epidemiological link with the high PrV presence in wild boars and the possibility of infection in wolves through consumption of infected wild boar carcasses or indirect transmission. To the best of our knowledge, this study is the first detection of Pseudorabies virus in free-ranging Italian wolves in northern and central Italy.

Streptococcus suis serotype 9 in Italy: genomic insights into high-risk clones with emerging resistance to penicillin.

BACKGROUND: Streptococcus suis is an important pig pathogen and an emerging zoonotic agent. In a previous study, we described a high proportion of penicillin-resistant serotype 9 S. suis (SS9) isolates on pig farms in Italy. OBJECTIVES: We hypothesized that resistance to penicillin emerged in some SS9 lineages characterized by substitutions at the PBPs, contributing to the successful spread of these lineages in the last 20 years. METHODS: Sixty-six SS9 isolates from cases of streptococcosis in pigs were investigated for susceptibility to penicillin, ceftiofur and ampicillin. The isolates were characterized for ST, virulence profile, and antimicrobial resistance genes through WGS. Multiple linear regression models were employed to investigate the associations between STs, year of isolation, substitutions at the PBPs and an increase in MIC values to ß-lactams. RESULTS: MIC values to penicillin increased by 4% each year in the study period. Higher MIC values for penicillin were also positively associated with ST123, ST1540 and ST1953 compared with ST16. The PBP sequences presented a mosaic organization of blocks. Within the same ST, substitutions at the PBPs were generally more frequent in recent isolates. Resistance to penicillin was driven by substitutions at PBP2b, including K479T, D512E and K513E, and PBP2x, including T551S, while reduced susceptibility to ceftiofur and ampicillin were largely dependent on substitutions at PBP2x. CONCLUSIONS: Here, we identify the STs and substitutions at the PBPs responsible for increased resistance of SS9 to penicillin on Italian pig farms. Our data highlight the need for monitoring the evolution of S. suis in the coming years.

SARS-CoV-2 Delta VOC in a Paucisymptomatic Dog, Italy.

Since the very beginning of the COVID-19 pandemic, SARS-CoV-2 detection has been described in several animal species. A total of 625 outbreaks in animals have been reported globally, affecting 17 species in 32 countries and the human source of infection has been recognized including pet owners, zookeepers, and farmers. In this report, we describe the case of a paucisymptomatic dog in Italy infected with SARS-CoV-2 from a household with three confirmed human cases of COVID-19 living in Pesaro (Marche region, Italy). The dog showed high viral RNA titers in the nasal and oropharyngeal swabs. In the nasal swab, SARS-CoV-2 RNA lasted for a least a week. By sequencing, the strain was assigned to the AY.23 lineage (PANGO), one of the sub-lineages of the major SARS-CoV-2 Delta variant of concern (VOC). Although we did not process the swabs of the three human cases, we strongly suspect a human origin for the dog infection. In this regard, AY.23 sequences, although never released thus far in the Marche region, were detected in the neighboring regions. Our findings highlight once more the need for a One Health approach for SARS-CoV-2 surveillance, management, and control, thus preventing viral spillover from animals to humans.

New Sequence Types and Antimicrobial Drug-Resistant Strains of Streptococcus suis in Diseased Pigs, Italy, 2017-2019.

Streptococcus suis is a pathogen associated with severe diseases in pigs and humans. Human infections have a zoonotic origin in pigs. To assess circulating strains, we characterized the serotypes, sequence types, and antimicrobial susceptibility of 78 S. suis isolates from diseased farmed pigs in Italy during 2017-2019. Almost 60% of infections were caused by serotypes 1/2 and 9. All but 1 of the serotype 2 and 1/2 isolates were confined to a single cluster, and serotype 9 isolates were distributed along the phylogenetic tree. Besides sequence type (ST) 1, the serotype 2 cluster included ST7, which caused severe human infections in China in 1998 and 2005. A large proportion of serotype 9 isolates, assigned to ST123, were resistant to penicillin. The emergence of this clone threatens the successful treatment of S. suis infection. Characterizing S. suis isolates from pigs will promote earlier detection of emerging clones.

Detection of Oxazolidinone Resistance Genes and Characterization of Genetic Environments in Enterococci of Swine Origin, Italy.

One hundred forty-five florfenicol-resistant enterococci, isolated from swine fecal samples collected from 76 pig farms, were investigated for the presence of optrA, cfr, and poxtA genes by PCR. Thirty florfenicol-resistant Enterococcus isolates had at least one linezolid resistance gene. optrA was found to be the most widespread linezolid resistance gene (23/30), while cfr and poxtA were detected in 6/30 and 7/30 enterococcal isolates, respectively. WGS analysis also showed the presence of the cfr(D) gene in Enterococcus faecalis (n = 2 isolates) and in Enterococcus avium (n = 1 isolate). The linezolid resistance genes hybridized both on chromosome and plasmids ranging from ~25 to ~240 kb. Twelve isolates were able to transfer linezolid resistance genes to enterococci recipient. WGS analysis displayed a great variability of optrA genetic contexts identical or related to transposons (Tn6628 and Tn6674), plasmids (pE035 and pWo27-9), and chromosomal regions. cfr environments showed identities with Tn6644-like transposon and a region from p12-2300 plasmid; cfr(D) genetic contexts were related to the corresponding region of the plasmid 4 of Enterococcus faecium E8014; poxtA was always found on Tn6657. Circular forms were obtained only for optrA- and poxtA-carrying genetic contexts. Clonality analysis revealed the presence of E. faecalis (ST16, ST27, ST476, and ST585) and E. faecium (ST21) clones previously isolated from humans. These results demonstrate a dissemination of linezolid resistance genes in enterococci of swine origin in Central Italy and confirm the spread of linezolid resistance in animal settings.

Detection of a gE-deleted Pseudorabies virus strain in an Italian red fox.

This study describes an Aujeszky's disease case in an adult male red fox found in an urban area in Central Italy, that exhibited a fatal infection with neurological lesions, but neither itching nor skin lesions. Diagnostic examinations included histology, and parasitological, bacteriological and virological analyses. Detection of parasitic enteric pathogens, bacteria, E. coli, Leptospira spp., rabies, canine distemper virus, parvovirus, hepatitis E virus and pseudorabies virus (PrV) was performed. Results showed the presence of a gE-deleted PrVthat was closely related to the NIA-3 strain but differed from the PrV strains currently circulating in wild boars and domestic pigs in Italy. All the results led to the conclusion that the fox suffered from Aujeszky's disease caused by a gE-deleted PrV strain closely related to a vaccine strain. The epidemiological link between the PrV vaccine strain and fox infection remains unclear. It could involve vaccinated pigs as a primary source of infection by direct or indirect contact with the red fox or less likely it could be related to improper use of the vaccine in the fox.

Transport to the Slaughterhouse Affects the Salmonella Shedding and Modifies the Fecal Microbiota of Finishing Pigs.

Contaminated pork is a significant source of foodborne Salmonellosis. Pork is contaminated at the slaughterhouse and the intestinal content is the predominant source of Salmonella for carcass contamination. The prevalence of Salmonella-positive pigs increases significantly when the time of transport to the slaughterhouse is longer than two hours. The hypothesis behind this study is that transport to the slaughterhouse increases the load of Salmonella in feces and determines a shift of the fecal microbiota in finishing pigs. Fecal samples were collected in a pig herd positive for Salmonella spp., the day before the transport and at the slaughterhouse. Salmonella loads were estimated by the most probable number (MPN) technique, according to the ISO/TS 6579-2:2012/A1. Moreover, the fecal bacteria composition was assessed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Our study showed that the load of Salmonella increases after transport, confirming that this phase of the production chain is a critical point for the control of Salmonella contamination. A lower richness and an increased beta-diversity characterized the fecal microbiota composition of Salmonella-positive animals after transport. In this stage, a natural Salmonella infection causes a disruption of the fecal microbiota as observed in challenge studies.

Detection of Echinococcus granulosus G3 in a Wild Boar ( Sus scrofa ) in Central Italy using PCR and Sequencing.

We report cystic echinococcosis in a free-living wild boar ( Sus scrofa ) in Europe. Parasites were identified by histopathology and molecular techniques, revealing Echinococcus granulosus of the G3 genotype.