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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. • pleuropneumoniae isolates resistant to florfenicol carrying the floR gene in Italy. • The floR gene was located on a new 5588 bp plasmid (named pAp-floR). • pAp- floR was stably maintained in A. pleuropneumoniae isolates. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 β-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., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams ( n  = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL-/AmpC-producing Escherichia spp. ( n  = 11 E. coli , n  = 1 E. marmotae , n  = 1 E . ruysiae ) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the bla CTX-M-15 gene (3/11) and bla CTX-M-14 and bla CTX-M-1 (2/11 each). The AmpC β-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli , as an index organism for ESBL-producing E. coli isolates in bivalves., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Leoni, Sacchini, Pieralisi, Angelico, Magistrali, Cucco, Massacci, Albini, Duranti, Cammà, Secondini, Rinaldi and Barchiesi.)

Objectives: To investigate the optrA-carrying genetic elements and their transferability in two linezolid-resistant Streptococcus dysgalactiae subsp. equisimilis (SDSE) strains of swine origin., Methods: SDSE strains (V220 and V1524) were phenotypically and genotypically characterized. Transferability of oxazolidinone resistance genes (filter mating), genetic elements and relatedness between isolates (WGS) were analysed. Excision of the genetic elements was assayed by inverse PCR., Results: SDSE isolates were resistant to chloramphenicol, florfenicol and linezolid, but susceptible to tedizolid and both carried the optrA gene.In SDSE V220 optrA was located on a 72.9-kb ICESdyV220 inserted in the 3' end of the chromosomal rum gene. It was 94%-96% identical (coverage, from 31% to 61%) to other optrA-carrying ICEs. In-depth ICESdyV220 sequence analysis revealed that optrA was carried by an IMESdyV220 (17.9 kb), also containing the tet(O/W/32/O) gene. Inverse PCR assays excluded the ICESdyV220 mobility. In SDSE V1524, optrA was carried by the ΦSdyV1524 prophage, integrated near the 5' end of the chromosomal had gene, showing a genetic organization similar to that of other streptococcal phage. Conjugation and transduction assays failed to demonstrate the optrA transferability to streptococcal recipients. V220 and V1524 belonged to two novel sequence types (ST704 and ST634, respectively)., Conclusions: To the best of our knowledge, this is the first identification of the optrA gene on a prophage and an ICE in SDSE isolates from swine brain.These findings are consistent with the current belief in the key role of bacteriophages and ICEs in the streptococcal evolution and adaptation., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Poultry is the most likely source of livestock-associated Extended Spectrum Beta-Lactamase (ESBL) and plasmid-mediated AmpC (pAmpC)-producing E. coli (EC) for humans. We tested the hypothesis that farming methods have an impact on the load of ESBL/pAmpC-EC in the gut of broilers at slaughter. Isolates (n = 156) of antibiotic-free (AF), organic (O), and conventional (C) animals were characterized for antibiotic susceptibility and antibiotic resistance genes. Thirteen isolates were whole-genome sequenced. The average loads of ESBL/pAmpC-EC in cecal contents were 4.17 Log CFU/g for AF; 2.85 Log CFU/g for O; and 3.88 Log CFU/g for C type (p < 0.001). ESBL/pAmpC-EC isolates showed resistance to antibiotic classes historically used in poultry, including penicillins, tetracyclines, quinolones, and sulfonamides. Isolates from O and AF farms harbored a lower proportion of resistance to antibiotics than isolates from C farms. Among the determinants for ESBL/pAmpC, CTX-M-1 prevailed (42.7%), followed by TEM-type (29%) and SHV (19.8%). Avian pathogenic E. coli (APEC), belonging to ST117 and ST349, were identified in the collection. These data confirm the possible role of a broiler as an ESBL/AmpC EC and APEC reservoir for humans. Overall, our study suggests that antibiotic-free and organic production may contribute to a reduced exposure to ESBL/AmpC EC for the consumer.

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.

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 (Tn 6628 and Tn 6674 ), plasmids (pE035 and pWo27-9), and chromosomal regions. cfr environments showed identities with Tn 6644 -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 Tn 6657 . 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.

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.

Competing Interests: Competing interests: None declared.

In rabbit, P. multocida is considered a predominant pathogenic agent; despite this, few data on the molecular epidemiology are available so far. The aim of this work was to characterize P. multocida isolates from rabbit affected by various diseases in Italy. Comparison was made to reference strains from other countries. Thirty-nine isolates were tested using PCRs to detect the genes coding capsular antigens, virulence factors and lipopolysaccharide structures (LPS). Multilocus sequence typing (MLST) was performed and 19 STs registered that belonged to 9 clonal complexes. Italian isolates were all related to P. multocida subsp. P. multocida. Three sequence types dominated (ST9, ST50 and ST74). The isolates were assigned to capsular types A (20/39), D (9/39) and F (10/39), to virulence genes pfhA (13/39), hgbB (21/39) and pfhA+hgbB (4/39) (one without virulence factors) and the isolates either belonged to the LPS genotypes 3 (22/39) or 6 (17/39). The clonal relationships of the Italian strains from rabbit had similarity to previously reported rabbit isolates that belonged to ST9, ST74, ST204 and ST206, however, they differed from other rabbit references strains that belonged to six other STs. In particular, ST9 with capsular type F has been previously reported from diseased rabbit in Czech Republic and ST74 has been observed for older rabbit isolates. ST50 has probably been reported from Spain. ST9 and ST50 have previously also been reported from birds and pig, respectively, whereas ST74 has exclusively been reported from pig. It remains to be investigated if the isolates obtained from diseased rabbit in Italy represent introductions from other host or they are primarily of rabbit origin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pasteurella multocida is a widespread pathogen associated with major animal diseases of economic significance. Despite this, little is known about the capsular types, virulence gene pattern, and antimicrobial susceptibility of isolates from hosts affected by different diseases, and no data are available in Italy. One hundred eighty six isolates of P. multocida, were taken from different species in different states of health in several Italian regions, and were tested for genes encoding for capsular types (cap) and major virulence factors (tbpA, toxA, hgbB and pfhA). Antimicrobial susceptibility was investigated with the agar diffusion test. The majority of isolates was capA+. However, the distribution differed according to species and disease of origin, with a greater heterogeneity in isolates from rabbits; capE was never found, while capB was detected once. Only capA+ and capF+ strains tested positive for pfhA. Conversely, almost all capD+ isolates were hgbB+. In bovine respiratory disease, pfhA+/tbpA+/capA+ isolates predominated, while tbpA+/toxA+/capD+ isolates predominated in sheep. Overall, low levels of resistance were found, with full susceptibility to ceftiofur and florfenicol. Lower susceptibility to older antimicrobials was recorded, since only approximately 1/3 of the isolates showed susceptibility to tylosin and erythromycin, and resistance to tetracycline (7.5%), and trimethoprim - sulphametoxazole (4.8%) was also observed.

Salmonella Typhimurium (S. Typhimurium) is responsible for foodborne zoonotic infections that, in humans, induce self-limiting gastroenteritis. The aim of this study was to evaluate whether the wild-type strain S. Typhimurium (STM14028) is able to exploit inflammation fostering an active infection. Due to the similarity between human and porcine diseases induced by S. Typhimurium, we used piglets as a model for salmonellosis and gastrointestinal research. This study showed that STM14028 is able to efficiently colonize in vitro porcine mono-macrophages and intestinal columnar epithelial (IPEC-J2) cells, and that the colonization significantly increases with LPS pre-treatment. This increase was then reversed by inhibiting the LPS stimulation through LPS antagonist, confirming an active role of LPS stimulation in STM14028-intracellular colonization. Moreover, LPS in vivo treatment increased cytokines blood level and body temperature at 4 h post infection, which is consistent with an acute inflammatory stimulus, capable to influence the colonization of STM14028 in different organs and tissues. The present study proves for the first time that in acute enteric salmonellosis, S. Typhimurium exploits inflammation for its benefit in piglets.

The aim of this study is to describe the prevalence and risk factors of Clostridium difficile shedding in six farms belonging to two companies in Northern Italy. Four hundred and twenty veal calves, randomly selected and individually identified, were sampled three times: at 0-16, 90-120, and 150 days after introduction. C. difficile was isolated at least once from 87 out of the 420 calves (20.7%). The prevalence of shedding was 20.24% at the first sampling and dropped to 0.72% at the second sampling. None of the samples obtained at 150 days tested positive. Sampling of cecal contents and carcass swabs at slaughter was stratified according to the herd of origin of the animals. C. difficile was never isolated at slaughter, excluding a prevalence higher than 3.5% on the basis of previous investigations. Therefore, in this work, the veal calf could not be confirmed as a potential source of C. difficile for the consumer. Eight different ribotypes (RT) have been described, but the vast majority of the isolates (87.8%) belonged to three ribotypes only: RT-078, RT-012 and RT-126, which are also among the most common of the ribotypes detected in humans in Europe. Most isolates, and all the RT-078 isolates, harbored genes coding for toxins A and B, the binary toxin, and showed a deletion in the gene encoding toxin C, suggesting that the veal calf was a reservoir for epidemic hyper-virulent strains. A correlation between age and shedding was found: the odds ratio (OR) ranged from 2.79 for 36-45 days of age to 4.57 for 13-28 days of age. The presence of diarrhea at first sampling was significantly associated with the recovery of C. difficile in feces (OR 3.26). A correlation was found between the administration of antimicrobials and shedding: an increased risk was shown when the number of antimicrobials used was higher than 4 (OR 4.02) or 5-6 (OR 5.83) or when polymyxin E or beta-lactams were administered., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Medically unexplained symptoms are an important mental health problem in primary care and generate a high cost in health services.Cognitive behavioral therapy and psychodynamic therapy have proven effective in these patients. However, there are few studies on the effectiveness of psychosocial interventions by primary health care. The project aims to determine whether a cognitive-behavioral group intervention in patients with medically unexplained symptoms, is more effective than routine clinical practice to improve the quality of life measured by the SF-12 questionary at 12 month., Methods/design: This study involves a community based cluster randomized trial in primary healthcare centres in Madrid (Spain). The number of patients required is 242 (121 in each arm), all between 18 and 65 of age with medically unexplained symptoms that had seeked medical attention in primary care at least 10 times during the previous year. The main outcome variable is the quality of life measured by the SF-12 questionnaire on Mental Healthcare. Secondary outcome variables include number of consultations, number of drug (prescriptions) and number of days of sick leave together with other prognosis and descriptive variables. Main effectiveness will be analyzed by comparing the percentage of patients that improve at least 4 points on the SF-12 questionnaire between intervention and control groups at 12 months. All statistical tests will be performed with intention to treat. Logistic regression with random effects will be used to adjust for prognostic factors. Confounding factors or factors that might alter the effect recorded will be taken into account in this analysis., Discussion: This study aims to provide more insight to address medically unexplained symptoms, highly prevalent in primary care, from a quantitative methodology. It involves intervention group conducted by previously trained nursing staff to diminish the progression to the chronicity of the symptoms, improve quality of life, and reduce frequency of medical consultations., Trial Registration: The trial was registered with ClinicalTrials.gov, number NCT01484223 [http://ClinicalTrials.gov].