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Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including β-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis’ OMVs, the aim of the work was to collect OMVs from five S. Infantis β-lactam resistant strains isolated from a broiler meat production chain and to investigate whether β-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of β-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of β-lactamase enzymes within the OMVs. This suggests that β-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

This work aimed to evaluate phenotypically and genotypically the colistin susceptibility of 85 Salmonella Infantis strains isolated in Italy from the broiler production chain, and to apply a whole-genome approach for the determination of genes conferring antimicrobial resistance (AMR). All isolates were tested by the broth microdilution method to evaluate the colistin minimum inhibitory concentrations (MICs). A multiplex PCR was performed in all isolates for the screening of mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes and whole-genome sequencing (WGS) of six S. Infantis was applied. Three out of 85 (3.5%) S. Infantis strains were colistin resistant (MIC values ranged from 4 to 8 mg/L) and mcr-1 positive. The mcr-1.1 and mcr-1.2 variants located on the IncX4 plasmid were detected in three different colistin-resistant isolates. The two allelic variants showed identical sequences. All six isolates harbored blaCTXM-1, aac(6′)-Iaa and gyrA/parC genes, mediating, respectively, beta-lactam, aminoglycoside and quinolone resistance. The pESI-megaplasmid carrying tet(A) (tetracycline resistance), dfrA1, (trimethoprim resistance) sul1, (sulfonamide resistance) and qacE (quaternary ammonium resistance) genes was found in all isolates. To our knowledge, this is the first report of the mcr-1.2 variant described in S. Infantis isolated from broilers chickens. Our results also showed a low prevalence of colistin- resistance, probably due to a reduction in colistin use in poultry. This might suggest an optimization of biosecurity control both on farms and in slaughterhouses.

This work aimed to evaluate the antimicrobial susceptibility of 87 Salmonella Infantis strains isolated in Italy from 2016 to 2019 along the food chain of broiler meat production and in humans and to determine the genetic profiles of the strains in order to establish a possible correlation with the antimicrobial pattern. All isolates were tested by the disk diffusion method to evaluate antimicrobial susceptibility toward sixteen antimicrobials, and the broth microdilution method was used to confirm extended spectrum &beta, lactamase (ESBL) production. PCR and pulsed field gel electrophoresis (PFGE) were applied to characterize ESBL-encoding and AmpC &beta, lactamase genes and to analyze the S. Infantis strains genetic profiles respectively. S. Infantis isolates showed high prevalence of resistance, in particular toward nalidixic acid (97.7%), tetracycline (96.5%), sulphamethoxazole/trimethoprim (91%) and cefepime (72.4%). The 80.5% of isolates were ESBL, cefotaxime-resistant, carrying the blaCTX-M1 gene. The most prevalent PFGE profile was XbaI.0126 (35.6%). The remaining strains had a genetic homology from 81% to 97% with the XbaI.0126 profile. The strains belonging to these profiles were isolated from different matrices collected along the broiler food chain independently on the year and from the region and there was no correlation between the PFGE profiles and resistance patterns. We found two ESBL-producing S. Infantis strains with the same XbaI.2621 profile isolated from humans and from poultry feces, not yet reported in Italy. Our findings confirmed the diffusion of ESBL-multi drug resistant (MDR) S. Infantis along the broiler food chain and in humans and underlined the importance of continuous monitoring to control and to reduce the prevalence of this bacterium, applying a global One Health approach.

The spread of resistant bacteria from livestock to the food industry promoted an increase of alternative poultry production systems, such as organic and antibiotic-free ones, based on the lack of antimicrobial use, except in cases in which welfare is compromised. We aimed to investigate the antibiotic susceptibility of commensal Escherichia coli isolated from organic, antibiotic-free, and conventional broiler farms and slaughterhouses toward several antimicrobials critically important for human health. To assess antimicrobial susceptibility, all E. coli isolates and extended spectrum beta-lactamase (ESBL) E. coli were analysed by the microdilution method. The prevalence of tigecycline, azithromycin and gentamicin E. coli-resistant strains was highest in organic samplings. Conversely, the lowest prevalence of resistant E. coli strains was observed for cefotaxime, ceftazidime and ciprofloxacin in organic systems, representing a significant protective factor compared to conventional systems. All E. coli strains were colistin-susceptible. Contamination of the external environment by drug-resistant bacteria could play a role in the presence of resistant strains detected in organic systems. Of interest is the highest prevalence of cephalosporin resistance of E. coli in conventional samplings, since they are not permitted in poultry. Our results suggest that monitoring of antibiotic resistance of the production chain may be helpful to detect “risks” inherent to different rearing systems.

The objectives of this work were to evaluate β-lactamase-mediated β-lactam resistance in Campylobacter coli and Campylobacter jejuni isolates obtained from broiler chickens, expression of the blaOXA-61 gene in relation to β-lactamase production, and the possible association between blaOXA-61 gene expression and the action of inhibitors when combined with β-lactams. All strains were tested by disk diffusion and nitrocefin methods to assess antibiotic susceptibility and β-lactamase production, respectively. PCR and qPCR amplification were performed to evaluate qualitative and quantitative blaOXA-61 expression. Campylobacter spp. showed a high level of resistance to the most of antimicrobials tested. C. coli strains were ampicillin resistant and blaOXA-61 positive, and 59 out of 60 isolates were positive in the nitrocefin test. Twenty C. jejuni isolates were positive for blaOXA-61 and the nitrocefin test, although two isolates were ampicillin sensitive. Amoxicillin/clavulanic acid and ticarcillin/clavulanic acid do not seem to be active against C. coli, as 73.3 %, and 88.3 % of isolates were resistant to amoxicillin/clavulanic acid and ticarcillin/clavulanic acid, respectively. C. jejuni was not susceptible to amoxicillin/clavulanic acid, with 90 % of the strains showing resistance, whereas ticarcillin associated with clavulanic acid was significantly more efficient than ticarcillin alone (P < 0.01), with 90 % of the strains found to be susceptible. An association between blaOXA-61 expression and amoxicillin/clavulanic acid and ticarcillin/clavulanic acid resistance (P = 0.0001) was seen in C. coli, as well as in C. jejuni for ampicillin/sulbactam (P = 0.0001). Our results suggest that the clavulanic acid only shows an inhibitory effect on C. jejuni when combined with ticarcillin and that the inhibitors action is lower if the blaOXA-61 gene is highly expressed.

Testudinid herpesviruses (TeHVs) have a worldwide distribution among tortoises. However, information such as risk factors promoting the occurrence or the recrudescence of the associated disease and the mid-term sequelae of an outbreak comprising the extent and dynamic of the viral shedding have been only minimally investigated. Critical management information is also lacking or anecdotal. Furthermore, major aspects of the viral pathogenesis including the likelihood of vertical transmission of the virus are virtually unknown. The present study describes the occurrence and the management of an outbreak of Testudinid herpesvirus genotype 3 (TeHV3) in a large, private collection of chelonians. Clinical, pathological, molecular and serological characterization of the outbreak were carried out. Seventy-five percent of the infected tortoises died. Complementation of molecular and serological testing was a critical point for successful management implementations. A case-control study was performed to analyze possible risk factors associated with the infection. Furthermore, a subgroup of six asymptomatic infected tortoises was monitored for two consecutive seasons after the outbreak: all the infected tortoises were determined to be intermittent shedders, except one, which was a persistent shedder. Post-hibernation was associated with the highest number of shedders. Finally, evidence of the most likely vertical transmission of the virus was obtained for the first time.

Helicobacter suis is the second most prevalent Helicobacter species in the stomach of humans suffering from gastric disease. This bacterium mainly inhabits the stomach of domesticated pigs, in which it causes gastric disease, but it appears to be absent in wild boars. Interestingly, it also colonizes the stomach of asymptomatic rhesus and cynomolgus monkeys. The origin of modern human-, pig- or non-human primate-associated H. suis strains in these respective host populations was hitherto unknown. Here we show that H. suis in pigs possibly originates from non-human primates. Our data suggest that a host jump from macaques to pigs happened between 100 000 and 15 000 years ago and that pig domestication has had a significant impact on the spread of H. suis in the pig population, from where this pathogen occasionally infects humans. Thus, in contrast to our expectations, H. suis appears to have evolved in its main host in a completely different way than its close relative Helicobacter pylori in humans

Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.

Ascending infections of equine uterus frequently result in placentitis and abortions; most of these infections are bacterial and are less commonly due to fungi. This report describes an abortion case in an Arab mare due to Candida guilliermondii that was diagnosed via cytological, histological, cultural and biomolecular assays. The histological lesions found were severe necrotizing placentitis associated with fetal pneumonia. To our knowledge this is the first case of C. guilliermondii abortion reported in equine species.

The major bacteria colonizing the intestinal tract (ileum and caecum) of organic (O) and conventional (C) chickens were counted, isolated and identified by conventional methods. Chickens were obtained from 7 conventional and 7 organic chicken farms (n=203). Intestinal sampling was performed at different ages, every 10 days, starting at 20 days until 40 and from 20 days to 80 days of age, respectively, for conventional and organic birds. Statistical analysis was performed on two separate data sets (40 days of age and all ages).The comparison of C vs O systems was analyzed with univariate and multivariate procedures. There were large differences in bacterial counts in relation to the portion of intestine, the rearing system and the farms. In the ileum of conventional birds Enterobacteria were higher than in organic birds (7.03 vs 6.09 CFUxlog/g; P

In recent years, there has been increasing evidence of the potential pathogenic significance of equine gammaherpesviruses in the horse. In humans, cattle and mice, gammaherpesviruses have already been associated with uterine infection. The aim of the present study was to investigate the presence of gammaherpesviruses in uterine flushings of mares with reproductive problems and to evaluate if there was a possible statistical association with clinical and laboratory findings in these cases. A total of 80 uterine flushings were collected from 61 mares with different reproductive problems and these were tested for equine herpesviruses (EHV) 1–5 by PCR. In the case of each mare in the study, the age, history of infertility, presence of anatomical defects in the reproductive tract, presence of systemic or local disease at time of sampling, phase in the oestrous cycle, post-partum interval, nature of uterine lavage performed (low versus large volume lavage), cytological and bacteriological examination results from the uterine flushing, and PCR herpesvirus results were recorded. Univariate analysis and multivariable logistic regression models were used to identify possible statistical associations and risk factors. Nine out of 61 mares (14.7%) had EHV-5 DNA in their uterine flushings. Co-infections with EHV-1 and EHV-2 were present in two cases. Of all the variables analyzed, only the cytological examination findings were associated with EHV-5 PCR positive results, both on univariate and multivariable analysis, especially in cases with an inflammation score of 3. It is postulated that presence of EHV-5 infection in the non-pregnant uterus may have a role to play in reproductive dysfunction and have a negative consequence on the pregnant uterus. Additional studies involving both healthy mares and mares with reproductive problems need to be performed, however, to elucidate whatever role equine gammaherpesviruses may play in the reproductive tract. This would be very worthwhile, since reproductive problems can have a significant impact on the equine breeding industry. Gaining a greater understanding of its causes could lead to new approaches for prevention and treatment.

The role of the horse in Coxiella burnetii infection has not been defined. Accordingly, a twofold approach was taken to further our knowledge on this topic: (1) conduct a systematic review of the literature to establish available evidence of C. burnetii infection in the horse; (2) undertake a biomolecular investigation of 122 cases of equine abortion, stillbirth and neonatal foal death, for the presence of C. burnetii using a PCR test targeting the IS1111 gene of C. burnetii. A review of the literature turned up seven studies that identified C. burnetii DNA in equine specimens, especially aborted fetuses, while an additional 34 studies sought to determine seroprevalence of the infection in the horse. A meta-analytical approach was taken to calculate a pooled mean seroprevalence in equines based on published studies. A seroprevalence of 15.8% (95% confidence interval: 9.6–23.0%) was obtained. This figure is comparable to those previously reported in other species, especially ruminants. None of the 122 cases of equine abortion, stillbirth or neonatal foal death were positive for C. burnetii DNA. C. burnetii has rarely been looked for in equine specimens in previous studies. Cases of equine abortion should be comprehensively investigated to assess the risk of abortion in a pregnant mare infected with C. burnetii. Consideration should also be given to the possible role of the horse as a source of the organism for other animal species including humans.

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.

This paper describes an outbreak of disease caused by Hafnia alvei in pullets. Cloudy swelling and the fatty degeneration of liver associated with splenic lymphocytic depletion were the most preminent lesions. The organism was identified by biochemical tests and by a Hafnia-specific bacteriophage test. Laying hens and pullets were infected experimentally with the organism by the oral and intraperitoneal route, and the clinical and pathological effects were similar to those observed in naturally infected subjects. The use of more sensitive diagnostic tests is suggested to avoid the possibility of a misdiagnosis due to similarities between this organism and Salmonella species. Resume Infection a Hafnia alvei chez des poulettes en Italie Cet article decrit un cas de maladie causee par Hafnia alvei chez des poulettes. Les lesions les plus preeminentes ont ete une degenerescence graisseuse du foie qui avait une apparence gonflee et marbree, et une depletion lymphocytaire au niveau de la rate. Le germe a ete id...

The aim of this study was to evaluate the possible differences in the intestinal microflora composition among the different rearing systems (conventional vs organic) and the Salmonella diffusion using bacteriological techniques. The results showed that the differences between the two groups at the same age, expressed by the bacterial count, are not conclusive in showing an influence of the rearing systems. Salmonella Hadar was isolated once in caeca of conventional and once in caeca of organic ones. Though the results are preliminary and referred to a well defined geographic area in Central Italy, Salmonella detection does not seem to be common in conventional and organic chicken farms.