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The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18–22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.

This study investigated the presence, distribution, and antimicrobial resistance profiles of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in a dairy herd located in Northern Italy. The feces of clinically healthy calves, their mothers, and the cows treated for mastitis, as well as water, environmental samples, and waste milk were collected and subjected to bacteriological culture on CHROMagarTM ESBL plates. A questionnaire was administered to identify risk factors. The isolates were identified as E. coli by MALDI-TOF MS and subjected to the double-disk synergy test (DDST) and minimal inhibitory concentration (MIC) assay. As a result, ESBL E. coli was isolated from the feces of 28 of 37 (75.67%) calves, the feces of 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and waste milk. All ESBL isolates showed multiple resistances and were categorized as multidrug-resistant (MDR). Several risk factors for ESBL E. coli selection and diffusion were identified, including lack of routine cleaning of calf feeding and housing equipment, administration of waste milk to male calves, and blanket dry cow therapy. In conclusion, this study highlighted the presence of MDR, ESBL E. coli in the feces of most dairy calves, and their association with different sample sources. Accordingly, adding to the prudent use of antibiotics, the adoption of adequate farm hygiene and biosecurity measures might also help prevent the spread and transmission of ESBL E. coli within the herd.

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.

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.

Stress represents a major risk factor for psychiatric disorders, including post-traumatic stress disorder (PTSD). Recently, we dissected the destabilizing effects of acute stress on the excitatory glutamate system in the prefrontal cortex (PFC). Here, we assessed the effects of single subanesthetic administration of ketamine (10 mg/kg) on glutamate transmission and dendritic arborization in the PFC of footshock (FS)-stressed rats, along with changes in depressive, anxious, and fear extinction behaviors. We found that ketamine, while inducing a mild increase of glutamate release in the PFC of naïve rats, blocked the acute stress-induced enhancement of glutamate release when administered 24 or 72 h before or 6 h after FS. Accordingly, the treatment with ketamine 6 h after FS also reduced the stress-dependent increase of spontaneous excitatory postsynaptic current (sEPSC) amplitude in prelimbic (PL)-PFC. At the same time, ketamine injection 6 h after FS was found to rescue apical dendritic retraction of pyramidal neurons induced by acute stress in PL-PFC and facilitated contextual fear extinction. These results show rapid effects of ketamine in animals subjected to acute FS, in line with previous studies suggesting a therapeutic action of the drug in PTSD models. Our data are consistent with a mechanism of ketamine involving re-establishment of synaptic homeostasis, through restoration of glutamate release, and structural remodeling of dendrites.

Posttraumatic stress disorder (PTSD), the fifth most prevalent mental disorder in the United States, is a chronic, debilitating mental illness with as yet limited options for treatment. Hallmark symptoms of PTSD include intrusive memory of trauma, avoidance of reminders of the event, hyperarousal and hypervigilance, emotional numbing, and anhedonia. PTSD is often triggered by exposure to a single traumatic experience, such as a traffic accident, a natural catastrophe, or an episode of violence. This suggests that stressful events have a primary role in the pathogenesis of the disorder, although genetic background and previous life events are likely involved. However, pathophysiology of this mental disorder, as for major depression and anxiety disorders, is still poorly understood. In particular, it is unknown how can a single traumatic, stressful event induce a disease that can last for years or decades. A major shift in the conceptual framework investigating neuropsychiatric disorders has occurred in recent years, from a monoamine-oriented hypothesis (which dominated pharmacological research for over half a century) to a neuroplasticity hypothesis, which posits that structural and functional changes in brain circuitry (largely in the glutamate system) mediate psychopathology and also therapeutic action. Rodent stress models are very useful to understand pathophysiology of PTSD. Recent studies with acute or subacute stress models have shown that exposure to short-time stressors (from several minutes to a few hours) can induce not only rapid, but also sustained changes in synaptic function (glutamate release, synaptic transmission/plasticity), neuroarchitecture (dendritic morphology, synaptic spines), and behavior (cognitive functions). Some of these changes, e.g., stress-induced increased glutamate release and dendrite retraction, are likely connected and occur more rapidly than previously thought. We propose here to use a modified version of a simple and validated protocol of footshock stress to explore different trajectories in the individual response to acute stress. This new conceptual framework may enable us to identify determinants of resilient versus vulnerable response as well as new targets for treatment, in particular for rapid-acting antidepressants. It will be interesting to investigate the putative prophylactic action of ketamine toward the maladaptive effects of acute stress in this new protocol.