Wild Micromammals as Bioindicators of Antibiotic Resistance in Ecopathology in Northern Italy

Simple Summary: In recent years, animal and human health have been linked in a “One Health” approach. Wildlife could act as a reservoir for different antibiotic-resistant pathogens, representing an issue for human and domestic animal health. The aim of this study was to evaluate the presence and circulation of antimicrobial-resistant bacterial species in wild micromammals in the province of Parma, Northern Italy. Multi-drug-resistant strains and a high prevalence of resistance to critically important antibiotics were detected. Furthermore, resistance to commonly used antibiotics was detected in a large percentage of isolates. Considering that micromammals are good bioindicators, obtained results highlighted a high prevalence of strains resistant to antimicrobials of critical importance for human and animals in the investigated areas, thus representing a public health hazard. Abstract: Antimicrobial resistance (AMR) is an increasing threat to human health and an important issue also in the natural environment. For this study, an ecopathological approach was applied to the monitoring of the antimicrobial resistance in the province of Parma, Northern Italy. Fourteen monitoring sites and seventy-four faecal samples from four species of wild micromammals (Apodemus sylvaticus, Microtus savii, Mus domesticus and Suncus etruscus) were collected. Samples were subjected to bacteriological examination and antimicrobial susceptibility testing. Antibiotics belonging to 13 different antibiotic classes were tested. Collected data showed a prevalence of multi-drug resistant (MDR) strains of 55.13% and significant differences in the prevalence of MDR strains among the different micromammal species, while sex, age and anthropization level did not significantly affected MDR strains prevalence. Moreover, a high prevalence of bacterial strains resistant to colistin (95%), gentamicin (87%) and amikacin (83%) was observed. To our knowledge, this is the first report on antibiotic resistance in wild micromammals in the province of Parma

in vitro antimicrobial activity of a gel containing antimicrobial peptide amp2041 chlorhexidine digluconate and tris edta on clinical isolates of pseudomonas aeruginosa from canine otitis

Background Pseudomonas aeruginosa (PA) may cause suppurative otitis externa with severe inflammation and ulceration in dogs. Multidrug resistance is commonly reported for this organism, creating a difficult therapeutic challenge. Objective The aim of this study was to evaluate the in vitro antimicrobial activity of a gel containing 0.5 μg/mL of antimicrobial peptide AMP2041, 0.07% chlorhexidine digluconate (CLX), 0.4% Tris and 0.1% EDTA on 30 clinical isolates of PA from canine otitis externa. Materials and Methods Antimicrobial activity was evaluated through minimal bactericidal concentration (MBC). Standardized bacterial suspensions were incubated with different concentrations of the gel at 37°C for 30 min and plated for colony forming unit (CFU) counts. Time-to-kill kinetics were evaluated with the undiluted product and at MBC for each PA strain at 30 s, 1, 5, 10, 15, 30 min, 24 and 48 h. Results The MBC was 1:64 for two of 30 strains, 1:128 for 15 of 30 strains and 1:256 for 13 of 30 strains. The geometric mean was 1:165, equivalent to a concentration of 0.003 μg/mL AMP2041 + 0.0004% CLX + 0.0024%Tris + 0.0006% EDTA. Time-to-kill assays with the undiluted product showed complete bactericidal effect within 30 s for all isolates, whereas at the MBC this effect was reached within 5 min for 20 of 30 isolates and within 30 min for all isolates. Bactericidal activity was maintained after 48 h for all isolates. Conclusion This gel has shown rapid, complete and long-lasting activity against a panel of 30 PA isolates from cases of canine otitis externa

Activity of AMP2041 against human and animal multidrug resistant Pseudomonas aeruginosa clinical isolates

Background: Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides. Methods: The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD90), while the activity over time was performed by time-kill assay with 12.5 μg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy. Results: AMP2041 showed a dose-dependent activity, with a mean (SEM) LD90 of 1.69 and 3.3 μg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD90 = 0.3 μg/ml) to the mucoid phenotype (LD90 = 10.4 μg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD90 was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041. Conclusions: The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability

Detection of Extended- Spectrum Beta-Lactamase producing Escherichia coli from mesenteric lymph nodes of wild boars (Sus scrofa)

Wild boars (Sus scrofa) are increasing in several European countries, including Italy. In areas with intensive animal farming, like the Italian Emilia-Romagna region, they are likely to be exposed to antimicrobialresistant (AMR) bacteria of livestock origin. In 2017-2018, 108 mesenteric lymph nodes samples were collected from 108 wild boars hunted in Parma province, Emilia-Romagna region, to be tested for ESBL- and carbapenemase-producing Escherichia coli. One isolate (WB-21L) out of 108 (0.9%) was phenotypically confirmed as ESBLproducing E. coli. The strain WB-21L was tested by PCR for the genes blaSHV, blaCTX-M, blaTEM, blaAmpC, blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48, blaSPM, blaBIC, blaSIM, blaDIM, blaGIM, blaAIM, resulting positive for TEM β-lactamase. Resistance to ampicillin, amoxicillin/clavulanic acid, streptomycin, sulfasomidine, tetracycline and trimethoprim confirmed the multi-resistance nature of the strain WB-21L. Nine E. coli isolates showed resistance to meropenem by the Kirby Bauer test but none of them showed Meropenem MIC values indicative of resistance. In conclusion, the present study shows the presence of ESBL E. coli in wild boars and the possible risk of transfer to game meat handlers and consumers. Future studies are needed to better evaluate the sources of AMR bacteria in wildlife

Antimicrobial activity of a standardized medical honey on bacterial isolates from infected skin lesions of non-traditional companion animals

: In recent years, due to the growing phenomenon of antimicrobial resistance, the search for alternative strategies to antibiotic treatments is increasing and a considerable interest for the use of medical honey in clinical practice has emerged. Honey has been used for the treatment of skin lesions, in both humans and animals. However, knowledge concerning the use of medical honey in non‑traditional companion animals is scarce. The aim of this study was to assess the antibacterial activity of a standardized medical honey (Revamil, BFactory) against bacterial strains isolated from skin lesions of non‑traditional companion animals. The minimum bactericidal concentration (MBC) of Revamil honey against seventeen clinical isolates and three reference strains was established.The medical honey showed antimicrobial activity against both Gram‑positive and Gram‑negative bacteria. Growth was inhibited for all the strains at concentrations of medical honey ranging from 10 to 40%. Pseudomonas oryzihabitans and Alcaligenes faecalis showed the lowest MBC (10%). The reference strain Staphylococcus aureus ATCC25923 showed a higher sensitivity to 20% honey compare to the corresponding clinical isolate (P = 0.001). The observed results suggest that Revamil could represent an effective therapeutic aid, useful for the reduction of antibiotic use, in case of pathological skin infections in non‑traditional companion animals

Survey on carbapenem-resistant bacteria in pigs at slaughter and comparison with human clinical isolates in Italy

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts