Occurrence of Antimicrobial-Resistant Enterococcus spp. in Healthy Chickens Never Exposed to Antimicrobial Agents in Central Italy.

Enterococci are part of the natural flora of the gastrointestinal tract of mammals, including humans, birds and invertebrates. They can cause infection, mainly among hospitalized patients, as well as acquire and transfer antimicrobial resistance genes. The present study allowed the isolation of 98 Enterococcus (73.47% E. faecium, 23.47% E. faecalis, 3.06% E. avium) strains from 120-day-old healthy chickens that had never been treated with antimicrobials. Their antimicrobial resistance was evaluated by the agar disk diffusion method; high-level aminoglycoside (streptomycin and gentamicin) and vancomycin resistance were established using the microbroth dilution method. The highest percentages of resistant isolates were detected with quinupristin-dalfopristin (88.78%), rifampicin (64.29%), tetracyclines (45.92%), and enrofloxacin (41.84%). High percentages of susceptible strains were found with teicoplanin (100%), amoxicillin-clavulanic acid (97.96%), nitrofurantoin (94.90%), ampicillin (92.86%), chloramphenicol (90.82%), and linezolid (88.78%). About 60% of the strains were classified as MDR (multidrug-resistant). Moreover, PCR was carried out to investigate genes encoding for tetracyclines resistance determinants: tet(M), tet(L), tet(O), tet(K), and Int-Tn. Genes were detected in 68 (69.38%) strains: 36 were shown to be resistant with the agar disk diffusion method, while 28 were intermediate, and 2 were susceptible. The present study showed that chickens never treated with antimicrobials potentially harbor enterococci having phenotypic and genotypic characters of antimicrobial resistance.

Antimicrobial Resistant Coagulase-Negative Staphylococci Carried by House Flies (Musca domestica) Captured in Swine and Poultry Farms.

House flies (Musca domestica) are very diffuse insects attracted by biological materials. They are abundantly present in farm environments and can frequently come in contact with animals, feed, manure, waste, surfaces, and fomites; consequently, these insects could be contaminated, carry, and disperse several microorganisms. The aim of this work was to evaluate the presence of antimicrobial-resistant staphylococci in house flies collected in poultry and swine farms. Thirty-five traps were placed in twenty-two farms; from each trap, 3 different kinds of samples were tested: attractant material present in the traps, the body surface of house flies and the body content of house flies. Staphylococci were detected in 72.72% of farms, 65.71% of traps and 43.81% of samples. Only coagulase-negative staphylococci (CoNS) were isolated, and 49 isolates were subjected to an antimicrobial susceptibility test. Most of the isolates were resistant to amikacin (65.31%), ampicillin (46.94%), rifampicin (44.90%), tetracycline (40.82%) and cefoxitin (40.82%). Minimum Inhibitory concentration assay allowed to confirm 11/49 (22.45%) staphylococci as methicillin-resistant; 4 of them (36.36%) carried the mecA gene. Furthermore, 53.06% of the isolates were classified as multidrug-resistant (MDR). Higher levels of resistance and multidrug resistance were detected in CoNS isolated from flies collected in poultry farms than in swine farms. Therefore, house flies could carry MDR and methicillin-resistant staphylococci, representing a possible source of infection for animals and humans.

House Flies (Musca domestica) from Swine and Poultry Farms Carrying Antimicrobial Resistant Enterobacteriaceae and Salmonella.

The house fly (Musca domestica) is a very common insect, abundantly present in farm settings. These insects are attracted by organic substrates and can easily be contaminated by several pathogenic and nonpathogenic bacteria. The aim of this survey was to evaluate the presence of Salmonella spp. and other Enterobacteriaceae in house flies captured in small-medium size farms, located in Northwest Tuscany, Central Italy, and to evaluate their antimicrobial resistance; furthermore, isolates were tested for extended spectrum ß-lactamase and carbapenems resistance, considering the importance these antimicrobials have in human therapy. A total of 35 traps were placed in seven poultry and 15 swine farms; three different kinds of samples were analyzed from each trap, representing attractant substrate, insect body surface, and insect whole bodies. Enterobacteriaceae were isolated from 86.36% of farms, 82.87% of traps, and 60.95% of samples; high levels of resistance were detected for ampicillin (61.25% of resistant isolates) and tetracycline (42.5% of resistant isolates). One extended spectrum ß-lactamase producer strain was isolated, carrying the blaTEM-1 gene. Salmonella spp. was detected in 36.36% of farms, 25.71% of traps, and 15.24% of samples. Five different serovars were identified: Kentucky, Kisarawe, London, Napoli, and Rubislaw; some isolates were in R phase. Resistance was detected mainly for ampicillin (31.21%) and tetracycline (31.21%). House flies could represent a serious hazard for biosecurity plans at the farm level, carrying and sharing relevant pathogenic and antimicrobial resistant bacteria.

In Vitro Antimicrobial Activity of Thymus vulgaris, Origanum vulgare, Satureja montana and Their Mixture against Clinical Isolates Responsible for Canine Otitis Externa.

Otitis externa is a frequent inflammation among dogs, mainly caused by bacteria and yeasts that are often resistant to conventional drugs. The aim of the present study was to evaluate the in vitro antibacterial and antifungal activities of commercial essential oils (EOs) from Origanum vulgare, Satureja montana, and Thymus vulgaris, as well as a mixture of these three components, against 47 clinical bacterial strains (Staphylococcus sp., Streptococcus sp., Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens) and 5 Malassezia pachydermatis strains, previously cultured from the ears of dogs affected by otitis externa. The tested Gram-positive bacteria were sensible to the analysed EOs with MICs ranging from 1.25% (v/v) to <0.0195% (v/v); Gram-negative isolates, mainly P. aeruginosa, were less sensitive with MICs from >10% (v/v) to 0.039% (v/v). M. pachydermatis isolates were sensitive to all EOs with MICs from 4.25% (v/v) to 2% (v/v). However, the mixture was active against all bacterial (except one P. aeruginosa strain) and fungal tested isolates. The three EOs and their mixture seem to be an interesting alternative for treating canine otitis externa when conventional antimicrobials are not active.

Retrospective Molecular Survey on Bacterial and Protozoan Abortive Agents in Roe Deer (Capreolus capreolus) from Central Italy

Bacterial and protozoan agents can determine abortion and other reproductive disorders in domestic ruminants, but data regarding their occurrence in wild ruminants are scanty worldwide, including in Italy. The aim of this retrospective study was to verify the occurrence of the main bacterial and protozoan abortive agents in 72 spleen samples previously collected from roe deer (Capreolus capreolus) living in mountain areas of Central Italy. All samples were collected and submitted to DNA extraction for other investigations. Molecular analyses were carried out on the DNA samples to detect Brucella spp., Chlamydia abortus, Coxiella burnetii, Salmonella enterica, Listeria monocytogenes, Neospora caninum, and Toxoplasma gondii. Three (4.16%) roe deer resulted PCR positive for C. burnetii and one (1.38%) for T. gondii. These findings suggest that roe deer living in the investigated areas do not act as important reservoirs of the searched agents. However, the tested animals lived in a closed area without contact with domestic animals that are usually involved in the epidemiology of the investigated pathogens. Monitoring of wild ruminants is pivotal to verify changes in the epidemiological scenario from a One Health perspective, too.

Occurrence of Bacterial and Protozoan Pathogens in Red Foxes (Vulpes vulpes) in Central Italy.

Most surveys of pathogens in red foxes (Vulpes vulpes) have focused on particular agents. The aim of this study was to verify, with bacteriological and molecular analyses, the occurrence of the main bacterial and protozoan pathogens that are able to infect canids, in red foxes regularly hunted in Central Italy. Spleen, brain, kidney and fecal samples from red foxes were submitted to bacteriological and/or molecular analyses to detect Salmonella spp., Yersinia spp., Listeria monocytogenes, Brucella spp., Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Leptospira spp., Neospora caninum, Hepatozoon canis, Babesia spp. and microsporidia. Two (9.1%) strains of Yersinia enterocolitica biotype 1 and 2 (9.1%) of Yersinia frederiksenii were isolated from 22 fecal samples. Among the 22 spleen samples, seven (31.8%) were PCR-positive for H. canis and 3 (13.6%) for Babesia vulpes. Kidneys from two (2.9%) foxes, among 71 tested, were PCR-positive for L. interrogans. Even though the analyses were carried out on a small number of animals, the results suggested that red foxes from the selected geographic area may act as reservoirs of some investigated pathogens.

Virulence Genes of Pathogenic Escherichia coli in Wild Red Foxes (Vulpes vulpes).

Different pathotypes of Escherichia coli can cause severe diseases in animals and humans. Wildlife may contribute to the circulation of pathogenic pathotypes, including enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC). This study analyzed 109 DNA samples previously extracted from fecal specimens collected from red foxes (Vulpes vulpes) to detect E. coli virulence genes eaeA, hlyA, stx1, and stx2, that characterize the EPEC, STEC, and EHEC strains. Thirty-one (28.4%) samples were positive for at least one investigated virulence gene: eaeA gene was detected in 21 (19.2%) samples, hlyA in 10 (9.1%), stx1 in 6 (5.5%), and stx2 in 4 (3.6%). Nine DNA samples resulted positive for two or three virulence genes: five (4.6%) samples were positive for eaeA and hlyA genes, two (1.8%) for eaeA and stx1, one (0.9%) for hlyA and stx1, one (0.9%) for eaeA, hlyA and stx2. Red foxes seem to be involved in the epidemiology of these infections and their role could be relevant because they may be source of pathogenic E. coli for other wild animals, as well as domestic animals and humans.

Antimicrobial-Resistant Enterococcus spp. in Wild Avifauna from Central Italy.

Bacteria of the genus Enterococcus are opportunistic pathogens, part of the normal intestinal microflora of animals, able to acquire and transfer antimicrobial resistance genes. The aim of this study was to evaluate the possible role of wild avifauna as a source of antimicrobial-resistant enterococci. To assess this purpose, 103 Enterococcus spp. strains were isolated from the feces of wild birds of different species; they were tested for antimicrobial resistance against 21 molecules, vancomycin resistance, and high-level aminoglycosides resistance (HLAR). Furthermore, genes responsible for vancomycin, tetracycline, and HLAR were searched. E. faecium was the most frequently detected species (60.20% of isolates), followed by E. faecalis (34.95% of isolates). Overall, 99.02% of the isolated enterococci were classified as multidrug-resistant, with 19.41% extensively drug-resistant, and 2.91% possible pan drug-resistant strains. Most of the isolates were susceptible to amoxicillin/clavulanic acid (77.67%) and ampicillin (75.73%), with only 5.83% of isolates showing an ampicillin MIC ≥ 64 mg/L. HLAR was detected in 35.92% of isolates, mainly associated with the genes ant(6)-Ia and aac(6')-Ie-aph(2″)-Ia. Few strains (4.85%) were resistant to vancomycin, and the genes vanA and vanB were not detected. A percentage of 54.37% of isolates showed resistance to tetracycline; tet(M) was the most frequently detected gene in these strains. Wild birds may contribute to the spreading of antimicrobial-resistant enterococci, which can affect other animals and humans. Constant monitoring is essential to face up to the evolving antimicrobial resistance issue, and monitoring programs should include wild avifauna, too.

Molecular survey on the presence of arthropod-borne bacteria and protozoans in roe deer (Capreolus capreolus) and ticks from Central Italy.

Environmental changes, due to climatic emergency and to anthropogenic activities severely impact on the epidemiology of vector borne diseases, mostly when transmitted by ticks. The data about the distribution of microorganisms responsible for them in roe deer (Capreolus capreolus) population living in Italy are scanty and completely lacking in Tuscany, so a molecular survey was carried out to estimate the prevalence of some zoonotic tick-borne pathogens in roe deer, and ticks removed from them, living in areas of Central Italy with high risk of arthropod exposure. Spleen samples from 72 roe deer were tested by PCR for Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Francisella tularensis and piroplasms. Moreover, 345 ticks were removed from 65 roe deer, morphologically or molecularly identified and grouped into 162 pools that were submitted to PCR for detecting the same pathogens. Forty-six (63.88%) roe deer were positive for at least one investigated pathogen: 43 (59.72%) for A. phagocytophilum, 2 (2.78%) for Babesia capreoli, 1 (1.39%) for B. burgdorferi, and 1 (1.39%) for Babesia sp. No animals were PCR positive for F. tularensis.  All ticks were identified as Ixodes ricinus. Seventy-six (46.91%) tick pools showed DNA of one or more pathogens: 66 (40.74%) were positive for A. phagocytophilum, 22 (13.58%) for B. burgodorferi s.l., 6 (3.70%) for B. venatorum and 3 (1.85%) for B. capreoli. No pools were positive for F. tularensis. Two or three pathogens were detected in 23 (14.19%) pools.

Low Level of Colistin Resistance and mcr Genes Presence in Salmonella spp.: Evaluation of Isolates Collected between 2000 and 2020 from Animals and Environment.

Salmonellosis is one of the most important zoonoses in Europe and the world. Human infection may evolve in severe clinical diseases, with the need for hospitalization and antimicrobial treatment. Colistin is now considered an important antimicrobial to treat infections from multidrug- resistant Gram-negative bacteria, but the spreading of mobile colistin-resistance (mcr) genes has limited this option. We aimed to evaluate colistin minimum inhibitory concentration and the presence of mcr (mcr-1 to mcr-9) genes in 236 Salmonella isolates previously collected from different animals and the environment between 2000 and 2020. Overall, 17.79% of isolates were resistant to colistin; no differences were observed in relation to years of isolation (2000-2005, 2009-2014, and 2015-2020), Salmonella enterica subspecies (enterica, salamae, diarizonae, and houtenae), origin of samples (domestic animals, wildlife, and environment), or animal category (birds, mammals, and reptiles); only recently isolated strains from houseflies showed the most resistance. Few isolates (5.93%) scored positive for mcr genes, in particular for mcr-1, mcr-2, mcr-4, mcr-6, and mcr-8; furthermore, only 2.54% of isolates were mcr-positive and colistin-resistant. Detected resistance to colistin was equally distributed among all examined Salmonella isolates and not always related to the presence of mcr genes.

Characterization and Comparison of Enterococcus spp. Isolates from Feces of Healthy Dogs and Urine of Dogs with UTIs.

Enterococcus spp. are opportunistic pathogens of both humans and animals characterized by high resistance to antimicrobials. Dogs could be intestinal carriers or suffer from Enterococcus infections, mainly urinary tract infections (UTIs). This study aimed to analyze and compare Enterococcus spp. isolated from healthy dog stools and sick dog urine. Overall, 51 isolates (29 from stools and 22 from UTI) were characterized at species level and tested for antimicrobial resistance, biofilm production and presence of resistance and virulence genes. E. faecium and E. faecalis resulted as equally distributed in stools samples, while E. faecalis predominated among UTI isolates. HLAR phenotype was detected in 47.1% isolates; 64.7% isolates were resistant to ampicillin (47.1% with a MIC ≥ 64 µg/mL). High levels of resistance were recorded for fluoroquinolones (enrofloxacin 74.5%, ciprofloxacin 66.7%), clindamycin (84.3%), tetracycline (78.4%) and quinupristin-dalfopristin (78.4%). No vancomycin resistant strains were detected. All but one isolate were multidrug-resistant. Most detected resistance genes were tetM (70.5%), pbp4 (52.9%) and aph(3')-IIIa (39.2%). All isolates were able to produce biofilm, but isolates from UTIs and belonging to E. faecalis more frequently resulted in strong biofilm producers. Most detected virulence genes were asa1 (52.9%), gelE (41.2%), cylA (37.3%) and esp (35.3%); all of them resulted as more frequently associated to E. faecalis. No particular differences emerged between isolates from feces and UTI, considering all evaluated aspects. Our results confirm pet dogs as carriers of multidrug-resistant enterococci; stool microflora could be considered as the most probable source of enterococcal UTI and E. faecalis carried by dogs seems to be more virulent than E. faecium, justifying its more frequent involvement in urinary tract infections.

Virulence and Antimicrobial Resistance in Canine Staphylococcus spp. Isolates.

Dogs are reservoirs of different Staphylococcus species, but at the same time, they could develop several clinical forms caused by these bacteria. The aim of the present investigation was to characterize 50 clinical Staphylococcus isolates cultured from sick dogs. Bacterial species determination, hemolysins, protease, lipase, gelatinase, slime, and biofilm production, presence of virulence genes (lukS/F-PV, eta, etb, tsst, icaA, and icaD), methicillin resistance, and antimicrobial resistance were investigated. Most isolates (52%) were Staphylococcus pseudointermedius, but 20% and 8% belonged to Staphylococcusxylosus and Staphylococcus chromogenes, respectively. Gelatinase, biofilm, and slime production were very common characters among the investigated strains with 80%, 86%, and 76% positive isolates, respectively. Virulence genes were detected in a very small number of the tested strains. A percentage of 14% of isolates were mecA-positive and phenotypically-resistant to methicillin. Multi-drug resistance was detected in 76% of tested staphylococci; in particular, high levels of resistance were detected for ampicillin, amoxicillin, clindamycin, and erythromycin. In conclusion, although staphylococci are considered to be opportunistic bacteria, the obtained data showed that dogs may be infected by Staphylococcus strains with important virulence characteristics and a high antimicrobial resistance.

(Epi)genetic profiling of extraembryonic and postnatal tissues from female monozygotic twins discordant for Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder caused by defects at the 11p15.5 imprinted region. Many cases of female monozygotic (MZ) twins discordant for BWS have been reported, but no definitive conclusions have been drawn regarding the link between epigenetic defects, twinning process, and gender. Here, we report a comprehensive characterization and follow-up of female MZ twins discordant for BWS. METHODS: Methylation pattern at 11p15.5 and multilocus methylation disturbance (MLID) profiling were performed by pyrosequencing and MassARRAY in placental/umbilical cord samples and postnatal tissues. Whole-exome sequencing was carried out to identify MLID causative mutations. X-chromosome inactivation (XCI) was determined by HUMARA test. RESULTS: Both twins share KCNQ1OT1:TSS-DMR loss of methylation (LOM) and MLID in blood and the epigenetic defect remained stable in the healthy twin over time. KCNQ1OT1:TSS-DMRLOM was nonhomogeneously distributed in placental samples and the twins showed the same severely skewed XCI pattern. No MLID-causative mutations were identified. CONCLUSION: This is the first report on BWS-discordant twins with methylation analyses extended to extraembryonic tissues. The results suggest that caution is required when attempting prenatal diagnosis in similar cases. Although the causative mechanism underlying LOM remains undiscovered, the XCI pattern and mosaic LOM suggest that both twinning and LOM/MLID occurred after XCI commitment.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.

Human teratogens and genetic phenocopies. Understanding pathogenesis through human genes mutation.

Exposure to teratogenic drugs during pregnancy is associated with a wide range of embryo-fetal anomalies and sometimes results in recurrent and recognizable patterns of malformations; however, the comprehension of the mechanisms underlying the pathogenesis of drug-induced birth defects is difficult, since teratogenesis is a multifactorial process which is always the result of a complex interaction between several environmental factors and the genetic background of both the mother and the fetus. Animal models have been extensively used to assess the teratogenic potential of pharmacological agents and to study their teratogenic mechanisms; however, a still open issue concerns how the information gained through animal models can be translated to humans. Instead, significant information can be obtained by the identification and analysis of human genetic syndromes characterized by clinical features overlapping with those observed in drug-induced embryopathies. Until now, genetic phenocopies have been reported for the embryopathies/fetopathies associated with prenatal exposure to warfarin, leflunomide, mycophenolate mofetil, fluconazole, thalidomide and ACE inhibitors. In most cases, genetic phenocopies are caused by mutations in genes encoding for the main targets of teratogens or for proteins belonging to the same molecular pathways. The aim of this paper is to review the proposed teratogenic mechanisms of these drugs, by the analysis of human monogenic disorders and their molecular pathogenesis.

Insights into 6q21-q22: Refinement of the critical region for acro-cardio-facial syndrome.

Deletions on chromosome 6q are rarely reported in the literature, and genotype-phenotype correlations are poorly understood. We report a child with a deletion of the 6q21-q22 chromosomal region, providing some intriguing results about the correlation between this region and acro-cardio-facial syndrome, congenital heart disease, split hand and foot malformation, and epilepsy.