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•High-risk CRKP are responsible for difficult-to-treat, hospital-acquired infections.•The percentage of NDM-CRKP increased from 0.1% to 22% in Southern Italy in BSI.•A prolonged outbreak of NDM-1-CRKP was identified during the COVID-19 pandemic.•WGS revealed the presence of hypervirulent XDR NDM-1 ST147 KP strains.•Genomic surveillance is crucial to monitor high-risk CRKP in hospital settings.

Study design: Meta-analysis Objectives: Denervation and androgen deficiency, peculiar to individuals with chronic spinal cord injury (SCI), could hinder, to some extent, both prostate growth and activity. To comprehensively assess the relationship between SCI and prostate volume, we carried out a meta-analysis of the available case-control studies. Methods: A thorough search of MEDLINE, Scopus and Web of Science was carried out to identify studies comparing prostate volume in men with and without SCI. Quality of the studies was assessed using the Newcastle–Ottawa Scale (NOS). Mean differences (MDs) in prostate volume were combined using a random effect model. Funnel plot was used to assess publication bias. Results: Four studies met the inclusion criteria and provided information on 278 men with SCI and 1385 able-bodied controls. The overall difference in prostate volume between the two groups reached the statistical significance (pooled MD: −14.85 ml, 95% CI: −27.10 to −2.61, p= 0.02). In a subgroup analysis including only the studies with the highest NOS score, the pooled MD remained significant (pooled MD: −18.56, 95% CI: −33.14 to −3.99, p= 0.01). The shape of funnel plot did not allow to rule out a possible publication bias. Conclusions: This meta-analysis suggests that in men with SCI, prostate volume tends to be smaller than in age-matched able-bodied men. Longitudinal studies of men with long-lasting SCI in advanced age are warranted to clarify whether this condition is associated with a lower risk of age-related prostate proliferative diseases.

Due to the increasing number of studies reporting the detection of antimicrobial-resistant isolates of Listeria monocytogenes, we sought to determine the antimicrobial susceptibility of L. monocytogenesisolates collected in Italy and find potential correlations to their serotypes and multilocus sequence types (MLST). The antimicrobial susceptibility of 317 L. monocytogenesisolates collected from food, humans, and the environment from 1998 to 2009 was assessed by minimum inhibitory concentration (MIC). Serotyping and MLST was also performed on all isolates. Potential correlations among antimicrobial resistance profiles, serotyping, and MLST were statistically evaluated. Twenty-four percent of L. monocytogenesisolates were resistant to oxacillin, 28.7% intermediate to clindamycin, and 24.3% to ciprofloxacin. The majority of isolates with elevated MIC to oxacillin was of environmental origin and belonged to serotype 4b/4e and ST2. Isolates with intermediate MIC values to clindamycin and ciprofloxacin were mostly of food and human origin and belonged to serotype 4b/4e and ST9. Regarding the time frame of isolate collection, comparing the last 3 years (2007–2009) to previous years (1998–2006), an increase was observed in the percentage of resistant and intermediate isolates per year. This trend strongly suggests the need for increasing attention on the prevalence of antimicrobial resistance in L. monocytogenesin Italy. To predict future resistance trends, the monitoring of clinical intermediate resistance might represent a useful tool especially for antibiotics associated to multiple-step mechanisms of acquired resistance. A specific focus should be addressed to antimicrobial-resistant isolates of serotype 4b, repeatedly associated with food-borne outbreaks.

Antimicrobial resistance in bacteria represents one of the most important challenges for public health worldwide. Human infections from antimicrobial-resistant bacteria can be transmitted from person to person, via the environment (especially in the hospital environment), or via handling or eating contaminated foods. Colistin is well known as a last-resort antibiotic for the treatment of human infections; a recent study performed in the People's Republic of China has revealed that colistin resistance is also conferred by the plasmid-mediated mcr-1gene in Escherichia coli. After that discovery, further plasmid-mediated, colistin resistance genes have been detected. However, to date, only reports on E. colicarrying the mcr-1gene (E. coli mcr-1+) in foodstuff are available. E. coli mcr-1+has been isolated from food of animal origin and vegetables; this discovery has opened a debate among food safety experts. This review aims to provide a critical overview of the currently available scientific literature on the presence of the plasmid-mediated, colistin resistance gene E. coli mcr-1in foodstuffs, focusing on the main implications and future perspectives for food safety.

AbstractThe epidemiology of methicillin-resistant Staphylococcus aureus(MRSA) in horses and its zoonotic potential is poorly understood. The objective of this study is to provide data on the prevalence and genetic characteristics of MRSA isolated from horses on farms, at racecourses, and at slaughterhouses in Italy, using standard and molecular methods. In addition, we report the prevalence of MRSA in horse handlers. Among 388 horses tested by nasal swabs, 27 (7%) were positive for MRSA ST398 (t011, t899, t1255) and ST1 (t127). The prevalence of MRSA in horses tested at slaughterhouses was significantly higher (p< 0.001) compared with those tested on farms and racecourses. Five (7%) out of 67 staff members working in close contact with horses (2 from slaughterhouse, 2 from riding stable, and 1 from racecourse) were carriers of MRSA ST398 (t011, t034) and ST1 (t127). The isolates from horses and humans carried SCCmecIVa or V and were pvlnegative and piapositive. All the isolates from both horses and humans were resistant to at least two antimicrobial classes. The circulation of MRSA in horses and in humans working in close contact with them should be considered an emerging public health issue. In fact, it represents a potential risk for people who work in close contact with horses, and for horse meat consumers.

AbstractRabbit meat has outstanding dietetic and nutritional properties. However, few data on microbiological hazards associated with rabbit productions are available. In this study, the presence of Listeria monocytogeneswas determined in 430 rabbit carcasses, 256 rabbit meat cuts and products, and 599 environmental sponges collected from four Italian rabbit slaughterhouses over a period of 1 year. Prevalence of L. monocytogenesamong the 1285 rabbit meat and environmental samples was 11%, with statistically significant differences between slaughterhouses. The highest prevalence (33.6%) was observed in rabbit meat cuts and products; the majority of positive environmental samples were collected from conveyor belts. Overall, 27.9% and 14.3% of rabbit cuts and carcasses, respectively, had L. monocytogenescounts higher than 1 colony-forming unit (CFU)/10 g. A selection of 123 isolates from positive samples was genotyped and serotyped to determine genetic profiles and diversity among L. monocytogenesisolates contaminating different slaughterhouses and classes of products investigated. Discriminatory power and concordance among the results obtained using multilocus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), automated EcoRI ribotyping, and serotyping were assessed. The isolates selected for typing were classified into serotypes 1/2a (52.8%), 1/2c (32.5%), and 1/2b (14.6%). The majority of the isolates were classified as ST14 (34.1%), ST9 (35.5%), ST121 (17.9%), and ST224 (14.6%). The greatest discriminatory power was observed with the MLVA typing, followed by MLST, PFGE, and ribotyping. The best bidirectional concordance was achieved between PFGE and MLST. There was 100% correlation between both MLST and MLVA with serotype. Moreover, a high unidirectional correspondence was observed between MLVA and both MLST and PFGE, as well as between PFGE and both MLST and serotyping. The results of this study show for the first time in Italy prevalence and genetic profiles of L. monocytogenesisolated in rabbit products and slaughterhouses.

ABSTRACTAspergillussection Nigriincludes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillusstrains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitrotriazole susceptibilities investigated. Aspergillus tubingensiswas isolated from humans, animals, and environmental settings, whereas Aspergillus awamoriand Aspergillus nigerwere isolated exclusively from humans. Phylogenetic analyses of ß-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensisfrom A. awamoriand A. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensisstrain was resistant to itraconazole and PSZ and one A. nigerstrain to PSZ. Sequence analysis of the cyp51Agene revealed different sequence types within a species, and A. tubingensisstrains were also phylogenetically distinct from A. awamori/A. nigerstrains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51Asequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillusisolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51Asequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.

AbstractIn this retrospective study, typing ability, discriminatory power, and concordance between typing results obtained on 123 Campylobacter jejuniturkey isolates, collected in 1998, within 14 different farms, applying multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), antibiotic resistance profile, and virulence gene pattern, were assessed and compared. Overall, 33 sequence types, 28 pulsotypes, 10 resistotypes, and 5 pathotypes were identified. MLST and PFGE showed the better discriminatory ability (i.e., Simpson's diversity index >0.90) as well as unidirectional (i.e., Wallace and adjusted Wallace coefficients >0.86) and bidirectional (i.e., adjusted Rand coefficient >0.60) concordance. Moreover, both methods showed a good unidirectional and bidirectional concordance with the resistotype. On the contrary, the congruence of both genotyping methods and resistotype with the pathotype seemed due to chance alone. A clonal relationship was identified among 66.7% of the isolates. Furthermore, 59.7% of the investigated isolates were resistant to two or more antimicrobials and 92% to tetracycline. All the isolates harbored cadFand pldAgenes, whereas a flaA gene product and a cdtB gene product were amplified from 85.4% and 79.7% of the isolates, respectively, using the primers designed by Bang et al. (2003). The results of this study clarify the level of genetic diversity among the C. jejunioriginating from turkeys. MLST level of correlation with PFGE, resistotype, and pathotype is assessed. This result supports the selection of type and number of typing methods to use in epidemiological studies. Finally, the identification of clonal complexes (i.e., groups of profiles differing by no more than one gene from at least one other profile of the group using the entire CampylobacterMLST database) shared between turkey and human isolates suggests that turkeys could be a possible source of Campylobacterinfection.

Salmonella entericasubsp. entericaserovar Abortusequi is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries, but only sporadically in Europe and the United States. We report an outbreak of S.Abortusequi in foals in Italy, characterized by high mortality. In a herd of Murgese horses, 10 of 34 newborns died at birth and a further 7 died, after developing severe clinical signs, during the first 10 d of life. Tissue specimens from different organs of 2 dead foals, synovial fluids from 4 sick foals, and vaginal and rectal swabs from their dams were cultured. A total of 16 isolates, all as pure cultures, were obtained and identified as Salmonella. The isolates exhibited the same antimicrobial resistance pattern and the same sequence type, ST251, a type that has been associated with S.Abortusequi. Six of 16 isolates were serotyped and found to be S.Abortusequi 4,12:-:e,n,x. Equine practitioners should be aware of S.Abortusequi infection as a cause of neonatal mortality in foals.

ABSTRACTA quantitative comparison between discriminatory indexes and concordance among multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and phage typing has been performed, testing 238 Salmonella entericaserotype Enteritidis isolates not epidemiologically correlated. The results show that MLVA is the best choice, but each typing method provides a piece of information for establishing clonal relationships between the isolates.

Astroviruses are important pathogens of human beings and animals. Feline astroviruses have been identified by electron microscopy in the feces of either asymptomatic or symptomatic cats, and experimental infection with one isolate was shown to induce enteric clinical signs and virus excretion, thus suggesting a possible role as enteric pathogen. However, due to the lack of specific diagnostic assays, feline astroviruses are not included in the diagnostic algorithms of feline infectious diseases, and their role as feline pathogens remains unclear. The present report describes a dual infection by Feline astrovirusand Feline panleukopenia virusin a 4-month-old cat with severe gastroenteritis and panleukopenia. Upon sequence analysis, the Feline astrovirusstrain was found to be genetically related to astroviruses identified in human beings and felids.

Dimethyl sulfoxide (DMSO) is an organic compound widely used as solvent in biological studies and as vehicle for drug administration. DMSO has been shown to possess several biological effects, including antioxidant, anti-inflammatory, antinociceptive effects, and it has been proposed to be therapeutic in several disorders, such as gastrointestinal diseases, rheumatologic diseases, and for the treatment of several manifestations of amyloidosis. To better define the biological profile of DMSO, we investigated its effect on an in vivo model of acute myocardial infarction in rats, caused by left anterior descending coronary artery ligation. Our results show that pretreatment of rats with intraperitoneal (ip) DMSO (500 μL/Kg) for 3 consecutive days before left anterior descending coronary artery ligation significantly (P< 0.05) reduced cardiac damage from 18.75 ± 4.88% (n = 12) to 4.46 ± 2.01% (n = 8); serum levels of troponin I from 29.35 ± 12.32 ng/mL (n = 8) to 2.95 ± 1.32 ng/mL (n = 4); and serum levels of myoglobin from 46.86 ± 10.35 ng/mL (n = 7) to 13.75 ± 0.85 ng/mL (n = 4). Our data demonstrate that DMSO has a protective effect in a model of acute myocardial infarction in rats.

Escherichia coliO26 is known as a verocytotoxin-producing E. coli(VTEC) organism that causes severe foodborne diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Although cattle are the most important reservoir of VTEC, only a few reports on the role of water buffalo (Bubalus bubalis) as a reservoir of VTEC and on the presence of these organisms in their milk are available. However, in Southern Italy, where water buffalo are intensively reared, an outbreak of hemolytic uremic syndrome due to E. coliO26 has recently been reported, in which the consumption of typical dairy products was considered to be a common risk factor. The aims of this work were to assess the prevalence of E. coliO26 in raw water buffalo milk, to characterize the virulence gene profiles of the isolates, and to evaluate their phenotypic antimicrobial resistance pattern. Of 160 analyzed samples, 1 (0.6%) tested positive for E. coliO26, and the isolate showed the stx1+/stx2+/eae−/hlyA+genotypic profile. The strain showed resistance against glycopeptides, macrolides, and penicillins. The presence of VTEC organisms in raw water buffalo milk could be considered to be a potential threat to consumers; however, the strict adherence to the processes used in the preparation of the most common buffalo dairy products could strongly mitigate the foodborne risk. To our knowledge, this article reports the first isolation and characterization of E. coliO26 VTEC in raw water buffalo milk.

Several strategies have been in place in food animal production to reduce the unnecessary use of antimicrobial agents. Beyond the monitoring of their use, the evaluation of the effect of these strategies on the occurrence and types of antimicrobial resistance (AMR) associated genes is crucial to untangle the potential emergence and spread of AMR to humans through the food chain. In the present study, the occurrence of these genes was evaluated in commensal Escherichia coliisolated from broiler carcasses “produced without the use of antibiotics” in 3 antibiotic-free (AB-free) farms in Italy in 2019. Sequenced data were analyzed along with publicly available genomes of E. colicollected in Italy from the broiler food chain from previous years (2017–2018). The genetic relationships among all 93 genomes were assessed on de novoassemblies by in silicoMLST and SNP calling. Moreover, the resistomes of all genomes were investigated. According to SNP calling, genomes were gathered in three clades. Clade A encompassed, among others, ST117, ST8070, and ST1011 genomes. ST10 belonged to clade B, whereas Clade C included ST58, ST297, ST1101, and ST23 among others. Regarding the occurrence of AMR genes, a statistically significant lower occurrence of these genes in the genomes of this study in comparison to the public genomes was observed considering the whole group of genes as well as genes specifically conferring resistance to aminoglycosides, β-lactams, phenicols, trimethoprim, and lincosamides. Moreover, significant reductions were observed by comparing the whole group of AMR associated mutations, as well as those specifically for fluoroquinolones and fosfomycin resistance. Although the identification of 3° generation cephalosporin resistance associated genes in AB-free E. coliis a concern, this study provides a first indication of the impact of a more prudent use of antimicrobial agents on the occurrence of AMR genes in Italian broiler production chain. More studies are needed in next years on a higher number of genomes to confirm this preliminary observation.

Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortus antibodies (by the agglutination and complement fixation tests) and the Nramp1 genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and the Nramp1 genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A+ (Nramp1AA or Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A– (Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; χ2, 11.65 for 1 degree of freedom). Monocytes from Nramp1BB subjects displayed significantly (P < 0.01) higher levels of Nramp1 mRNA than Nramp1AA subjects and also a significantly (P < 0.01) higher ability in controlling the intracellular replication of several Brucella species in vitro. Thus, selection for the Nramp1BB genotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.

ABSTRACTBrucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortusantibodies (by the agglutination and complement fixation tests) and the Nramp1genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and the Nramp1genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A+(Nramp1AAor Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A−(Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; χ2, 11.65 for 1 degree of freedom). Monocytes from Nramp1BBsubjects displayed significantly (P< 0.01) higher levels of Nramp1mRNA than Nramp1AAsubjects and also a significantly (P< 0.01) higher ability in controlling the intracellular replication of several Brucellaspecies in vitro. Thus, selection for the Nramp1BBgenotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.

Natural enemies of ticks include the parasitoid wasp Ixodiphagus hookeri(Hymenoptera: Encyrtidae). The aim of this preliminary study was to investigate the occurrence of I. hookeriDNA in a community of ticks (Ixodes ricinus, Dermacentor marginatus, Hyalomma marginatum, Haemaphysalis inermisand Rhipicephalus turanicus). From May 2010 to March 2012, ticks were collected monthly by dragging and flagging, identified, and 481 adults and 305 nymphs screened molecularly for infection with I. hookeri. Of the samples tested (n=786), 3.1% (n=25) were positive for I. hookeriDNA, 7.2% (n=22) in nymphs and 0.6% (n=3) in adults. I. hookeriDNA was only detected in I. ricinus. This study shows that I. hookeriinfests I. ricinusin southern Italy, with nymphs being the main developmental stage affected by this wasp.

Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide.

ABSTRACTA clinical outbreak of bovine piroplasmosis was reported in Italy. The etiological agent was characterized as Babesia occultans, a parasite regarded as apathogenic and never detected before in continental Europe. This report paves the way for further studies to assess the occurrence of this tick-transmitted protozoan in other European regions.