Correlation Between Microbial Community and Hatching Failure in Loggerhead Sea Turtle Caretta caretta

Microbial communities provide essential information about host ecology and could be helpful as a tool to improve species conservation efforts. However, microbes can also infect and compromise the host development process and viability. Caretta caretta is the most widespread marine turtle species in the Mediterranean basin and is the only species of sea turtle nesting along the Italian coasts. Little is known about the microbiota composition of the nest of sea turtles and its correlation with hatching failures. In this study, the microbial composition of two nests of C. caretta featuring different rates of hatching success from a nesting beach in Lampedusa (Italy) was analyzed and compared. The bacterial community was determined using culture-dependent methods and next-generation sequencing based on 16S rRNA gene metabarcoding analysis. Our results showed five dominant bacterial phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes) and indicated different bacterial families (Pseudomonadaceae and Brucellaceae) as likely causes of hatching failures. Besides, our findings demonstrated the nests' active role in modulating the sand's bacterial communities. This study suggests microbiological analysis could be a valuable tool in monitoring nests to take preventive actions and reduce hatching failures

First data on microflora of loggerhead sea turtle (Caretta caretta) nests from the coastlines of Sicily

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C. caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species

Prevalenza e profili di antimicrobicoresistenza in Salmonella spp isolata da alimenti nel triennio 2019-2021

Summary Salmonella spp. is among the most frequent causes of foodborne diseases, and the increasing occurrence of MDR strains is an additional cause for concern. In the three-year period 2019-2021, we collected Salmonella spp. strains isolated from different food categories analyzed in the context of Regulation (EC) No 2073/2005, to assess their antibiotic susceptibility profile and ESBLs production. To determine the susceptibility profile and identify MDR strains, we performed the Kirby-Bauer method by testing 17 antibiotics belonging to different classes, moreover the double disc test therefore allowed us to evaluate ESBL production. Phenotypic tests showed that 36/67 strains are MDR and 53% of these are ESBLs producers. Our results confirmed the prevalence of S. Infantis MDR and ESBL producer in chicken meat, and that further studies on the prevalence of food-borne MDR strains are needed, especially from a One Health perspective. Introduzione: Salmonella spp. è comunemente causa di infezioni gastrointestinali e di focolai di origine alimentare nell’uomo. La principale via di infezione è l'ingestione di cibo o acqua contaminati e S. Enteritidis, S. Typhimurium, S. Typhimurium monofasica, S. Infantis e S. Derby rappresentano sono i cinque sierotipi più comunemente coinvolti nelle infezioni umane. Nell'UE, i controlli microbiologici sugli alimenti effettuati nel contesto del regolamento (CE) n. 2073/2005 hanno rilevato le percentuali più elevate di campioni positivi alla salmonella nei prodotti a base di uova, carne di pollame e prodotti a base di pollame, che risultano essere le maggiori fonti di trasmissione di Salmonella spp. per l'uomo [2]. L'uso eccessivo di antibiotici ha contribuito alla selezione di ceppi di Salmonella MDR (Multi Drug Resistant), resistenti a tre o più classi di antibiotici [3]. La diffusione di Salmonella MDR rappresenta un problema sanitario rilevante, in quanto causa ospedalizzazioni più lunghe, malattie prolungate e tassi di mortalità più elevati [7, 5]. Inoltre il meccanismo di resistenza è potenziato dalla presenza di beta-lattamasi a spettro esteso (ESBL), in grado di idrolizzare penicilline e cefalosporine di prima, seconda e terza generazione. Lo scopo di questo studio è stato quello di individuare i sierotipi di Salmonella spp. presenti in diverse matrici alimentari analizzate nel contesto del regolamento (CE) n. 2073/2005 [2] con particolare riguardo alla diffusione di Salmonella MDR produttore di ESBL. Materiali e metodi: Da gennaio 2019 a dicembre 2021 sono stati analizzati 145 carni di pollame, 106 di maiale, 54 di manzo, 109 molluschi bivalvi, 43 uova e 36 semi germogliati, per un totale di 493 matrici alimentari. L’isolamento di Salmonella spp è stato eseguito secondo la norma ISO 6579-1:2017, i ceppi sono stati identificati mediante saggi biochimici- enzimatici e sierotipizzazione, secondo lo schema Kauffmann - White - Le Minor [6]. Il fenotipo di suscettibilità agli antibiotici è stato determinato mediante il metodo Kirby-Bauer, testando 17 antibiotici appartenenti a 6 classi differenti. L'interpretazione delle zone di inibizione e la classificazione degli isolati come sensibili (S), intermedi (I) o resistenti (R) sono state effettuate secondo le linee guida CLSI [1]. Su 36 ceppi è stata testata la produzione di ESBL mediante Double Disk test (DDT), secondo le linee guida EUCAST [4] e su 19 dei ceppi testati è stata effettuata la caratterizzazione molecolare in PCR per la ricerca dei geni beta-lattamasi produttori. Risultati: Nel triennio 2019-2021, sul totale delle matrici alimentari esaminate, sono stati isolati 67 ceppi di Salmonella spp. (15/172 nel 2019, 17/132 nel 2020 e 35/189 nel 2021); inoltre, nel triennio considerato, sono state riscontrate le seguenti percentuali di isolamento (Figura 1): carne di pollame (40%, 52,9% e 71,4%), carne di maiale (20%, 23,5% e 5,7%), carne bovina (2,9% soltanto nel 2021), molluschi bivalvi (26,7% nel 2020 e 8,6% nel 2021), uova (11,8% soltanto nel 2020) e semi germogliati (13,3%, 11,8%, 11,4%). Tra i vari sierotipi isolati, S. Infantis è stato quello predominante (48%), isolato in 32 campioni di carne di pollame; i sierotipi di S. Typhimurium (9%), S. Derby (6%) e S. Enteritidis (3%) hanno mostrato una bassa prevalenza (Figura 2). Riguardo i profili di antimicrobicoresistenza, 43/67 ceppi testati sono risultati resistenti ad almeno un antibiotico, con percentuali del 31,3% alla kanamicina, 43,2% alle sulfonamidi, 47,7% all'acido nalidixico, 49,2% all'ampicillina e 50,7% alla tetraciclina. Pochi ceppi sono risultati resistenti a levofloxacina (5%) e cloramfenicolo (6%); tutti sono risultati sensibili a imipenem, ciprofloxacina ed enrofloxacina. Un profilo MDR è stato trovato in 36/67 ceppi (54%), che hanno mostrato resistenza a tre (n=4), quattro (n=22) e cinque (n=10) classi di antibiotici. I profili MDR più frequenti sono stati: R ad aminoglicosidi, beta-lattamici, chinolonici, sulfamidici e tetracicline (in 8 S. Infantis, 1 S. Salamae e 1 S. Kentucky) e R a beta-lattamici, chinolonici, sulfamidici e tetracicline (in 9 S. Infantis e 1 S. Cerro). Il DDT ha consentito di rilevare la produzione di ESBL in 19/36 ceppi MDR (53 %) (Figura 3). Tale risultato fenotipico è stato confermato rilevando in tutti i ceppi la presenza dei geni beta-lattamasi risultati, con maggiore frequenza, blaSHV (68.4%) e blaCTX- M (47.3%) Conclusioni: I nostri dati evidenziano come sia elevato il rischio di infezione da Salmonella MDR, in particolare produttore di ESBL, per i consumatori; inoltre confermano quanto raccomandato dall’EFSA rispetto alla necessità di un monitoraggio efficace dell’AMR anche negli alimenti, soprattutto in un’ottica One Health, che riconosce la circolarità della salute umana, animale ed ambientale

Large-scale survey for canine vector-borne parasites in free-ranging dogs and foxes from six diverse bioclimatic regions of Chile

Chile is a large country with a marked range of climate conditions that make it an ideal scenario for the study of vector-borne parasites (VBPs); however, knowledge about their distribution is limited to a few confined areas of this country. The presence of Hepatozoon spp., piroplasmids, Leishmania spp. and filarioids was investigated through molecular and serological methods in blood and serum samples of 764 free-ranging rural dogs, 154 Andean foxes (Lycalopex culpaeus), and 91 South American grey foxes (Lycalopex griseus) from six bioclimatic regions across Chile. Hepatozoon spp. DNA was exclusively detected in foxes (43% prevalence), including sequences closely related to Hepatozoon felis (24.1%; only Andean foxes), Hepatozoon americanum (16.2%; only grey foxes), and Hepatozoon canis (1.25%; in one grey fox). Risk factor assessment identified a higher probability of Hepatozoon infection in juvenile foxes. DNA of piroplasmids was detected in 0.7% of dogs (Babesia vogeli) but in no fox, whilst antibodies against Babesia sp. were detected in 24% of the dogs and 25% of the foxes, suggesting a wider circulation of canine piroplasmids than previously believed. A positive association between the presence of antibodies against Babesia and high Rhipicephalus sanguineus sensu lato burden was observed in dogs. Leishmania spp. DNA and antibodies were detected in 0.8% and 4.4% of the dogs, respectively. Acanthocheilonema reconditum was the only blood nematode detected (1.5% of the dogs and no fox). Differences in prevalence among bioregions were observed for some of the VBPs. These results expand our knowledge about the occurrence of vector-borne parasites in Chile, some of which are firstly reported herein. This information will facilitate the diagnosis of vector-borne diseases in domestic dogs and improve the control measures for both domestic and wild canids

Occurrence of Antibiotic Resistance in the Mediterranean Sea

Seawater could be considered a reservoir of antibiotic-resistant bacteria and antibiotic resistance genes. In this communication, we evaluated the presence of bacterial strains in seawater collected from different coasts of Sicily by combining microbiological and molecular methods. Specifically, we isolated viable bacteria that were tested for their antibiotic resistance profile and detected both antibiotic and heavy metal resistance genes. Both antibiotic-resistant Gram-negative bacteria, Vibrio and Aeromonas, and specific antibiotic resistance genes were found in the seawater samples. Alarming levels of resistance were determined towards cefazolin, streptomycin, amoxicillin/clavulanic acid, ceftriaxone, and sulfamethoxazole/trimethoprim, and mainly genes conferring resistance to β-lactamic and sulfonamide antibiotics were detected. This survey, on the one hand, presents a picture of the actual situation, showing the pollution status of the Tyrrhenian coast of Sicily, and, on the other hand, can be considered as a baseline to be used as a reference time for future analysis

Can Human Handling Increase the Presence of Multidrug Resistance (MDR) in Salmonella spp. Isolated from Food Sources?

The spread of antibiotic resistance (AR) among zoonotic pathogens is a serious health problem, especially because in the last decade the massive use of antibiotics has favored the emergence of Multidrug Resistance (MDR) strains. Some species of the Salmonella genus are among the major causes of foodborne infections worldwide and could represent reservoirs of AR. For these reasons, the susceptibility to six antibiotic classes of 63 strains isolated from animals and food was determined to assess the presence of MDR strains. In addition, the detection of resistance genes was done for strains that resulted in MDR. A statistically significant difference was found when comparing the presence of Salmonella spp. MDR strains between strains isolated from animals and strains isolated from food. Our data seem to indicate that MDR occurs mostly in Salmonella strains isolated from food

Occurrence of Antibiotic Resistance in the Mediterranean Sea

Seawater could be considered a reservoir of antibiotic-resistant bacteria and antibiotic resistance genes. In this communication, we evaluated the presence of bacterial strains in seawater collected from different coasts of Sicily by combining microbiological and molecular methods. Specifically, we isolated viable bacteria that were tested for their antibiotic resistance profile and detected both antibiotic and heavy metal resistance genes. Both antibiotic-resistant Gram-negative bacteria, Vibrio and Aeromonas, and specific antibiotic resistance genes were found in the seawater samples. Alarming levels of resistance were determined towards cefazolin, streptomycin, amoxicillin/clavulanic acid, ceftriaxone, and sulfamethoxazole/trimethoprim, and mainly genes conferring resistance to β-lactamic and sulfonamide antibiotics were detected. This survey, on the one hand, presents a picture of the actual situation, showing the pollution status of the Tyrrhenian coast of Sicily, and, on the other hand, can be considered as a baseline to be used as a reference time for future analysis

Adding semantic to solve ‘PP attachment’ in Spanish

This paper describes a semantic module called Disambiguator of prepositions (DISPRE) in English, DESambiguador de PREposiciones in Spanish (hereinafter, DESPRE), that solves the prepositional phrase attachment for Spanish, and complements the syntactic analysis (based on tags and syntactic rules) of project FreeLing. Syntactic analysis is not enough to correctly disambiguate prepositions and attach each prepositional phrase to the appropriate head. This is because, depending on the context, the same preposition conveys different meanings. DESPRE analyzes the preposition and its surroundings, assigning it to its correct sense; once the preposition sense is disambiguated, parsing rules can easily determine which is the right head, thus building the correct dependency tree. In this manner, with the help of the preposition semantics, the sentence is disambiguated, and the prepositional phrase is correctly attached. DESPRE uses an ontology with semantic frames (for each prepositional sense) to disambiguate and solve prepositional phrase attachment. New rules have been added to the FreeLing dependency parser to handle the different preposition senses. Thus, DESPRE, the modified version of FreeLing, uses syntactic and semantic analysis. Several examples show the effectiveness of DESPRE, which was able to attach correctly prepositional phrases that the syntactic analysis of Freeling alone could not do.Peer ReviewedPostprint (published version

Preliminary Results on the Prevalence of Salmonella spp. in Marine Animals Stranded in Sicilian Coasts: Antibiotic Susceptibility Profile and ARGs Detection in the Isolated Strains

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach

Helminth Infection of the Loggerhead Sea Turtle Caretta caretta along the Coasts of Sicily and the North West Adriatic Sea

We provide new data on the presence of helminth parasites in 64 individual loggerhead sea turtles Caretta caretta stranded along the coasts of Sicily and the northwest Adriatic Sea between June 2014 and August 2016. The necropsy examination revealed 31 individuals (48.4%) positive for endoparasites, showing a greater prevalence of trematodes than nematodes. In particular, seven species and a single genus of Trematoda (Hapalotrema) and a single species and genus of Nematoda (Kathlania) were identified. Among the Digenea flukes the species with the highest prevalence of infection were Rhytidodes gelatinosus (34.6%) and Hapalotrema sp. (33.3%), while among the Nematoda they were Kathlania sp. (33.3%) and Sulcascaris sulcata (33.3%). Analysis of variance (ANOVA) was applied among the recovery sites of the stranded loggerhead sea turtles and prevalence of endoparasites was used to highlight any relationship between the parasites and the origin of the hosts. ANOVA showed significant differences (p < 0.001) among the data used