Gastrointestinal investigation of parasites and Enterobacteriaceae in loggerhead sea turtles from Italian coasts.

BACKGROUND: Caretta caretta is the most abundant sea turtle species in the Mediterranean, and studies on this species have vastly expanded during recent years, including those investigating gut bacterial and parasitic communities. Members of these communities have been reported with variable prevalence and pathogenicity, mainly depending on their host and environment (e.g. lifespan, distribution, habitat, diet, health status and stressors). Indeed, many species commonly inhabiting the sea turtle gastrointestinal tract exhibit an opportunistic behaviour. This study aimed to provide baseline data on enterobacterial and parasitic composition, through bacteriological culture-based methods and the FLOTAC parasitological technique, in cloacal and faecal samples of 30 live Caretta caretta, examined upon their arrival at the Marine Turtle Research Centre (Portici, Italy). RESULTS: Enterobacteriaceae were isolated in 18/23 cloacal samples (78.3%), with Citrobacter and Morganella as the most common genera, followed by Proteus, Enterobacter, Providencia, and Hafnia. Parasitic elements were detected in 11/30 faecal samples (36.7%), with Enodiotrema, Rhytidodes, and Eimeria as most common genera, followed by Pachypsolus and Cymatocarpus. Additionally, Angiodyctium is reported for the first time in this host. The majority (47.8%) of sea turtles hosted exclusively Enterobacteriaceae, whereas 30.4% hosted both parasites and Enterobacteriaceae; the remaining 21.8% hosted neither of the agents. CONCLUSIONS: Bacteria and parasites evaluated in the present study are common in Mediterranean loggerhead sea turtles, with slight differences between the western and eastern basin. Although naturally present in the gastrointestinal system of free-living sea turtles, their relationship with these hosts might range from mutualism to parasitism. Indeed, members of the gut community might express their pathogenic potential in immune-compromised animals, such as those in rehabilitation facilities. Therefore, it is advisable to include in the standard work-up of rescued sea turtles a screening procedure for such opportunistic agents, in order to better evaluate the animal's health status and achieve timely intervention with appropriate treatment, thus improving rehabilitation. Furthermore, data collected from free-living sea turtles represent a starting point for investigating wild populations. However, further studies are needed to clarify the differences between sea turtle's normal gut microbiome and pathobiome.

Aeromonas induced polyostotic osteomyelitis in a juvenile loggerhead sea turtle Caretta caretta.

Bacterial bone infections have been occasionally reported in wild sea turtles. This study reports on a sub-adult Caretta caretta affected by Aeromonas hydrophila osteomyelitis, with extensive involvement of cranial and caudal flippers. The turtle was severely debilitated, had limited flipper mobility and showed signs of pain in reaction to manipulation. Radiographs and computed tomography revealed multiple lytic bone lesions. Since an infectious polyostotic osteomyelitis was suspected, the turtle was subjected to echo-assisted fine needle aspiration to characterize the etiology of the disease. Bacterial cultures and antibiotic susceptibility testing led to the isolation of Aeromonas hydrophila responsive to amikacin and doxycycline. Therefore, the turtle was treated with these antibiotics and monitored through repeat bacterial cultures and diagnostic imaging. The turtle was released 17 mo after admission, upon resolution of clinical signs. The documentation of this case provides a treatment approach that may improve the outcome of Aeromonas-associated osteomyelitis, especially in endangered wildlife species.

Antibiotic Resistance of Bacteria Isolated from Clinical Samples and Organs of Rescued Loggerhead Sea Turtles (Caretta caretta) in Southern Italy.

Antimicrobial resistance affects all environments, endangering the health of numerous species, including wildlife. Increasing anthropic pressure promotes the acquisition and dissemination of antibiotic resistance by wild animals. Sea turtles, being particularly exposed, are considered sentinels and carriers of potential zoonotic pathogens and resistant strains. Therefore, this study examined the antibiotic resistance profiles of bacteria isolated from loggerhead sea turtles hospitalised in a rescue centre of Southern Italy over a 9-year period. Resistance to ceftazidime, doxycycline, enrofloxacin, flumequine, gentamicin, oxytetracycline and sulfamethoxazole-trimethoprim was evaluated for 138 strains isolated from the clinical samples or organs of 60 animals. Gram-negative families were the most isolated: Vibrionaceae were predominant, followed by Shewanellaceae, Pseudomonadaceae, Enterobacteriaceae and Morganellaceae. These last three families exhibited the highest proportion of resistance and multidrug-resistant strains. Among the three Gram-positive families isolated, Enterococcaceae were the most represented and resistant. The opportunistic behaviour of all the isolated species is particularly concerning for diseased sea turtles, especially considering their resistance to commonly utilised antibiotics. Actually, the multiple antibiotic resistance was higher when the sea turtles were previously treated. Taken together, these findings highlight the need to improve antimicrobial stewardship and monitor antibiotic resistance in wildlife, to preserve the health of endangered species, along with public and environmental health.

Research Note: Cecal microbiota harbored by free-range chickens may influence the reduction of Helicobacter pullorum relative abundance.

Helicobacter pullorum is recognized as an emerging food-borne pathogen that may colonize the intestinal tract and the liver of avian species and humans causing several gastrointestinal and liver diseases. However, not all strains are reported to be capable of causing clinical disease, thus making poultry as reservoir for the zoonotic transmission of the infection through carcass contamination of broilers at slaughter. In poultry, the prevalence of this bacterium could be underestimated and the available data mainly refer to conventional rearing systems, whereas free-range and organic breedings have been poorly investigated. Therefore, this study was aimed to characterize the caecal microbiota community of free-range grown chickens and determine the presence and the relative abundance of H. pullorum by using NGS-based 16S rDNA sequencing. A total of 18 chickens reared for 56 d on a semi-extensive management system were euthanized at two time points: 9 birds at 28 d of age (before have access to outdoor; I = Indoor) and other 9 birds at 56 d of age (before slaughter; O = Outdoor). Cecal contents were collected for microbiota analyses. H. pullorum was detected in the cecum of 16/18 samples and its proportion in indoor was significantly higher than outdoor chickens (2.46 and 0.52%, respectively; P < 0.05), showing 78.8% of decrease with the outdoor access of the chickens. Therefore, it may be assumed that the potential for zoonotic infection is less likely. Moreover, H. pullorum was negatively correlated with 17 bacterial species as significantly more abundant in Outdoor microbial caecal communities. Among these, we highlighted the presence of Mucispirillium schaedleri and Oscillospira, already previously associated with a healthy gut and thus representing promising gut bacterial markers for host health. Our findings suggest that alternative production systems with outdoor access, may play a crucial role in the establishment of a healthy gut microbiota, which in turn might prevent colonization of harmful bacteria such as Helicobacter pullorum.

Prevalence and Phenotypic Antimicrobial Resistance among ESKAPE Bacteria and Enterobacterales Strains in Wild Birds.

Antimicrobial resistance (AMR) is a current public health issue globally. To counter this phenomenon and prioritize AMR in the health sector, the World Health Organization (WHO) published a list of bacterial pathogens against which the development of new antimicrobial agents is urgently needed, designating the ESKAPE pathogens (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) with a 'priority status'. Moreover, the One Health High Level Expert Panel (OHHLEP) states that human health is closely linked to animal and environmental health, thus promoting a holistic One Health approach in order to be prepared to address possible emerging health threats from the human-animal-environment interface. Wild birds may host and spread pathogens, integrating the epidemiology of infectious diseases. The aim of this study was to examine the role of wild birds as a source of ESKAPE bacteria and other antibiotic-resistant enterobacterales. A total of fifty strains within the ESKAPE group were detected in 40/163 cloacal samples of examined birds (24.5%). Additionally, different strains of enterobacterales were detected in 88/163 cloacal samples (53.9%). Isolated strains exhibited antimicrobial resistance, including towards critically important antibiotics (e.g., third, fourth, fifth generation cephalosporins, fluoroquinolones) for human medicine. Our results confirm that wild birds are potential reservoirs of several pathogens and antimicrobial-resistant bacteria and that they could be involved in the dissemination of those bacteria across different environments, with resulting public health concerns.

Detection of Chlamydial DNA from Mediterranean Loggerhead Sea Turtles in Southern Italy.

Chlamydiae are obligate intracellular bacteria that include pathogens of human and veterinary importance. Several reptiles were reported to host chlamydial agents, but pathogenicity in these animals still needs clarification. Given that only one report of chlamydiosis was described in sea turtles, and that chlamydiae might also be detected in hosts without clinical signs, the current study examined asymptomatic Mediterranean loggerhead sea turtles for the presence of chlamydial DNA. Twenty loggerhead sea turtles, rehabilitated at the Marine Turtle Research Centre (Portici, Italy), were examined collecting ocular-conjunctival, oropharyngeal and nasal swabs. Samples were processed through quantitative and conventional PCR analyses to identify Chlamydiales and Chlamydiaceae, with particular attention to C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis. Although it was not possible to determine the species of chlamydiae involved, the detection of chlamydial DNA from the collected samples suggests that these microorganisms might act as opportunistic pathogens, and underlines the role of sea turtles as potential carriers. This study highlights the presence of chlamydial agents in sea turtles, and encourages further research to fully characterize these microorganisms, in order to improve the management of the health and conservation of these endangered species, and prevent potential zoonotic implications.

An Outdoor Access Period Improves Chicken Cecal Microbiota and Potentially Increases Micronutrient Biosynthesis.

Characterizing the gut microbiota of free-range and alternative poultry production systems provides information, which can be used to improve poultry welfare, performance, and environmental sustainability. Gut microbiota influence not only the health and metabolism of the host but also the presence of zoonotic agents contaminating food of animal origin. In this study, the composition and diversity of the cecal microbiota community of free-range grown chickens were characterized by 16S rDNA high-throughput Illumina sequencing. Significant differences were observed in the composition of chicken cecal microbiota at the time points of 28 days of age (Indoor group) and 56 days of age (Outdoor group), i.e., before and after the outdoor access period of chicken groups. The Outdoor group showed a richer and more complex microbial community, characterized by the onset of new phyla such as Deferribacterota and Synergistota, while the Indoor group showed an increase in Campylobacterota. At the species level, it is noteworthy that the occurrence of Mucispirillum schaedleri in Outdoor group is known to potentially stimulate mucus layer formation in the distal intestinal tract, thus being associated with a healthy gut. We also report a significant decrease in the Outdoor group of Helicobacter pullorum, highlighting that the lower abundance at the age of slaughter reduced the possibility to contaminate chickens' carcasses and, consequently, its zoonotic potential. As revealed by a mutual exclusion study in network analysis, H. pullorum was present only if Bacteroides barnesiae, an uncultured organism of the genus Synergistes, and Bacteroides gallinaceum were absent. Finally, microbiome predictive analysis revealed an increase of vitamins and micronutrient biosyntheses such as queuosine (Q) and its precursor pre Q0, in the Outdoor group, suggesting that the outdoor evolved microbiota of chickens do contribute to the vitamin pool of the gut and the biosynthesis of micronutrients involved in vital cell processes.

Unveiling the egg microbiota of the loggerhead sea turtle Caretta caretta in nesting beaches of the Mediterranean Sea.

Microbes have central roles in the development and health of animals, being the introduction of specific microbial species a potential conservation strategy to protect animals from emerging diseases. Thus, insight into the microbiota of the species and their habitats is essential. In this manuscript, we report for the first time the bacterial composition of all the components (eggshells of hatched and unhatched eggs, internal content of unhatched eggs, intestinal content of hatchling and pipping sea turtles, and sand) of three nesting beaches of Caretta caretta along the Italian coasts of the Mediterranean Sea. The analysis of 26 amplicon samples was carried out using next-generation sequencing analysis, targeting V3-V4 regions of the bacterial 16S rRNA gene. Samples featured mainly Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, whose percentages depended on the sample type. Our results showed that, although from different sampling sites, the internal content of the unhatched eggs, intestinal content of hatchling and pipping sea turtles share the microbiota, which was yet different from that of eggshells and sand of the same nesting beach. This study suggests the maternal and environmental influence alongside a protective role of eggshells in shaping the egg microbiota of Caretta caretta sea turtles.

Symblepharon, Ankyloblepharon, and Salt Gland Dysfunction in a Loggerhead Sea Turtle (Caretta caretta).

Adhesions involving the bulbar and the palpebral conjunctiva (Symblepharon) may interfere with tear drainage, cause chronic conjunctivitis, and reduce ocular motility. This condition may be associated with adhesion of the edges of the upper and lower eyelids (ankyloblepharon). The present case describes bilateral symblepharon, ankyloblepharon and salt gland dysfunction in a juvenile Caretta caretta. The loggerhead presented both eyelids swollen, ulcerated, and not separable when rescued. Eye examination was not possible, but ultrasonography showed right bulbar integrity, while the left eye was smaller, with a thicker cornea that had lost its normal doubled lined structure. Surgical dissection of the fibrous adhesions between the palpebral and bulbar conjunctiva, cornea, and third eyelid was performed, and large dacryoliths were removed. The microscopic findings were consistent with chronic keratoconjunctivitis. Ultrastructurally, no virus-like particles were observed. In addition, tissue samples were negative for herpesvirus by qualitative PCR. The eyelids of both eyes and the corneal epithelium of the right eye healed; moreover, the vision was restored in the right eye. There were no recurrences after 12 months of follow-up, and the turtle was released 16 months after the end of treatments on the southern Tyrrhenian coast in the western Mediterranean Sea. To the authors' knowledge, this is the first report of symblepharon with ankyloblepharon and salt gland dysfunction in Caretta caretta turtle. Ocular ultrasonography was helpful in the preliminary diagnostic work-up.

Presence of Campylobacterjejuni and C. coli in Dogs under Training for Animal-Assisted Therapies.

This study was conducted to evaluate the presence of Campylobacter (C.) jejuni and C. coli in dogs at five dog training centers in Southern Italy. A total of 550 animals were sampled by collecting rectal swabs. The samples were processed to detect thermotolerant Campylobacter spp. by culture and molecular methods. Campylobacter spp. were isolated from 135/550 (24.5-95% confidence interval) dogs. A total of 84 C. jejuni (62.2%) and 51 C. coli (37.8%) isolates were identified using conventional PCR. The dog data (age, sex, breed, and eating habits) were examined by two statistical analyses using the C. jejuni and C. coli status (positive or negative) as dependent variables. Dogs fed home-cooked food showed a higher risk of being positive for C. jejuni than dogs fed dry or canned meat for dogs (50.0%; p < 0.01). Moreover, purebred dogs had a significantly higher risk than crossbred dogs for C. coli positivity (16.4%; p < 0.01). This is the first study on the prevalence of C. jejuni and C. coli in dogs frequenting dog training centers for animal-assisted therapies (AATs). Our findings emphasize the potential zoonotic risk for patients and users involved in AATs settings and highlight the need to carry out ad hoc health checks and to pay attention to the choice of the dog, as well as eating habits, in order to minimize the risk of infection.

Insect Derived Lauric Acid as Promising Alternative Strategy to Antibiotics in the Antimicrobial Resistance Scenario.

Antibiotic misuse is greatly contributing to an increase in antimicrobial resistance (AMR) in humans and animals. Natural and synthetic alternative strategies are being investigated in human and veterinary medicine, but little attention is paid to the antimicrobial effects of edible lipids, such as medium-chain fatty acids (MCFAs) and monoglycerides. Among MCFAs, lauric acid (LA) and its monoglyceride derivative, glycerol monolaurate (GML), exhibit the strongest antimicrobial activity. Coconut and palm kernel oils are considered the main sources of LA. On the other hand, some edible insects (e.g., Hermetia illucens) are gaining interest as novel feed ingredients, due to the high amount of LA they contain as well as their numerous bioactive components, which provide many additional benefits to animal health. Although the beneficial effect of both MCFAs and LA is gradually being recognized, their high content within insects and, consequently, their possible role as antimicrobials, has not been well-reported. This mini review focuses on the anti-infective effects of the insect-derived MCFAs LA and its derivatives. We emphasize the potential of insect lipids, compared to the other vegetable sources, in the current global scenario where a sustainable and circular economy is required. Finally, we critically discuss the use and the benefits of edible insects such as favorable options as feed and food from the perspective of animal and human nutrition.

Prevalence and Antimicrobial Resistance of Enteropathogenic Bacteria in Yellow-Legged Gulls (Larus michahellis) in Southern Italy.

Wild birds may host and spread pathogens, integrating the epidemiology of infectious diseases. Particularly, Larus spp. have been described as responsible for the spread of many enteric diseases, primarily because of their large populations at landfill sites. The aim of this study was to examine the role of yellow-legged gulls as a source of enteropathogenic bacteria such as Campylobacter spp., Salmonella spp., Shiga toxin-producing Escherichia coli and Yersinia spp., with particular attention to antibiotic-resistant strains. Enteropathogenic bacteria were isolated from 93/225 yellow-legged gulls examined from April to July, during a four-year period (2016-2019). Specifically, Campylobacter spp. was isolated from 60/225 samples (26.7%), and identified as C. coli (36/60) and as C. jejuni (24/60). Salmonella spp. was isolated from 3/225 samples (1.3%), and identified as Salmonella arizonae. Shiga toxin-producing E. coli were isolated from 30/225 samples (13.3%) samples, and serotyped as E. coli O128 (12/30) O26 (9/30), O157 (6/30) and O11 (3/30); Yersinia spp. was never detected. Isolated strains exhibited multidrug resistance, including vitally important antibiotics for human medicine (i.e., fluoroquinolones, tetracyclines). Our study emphasizes the importance of yellow-legged gulls as potential reservoirs of pathogenic and resistant strains and their involvement in the dissemination of these bacteria across different environments, with resulting public health concerns.

Fecal Sample Collection Method for Wild Birds-Associated Microbiome Research: Perspectives for Wildlife Studies.

Gut microbial communities play important roles in host health, modulating development, nutrient acquisition, immune and metabolic regulation, behavior and diseases. Wildlife microbiome studies and host-microbe interaction and exploration might be an important goal for evolutionary biology, conservation, and ecology. Therefore, collection and sampling methods must be considered before choosing a microbiome-based research plan. Since the fecal microbial community reflects the true gut community better than that of cloacal swab samples and only few nondestructive methods have been described, we propose an easy-to-build box for a noninvasive fecal collection method. The main components of the collection box include a plastic storage box, a plastic tray, a vinyl-coated hardware cloth, and a 10% bleach solution. In the plastic box, the tray is positioned under the raised grate, where the bird is placed, to reduce the risk of contamination of the fecal samples. This procedure could simplify handling and processing phases in wild birds or other animals. It might represent a cheap and useful method for research studies, wildlife rescue center activities, veterinary practices, and conservation practitioners.

Antimicrobial Resistance of Escherichia coli and Pseudomonas aeruginosa from Companion Birds.

Antimicrobial resistance is a public health concern worldwide and it is largely attributed to the horizontal exchange of transferable genetic elements such as plasmids carrying integrons. Several studies have been conducted on livestock showing a correlation between the systemic use of antibiotics and the onset of resistant bacterial strains. In contrast, although companion birds are historically considered as an important reservoir for human health threats, little information on the antimicrobial resistance in these species is available in the literature. Therefore, this study was aimed at evaluating the antimicrobial resistance of Escherichia coli and Pseudomonasaeruginosa isolated from 755 companion birds. Cloacal samples were processed for E. coli and P. aeruginosa isolation and then all isolates were submitted to antimicrobial susceptibility testing. P. aeruginosa was isolated in 59/755 (7.8%) samples, whereas E. coli was isolated in 231/755 (30.7%) samples. Most strains showed multidrug resistance. This study highlights that companion birds may act as substantial reservoirs carrying antimicrobial resistance genes which could transfer directly or indirectly to humans and animals, and from a One Health perspective this risk should not be underestimated.

Diagnosis of Centrocestus formosanus Infection in Zebrafish (Danio rerio) in Italy: A Window to a New Globalization-Derived Invasive Microorganism.

: Centrocestus formosanus is a digenetic trematode with a complex life cycle, involving invertebrate and vertebrate hosts, humans included. In particular, it causes gill lesions and mortality in freshwater fish species, and gastrointestinal symptoms in infected humans. Here, we describe the occurrence of C. formosanus infection in zebrafish imported in Italy and propose a newly designed species-specific primer pair to ameliorate the diagnostic investigations for C. formosanus. Gill arches of 30 zebrafish were examined for the presence of encysted metacercariae under a stereomicroscope and processed through molecular analyses targeting the ribosomal internal transcribed sequence 2 (ITS2). Although C. formosanus distribution was originally restricted to Asia, it has been subsequently reported in new countries, revealing itself as an invasive species and raising important concerns for biodiversity, economy, scientific research, as well as animal and public health. Given the crucial role played by the ornamental fish industry in spreading this parasite, there is an urgent need for control measures to prevent the introduction and establishment of C. formosanus in non-endemic areas, including Europe. We also suggest developing new strategies in microbiology and epidemiology to better explore this new globalization-derived invasive species.

Loggerhead sea turtles as sentinels in the western Mediterranean: antibiotic resistance and environment-related modifications of Gram-negative bacteria.

Sea turtles possess relevant characteristics to serve as sentinel species for monitoring the health of marine ecosystems, which is currently threatened. This study examined 35 loggerhead turtles from the western Mediterranean, focusing on the oral and cloacal prevalence of aerobic Gram-negative bacteria, their antibiotic resistance and the influence of several variables linked both to the animal and the environment (i.e. estimated life stage; area, season and cause of recovery; plastic ingestion). Conventional bacteriology methods led to the isolation of bacterial families commonly regarded as opportunistic pathogens (i.e. Aeromonadaceae; Enterobacteriaceae; Pseudomonadaceae; Shewanellaceae; Vibrionaceae), but pointing out sea turtles as carriers of potential zoonotic agents. The high rates of antibiotic resistance, here detected, raise important concerns on the dissemination of this phenomenon in marine environments. Moreover, several of the examined variables showed a significant influence on the prevalence of bacterial families, strengthening the role of sea turtles as mirrors of their ecosystems.

Campylobacter coli infection in pet birds in southern Italy.

Avian species are considered as the main reservoir of Campylobacter spp. However, few data are available on the presence of this microorganism in pet birds. This study was therefore performed to determine the prevalence of Campylobacter spp. in pet birds bred in southern Italy. Faecal samples were collected from 88 cages housing different species of pet birds and examined by bacteriological culture and polymerase chain reaction. A total of 13.6% of the cage samples were positive for Campylobacter coli. Other Campylobacter spp. were not found. The study shows that C. coli can be isolated from the cages of apparently healthy pet birds, which should therefore be considered as potential carriers of C. coli and a possible source of infection for humans and companion animals.

Microbiological survey of birds of prey pellets.

A microbiological survey of 73 pellets collected from different birds of prey species housed at the Wildlife Rescue and Rehabilitation Center of Napoli (southern Italy) was performed. Pellets were analyzed by culture and biochemical methods as well as by serotyping and polymerase chain reaction. We isolated a wide range of bacteria some of them also pathogens for humans (i.e. Salmonella enterica serotype Typhimurium, Campylobacter coli, Escherichia coli O serogroups). This study highlights the potential role of birds of prey as asymptomatic carriers of pathogenic bacteria which could be disseminated in the environment not only through the birds of prey feces but also through their pellets.