Distinct Effects of Moxifloxacin and Bedaquiline on Growing and 'Non-Culturable' Mycobacterium abscessus.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

CanB, a Druggable Cellular Target in Mycobacterium tuberculosis.

Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis ß-carbonic anhydrases that catalyze the reversible reaction of CO2 hydration to form HCO3- and H+. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.

Mycobacterium abscessus Infections in Cystic Fibrosis Individuals: A Review on Therapeutic Options.

Mycobacterium abscessus is an opportunistic pathogen that mainly colonizes and infects cystic fibrosis patients' lungs. M. abscessus is naturally resistant to many antibiotics such as rifamycin, tetracyclines and ß-lactams. The current therapeutic regimens are not very effective and are mostly based on repurposed drugs used against Mycobacterium tuberculosis infections. Thus, new approaches and novel strategies are urgently needed. This review aims to provide an overview of the latest ongoing findings to fight M. abscessus infections by analyzing emerging and alternative treatments, novel drug delivery strategies, and innovative molecules.

Subclinical Ketosis in Dairy Herds: Impact of Early Diagnosis and Treatment.

Clinical and subclinical ketosis (SCK) in dairy cows occurs during the lactation period frequently in many herds, causing a reduction in milk yield and alterations in milk quality with significant economic losses for farmers. SCK is defined as a preclinical stage of ketosis characterized by an elevated ketone body level without clinical signs. Often many cows develop an elevated ketone body level during the first weeks of lactation even though it never goes up to a critical point causing clinical signs. This study aimed to evaluate the prevalence of SCK in Sicily and assess the effect of a treatment with propylene glycol (PG) to control the SCK, thus, reducing the negative effect on milk quality yield. This cross-sectional study was carried out on 22 farms located south-east of Sicily and 1,588 cows in lactation. A total of 3,989 individual milk samples were collected from calving to 80 subsequently days to check the ß-hydroxybutyrate (BHB) values in order to establish the SCK status by the Fourier Transform Infrared Spectroscopy. Moreover, the contents of fat, protein, lactose, casein, urea, somatic cell count and acetone were evaluated to identify a correlation between SCK and milk quality. A total of 1,100 cows showed BHB values higher than 0.10 mmol/L. These cows were considered SCK positive, were separated from the rest of the herd, and treated with PG (400 g/head per day), all SCK cows were treated with PG and cows without SCK were not treated. The results showed a prevalence of 41.5% of SCK-positive cows during the first 9 days of lactation. The comparison among the cure rate of treated cows shows that the treatment was most effective in the first 7 days of lactation (76.5% of treated cows) than in the following days. PG positively influenced the milk quality parameters, except for the fat proportion. Moreover, the animals treated with PG showed also an increase in milk yield, supporting the economical sustainability of treatment.

Investigation of an Autochthonous Outbreak of Bovine Besnoitiosis in Northwestern Sicily.

Bovine besnoitiosis is a parasitic disease caused by the protozoan Besnoitia besnoiti, leading to infertility in bulls and abortions in cows. In Italy, it is considered an emerging disease, recently introduced by the importation of animals from Spain and France. In the last decade, many outbreaks have been reported and confirmed in native cattle, mostly in northern and central Italy. This study reports on an autochthonous outbreak of bovine besnoitiosis in two nearby farms located in northwestern Sicily. A 15-month-old Limousine bull born on the farm showed typical clinical signs of the chronic disease phase, such as edema of the scrotum with subsequent hyperkeratosis associated with eschars and thickening of the skin. A histopathological examination revealed the presence of Besnoitia tissue cysts containing bradyzoites in the eyes, tendons, testicles, dermis, and nictitating membrane. A serological investigation using a commercial ELISA kit revealed a high seroprevalence of the antibody anti-B. besnoiti (79.2% for the farms in this study). Clinical disease showed low prevalence (1.5%) despite the high seroprevalence of specific antibodies in the herd, confirming that bovine besnoitiosis is an emergent endemic pathogen in Sicily, but its clinical behavior still remains sporadic.

Analysis of the Microbial Intestinal Tract in Broiler Chickens during the Rearing Period.

Gut microbiota contributes to animal health. However, identifying which microorganisms or associated functions are involved remains, still, difficult to assess. In the present study, the microbiota of healthy broiler chickens, under controlled diet and farm conditions, was investigated by 16S rRNA gene sequencing in four intestine segments and at four ages. In detail, 210 Ross-308 male chickens were raised according to the EU guidelines and fed on a commercial diet. The duodenum, jejunum, ileum, and caecum microbiota were analyzed at 11, 24, 35, and 46 days of life. Although the microbial composition was revealed as homogeneous 11 days after chicks hatched, it was found to be similar in the proximal intestine segments and different in ileum and caecum, where almost the same genera and species were detected with different relative abundances. Although changes during the later growth stage were revealed, each genus remained relatively unchanged. Lactobacillus mostly colonized the upper tract of the intestine, whereas the Escherichia/Shigella genus the ileum. Clostridium and Bacteroides genera were predominant in the caecum, where the highest richness of bacterial taxa was observed. We also analyze and discuss the predicted role of the microbiota for each intestine segment and its potential involvement in nutrient digestion and absorption.

New insights on Avian orthoreovirus and Chicken astrovirus co-infection in an Italian broiler flock: preliminary biomolecular and pathological results.

Common pathogens of intensive poultry farms, either parasitic or bacterial, such as Coccidiaor Salmonella, are well known and strictly controlled by veterinary management. This case study reports an unusual case of runting stunting syndrome (RSS) observed on a Sicilian poultry farm of broiler chickens during 2019. The investigation was carried out on five chickens which present delayed in body weight and growth performance. Animals showed also difficulty in deambulation and diarrhea. At necropsy, intestinal lesions were detected in three of the five clinical cases. Gut samples were collected and analyzed to identify potential pathogens responsible for the RSS. Presence of viruses was detected by using quantitative reverse transcription PCR (RT­qPCR), while selected tissues were fixed and embedded in paraffin wax according to routine procedures. All histological sections were stained with hematoxylin­eosin. RT­qPCR successfully detected both Chicken astrovirus (CAstV) and Avian orthoreovirus (ARV). Histology evidenced severe specific lesions on the intestinal mucosa in liver and kidneys. Chicken astrovirus and Avian orthoreovirus RNA was also detected in cecal tonsils, kidney and liver, thus implying their possible primary role in inducing the disease. Further studies are needed to evaluate the role of other possible factors (low biosecurity measures, e.g.) and, most of all, the consequences in terms of economic losses and animal health impairment.

Morphological and Immunohistochemical Examination of Lymphoproliferative Lesions Caused by Marek's Disease Virus in Breeder Chickens.

Marek's disease is widely controlled by vaccination programs; however, chickens are not totally protected, especially immediately after the vaccination when a strong challenge could interfere with the effectiveness of vaccination in the absence of proper biosecurity practice. This case report describes the occurrence of Marek's disease (MD) observed in a breeder chicken flock reared southeast of Sicily. MD outbreak occurred from 32 to 47 weeks with an increase in weekly mortality rate (+0.4-0.6%). Overall, mortality rate related to Marek's disease was about 6% at the end of the cycle. Carcasses of chickens found during the occurrence of disease underwent necropsy, and tissues were collected to confirm the infection. Gizzard, cecal tonsil, intestine, spleen and tumor mass were collected and analyzed from a carcass of one hen, 32 weeks old and apparently asymptomatic. Multiplex real-time PCR performed on spleen tissues detected the presence of MD virus pathogenic strain. Macroscopic and microscopic evaluation of the rest of the samples confirmed the neoplastic disease. Moreover, the immunophenotype of the tumor cells was identified as CD3 positive by immunohistochemical (IHC) staining. The vaccinated flock had become rapidly infected with the MD virus, which proves that the challenge of the MD virus was too strong in the rearing house at the beginning of the cycle, causing the outbreak.

Effects of Microencapsulated Blend of Organic Acids and Essential Oils as a Feed Additive on Quality of Chicken Breast Meat.

The present study aims to investigate the effect of dietary supplementation based on a blend of microencapsulated organic acids (sorbic and citric) and essential oils (thymol and vanillin) on chicken meat quality. A total of 420 male Ross 308 chicks were randomly assigned to two dietary treatments: the control group was fed with conventional diet (CON), while the other group received the control diet supplemented with 0.5% of a microencapsulated blend of organic acids and essential oils (AVI). In breast meat samples, intramuscular fat content and saturated/polyunsaturated fatty acids ratio were reduced by AVI supplementation (p < 0.05). Moreover, atherogenic (p < 0.01) and thrombogenic (p < 0.05) indices were lower in AVI than CON treatment. AVI raw meat showed a lower density of psychrotrophic bacteria (p < 0.05) at an initial time, and higher loads of enterococci after 4 days of refrigerated storage (p < 0.05). No contamination of Listeria spp., Campylobacter spp., and Clostridium spp. was found. TBARS values of the cooked meat were lower in the AVI treatment compared to CON (p < 0.01). Among colour parameters, a*, b* and C* values increased between 4 and 7 days of storage in AVI cooked meat (p < 0.05). Overall, organic acids and essential oils could improve the quality and shelf-life of poultry meat.

Effects of Microencapsulated Blends of Organics Acids (OA) and Essential Oils (EO) as a Feed Additive for Broiler Chicken. A Focus on Growth Performance, Gut Morphology and Microbiology.

The goal of the trial was testing the effects of a blend of organic acids and essential oils dietary supplementation on growth performance and gut healthiness in broiler chickens. In total, 420 male Ross 308 chicks (1-day old) were randomly assigned to two dietary treatments: basal (BD) and organic acids and essential oils (OA&EO) diets (three replicates/treatment; 70 broilers/replicate). BD group received commercial diets whereas OA&EO group basal diets + 5 g/kg of microencapsulated organic acids and essential oils. OA&EO treatment improved the average daily gain (p < 0.01) and feed conversion ratio at 37-47 days compared to BD treatment. OA&EO treatment improved gut morphology mostly at ileum and duodenum levels in terms of villi height, crypt depth, number of villi, mucosa thickness and villi area at 24 and 34 sampling days. A certain selective action against Clostridium perfringens in ileum of OA&EO group was shown at 33 (p = 0.053) and 46 days (p = 0.09) together with lower median values for Enterobacteriaceae, Enterococci, Mesophilic bacteria and Clostridium perfringens at ceca level. Overall, organic acids and essential oils supplementation improved growth performance in the final growth stage and some morphological gut traits and reduced to a certain extent Clostridium perfringens count in ileum.

The Wide Range of Antibiotic Resistance and Variability of Genotypic Profiles in Escherichia coli from Domestic Animals in Eastern Sicily.

The emergence of multidrug resistance among Enterobacteriaceae in livestock poses a serious public health threat. Escherichia coli, a usual host of intestinal microbiota, is recognized also as etiological agent of numerous infections widespread in both humans and animals. The colibacillosis is one of the most reported zoonoses worldwide, typically treated with antibiotics in the primary stages. This strategy has promoted the onset of antibiotic-resistant serotypes of E. coli, reducing the effectiveness of therapeutic treatments and contributing to antibiotic resistance spread. The current study focused on biodiversity, pathogenicity, and antibiotic resistance profile of 104 E. coli strains isolated from domestic animals in Eastern Sicily. The strains were isolated from sick animals and carcasses of six different animal species and screened for resistance against 16 antibiotic molecules, as recommended by WHO and OIE. The antibiotic resistance patterns highlighted that all strains were multi-resistant, showing resistance to at least three antibiotic classes. The highest incidence of resistance was observed against amoxicillin (100%), tylosin (97%), sulfamethoxazole (98%), and erythromycin (92%), while the lowest for colistin (8%). The pathotype characterization identified two EPEC strains and the study of genetic linkage (PFGE) showed a wide variety of profiles. The current study emphasized the wide range of multidrug resistance and genotyping profiles in E. coli isolated in Easter Sicily.