Extensive Genome Exploration of Clostridium botulinum Group III Field Strains.

In animals, botulism is commonly sustained by botulinum neurotoxin C, D or their mosaic variants, which are produced by anaerobic bacteria included in Clostridium botulinum group III. In this study, a WGS has been applied to a large collection of C. botulinum group III field strains in order to expand the knowledge on these BoNT-producing Clostridia and to evaluate the potentiality of this method for epidemiological investigations. Sixty field strains were submitted to WGS, and the results were analyzed with respect to epidemiological information and compared to published sequences. The strains were isolated from biological or environmental samples collected in animal botulism outbreaks which occurred in Italy from 2007 to 2016. The new sequenced strains belonged to subspecific groups, some of which were already defined, while others were newly characterized, peculiar to Italian strains and contained genomic features not yet observed. This included, in particular, two new flicC types (VI and VII) and new plasmids which widen the known plasmidome of the species. The extensive genome exploration shown in this study improves the C. botulinum and related species classification scheme, enriching it with new strains of rare genotypes and permitting the highest grade of discrimination among strains for forensic and epidemiological applications.

Genomic analysis of extra-intestinal Campylobacter jejuni and Campylobacter coli isolated from commercial chickens.

Campylobacter jejuni and Campylobacter coli have commonly been considered harmless commensal inhabitants of the chicken gut; however, these Campylobacter spp. are known to be able to multiply in the gut and invade other tissues, negatively affecting host health and performance. In this study, fourteen Campylobacter spp. were isolated from chickens showing foci of necrosis on the liver surface resembling lesions observed in cases of avian vibrionic hepatitis/spotty liver disease. The whole genome sequences of the fourteen isolates were analysed and their virulomes compared to those of Campylobacter reference sequences, aiming to investigate the possible association between virulence genes and the observed pathological lesions. Nine C. jejuni and five C. coli were studied. These Campylobacter shared twelve virulence factors with other isolates originated from chicken livers and hosted a higher number of virulence-associated genes in comparison to the reference genomes, including genes encoding for factors involved in adherence to and invasion of the intestinal epithelial cells. Our findings seem to point out that these twelve common virulence-associated genes, together with the presence of a high number of virulence factors involved in adherence, invasion and motility, might be responsible for the extra-intestinal spread of our isolates and the colonization of parenchymatous tissues, possibly causing the pathological lesions observed.

Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay.

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66-100%), 96.08% (95% CI: 86.54-99.52%), and 93.33% (95% CI: 78.25-98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15-99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

Death Associated to Methicillin Resistant Staphylococcus aureus ST8 Infection in Two Dolphins Maintained Under Human Care, Italy.

The present study describes the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) from respiratory tract of 2 dolphins of different origin, a stranded juvenile Risso's dolphin (Grampus griseus) and a captive born common bottlenose dolphin (Tursiops truncatus) calf, which died in the same institution at 1-month distance from the other. A complete microbiological and genetic investigation confirmed the presence of MRSA clone-complex 8, sequence type (ST) 8, spa-type t008 in both individuals. This strain differs from the one previously reported in walruses and dolphins and has never been described in dolphins before, but it is randomly isolated from Italian human patients. Vertical transmission of the infection may also occurs in other species and considering the description and location of the pathological lesions, this seems to be the most likely route of transmission implied in the young bottlenose dolphin. Staphylococcus aureus is known as an opportunistic agent, usually secondary to other pathogens, but its multiple antibiotic resistance and its zoonotic implications suggest a thorough and strict application of animal management hygiene protocols.

Detection of Clostridium tetani Neurotoxins Inhibited In Vivo by Botulinum Antitoxin B: Potential for Misleading Mouse Test Results in Food Controls.

The presence of botulinum neurotoxin-producing Clostridia (BPC) in food sources is a public health concern. In favorable environmental conditions, BPC can produce botulinum neurotoxins (BoNTs) outside or inside the vertebrate host, leading to intoxications or toxico-infectious forms of botulism, respectively. BPC in food are almost invariably detected either by PCR protocols targeted at the known neurotoxin-encoding genes, or by the mouse test to assay for the presence of BoNTs in the supernatants of enrichment broths inoculated with the tested food sample. The sample is considered positive for BPC when the supernatant contains toxic substances that are lethal to mice, heat-labile and neutralized in vivo by appropriate polyclonal antibodies raised against purified BoNTs of different serotypes. Here, we report the detection in a food sample of a Clostridium tetani strain that produces tetanus neurotoxins (TeNTs) with the above-mentioned characteristics: lethal for mice, heat-labile and neutralized by botulinum antitoxin type B. Notably, neutralization occurred with two different commercially available type B antitoxins, but not with type A, C, D, E and F antitoxins. Although TeNT and BoNT fold very similarly, evidence that antitoxin B antiserum can neutralize the neurotoxic effect of TeNT in vivo has not been documented before. The presence of C. tetani strains in food can produce misleading results in BPC detection using the mouse test.

Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

Molecular characterization and antimicrobial susceptibility of Clostridium difficile isolated from rabbits raised for meat production.

Clostridium difficile is an important cause of enteric disease in humans and animals. Recent studies demonstrated a genetic overlap between C. difficile isolated from animals and humans suggesting animals as possible reservoir for human pathogenic strains. This study was a preliminary investigation on the occurrence of C. difficile in rabbits raised in industrial holdings for food production and aimed to characterise isolates and estimate their antimicrobial susceptibility. C. difficile isolates were characterized by toxin profiles, toxinotyping and PCR-ribotyping. The MICs of six antibiotics were determined using E-test. Between 2007 and 2013, 285 industrial holdings (representing 40% of the national census) submitted rabbits to our laboratory for diagnostic purposes, among these holdings, groups of three to five post-weaned rabbits were sampled once by convenience. 1279 samples of caecal content were collected. The overall isolation rate of C. difficile from the enteric specimen was 3% (38/1279), with no difference among animals affected or not by enteric disorders. Among isolates 66% (25/38) were toxigenic. Sixteen different PCR-ribotypes (RTs) were identified. Among the toxigenic strains RT-014/020, RT-078 and RT-012 were found in at least three rabbit holdings. According to the ECOFF threshold, 82% (31/38) C. difficile isolates displayed a reduced susceptibility to at least one and 18% (7/38) to three tested antimicrobials. Rabbits are colonized by heterogeneous C. difficile ribotypes many of which are commonly isolated in humans. One third of isolates displayed a reduced susceptibility to MTZ, the first choice antimicrobial for human CDI treatment. According to our findings rabbits are a potential source of C. difficile for humans.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains.

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred. Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain. An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study. Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds. Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off. We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Antibiotic resistance patterns and PCR-ribotyping of Clostridium difficile strains isolated from swine and dogs in Italy.

Recent studies suggest animals, in particular farm and companion animals, as possible reservoir for Clostridium difficile human pathogenic strains. The aim of this study was to give a first characterization of C. difficile isolates from Italian swine and dogs. In total, 10 different PCR-ribotypes were identified among porcine strains and six among canine strains. The predominant type found among porcine strains was 078 (50%), whereas the most frequently detected among canine strains was the non-toxinogenic 010 (64%). Considering the CLSI breakpoints, 60% of porcine isolates was resistant to ERY, 35% to MXF, 15% to CLI, 5% to RIF, and none to MTZ or VAN. Among dogs, 51% of strains was resistant to CLI, 46% to ERY, 21% to MTZ and 5% to MXF or RIF, and none to VAN. Five porcine strains (10%) and 9 canine isolates (41%) were MDR. Interestingly, 8 MDR canine strains were highly resistant to MTZ, with MICs ≥32 mg/L. Considering the EUCAST cut-off for MTZ (MIC >2 mg/L), 13 canine isolates and one porcine strain were found with reduced susceptibility to MTZ (MICs ranging from 3 to ≥256 mg/L). Swine and canine strains showing resistance or reduced susceptibility to MTZ belonged to PCR-ribotype 010 and 078. These PCR-ribotypes have been associated to reduced susceptibility to MTZ also in human, suggesting a potential risk for the emergence of C. difficile strains resistant to the current first-line antibiotic for CDI treatment. The agar incorporation method (AIM) was confirmed as the best method to detect C. difficile strains with this phenotype also after strains manipulations. The results obtained add further evidences about the possible role of animals as source of MDR C. difficile strains and reservoir of antibiotic resistance determinants.

First reporting of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in an industrial rabbit holding and in farm-related people.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been described in food-producing animals and farm or slaughterhouse workers involved in the primary industrial production of swine, bovine and poultry. This communication describes the first case of LA-MRSA (ST398, spa types t034 and t5210) occurring in rabbits raised intensively for meat production and involving farm workers or their family members. In 2012-2013, in a study involving 40 rabbit industrial holdings in Italy, one farm was found to have rabbits colonized or infected with MRSA. Four farm workers and one of their relatives were found to be carrying MRSA. In this case holding, rabbits, people and the holding environment were further investigated and followed up by a second sampling five months later. MRSA was found in 48% (11/23) and 25% (15/59) of the rabbits carrying S. aureus at first and second samplings, respectively. Five months after first detection, some farm workers or family members were still MRSA carriers. Surface samples (2/10) and air samples (2/3) were contaminated with MRSA. Air samples yielded MRSA counts of 5 and 15CFU/m(3). MRSA from rabbits and people collected at first sampling were spa types t034 and t5210 belonging to ST398. The MRSA isolates from rabbits and persons tested at second sampling were t034 and t5210, but spa types t1190 and t2970 were also detected in MRSA isolates from rabbits. Tracing the epidemiological pattern earlier may prevent further spread of LA-MRSA in these food producing animals.