A Longitudinal Case Study on Dissemination of ST398 Methicillin-Resistant Staphylococcus aureus Within a Dairy Cow Herd.

The study was conducted to describe the dynamics of ST398 methicillin-resistant Staphylococcus aureus (MRSA) on a dairy herd in northeastern Italy. MRSA was first identified in this herd of 120 cows in 2016, after which the herd was sampled once every 3 months for 1 year (April 2016-May 2017). Samples collected included nasal swabs and milk samples from cows and nasal swabs from farmworkers. In addition, pen fencing and teat milk liners were swabbed and air samples from cow pens and the milking parlor were collected. All samples were tested for MRSA using a selective medium; positive isolates were confirmed by mecA PCR. A representative set of MRSA isolates was genotyped using spa typing and multilocus sequence typing. Overall, 34 (mean 23%, range 16-30%) milking cows were found harboring MRSA in the mammary gland and only 6 recovered from infection or colonization. The mean incidence rate was 14% (range 8-20%), mean cure rate was 23% (range 13-43%), and estimated basic reproduction number (R0) was 1.08. The average of positive quarters found was 35.1% and most of the positive quarters (82.4%) developed subclinical mastitis. The mean duration of MRSA colonization in quarters during the study was 247 days, but quarters affected by subclinical mastitis harbored MRSA for a longer time than healthy ones (285 days vs. 131 days). After the second sampling, the farmer segregated MRSA-positive cows from the uninfected cows and milked them last. Despite segregation, 25 newly infected or colonized cows were detected. MRSA isolates from cows, environment, and two farmworkers belonged to the same sequence type (ST398) and spa type (t034). This study highlights the ability of ST398 MRSA to cause a persistent infection of the mammary gland and to survive in the farm environment.

Contamination of hospital food with Clostridium difficile in Central Italy.

This study investigates the contamination of foods with Clostridium difficile in 3 hospitals of Central Italy. We used real-time PCR for tpi gene to analyse 350 food samples, including 296 cooked meals and 54 foods to be eaten raw. The molecular screening test was positive for 3 samples, but toxigenic C. difficile was isolated only in two of them. The prevalence in cooked food was 0,3% (1/296), while in uncooked processed foods was 1,9% (1/54). Data support the potential risk of food as a source of toxigenic C. difficile for hospitalized patients, but further investigations are needed.

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 of Clostridium perfringens strains isolated from diseased turkeys in Italy.

One hundred and six Clostridium perfringens field strains, isolated from diseased turkeys in Italy between 2006 and 2015, were toxinotyped by polymerase chain reaction. Strains were derived from intestines (87), livers (17) and subcutaneous tissues (2). In addition to the four major toxins, strains were also screened for NetB toxin, enterotoxin and beta2 toxin encoding genes. The intestinal gross lesions of turkeys with enteric disorders were statistically studied with respect to the presence of C. perfringens beta2 toxin encoding gene and coccidia in the gut. All the isolates belonged to the toxinotype A and were netB negative. Enterotoxin (cpe) and beta2 toxin (cpb2) encoding genes were detected in two (2.63%) and 76 (71.69%) strains, respectively. Toxinotype results agree with the few published reports concerning the genetic characterization of C. perfringens of turkey origin. On the contrary, the presence of netB and cpb2 genes differs from the results of a previous study where these genes were detected respectively in 6.6% and in 0.5% of the tested strains. Necrotic enteritis in turkeys was not statistically correlated either to the presence of cpb2 gene, or to the synergistic effect operated by coccidia, even though a high percentage of birds with these protozoa in the gut showed necrotic enteritis lesions (64.29%).

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

BACKGROUND: Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. RESULTS: Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains showed multidrug resistance profiles. CONCLUSIONS: Our results confirm that S. pseudintermedius is the most frequently isolated coagulase-positive staphylococcus from bitches' milk. The isolation of several different strains of MRSP with different genetic characteristics in the same kennel and the fact that two of the strains belonged to a sequence type (ST) described for the first time are noteworthy findings.

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.

Prevalence and risk factors associated with Clostridium difficile shedding in veal calves in Italy.

The aim of this study is to describe the prevalence and risk factors of Clostridium difficile shedding in six farms belonging to two companies in Northern Italy. Four hundred and twenty veal calves, randomly selected and individually identified, were sampled three times: at 0-16, 90-120, and 150 days after introduction. C. difficile was isolated at least once from 87 out of the 420 calves (20.7%). The prevalence of shedding was 20.24% at the first sampling and dropped to 0.72% at the second sampling. None of the samples obtained at 150 days tested positive. Sampling of cecal contents and carcass swabs at slaughter was stratified according to the herd of origin of the animals. C. difficile was never isolated at slaughter, excluding a prevalence higher than 3.5% on the basis of previous investigations. Therefore, in this work, the veal calf could not be confirmed as a potential source of C. difficile for the consumer. Eight different ribotypes (RT) have been described, but the vast majority of the isolates (87.8%) belonged to three ribotypes only: RT-078, RT-012 and RT-126, which are also among the most common of the ribotypes detected in humans in Europe. Most isolates, and all the RT-078 isolates, harbored genes coding for toxins A and B, the binary toxin, and showed a deletion in the gene encoding toxin C, suggesting that the veal calf was a reservoir for epidemic hyper-virulent strains. A correlation between age and shedding was found: the odds ratio (OR) ranged from 2.79 for 36-45 days of age to 4.57 for 13-28 days of age. The presence of diarrhea at first sampling was significantly associated with the recovery of C. difficile in feces (OR 3.26). A correlation was found between the administration of antimicrobials and shedding: an increased risk was shown when the number of antimicrobials used was higher than 4 (OR 4.02) or 5-6 (OR 5.83) or when polymyxin E or beta-lactams were administered.

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.

International Clostridium difficile animal strain collection and large diversity of animal associated strains.

BACKGROUND: Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. RESULTS: Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). CONCLUSIONS: This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

MALDI-TOF MS for the identification of veterinary non-C. neoformans-C. gattii Cryptococcus spp. isolates from Italy.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses.

Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial.

Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.

Association between ability to form biofilm and virulence factors of poultry extra-intestinal Campylobacter jejuni and Campylobacter coli.

Campylobacter species are known to be able to produce biofilm, which represents an ideal protective environment for the maintenance of such fragile bacteria. Since the genetic mechanisms promoting biofilm formation are still poorly understood, in this study we assessed the ability of C. jejuni (n = 7) and C. coli (n = 3) strains isolated from diseased poultry, and previously characterized by whole genome sequencing, to form biofilm. The in vitro analyses were carried out by using a microtiter based protocol including biofilm culturing and fixation, staining with crystal violet, and measurement of the optical density (OD570). The ability to form biofilm was categorized into four classes (no, weak, moderate, and strong producers). Potential correlations between OD570 and the presence/absence of virulence determinants were examined. The C. jejuni were classified as no (n = 3), weak (n = 2), and moderate (n = 2) biofilm producers; however, all possessed genes involved in chemotaxis, adhesion, and invasion to the host cells. No genes present exclusively in biofilm producers or in non-biofilm producers were identified. All C. coli were classified as weak producers and showed a similar set of virulence genes between each other. A trend of increased mean OD570 was observed in the presence of flaA and maf7 genes. No association between biofilm production classes and the explanatory variables considered was observed. The results of this study suggest that further investigations are needed to better identify and characterize the genetic determinants involved in extra-intestinal Campylobacter biofilm formation.

Neurotoxin gene profiling of clostridium botulinum types C and D native to different countries within Europe.

Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C (n = 12), C-D (n = 29), D (n = 5), and D-C (n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains (n = 20), non-BoNT-producing clostridia (n = 20), and other bacterial species (n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n = 108), poultry (n = 60), and bovines (n = 56). Among the 292 samples, 144 were positive for either the bont/C-D or the bont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

Genome Sequence of Campylobacter Strain 19-13652, Isolated from Breeding Pheasants.

We report the whole-genome sequence of a Campylobacter strain that was isolated from breeding pheasants presenting "bulgy eyes" in Italy. Traditional molecular typing methods did not return any reliable result. Whole-genome sequencing and sequence comparison with known genomes did not meet the criteria for assignment to an existing species.

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.

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.

Prevalence, Molecular Characterization and Antimicrobial Susceptibility of Clostridioides difficile Isolated from Pig Carcasses and Pork Products in Central Italy.

In the last decade, the incidence and severity of Clostridioides difficile infections (CDIs) in humans have been increasing and community-associated infections have been described. For these reasons, the interest in C. difficile in food and in food animals has increased, suggesting other possible sources of C. difficile acquisition. This study evaluated the presence of C. difficile on pig carcasses at the slaughterhouse and in pork products in Central Italy. The contamination rate on pig carcasses was 4/179 (2.3%). Regarding food samples, a total of 216 pork products were tested (74 raw meat preparations and 142 ready-to-eat food samples made by cured raw meat). The real-time PCR screening was positive for 1/74 raw meat preparation (1.35%) and for 1/142 ready-to-eat food samples (0.7%) C. difficile was isolated only from the raw meat preparation (pork sausage). All the isolated strains were toxigenic and susceptible to all the tested antibiotics. Strains isolated from carcass samples displayed A+B+CDTa+CDTb+ profile, were toxinotype IV and belonged to the same ribotype arbitrary named TV93, while the one isolated from food samples displayed A+B+CDTa-CDTb- profile and it was not possible to determine ribotype and toxinotype, because it was lost after freeze storage. It was concluded that the prevalence of C. difficile in the pork supply chain is very low.

Clostridioides difficile in Calves in Central Italy: Prevalence, Molecular Typing, Antimicrobial Susceptibility and Association with Antibiotic Administration.

The emergence of Clostridioides difficile as the main agent of antibiotic-associated diarrhoea has raised concerns about its potential zoonotic role in different animal species. The use of antimicrobials is a major risk factor for C. difficile infection. Here, we provide data on C. difficile infection in dairy and beef calves in Umbria, a region in central Italy. This cross-sectional study focuses on prevalence, risk factors, ribotypes, toxinotypes and antimicrobial resistance profiles of circulating ribotypes. A prevalence of 19.8% (CI95%, 12-27.6%) positive farms was estimated, and the prescription of penicillins on the farms was associated with C. difficile detection (OR = 5.58). Eleven different ribotypes were found, including the ST11 sublineages RT-126 and -078, which are also commonly reported in humans. Thirteen isolates out of 17 showed resistance to at least one of clindamycin, moxifloxacin, linezolid and vancomycin. Among them, multiple-drug resistance was observed in two isolates, belonging to RT-126. Furthermore, RT-126 isolates were positive for tetracycline resistance determinants, confirming that tetracycline resistance is widespread among ST11 isolates from cattle. The administration of penicillins increased the risk of C. difficile in calves: this, together with the recovery of multi-resistant strains, strongly suggests the need for minimising antibiotic misuse on cattle farms.

Selective resistance to different glucocorticoids in severe autoimmune disorders.

Resistance to glucocorticoids often occurs in patients with severe inflammatory disorders. Occasionally, this resistance could be overcome by switching to a different glucocorticoid, but the mechanisms of this selectivity are not clear. We studied this condition in three patients with severe inflammatory disorders, who responded satisfactorily to betamethasone, but could not be switched to equipotent doses of methylprednisolone or prednisone. While betamethasone displayed similar activity on lymphocyte proliferation in cells obtained from the three patients and controls, higher concentrations of methylprednisolone were needed to inhibit proliferation in patients' cells. In a competition study, the concentration of methylprednisolone that inhibited 50% of specific [(3)H]dexamethasone binding was increased in patients' lymphocytes. Higher Rhodamine-123 efflux was demonstrated in CD4 T cells from two patients, suggesting that an increased activity of membrane transporters could be responsible for the selective response to different glucocorticoids, even if P-glycoprotein and MRP1 expression was not increased.

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.