Application of Fourier Transform Infrared Spectroscopy to Discriminate Two Closely Related Bacterial Species: Bacillus anthracis and Bacillus cereus Sensu Stricto.

Fourier transform infrared spectroscopy (FTIRS) is a diagnostic technique historically used in the microbiological field for the characterization of bacterial strains in relation to the specific composition of their lipid, protein, and polysaccharide components. For each bacterial strain, it is possible to obtain a unique absorption spectrum that represents the fingerprint obtained based on the components of the outer cell membrane. In this study, FTIRS was applied for the first time as an experimental diagnostic tool for the discrimination of two pathogenic species belonging to the Bacillus cereus group, Bacillus anthracis and Bacillus cereus sensu stricto; these are two closely related species that are not so easy to differentiate using classical microbiological methods, representing an innovative technology in the field of animal health.

High Genetic Diversity and Virulence Potential in Bacillus cereus sensu lato Isolated from Milk and Cheeses in Apulia Region, Southern Italy.

The Bacillus cereus group includes species that act as food-borne pathogens causing diarrheal and emetic symptoms. They are widely distributed and can be found in various foods. In this study, out of 550 samples of milk and cheeses, 139 (25.3%) were found to be contaminated by B. cereus sensu lato (s.l.). One isolate per positive sample was characterized by Multilocus Sequence Typing (MLST) and for the presence of ten virulence genes. Based on MLST, all isolates were classified into 73 different sequence types (STs), of which 12 isolates were assigned to new STs. Virulence genes detection revealed that 90% and 61% of the isolates harboured the nheABC and the hblCDA gene cluster, respectively. Ninety-four percent of the isolates harboured the enterotoxin genes entS and entFM; 8% of the isolates possessed the ces gene. Thirty-eight different genetic profiles were identified, suggesting a high genetic diversity. Our study clearly shows the widespread diffusion of potentially toxigenic isolates of B. cereus s.l. in milk and cheeses in the Apulia region highlighting the need to adopt GMP and HACCP procedures along every step of the milk and cheese production chain in order to reduce the public health risk linked to the consumption of foods contaminated by B. cereus s.l.

Toxigenic Genes, Pathogenic Potential and Antimicrobial Resistance of Bacillus cereus Group Isolated from Ice Cream and Characterized by Whole Genome Sequencing.

Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes' detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety.

Campylobacter vulpis sp. nov. isolated from wild red foxes.

During a sampling of wild red foxes (Vulpes vulpes) for the detection of Epsilonproteobacteria, 14 strains were isolated from the caecal contents of 14 epidemiologically-unrelated animals. A genus-specific PCR indicated that the isolates belonged to the genus Campylobacter. Based on the results of a species-specific PCR, the isolates were initially identified as C. upsaliensis. However, multi-locus sequence typing (MLST) revealed that the isolates were significantly different from the C. upsaliensis present in the MLST database. A polyphasic study, including conventional biochemical and tolerance characteristics, morphology by transmission electron microscopy (TEM), MALDI-TOF analysis, and genetic comparisons based on partial 16S rDNA and atpA gene sequences, was undertaken. Finally, the complete genome sequence of the type strain 251/13T and the draft genome sequences of the other isolates were determined. Average nucleotide identity, average amino acid identity and in silico DNA-DNA hybridization analyses confirmed that the isolates represent a novel taxon for which the name Campylobacter vulpis sp. nov. is proposed, with isolate 251/13T (=CCUG 70587T = LMG 30110T) as the type strain. In order to allow a rapid discrimination of C. vulpis from the closely-related C. upsaliensis, a specific PCR test was designed, based on atpA gene sequences.

Fate of Methicillin-resistant Staphylococcus aureus (MRSA) under simulated acidic conditions of the human stomach.

A known amount (107 cfu/ml) of animal origin Methicillin-resistant Staphylococcus aureus (MRSA) ST398/t011/V and of human origin MRSA ST1/t127/IVa strains were individually inoculated into ricotta cheese and hamburger samples. The pH of each food matrix was gradually decreased from 6.0 down to 2.0 during a period of about 2 hr, under conditions simulating the mechanical digestion of the human stomach. Afterward, the MRSA strains were recovered by using a MRSA-specific plating medium. Although both strains showed a certain acidic resistance, they showed different responses at low pH values during the experiment: ST398 survived unharmed during the course of the experiments to the last stage at pH 2 where counts of 6.4 cfu/g for the hamburger and 7.5 log cfu/g for ricotta cheese assays were obtained. In contrast, the ST1 population was no longer detectable at pH 3 in the hamburger and at pH 2 in the ricotta cheese assays. To the best of our knowledge, this is the first study that investigates the ability of MRSA to overcome the acidic conditions of the human stomach and that adds new evidence that might contribute to expand the scientific knowledge on the significance of MRSA in the food safety debate.

Multi-locus sequence typing and virulence profile in Bacillus cereus sensu lato strains isolated from dairy products.

Members of Bacillus cereus group are important food contaminants and they are of relevant interest in food safety and public heath due to their ability to cause two distinct forms of food poisoning, emetic and diarrhoeal syndrome. In the present study, 90 strains of B. cereus isolated from dairy products, have been typed using Multilocus Sequence Typing (MLST) analysis and investigated for the occurrence of 10 enterotoxigenic genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM, entS and bceT) and one emetogenic gene (ces), to determine their genetic diversity. A total of 58 sequence types were identified and among these 17 were signalled as new profiles. Among the virulence genes, the majority of our strains carried the entS (92%), entFM (86%), nhe (82%) and cytK (72%) genes. All remaining genes were identified in at least one strain with different prevalence, stressing the genetic diversity, how even the different grade of pathogenicity of B. cereus isolated from dairy products.

Antimicrobial Susceptibility and Multilocus Sequence Typing of Listeria monocytogenes Isolated Over 11 Years from Food, Humans, and the Environment in Italy.

Due to the increasing number of studies reporting the detection of antimicrobial-resistant isolates of Listeria monocytogenes, we sought to determine the antimicrobial susceptibility of L. monocytogenes isolates collected in Italy and find potential correlations to their serotypes and multilocus sequence types (MLST). The antimicrobial susceptibility of 317 L. monocytogenes isolates collected from food, humans, and the environment from 1998 to 2009 was assessed by minimum inhibitory concentration (MIC). Serotyping and MLST was also performed on all isolates. Potential correlations among antimicrobial resistance profiles, serotyping, and MLST were statistically evaluated. Twenty-four percent of L. monocytogenes isolates were resistant to oxacillin, 28.7% intermediate to clindamycin, and 24.3% to ciprofloxacin. The majority of isolates with elevated MIC to oxacillin was of environmental origin and belonged to serotype 4b/4e and ST2. Isolates with intermediate MIC values to clindamycin and ciprofloxacin were mostly of food and human origin and belonged to serotype 4b/4e and ST9. Regarding the time frame of isolate collection, comparing the last 3 years (2007-2009) to previous years (1998-2006), an increase was observed in the percentage of resistant and intermediate isolates per year. This trend strongly suggests the need for increasing attention on the prevalence of antimicrobial resistance in L. monocytogenes in Italy. To predict future resistance trends, the monitoring of clinical intermediate resistance might represent a useful tool especially for antibiotics associated to multiple-step mechanisms of acquired resistance. A specific focus should be addressed to antimicrobial-resistant isolates of serotype 4b, repeatedly associated with food-borne outbreaks.

Global Emergence of Colistin-Resistant Escherichia coli in Food Chains and Associated Food Safety Implications: A Review.

Antimicrobial resistance in bacteria represents one of the most important challenges for public health worldwide. Human infections from antimicrobial-resistant bacteria can be transmitted from person to person, via the environment (especially in the hospital environment), or via handling or eating contaminated foods. Colistin is well known as a last-resort antibiotic for the treatment of human infections; a recent study performed in the People's Republic of China has revealed that colistin resistance is also conferred by the plasmid-mediated mcr-1 gene in Escherichia coli. After that discovery, further plasmid-mediated, colistin resistance genes have been detected. However, to date, only reports on E. coli carrying the mcr-1 gene (E. coli mcr-1+) in foodstuff are available. E. coli mcr-1+ has been isolated from food of animal origin and vegetables; this discovery has opened a debate among food safety experts. This review aims to provide a critical overview of the currently available scientific literature on the presence of the plasmid-mediated, colistin resistance gene E. coli mcr-1 in foodstuffs, focusing on the main implications and future perspectives for food safety.

Identification of virulence and antibiotic resistance factors in Arcobacter butzleri isolated from bovine milk by Whole Genome Sequencing.

Arcobacter butzleri is a pathogenic aerobic bacterium responsible for diarrhea and septicemia in humans. It is frequently isolated from food products of animal origin, including milk and dairy products. To data, few reports are currently available on the genetic characteristics and virulence profiles of A. butzleri. The aim of this study was to investigate the genetic diversity and to characterize the virulence and antibiotic resistance profiles of 10 A. butzleri strains isolated from bovine milk samples by Whole Genome Sequence (WGS). Multi-Locus Sequence Typing (MLST) revealed that three isolates belonged to the ST66, two to the ST420 and the remaining five strains to the ST627, ST629, ST630, ST633 and ST637, respectively. The 100% of the strains carried cadF, pldA, ciaB, cj1349, mviN and tlyA virulence factors genes; 60% iroE; 50% irgA; 10% hecB. Resistome prediction showed a multidrug resistance: 100% of isolates resulted resistant to fluoroquinolones and tetracycline; 90% of strains to rifampicin and cephalosporins and a minor percentage to other antibiotics. Furthermore, the 50% of strains harbored four mutations in Mycobacterium tuberculosis katG gene conferring resistance to isoniazid. The study provided interesting data on the virulence characteristics and on the genetic endowment related to the antimicrobial resistance of A. butzleri isolates from milk. The determination of the STs also added information concerning the genetic variability of this microorganism. To date, a very limited number of studies have been published on the typing of A. butzleri using WGS, so this paper proposes an innovative methodological approach that allows a rapid and complete characterization of pathogenic microorganisms.

MRSA in swine, farmers and abattoir workers in Southern Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important medical issue, since it causes serious and sometimes fatal infections in humans. Intensively reared swine may serve as reservoirs for MRSA that can infect swine workers, and also consumers (via contaminated meat). In this study, MRSA strains were isolated from 55 of the 85 (64.7%) intensive pig farms surveyed, and prevalence was greater on pig fattening farms than on breeding farms. In addition, we included in the study 63 foreign pigs imported for slaughter. Overall, the prevalence of MRSA in the 418 sampled swine was 59.1%; 12 genotypes were identified among the isolates; ST398 (96.4%) was most prevalent, followed by ST97 (2%), ST9 (0.8%) and ST1 (0.8%). MRSA isolates were also detected in 26 (17.3%) of the 150 operators included in the study; the genotypes detected were ST398 (85%), ST9 (7.6%), ST5 (3.8%) and ST1 (3.8%). All the strains were pvl negative and pia positive. Both swine and human strains displayed a multi-resistance pattern, and almost all were resistant to tetracycline. The results obtained in this study confirm the high prevalence of MRSA in swine reared and slaughtered in Italy, and underline the public health risk linked to the spread of antimicrobial-resistant Staphylococcus aureus among intensively reared pigs.

Dolphin Morbillivirus in Eurasian Otters, Italy.

We report biomolecular evidence of dolphin morbillivirus in 4 wild Eurasian otters (Lutra lutra) from southern Italy; 2 animals showed simultaneous immunohistochemical reactivity against morbilliviral antigen. These cases add further concern and support to the progressively expanding host range of dolphin morbillivirus in the western Mediterranean Sea.

Evaluation of in vitro antimicrobial susceptibility of Bacillus anthracis strains isolated during anthrax outbreaks in Italy from 1984 to 2017.

Anthrax, caused by Bacillus anthracis, is a non-contagious infectious disease that affects a wide range of animal species (primarily ruminants) including humans. Due to the often-fatal outcome in humans, quick administration of definitely effective antimicrobials is crucial either as prophylaxis or as a clinical case therapy. In this study, 110 B. anthracis strains, temporally, geographically, and genetically different, isolated during anthrax outbreaks in Italy from 1984 to 2017, were screened using a broth microdilution method to determine their susceptibility to 16 clinically relevant antimicrobial agents. The strains were isolated from various matrices (human, animal, and environmental samples) and were representative of thirty distinct genotypes previously identified by 15-loci multiple-locus variable-number of tandem repeats analysis. The antimicrobials tested were gentamicin, ceftriaxone, streptomycin, penicillin G, clindamycin, chloramphenicol, vancomycin, linezolid, cefotaxime, tetracycline, erythromycin, rifampin, amoxicillin, ciprofloxacin, doxycycline, and trimethoprim. All isolates were susceptible to most of the tested antimicrobials, with the exception of trimethoprim for which all of them showed high minimal inhibitory concentration values. An intermediate level of susceptibility was recorded for ceftriaxone and cefotaxime. Although the Centers for Disease Control and Prevention recommend the use of doxycycline, ciprofloxacin, penicillin G, and amoxicillin for treatment of human cases and for post-exposure prophylaxis to anthrax spores, this study shows a high degree of in vitro susceptibility of B. anthracis to many other antimicrobials, suggesting the possibility of an alternative choice for prophylaxis and therapy.

Evaluation of oxfendazole in the treatment of zoonotic Onchocerca lupi infection in dogs.

The genus Onchocerca encompasses parasitic nematodes including Onchocerca volvulus, causative agent of river blindness in humans, and the zoonotic Onchocerca lupi infecting dogs and cats. In dogs, O. lupi adult worms cause ocular lesions of various degrees while humans may bear the brunt of zoonotic onchocercosis with patients requiring neurosurgical intervention because of central nervous system localization of nematodes. Though the zoonotic potential of O. lupi has been well recognized from human cases in Europe, the United States and the Middle East, a proper therapy for curing this parasitic infection in dogs is lacking. To evaluate the efficacy of oxfendazole, 11 out of the 21 client-owned dogs (21/123; 17.1%) positive for skin-dwelling O. lupi microfilariae (mfs), were enrolled in the efficacy study and were treated with oxfendazole (50 mg/kg) per OS once a day for 5 (G2) or 10 (G3) consecutive days or were left untreated (G1). The efficacy of oxfendazole in the reduction of O. lupi mfs was evaluated by microfilarial count and by assessing the percentage of mfs reduction and mean microfilaricidal efficacy, whereas the efficacy in the reduction of ocular lesions was evaluated by ultrasound imaging. All dogs where subjected to follow-ups at 30 (D30), 90 (D90) and 180 (D180) days post-treatment. The percentage of reduction of mfs was 78% for G2 and 12.5% for G3 at D180. The mean microfilaricidal efficacy of oxfendazole in the treatment of canine onchocercosis by O. lupi at D30, D90 and D180 was 41%, 81% and 90%, in G2 and 40%, 65% and 70%, in G3, respectively. Retrobulbar lesions did not reduce from D0 to D180 in control group (dogs in G1), whereas all treated dogs (in G2 and G3) had slightly decreased ocular lesions. Percentage of reduction of ocular lesions by ultrasound examination was 50% and 47.5% in G2 and G3 at D180, respectively. Despite the decrease in ocular lesions in all treated dogs (G2 and G3), oxfendazole was ineffective in reducing ocular lesions and skin-dwelling O. lupi mfs in treated dogs (G2 and G3) in a six-month follow-up period. Here we discuss the need for more reliable diagnostic techniques and efficient treatment protocols to better plan future intervention strategies.

Arcobacter spp. in bovine milk: An emerging pathogen with potential zoonotic risk.

The aim of the present study was to assess the prevalence and genetic characteristics of Arcobacter spp. in bovine bulk tank milk produced in Apulia Region (Italy). Samples collected from 396 dairy farms, after enrichment in a selective broth, were subjected to an Arcobacter genus - specific Real Time PCR. Positive broths, previously filtered, were seeded on Karmali, MCCD and Columbia Blood Agar plates; presumptive Arcobacter spp. colonies were identified using an amplification and sequencing method and then characterized by Multi-Locus Sequence Typing (MLST). Prevalence of Arcobacter spp. in bovine milk samples was 5% (20/396); A. butzleri was the only isolated species, in agreement with previous studies that reported A. butzleri as the most commonly recovered species in milk and dairy products. MLST analysis of the 20 A. butzleri strains identified 81 alleles and 16 STs. Consistent with previous studies, MLST revealed a high level of heterogeneity between the A. butzleri isolates and confirmed the high discriminatory power of this method and its suitability for epidemiological investigations. This study confirmed the importance of raw milk as a possible source of Arcobacter spp. for humans.

High Occurrence of Methicillin-Resistant Staphylococcus aureus in Horses at Slaughterhouses Compared with Those for Recreational Activities: A Professional and Food Safety Concern?

The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in horses and its zoonotic potential is poorly understood. The objective of this study is to provide data on the prevalence and genetic characteristics of MRSA isolated from horses on farms, at racecourses, and at slaughterhouses in Italy, using standard and molecular methods. In addition, we report the prevalence of MRSA in horse handlers. Among 388 horses tested by nasal swabs, 27 (7%) were positive for MRSA ST398 (t011, t899, t1255) and ST1 (t127). The prevalence of MRSA in horses tested at slaughterhouses was significantly higher (p < 0.001) compared with those tested on farms and racecourses. Five (7%) out of 67 staff members working in close contact with horses (2 from slaughterhouse, 2 from riding stable, and 1 from racecourse) were carriers of MRSA ST398 (t011, t034) and ST1 (t127). The isolates from horses and humans carried SCCmec IVa or V and were pvl negative and pia positive. All the isolates from both horses and humans were resistant to at least two antimicrobial classes. The circulation of MRSA in horses and in humans working in close contact with them should be considered an emerging public health issue. In fact, it represents a potential risk for people who work in close contact with horses, and for horse meat consumers.

Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer.

Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu.

In vitro efficacy of ribavirin against canine distemper virus.

Despite vaccination, canine distemper virus (CDV) remains one of the important pathogen of dogs with worldwide distribution. Ribavirin (RIB) inhibits replication of measles virus (MV), a morbillivirus closely related to CDV, both in vitro and in vivo. In this report the antiviral activity of RIB against CDV in cell cultures was assessed. Quantitative real-time RT-PCR was used to measure viral RNA in VERO cells infected by CDV and to evaluate the inhibitory effects of RIB. RIB caused a dose- and time-dependent decrease in accumulation of CDV RNA when added after virus adsorption. RIB was highly effective in preventing CDV replication at low concentrations with 50% virus-inhibitory concentrations ranging from 0.02 to 0.05 mM. Such low values were comparable to values displayed by highly susceptible strains of MV. In addition, CDV was passaged sequentially in VERO cell monolayers in the presence of RIB to trigger viral extinction. The virus was no longer detected after three passages, suggesting that error catastrophe is one of the modes of action of RIB against CDV. These findings suggest RIB as a promising tool for the therapy of CD in dogs.