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.

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.

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.

Biofilm Formation and Its Relationship with the Molecular Characteristics of Food-Related Methicillin-Resistant Staphylococcus aureus (MRSA).

The capability to produce biofilm is an important persistence and dissemination mechanism of some foodborne bacteria. This paper investigates the relationship between some molecular characteristics (SCCmec, ST, spa-type, agr-type, cna, sarA, icaA, icaD, clfA, fnbA, fnbB, hla, hlb) of 22 food-related methicillin-resistant Staphylococcus aureus (MRSA) strains and their ability to form biofilm on stainless steel and polystyrene. Five (22.7%, 5/22) strains were able to synthesize biofilm on polystyrene, and one of these (4.5%, 1/22) strains was also able to synthesize biofilm on stainless steel. The largest amount of biofilm was formed on polystyrene by 2 MRSA strains isolated from cows' milk, thus raising concern about the dairy industry. The majority of MRSA biofilm producers carried SCCmec type IVa, suggesting that the presence of SCCmecIVa and/or agr type III could be related to the ability to form biofilm. In conclusion, in order to achieve an acceptable level of food safety, Good Hygiene Practices should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA biofilm-producing strains in the food industry. PRACTICAL APPLICATION: In this study, some assayed isolates of food-related MRSA demonstrated the capacity to form biofilm. Biofilm formation differed according to surface characteristics and MRSA strains. A relationship was observed between some molecular characteristics and the ability to form biofilms. Few studies have investigated the ability of MRSA to form biofilms, and the majority of these studies have investigated clinical aspects. This work was performed to investigate whether or not there is a difference between MRSA food isolates and MRSA clinical isolates in their ability to form biofilm. These initial findings could provide information that will contribute to a better understanding of these aspects.

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.

Draft Genome Sequences of Six Listeria monocytogenes Strains Isolated from Dairy Products from a Processing Plant in Southern Italy.

Here we announce the draft genome sequences of 6 Listeria monocytogenes strains from ricotta cheese produced in a dairy processing plant located in southern Italy and potentially involved in a multistate outbreak of listeriosis in the United States.

Amplified Fragment Length Polymorphism and Multi-Locus Sequence Typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment.

Standardized tools for typing Listeria monocytogenes isolates are required in epidemiological surveys investigating food-borne disease outbreaks and in the food-processing environment to identify the sources of contamination and routes by which the organisms are spread. In this survey Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) have been used to study 103 L. monocytogenes isolates from food and environmental sources. A total of 62 AFLP types and 66 MLST Sequence Types were identified. AFLP and MLST produced similar results in terms of discriminating power. The Discrimination Index calculated for the two techniques was 0.976 for AFLP and 0.972 for MLST. These values were appreciably higher compared to serotyping (0.739). A good congruence was observed between AFLP and MLST. The present study demonstrated that AFLP and MLST subtyping are suitable tools for studying the epidemiology of L. monocytogenes. The great advantage of MLST over AFLP and other molecular typing methods based on fragment fingerprinting lies in the unambiguity of sequence data while AFLP is less costly and highly processive. In conclusion the two methods can be perfectly integrated for high-resolution genotyping of L. monocytogenes.