Identification of risk factors and hotspots of antibiotic resistance along the food chain using next-generation sequencing.

Bacterial antimicrobial resistance (AMR) is considered to be very alarming following an upward trend and thus posing a primary threat to public health. AMR has tremendous adverse effects on humans, farm animals, healthcare, the environment, agriculture and, thus, on national economies. Several tools have been proposed and adopted by numerous countries after comprehending the need for antimicrobial stewardship and for a rational use of antibiotics. These tools include diagnostics for infections or AMR detection, for measuring and monitoring antibiotic consumption (e.g. surveillance tools) and for guiding medical doctors and veterinarians in selecting suitable antibiotics. In addition, it has been known that the food chain represents a leading vector for the transmission of pathogens to humans via various routes (direct or indirect). Considerable efforts have been made and are still in progress both at international and national levels in order to control and mitigate the spread of pathogens and thus ensure food safety. During the last decades, a new concern has risen regarding the food chain playing a potential major role in the transmission of resistant bacteria as well as resistance genes from the animal kingdom to humans. Several recent studies highlight the role of food processing environments as potential AMR hotspots contributing to this spread phenomenon. Next-generation sequencing (NGS) technologies are becoming broadly used in the AMR field, since they allow the surveillance of resistant microorganisms, AMR determinants and mobile genetic elements. Moreover, NGS is capable of providing information on the mechanisms driving and spreading AMR throughout the food chain. In the current work programme, the aim was to acquire knowledge and skills to track AMR genes and mobile genetic elements in the food chain through NGS methodologies in order to implement a quantitative risk assessment and identify hotspots and routes of transmission of AMR along the food chain.

#EUROmicroMOOC: using Twitter to share trends in Microbiology worldwide.

Twitter is one of the most popular social media networks that, in recent years, has been increasingly used by researchers as a platform to share science and discuss ongoing work. Despite its popularity, Twitter is not commonly used as a medium to teach science. Here, we summarize the results of #EUROmicroMOOC: the first worldwide Microbiology Massive Open Online Course taught in English using Twitter. Content analytics indicated that more than 3 million users saw posts with the hashtag #EUROmicroMOOC, which resulted in over 42 million Twitter impressions worldwide. These analyses demonstrate that free Microbiology MOOCs shared on Twitter are valuable educational tools that reach broad audiences throughout the world. We also describe our experience teaching an entire Microbiology course using Twitter and provide recommendations when using social media to communicate science to a broad audience.

Risk assessment of antimicrobial resistance along the food chain through culture-independent methodologies.

Antimicrobial resistance (AMR) represents a major challenge for Public Health and the scientific community, and requires immediate and drastic solutions. Acquired resistance to certain antimicrobials is already widespread to such an extent that their efficacy in the treatment of certain life-threatening infections is already compromised. To date, the emergence and spread of AMR has been attributed to the use, misuse or indiscriminate use of antibiotics as therapeutic drugs in human, animal and plant health, or as growth promoters in veterinary husbandry. In addition, there is growing concern over the possibility of AMR transmission via the food chain. Food processing environments could act as potential hotspots for AMR acquisition and spread. Indeed, biocide use and exposure to food-related stresses and food processing technologies could presumably act as selection pressures for increased microbial resistance against clinically relevant antibiotics. Global AMR surveillance is critical for providing the necessary information to form global strategies and to monitor the effectiveness of public health interventions as well as to detect new trends and emerging threats. Surveillance of AMR is currently based on the isolation of indicator microorganisms and the phenotypic characterisation of the strains isolated. However, this approach provides very limited information on the mechanisms driving AMR or on the presence and spread of AMR genes. Whole genome sequencing (WGS) of bacterial pathogens is a powerful tool that can be used for epidemiological surveillance, outbreak detection and infection control. In addition, whole metagenome sequencing (WMS) allows for the culture-independent analysis of complex microbial communities, providing useful information on the occurrence of AMR genes. Both approaches can be used to provide the information necessary for the implementation of quantitative risk assessment of AMR transmission routes along the food chain.

Microbiological, physicochemical and sensory parameters of dry fermented sausages manufactured with high hydrostatic pressure processed raw meat.

The aim of this trial was to describe physicochemical, microbiological and organoleptic characteristics of dry fermented sausages produced from high hydrostatic pressure (HHP) pre-processed trimmings. During ripening of the meat products pH, weight, water activity (aw), and several microbiological parameters were measured at zero, eight, fifteen days and after 6weeks. Sensory characteristics were estimated at day 15 and after six weeks by a test panel by using several sensory tests. Enterobacteriaceae were not detected in sausages from HHP-processed trimmings. Fermentation was little affected, but weight and aw of the HHP-processed sausages decreased faster during ripening. HHP-treated sausages were consistently less favoured than non HHP-treated sausages, but the strategy may be an alternative approach if the process is optimized.

Antibacterial activity and mode of action of a commercial citrus fruit extract.

AIMS: This study addresses the antibacterial activity and mechanism of action of BIOLL(+®), a commercial extract obtained from citrus fruits. METHODS AND RESULTS: Strong activities with minimum inhibitory concentrations (MIC) ranging from 10 ppm (for some Brachyspira hyodysenteriae strains) to 80 ppm (for various Salmonella enterica and Escherichia coli strains) were observed. Membrane integrity tests and Fourier transform infrared (FT-IR) spectroscopic analyses were performed to shed light on the effects caused on molecular structure and composition. Physical effects, with formation of pores and leakage of intracellular components, and chemical effects, which were dependent on the bacterial species, were evident on cellular envelopes. Whereas for S. enterica and E. coli, changes were focused on the carboxylic group of membrane fatty acids, for B. hyodysenteriae, the main effects were found in polysaccharides and carbohydrates of the cell wall. CONCLUSIONS: The great antibacterial activity shown by BIOLL(+®) and its proposed dual physico-chemical mode of action, with species-specific cellular targets, show its attractiveness as an alternative to antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic resistance is becoming a serious problem. Our study characterizes a novel antimicrobial extract, which could represent an alternative to antibiotics for treatment or prevention of bacterial infectious diseases.

Heterogeneity in resistance to food-related stresses and biofilm formation ability among verocytotoxigenic Escherichia coli strains.

This study assessed the resistance of ten verocytotoxigenic Escherichia coli (VTEC) isolates of commonly encountered serogroups/-types and two non-pathogenic E. coli strains to various food-related stresses (acid, alkaline, heat and high hydrostatic pressure treatments) and their biofilm formation ability. In addition, the global changes in the cellular composition in response to the exposure to these adverse environments were monitored by Fourier Transform Infrared (FT-IR) spectroscopy for two of the strains. Large inter-strain variations in stress resistance were observed. The most tolerant strains belonged to serogroup O157 which included both the O157:H7 type strain EDL933 and a representative isolate of the sorbitol fermenting O157:H- VTEC clone (strain MF3582). Strain C-600, a non-pathogenic laboratory strain, was sensitive to multiple stresses. Although wide variation in biofilm-forming ability was observed among VTEC isolates, no consistent relationships between biofilm-forming ability and capacity to withstand stress exposures were found. Analysis of the allelic status of the rpoS gene, involved in the general stress response of stationary-phase cells, allowed detection of loss-of-function mutations for two strains, E218/02 and MF2411, both of them showing as common features a high sensitivity to alkaline and heat treatments and a poor ability to form mature biofilms. Evidences found in this study confirm rpoS as a highly mutable gene in nature, and suggest its relevance not only for the mount of an active stress response but also for the establishment of mature biofilm communities. Our findings contribute to increase the knowledge on the resistance of VTEC to environmental stresses commonly encountered in the food chain, which can lead to improved strategies for preventing VTEC infections.

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria.

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.

Changes in ultrastructure and Fourier transform infrared spectrum of Salmonella enterica serovar typhimurium cells after exposure to stress conditions.

The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H2O2) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.

Changes in Fourier transform infrared spectra of Salmonella enterica serovars Typhimurium and Enteritidis after adaptation to stressful growth conditions.

The effects of growth conditions (temperature in the range 10-45 degrees C, sodium chloride concentration in the range 0-4%, aerobic vs. anaerobic growth and acidification of the growth medium, up to pH 4.5) on the Fourier transform infrared (FT-IR) spectra of Salmonella enterica serovars Typhimurium and Enteritidis were studied using multivariate statistical methods (Hierarchical Cluster Analysis and Factor Analysis). Although all environmental factors tested affected S. Typhimurium and S. Enteritidis FT-IR spectra to some extent, growth temperature was the most influential factor within the five spectral regions. The w(4) spectral region (1200 to 900 cm(-1)) was the most variable region, suggesting that S. Typhimurium and S. Enteritidis modulate their cell wall and cell membrane composition in response to shifts in growth temperature. Changes in membrane fluidity were determined by monitoring the vibrational modes of the acyl chain v(s)CH(2) symmetric stretching band by FT-IR spectroscopy. For cells grown in unsupplemented media an increase in growth temperature was linked to a decrease in membrane fluidity. Even though the effect of NaCl concentration, pH and atmosphere was considered of less importance, cells grown in acidified media also showed a reduction in their membrane fluidity, and the addition of sodium chloride to the culture medium was associated with an increase in the bacterial membrane fluidity. These findings can help interpret how important adaptive mechanisms for the survival of pathogenic bacteria in foods are, and show that FT-IR spectroscopy is a useful tool to understand how environmental conditions mimicking those in certain food products affect the cell. Also, FT-IR can be used to perform a rapid discrimination between bacterial phenotypes with different adaptive tolerance responses to environmental stress.