10.12.2019 SYNLAB develop NGS technique for in-depth investigation of Biofilms – The most robust and cost-effective method for Biofilm Critical Path Analysis No culturing prior to analysis. Quickly adaptable to any matrix Cost effective root cause analysis Rapid turnaround times (3-4 days for a full screen) Scalable to customers need Constantly evolving analytic platforms for review of historical data. Overview Biofilms form through the association of multiple bacterial species embedded in extracellular matrices, which grow ecosystems with very effective protection barriers. The development within biofilm provides numerous ecological advantages, notably an increased capability to attach to surfaces, reduced susceptibility to mechanical abrasion and an enhanced resistance to disinfectants like quaternary ammonium compound and other biocides. Once established, these communities can have critical negative impact on structural equipment by inducing corrosion, adversely effecting product organoleptic taint and odour or degradation via enzymatic secretions. However, a more significant concern when biofilms form in food production environments comes from human pathogens that can develop within this protected environment and can represent a substantial health risk by concealing dangerous bacteria. This is especially critical where biofilms colonise on stainless steel or glass surfaces within process equipment or plant, which is generally inaccessible or difficult to clean. The complexity of these communities gives them with an incredible plasticity and adaptability to rapidly changing conditions such as temperature variations or cleaning regimes: different populations will grow or become sub-clonal, with the ability to change their genetic and metabolic pathways. This makes the control or eradication of biofilms a very significant, and ever developing, challenge. The detection and eradication of biofilm from food production, chemical plants or medical equipment has necessitated the drive to develop methods to understand the composition and complexity of these biofilms in much greater detail. Conventional Biofilm Characterisation Traditional approaches to characterise the microflora in biofilm were based on culturing and agar plating. The main obstacles with these methods are as simple as missing fastidious organisms, viable but not culturable in laboratories environment (VBNC) like Listeria and Salmonella exposed to chlorine (1), to many groups of specific interest like biocide resistant sub-clones or adapted psychrophilic bacteria (cold environment). Because VBNC cells can be detected by using methodologies like PCR, an important role was given to the development of newer molecular technologies like SSU rRNA (16S) cloning libraries and Q-PCR which opened another window on this micro-world. Despite efficiently circumventing some issues, long turnaround time and high costs in conjunction with biases and limitations of delivering analysis using far from perfect “universal” primers, accentuate further the need for innovative solutions to understand what forms biofilms and their complex biological properties. The SYNLAB approach The SYNLAB approach to the precise identification of complex biofilm communities uses Next Generation Sequencing (NGS) as a highly efficient and cost-effective technique to deliver a depth of information so far almost inaccessible. The power of NGS resides in its ability to generate a massive amount of genetic information in a relatively simple and rapid manner, especially when combined with the most prevalently used marker to study microbiological communities (SSU rRNA). The analysis involves extensive parallel sequencing, where information is gathered from several sets of primers, targeting hypervariable regions and taking data from several sources simultaneously. The multiplicity of targets used in a single run, overlapping and complementing large amount of information, opens the possibility to investigate not only the VBNC, but also micro-populations which have so far been impossible to reach and analyse without a prior knowledge of their existence. Another valuable feature of the SYNLAB approach is the scalability of the technology, versatile enough for type species in a probiotic mix of known organisms to perform a most detailed survey of complex sewage sludge containing numerous diverse super-adapted bacterial genus, virtually unculturable in isolation. Conclusion Biofilms represent a major risk for the food industry, not only from an economic point of view but also as food safety issues: The implications on the commercial aspect (degradation of equipment and impact on the product organoleptic and integrity) are only part of the problem with increased concerns for the public health due to possible pathogens hidden in theses biofilms. Whilst there are many new and traditional methods to tackle the elimination of biofilms, the precise determination of their make-up can be crucial in delivering the most accurate and cost-effective root cause analysis to ensure long-term eradication. This can especially be the case in complex CIP systems, which can be subject to re-occurring issues, or in testing the efficacy of open plant hygiene cleaning regimes. This unique SYNLAB solution is a natural progression, evolved from many years as leaders in investigative microbiology using Sanger sequencing. Major advantages of the NGS platform are versatility and scalability. SYNLAB UK are currently using the platform in an array of applications covering accredited meat speciation, typing of probiotic mixes of known organisms to the detailed survey of complex sewage sludge containing diverse super-adapted bacterial genus.