Strategies of phage contamination prevention in industry

Bacteria-driven processes in industry are commonly employed in various fi elds, from traditional biotechnology, which mostly focuses on production of food and beverages, through modern biotechnology, including production of solvents, green chemistry, vitamins, amino acids etc., to the most refi ned fi elds of use, including biopharmaceutical production of highly valuable proteins and other active pharmaceutical ingredients. All of these processes are prone to irregularities caused by contaminants. The most potent contaminants are bacteriophages, however, the level of susceptibility of the process to contamination by phages and to their deleterious effects is strongly dependent on several factors. Although a lot of information about the impact of bacteriophages on production processes driven by bacteria is available, paradoxically, the smallest number of scientifi c papers is published about the processes where the potential impact of phages is the most severe.


Introduction
Bacteria-driven processes in industry are commonly employed in various fi elds, from traditional biotechnology, which mostly focuses on production of food and beverages, through modern biotechnology, including production of solvents, green chemistry, vitamins, amino acids etc., to the most refi ned fi elds of use, including biopharmaceutical production of highly valuable proteins and other active pharmaceutical ingredients. All of these processes are prone to irregularities caused by contaminants. The most potent contaminants are bacteriophages, however, the level of susceptibility of the process to contamination by phages and to their deleterious effects is strongly dependent on several factors. Although a lot of information about the impact of bacteriophages on production processes driven by bacteria is available, paradoxically, the smallest number of scientifi c papers is published about the processes where the potential impact of phages is the most severe.

In general, phage contaminations can be divided into two types:
Primary contamination, the source of which usually lays outside of the facility, and the phage got introduced into the facility in relatively low amount, for example by personnel or by contamination of raw materials.
Secondary contamination, the source of which comes from a previously contaminated process, from which a phage (usually in great quantity), was spread within the facility or installation.
Routes of process contamination in case of primary and secondary contaminations are usually different, and prevention usually requires distinct methods to be employed.
How does the complexity of the bacterial community driving the production process infl uence the potential phage impact?
In general, a rule of thumb says that the more complex the bacterial community, the less prone it is to the disturbance of the process by bacteriophages. The reason for that is that multiple bacterial strains and/or l species involved in the production process can, at least to some extent, substitute for each other in the conversion of a substrate into the desired product. This is the case in facilities such as biogas production facilities or certain fermented food production facilities, which depend on natural, extremely complex microbial communities that already include bacteriophages, and their operation is not dependent on aseptic process development. Instead, bacteriophages may even be considered an important component of the whole production microbiome [1]. The less complex the microbial community gets, the more damage to the process can be caused phages. Industrial biotechnology [2][3][4] and biopharma [5], very seldom release any information about phages attacking their processes. There are a few possible reasons for that.
Firstly, a phage attack, considered a failure, may not be seen as a valuable result to be published. Secondly, a company's interest may be to avoid any information disclosure, which may be crucial in keeping the know-how and in providing a competitive advantage. Another reason may lay in the fear of losing customers, who may be concerned by the failure to provide safety to the process and may question the competence of the whole team. The relative lack of information in scientifi c journals about phages attacking processes other than dairy production may create a false impression that bacterial-driven processes in industrial biotechnology and biopharma are safe, or at least much safer from phage attacks. This is not true, but each of the industries suffering from phage attacks has different ways of minimizing their occurrence and impact. The strategies of phage prevention are dependent not only on the ability of a phage to kill bacteria in the process, but also on the volume of the production and the ability to use replacement strains in subsequent production in case of a phage attack.

Strategies to prevent or minimize phage impact
Strain rotation and manipulation: Starter culture rotation is commonly employed in diary industry as a standard procedure preventing production irregularities. This industry relies mostly on utilization of multi-species starter cultures with various phage resistances patterns, with the starter cultures changed on a regular basis. This allows for the formation of the product even if phages are not eliminated entirely from the production environment [6][7][8]. Another aspect of this strategy is the rotation of starter culture in case of a single species use [9]. This strategy can be also applied in case of certain industrial biotechnology setups, but it is much more challenging than in the case of food production. The main reason is the diffi culty in obtaining a suffi cient number of strains of a desired producer that would show good enough kinetics of product formation and high enough fi nal concentration of the product to ensure the process is profi table. Since this industry usually operates on relatively thin profi t margins, the change in performance of a production strain caused by selection of a phage resistant mutant may have a fundamental impact on the process economy. Nevertheless, such attempts are sometimes made.
Usually, the approaches include isolation of phages from the vicinity of the facility, exposure of the production strain to those phages, and isolation of resistant mutants. This approach allows for isolation of a panel of production strains, giving hope that at least one production strain will be resistant matter what the situation, the production has to be conducted using the host susceptible to a phage, endangering the whole production process. Sometimes bacterial strains with engineered phage resistance to selected group of phages is used, e.g. TonA mutants of E. coli [10], but these manipulations are not effective for phages utilizing different receptors or metabolic pathways. In case of dairy industry use of such strains is more widely used, but to the date this strategy never resulted in production of ultimately resistant strain [11]. In case of biopharmaceutical facilities, major efforts have to be invested in process design, if possible, and in proper choice of equipment. Very important aspects in this case are facility design, facility hygiene, and appropriate procedures. bioreactors, as well as air fi ltration. The latter poses the biggest challenge and is also the most diffi cult to control [12,13].

Process design and choice of equipment
In case of fermented food production and dairy industry, removal of all phages from raw materials is often impossible [6,8], so the phages will be propagated despite the best design

Facility hygiene
Facility hygiene is an important aspect of prevention of both primary and secondary phage contaminations. It is most crucial in case of cleaning up a contamination, which has a potential to spread from the production line into the facility.
Contaminated bacterial culture may contain as many as 10 12 or even close to 10 13 phages/ml, thus a release of even a relatively small volume of the phage lysate may result in a massive contamination of the facility [13].
There are various approaches for keeping a facility clean, however, the most surprising observation is that a vast majority of facilities utilize cleaning agents and disinfectants with no proven action against bacteriophages. To make things worse, many of the facilities do not use a single effective antiphage agent, focusing more on the reduction of bacterial load in the facility environment. Additionally, declared viricidal effi ciency of disinfectants is based most frequently on studies on enveloped viruses, and thus they may be misleading. In fact, there is only a very limited array of effective disinfectants capable of inactivation of a wide spectrum of bacteriophages.
Among the most effective are strong oxidizing agents, but these in turn may be problematic to use on some surfaces

Summary
Different industries suffering from phage contamination adopted different strategies of handling potential problems arising from the deleterious effects of phages on bacterialdriven processes (Table 1).
Over the decades of operations, each industry optimized some aspects of bacteriophage problem prevention, while other aspects remained relatively poorly improved. Implemented improvements allowed the industries to operate in a profi table manner, however, in some cases there is room for improvement, which could help to better prevent phage contamination in production facilities. Details of selected methods that could be used for this purpose are described in previous reviews, including Primrose 1990, Bogosian 2006, Los et al. 2004, and Los 2012.