Hard to Kill Bugs

Strategies to combat bugs that resist radiation and disinfectants

Dangerous microorganisms are everywhere. Humans do their best to get rid of them, but this labor is, unfortunately, not always successful. Even though we do understand the importance of microorganisms in some contexts, we are extremely aware of the consequences their presence has in a manufacturing environment. Many regulations, including the latest publication of the EU GMP Annex 1, mention the importance of an effective Contamination Control Strategy (CCS) to control ingress, transport, and proliferation of these bugs.

Despite all the efforts, studies have demonstrated that there are some microorganisms that stubbornly resist radiation and disinfectants. To learn more about useful strategies to combat these bugs, we turned to one of Charles River’s experts, Sujan Timilsina, PhD. Sujan is a Bioinformatics Scientist II with the Microbial Solutions business and has over 10 years of experience in microbial detection and identification, and high throughput sequencing. Sujan is also knowledgeable about the development and application of bioinformatic pipelines that can be used to phylogenetically analyze microbial evolution and movement across a wide range of microorganisms.

What do you think are the best technologies or platforms to use to identify these resistant microorganisms? 

Resistance in microorganism is an associated phenotype and usually strain specific. Some are resistant to antibiotics, UV and high temperatures, though not all species strains carry that trait. The distribution of these organisms varies as each resistance bug may have different niches where they flourish and where selection pressure against other microorganisms is high. Nonetheless, for identification purposes, we use different phenotypic, proteotypic, and genotypic methods that help identify the organism at various levels. Identifying specific resistance will need in-depth analysis, but for general taxonomic identification purposes, we can use proteotypic and genotypic methods. The microbial identification methods need a robust database that strains can be compared against. For example, we can use sequencing platform to sequence genes used for identification, and if that organism of interest is not described in the database we are comparing against, we may not be getting the correct result. Among the platforms we use, these resistant organisms can be identified by comparing against a well curated microbial database, such as the one maintained by Accugenix^®.

What would be the right path to identify them? Are there any best practices you would like to share? 

In scenarios where we are interested in identifying the organisms and determining its source, a multi-prong approach will be valuable. As a first step, it is critical to identify the organism. Using 16S for prokaryotic bacteria and ITS gene sequences for eukaryotic fungal organisms and comparing them against a larger database, we can get a good idea about the organism of interest. As the resistance among organisms could be strain specific, it will be ideal to use strain typing methods that will help to piece together the puzzle on where the organism could have been potentially introduced. Initial organism identification is critical, as the subsequent process of the strain typing could be implemented accordingly by selectively using 4-10 housekeeping genes (genes associated with microbial metabolism) to determine the strain diversity. With the application of whole genome sequencing and utilizing the larger genomic region for comparison, we can further advance our capacity to effectively trace and track resistant microorganisms.

What makes some microorganisms so hard to identify and, ultimately, kill? 

Tricky question. There are a multitude of reasons why these organisms are hard to identify. Sometimes we don’t have any existing knowledge of the organism. Some organisms do not grow in a growth media, which limits our understanding of these organisms. We have greatly improved our ability to learn about these microbes after being able to sequence such organisms without the need of growing them in a growth media. New organisms are described very frequently, and this frequency has increased significantly, for prokaryotes since 2012 due to whole genome sequencing tools. 

For the ones, that we recognize and have studied the challenges are different. Some fungi may have unique reproductive and dormant structures that limit rapid growth or extraction of genetic material to help in species identification. For example, if we consider an Aspergillus that produces ascus spores, it can remain dormant for a considerable length of time, making the identification process challenging. 

Correct identification is important to evaluate methods for eliminating the organisms from their unwanted areas. Microorganisms are quick to adapt, and the population could be selected in a different direction depending on their exposure and selection pressure. Continuous exposure to a certain antibiotic or fumigation could lead to selection of strains that are relatively resistant to the methods we use to eradicate them. So, it is important to understand what we are dealing with in addition to applying general control procedures. We may have to implement organism or niche specific approaches to control them.

Based on your experience, are there specific regions where we “encounter” these organisms the most? 

Initial assumptions are that resistant organisms appear where there is high selection. Nonetheless, with more study and identification of organisms, we have found significant diversity in microbe populations and their strategies to adapt to unfavorable environment even when they are not exposed to selective conditions. Some organisms are ubiquitous in distribution and additional identifications are being made with the availability of central databases to compare and describe them phylogenetically and taxonomically. Deinococcus is one example. Some of the species of this genus are resistant to high radiation and have unique DNA repair mechanisms that help them to thrive in these conditions. It is likely that certain population within the species group might be favored in such harsh conditions. 

However, the species can be encountered frequently in areas outside the radiation zone. Same goes for multiple antibiotic resistant and heat tolerant organisms. Several of these species have been identified during the microbial identification process through Accugenix^®. This shows that these organisms are out there; the strains may not be resistant to these control mechanisms, but the species could be present in our surroundings.

Are there any other recommendations you would like to share?

In cases of microbial contamination preventative measures are always better. However, if contamination did occur, initial identification is the key to successful mitigation of microbial issues. Identifying the microorganisms and tracking the strains in production facilities becomes critical. My recommendation would be to use a validated library for identification and keep track of your organisms. Sudden changes in microbial populations could indicate contamination issues. Accugenix^® provides an in-depth evaluation of organisms reported from the facility through Tracking and Trending features via AccuPedia^®, which  could be a good starting resource for understanding these microbes.

For more details and interesting facts on these radio- and disinfectant- resistant microorganisms, watch our exclusive on-demand video series (only available for a limited time) presented by Ziva Abraham, President and Founder of Microrite, Inc. and Duncan Barlow, Technology and Market Development Manager for Charles River Laboratories’ Microbial Solutions division.

Source: www.criver.com