Hello, and welcome to this lecture. My name is Ana Rita Rebelo, and I will be talking to you about detection of colistin resistance. This lecture will be divided in three main parts. First, we will discuss colistin resistance itself, then we will discuss the phenotypic and genotypic methods that are used to detect colistin resistance. We will start with a brief explanation about colistin resistance. Colistin is an anti-microbial that belongs to the class of polymyxins. It was introduced for human medicine in the 1960s, but its use has been limited due to his very high neuro and nephrotoxicity. Nowadays, it is mainly used in veterinary medicine and in the production animal industry. However, in the last decade, we have observed a resurgence in the use of colistin in humans due to the emergence of multi-drug resistant bacteria. The European Medicines Agency published a report in 2016 estimating that in only four years from 2010 and 2014, the use of colistin doubled due to infections caused by this multi-drug resistant organisms. Colistin is a very important antimicrobial, it is a last resort anti-microbial and therefore, we must attempt to preserve its use. It is active intrinsically in internal bacteria CIA, but also in acinetobacter, pseudomonas aeruginosa, and haemophilus influenza, and some other species. Gram-positive bacteria are intrinsically resistant to these antimicrobial, and also proteus and serretia. Colistin acts through an interaction with a lipopolysaccharide of the bacterial cell membrane leading to the leakage of the cellular contents. Thus, any mechanism that is able to modify the lipopolysaccharide or the interaction of colistin with this target will maybe lead to colistin resistance. At this point in time, we have knowledge regarding two different mechanisms of colistin resistance. In particular, chromosomal point mutations and the MCR genes. Chromosomal point mutations exists in systems that are associated with the synthesis and maintenance of the lipopolysaccharide. Examples of such systems are the PhoPQ and PmrAB two-component systems. Mutations in these systems can likely lead to colistin resistance in the organisms. The MCR genes were discovered for the first time in 2015, and they increased our concern about colistin resistance because they are harbored in plasmids. As such, they have a very high potential of being transmitted between organisms and between different species. There are at this moment eight known MCR genes from Number 1 to Number 8, and some of them present a few too many variants. We will now discuss how can we effectively detect this resistance. We will start with the phenotypic methods that are usually used in microbiology laboratories. From the routine test methods, only dilution methods are recommended for testing for colistin resistance. This is due to several characteristics of the testing process. Some of these characteristics are related to the biochemical properties of colistin itself. For example, colistin can bind to the surface of the plastics that are frequently use in laboratory materials, and thus, we should always use polystyrene trays. We should never treat the laboratory materials that we are using with surfactants compounds such as polysorbate 80. Because it is suspected that this types of compounds can act synergistically with colistin, thus leading to falsely low MIC values. There are also characteristics of the LPS itself that can affect the performance of the testing. For example, it is known that the cations that are present in the culture media can change the MIC values that we are recording. Thus, it is recommended that we use cation-adjusted Mueller Hinton broth, but we should always pay close attention to the types of broths that we are using and confirm their composition. Additionally, the drug powder that we choose to perform the MIC testing is also very important. Colistin exists in two different types of powders, from which one of them is an inactive prodrug. If we were to use the inactive prodrug, we would obtain false high MIC values. So we should always use colistin sulfate to perform our broth microdilution tests. Further information about the recommendations for performing MIC for colistin susceptibility testing can be found in the EUCAST website, and I highly recommend that you read through them before proceeding with your testing. Other routine test methods such as diffusion methods are very unreliable for colistin susceptibility testing. Particularly because colistin diffuses very poorly in the agar. They produce a very high rate of false susceptibility. For example, in pseudomonas aeruginosa, it is supposed that they can produce up to 32 percent of very major errors, so a false susceptibility results. Other methods exist, such as selective agar that only allow for the growth of colistin resistant isolates. Another example is a Rapid Polymyxin NP tests, but these methods have not yet been validated for years in a routine laboratories. As with any type of laboratory tests, quality control is very important. Especially because colistin susceptibility testing has so many particularities. Usually, we perform quality control with strange that are susceptible to the antimicrobials and colistin is no exception. We should use either E. coli ATCC or pseudomonas aeruginosa ATCC as a susceptible control, but we should also use a resistant strain. In this case, a low level resistance strain are growing MCR one gene. Genotypic methods also exist, and they are based on the detection of the chromosomal or genetic mechanisms that lead to colistin resistance. In this particular case, a genotypic method will try to detect either MCI genes or chromosomal point mutations in the systems that are involved with lipopolysaccharide maintenance. It's very important that you remember that a negative genotypic test cannot be used as a prediction of susceptibility. In the same way, a positive genotypic test is likely but not warranted to be a reflection of colistin resistance. We have limited knowledge of the mechanisms that cause colistin resistance. There might be interactions between different genetic systems in play. So we can only make predictions, we can never use genotypic results to affirm that an isolate is either colistin susceptible or colistin resistant. We see an example of an E. coli that is phenotypically susceptible to colistin but presents one point mutation in the pmrB gene. We have an opposite example of an enterobacter isolate which is phenotypically resistant to colistin. However, it presents no known mechanisms of colistin resistance. You must remember that a result of a genotypic tests should always be reported as either presence or absence of the mechanism, never as susceptibility or resistance to the antimicrobial. One example of such method is a multiplex polymerase chain reaction that was developed at the EURL-AR laboratory, and it was developed for surveillance purposes only in enterobacteriaceae. It's not validated for other families. It is quite fast to perform, it is inexpensive, and as such it can be of use in certain circumstances where low resources are available. One of its limitations it's that it requires a confirmation or an update each time a new mcr gene is discovered. Another genotypic method for detection of colistin resistance is the screening of a whole genome sequence of an isolates against databases that are collections of the colistin resistance mechanisms, both a point mutations and the MCI genes. This type of approach has a very high potential for a continuous and easy update, you just introduced a new sequence that you want to investigate. Also, it might reveal new mechanisms of resistance if you find similar gene sequences to the ones that are currently known. It is important that when using this approach, you keep in mind that there might be currently unknown point mutations that lead to colistin resistance but have not been yet described. So you should always keep an eye out for new point mutations in the systems that are involved in lipopolysaccharide maintenance. In the same way, if you are screening for MCI genes, you should be very careful with the identity threshold that you choose. You should never choose a threshold close to 100 percent, because you might be missing new variants of the genes or even new genes that have an intermediate percentage of identity to the current known genes. The main messages that we would like you to take home are that broth microdilution is presently the only recommended methods for colistin susceptibility testing. Quality control is essential, you can never performed this types of testing without assuring that you can trust your results. Genotypic methods cannot predict susceptibility nor resistance, but they are very important for performing. For example, epidemiological studies, they let you analyze the transference of resistance between isolates. Efforts are currently ongoing to improve our ability to detect colistin resistance, and stay tuned for updates from the international agencies such as EUCAST and CLSI. I would like to thank you for your participation on behalf of the EURL-AR tip. I hope you enjoyed this lecture. More information on this topic can be found on our website antimicrobialresistance.dk. Thank you.
