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It's a pleasure to be here today. I'm told that nowadays I should always start with a list of disclosures. Uh I'm largely retired now and the list is steadily going downwards, but I do continue to do some consulting for the farmer and diagnostic industries. Uh have a few shareholdings. Uh None of what I say tonight is going to be particularly relevant to any of these. Um I hardly need to say to this audience that carbenin resistance among gram negative bacteria is a growing problem. Now, this slide is just cold from Pfizer's Atlas surveillance for 2022 and don't be beguiled by the very color scheme that light ochre color indicates that somewhere between 40 60% of ice that were resistant to carbon pens. So, Argentina Brazil, India, South Africa, Greece and then the, the, the light green, this this tone more than 20% Turkey, Italy, Nigeria carbine resistance is a growing problem particularly in Klebsiella. Also to a lesser degree in E coli. Now, if we drill down into a little bit more detail, people talk of the five big carbapenemases A PC ox 48 IV and ND N and different ones are prevalent in different parts of the world, in the Americas, China, also, Greece and Italy A PC is predominant in the Middle East ox of 48 and in the Indian subcontinent N DM. But as we'll see in just a moment, these distinctions, these, these distributions are getting more and more blurred with time. Now, I should also say that the bottom three there, IV and MDM are all metallic types. K PC and OXR 48 are not metalite types. They're sea inside. I don't want to go into the chemistry of that, but it is important for what we're going to talk of today because these, these carbapenemases, the KP CS sa 48 and the three metals are really very different from one another. A PC, very poor spectrum enzyme only really temocilline and Cedrol are stable and til is only available in a very few countries. It is inhibited by avibactam and vib bacta ox of 48. It's a penicillinase, not a cephalosporinase that also attacks carb dependence. It's weakly inhibited by Avibactam. Not that it really matters because ceftazidine is stable anyhow. So if you've got an ice that with K PC or OX of 48 it's typically susceptible to ceftazidine plus avibactam. If it's got K PC, it's susceptible to meropenem, vib bact and imipen velib as well. But if it's got a metal, it's resistant to those, shall I call them established? Newer betalactam inhibited combinations on the other hand, the metal producer is typically susceptible still to confederal and a tram is stable to metanol beta lactamases. Now, the catch is that most metanol betalactamase producers have also got ESB LS that make them resistant to AZ triac metals, not inhibitors, as I say by a bact vac or, or, or VAC. However, they are inhibited by zinc chelators and things that bind up the zinc enzymes that these metals depend upon EDTA and dipicolinic acid. Now, I've said that although the tunnel carbapenemase is only dominated in a few countries that are widespread elsewhere and here's the evidence. And I regret, I can't find slides that go be uh that maps that go beyond 2018. But you can see that even by then even pre panic metals and N DM in particular were distributed over a wide range of countries. And here's the map for N DM endemic in India Pakistan, Bangladesh. But outbreaks reported across most of the world and even when there hasn't been an outbreak recorded at least sporadic occurrence, these things are widespread, you're likely to encounter them. So when do you recognize that bacteria are carbapenemase producers? Whether they've got an MBL or uh uh or, or a non metallic enzyme such as K PC or OXY 48. The usual point is here, you have a susceptibility test and you look at the result and say, oh dear, this bug is resistant to carbapenem. Why is it resistant occasionally you'll see e evidence elsewhere. Sometimes, for example, you use screening media for carbin resistance, for example, on rectal swabs to inform infection control, you plate bacteria onto carin containing chromogenic media 24 hours after specimen, you recognize there were colonies coming up, the patients colonized or perhaps you did have a clinical specimen infected by carapin resistant organisms. But and just occasionally you might have run um a specimen, for example, respiratory secretions on a syndrome with PCR something like a curets, er Univera or a bio film array that looks for the genes that encode caras along with markers for various different pathogens directly that say the respiratory secretion. And I will briefly talk about this at the end. But the common situation, as I say is the middle one, you've got the specimen from the patient. You've grown a bug, you've done a susceptibility test and OD E you've got a bug that's resistant to carpal PMS based on a disc or a vitech or whatever test. No, if it's got a metallo carpene bases, it's usually very clearly resistant to carpene. Remember I've said that the commonest metallo is this one ND N, New Delhi metallo. And these are just the M I CS of meropenem for isolate and bacteral with various different carbapenemases. And you see that the red, the DM is up at the high M IC end of the scale. It's OXY 48 produces bacteria with a non metalloenzyme that are often the hard ones to recognize because they still say stay relatively susceptible. The ones with DM and other materials like VIM and, and I are usually pretty obviously resistant. Sometimes from the immediate susceptibility testing. You can get a strong pointer, what I call the rule in that the bacteria very likely have a maternal carinate. Remember I said that a tri was stable to uh metall car penas. So if you're looking at your disc results or your um vitech results, I'm showing you that the, that the bug by bug. I mean, here, I'll say a few words on pseudomonas, but it's more difficult if your bug is resistant to carbapenems and to modern generation CPS except Cedol, but susceptible to a Tron. Now, then you can be pretty certain it's got a metal carbapenemase producer. Unfortunately, though many MBL producers are also resistant to A three and M because besides the metallo enzyme, they've also got an ESB L. You also, if you're testing the newest antibody combinations, you might have got another pointer because if you enter bacter valis is resistant to Ceftazidine, avibactam, but susceptible to Ke Col and to Aztrin avibactam. Again, it's probably got an MBL. The point is that Ceftazidine is destroyed by MBL S and extended spectrum betalactamase and avibactam will only inhibit the ESB L. So the Ceftazidine II is still being destroyed by the metalloenzyme and the bugs resistant to Ceftazidine, avibactam as three M avibactam. Well the Avibactam has inhibited uh the ESB L, the M, the Metanol car pens, the MBL can't attack the S3 and M. So you've got susceptibility. So if either of these apply, then you can be pretty darn sure that you're looking at a Metanol car pens producer. On the other hand, there will rule out again if you're testing some of these newest beta laptops, if your bug was resistant to IPM or to meropenem, and it's become very susceptible to imipen melitta or meropenem vaborbactam. Those are really strong pointers to AK PC carbate and if it was resistant to carbapenems, but it was still susceptible to third generation cins as well as to A three and M, then that's a strong pointer to an ox of 48 carbapenemase without an ESB L or one of the really rare carbapenemases like IMI. But the common situation is you're not quite sure from the susceptibility tests, whether it's an MBL or some other CARB PMA S. So perhaps you want to do additional tests and I'll run through now, microbiological tests which is essentially carbapenem susceptibility with and without inhibitors polymerase chain reaction. Pcr immunochromatographic methods, later flow tests and biochemical methods. Some of these are home blue but most of them can be got commercially. So, microbiological confirmation of an MBL producer. Remember I've said that a metal betalactamase, an MBL requires zinc for its activity. So if you pull away the zinc with a chelator ethylenediaminetetraacetic acid or dipicolinic acid, then you inactivate the enzyme. So you can do disc tests one disc with IMIPEN alone and a second disc with Imipen Co EDTA. And you can buy these things as combination discs from various suppliers or um a AAA strip type test with IMIPEN alone at one end and IMIPEN plus inhibitor at the other end. And so here we've got IMIPEN alone, no zone, big zone with EDTA present. And here the M IC was what about 16 mg per liter, imipen alone and less than 1 mg per liter with the inhibitor that strong synergy in the presence of EDT or dipicolinic acid. Those are positive results. Um The benefits of this methodology, it's cheap. It's fairly convenient. It will detect any type of A MBL. The rare ones like Sao Paulo Nel, which is common in pseudomonas in Brazil but extremely rare elsewhere. Not just, it won't just detect the big big three Nitel types, detect any others as well. The catches if you're doing this subsequent to the main susceptibility testing, it's added a day to your turnaround and it doesn't work very well with pseudomonas, aeruginosa. The problem with pseudomonas is that its outer membrane is stabilized by calcium and magnesium irons and EDTA strips those away. It permeabilise the pseudomonas to the imipen. So you start getting false positive results for pseudomonas that haven't got a metalloenzyme and you sometimes get a weak false positive with Acin Bacter Baumannii where most carbapenem resistance is due to um serine enzymes up to 23 and such like which are stabilized by zinc. Zinc's not critical to their catalytic activity, but they're stabilized by it. So again, you can get a false positive result. So these things are fine with enterobacterales. But if you apply them to non fermenters, just be careful. Weak results are probably false positives. PCR, I'm sure I hardly need explain the principles of PCR to anybody. Nowadays, um Various PCR kits are, are marketed for Carpinea detection, mostly as aids to infection control using rectal swabs. But you can also apply them to the bacteria that, that you, you've grown er from the patient. They normally seek the big five carbapenemases, including the big three metal types that should read in. And MDM obviously you require a PCR machine, but those are increasingly available in laboratories and the good news is you get a result within two hours, they work for pseudomonas just as well as for enterobacterales. But of course, they only find the carbapenemases they seek and that might be a problem if for example, you're in Brazil where SPM the tunnel carbapenemase is prevalent in pseudomonas, you won't find it because the PCR isn't looking next one's immunochromatography. Now, everybody's familiar with these strips for looking for COVID virus in, in patients or, or in themselves. But the principle is perhaps less familiar. So just to explain it, we've got a suspension of the bacteria in a buffer. The um bacteria are lazed in the buffer, they're then dropped onto the end of the strip that little well, and then they move onto this pad here where there are antibodies to the carbapenemase labeled with a colored marker. Usually something that's, that's, that's a flow of four or, or, or just a color of four. And then the um the now labeled carbapenemases are drawn across a pad, there's a wick at this end that pulls the liquids across and it pulls across lines of antibodies to specific carbapenemases. So if the specific carbapenemase is pleasant in the sample, then you get a sharp colored line as here and I apologize for the fuzziness of the photo, but that should read M and here in this sample there was an MDM, New Delhi maternal car ma and we know the tests work properly because we've got a sharp control line as well. So that's the immunochromatographic lateral flow method. Final method you might be thinking of using is a biochemical test to detect carbon pinas. And the common one here is the acidometric or it's sometimes called the Carba NP test. After the folks who are the old acidometric test to apply specifically to detecting carpene what's going on is as our carin is hydrolyzed, the ring is opened that creates acidity which is sufficient to treat phenol lead indicator from lead to yellow. Now again, you can, you can get this commercialized as a, as a little test to detect carbapenemases in general lies the bacteria that carbapenem resistant. You pop them into the er carbapenem indicator mix and you look and see the indicator trich yellow. But if you do the test with EDTA present, then if it's a metal type, then you get a ne negative result with the EDTA present. But a positive result without the EDTA was if this was a A PC or an OX of 48 you'd get a PO positive result regardless of EDTA. So the test can be adapted to specifically detect the tunnel carpal pens. You can also and I'm not going to go into details, detect that hydrolysis product by moldy tough. And again, there are are kits marketed to do so. And again, you could add EDTA and see if it inhibits the hydrolysis or you can use as in the so called carbapenemase inactivation method. You can use a microbiological indicator to see if the carpene is inactivated or not. So, what you would do is you would make a suspension of bacteria, add a carpene disc, parallel suspension, Carbapenem disc with DT. And you see, and then after a couple of hours you transfer that carpene disc to agar seeded with indicator organism. If the carbapenem has been inactivated, your indicated bug will grow up to the disc. And if the EDTA stops the carbin being activated, then it's a metal of carbapenem. As for which tests best well, depends on cost, depends where you are what you have available. How important you rate speed. PCR and immunochromatography obviously give you the fastest results. Carbo NP is also pretty quick. Um Whether you able to guess the carb a type from the primary susceptibility test. Um What throughput's required? If you're doing a lot of tests, then an automated PCR is a lot more convenient and keep loading lateral flow strips. But if you've only got one or two to do the lateral flow is more convenient, it doesn't require specialized equipment. And again, whether you're concerned with rare carpet pens. Now, I wanted also to touch on much faster carbapenemase detection and the tunnel carbapenemase detection directly from clinical specimens. Two marketed systems here, the Curatus uno and the uh the emeria bio fire film of a pneumonia panel. What you're doing here if you take a respiratory specimen from the patient sputum, uh bronchial alveolar lavage, endotracheal asper, you load that onto a multiplex PCR which goes into the machine and within uh about two hours or less than two hours in this case and about six hours. In this case, you get a result out detecting markers for the common gens of hospital and ventilator pneumonia and also critical resistance genes including those for the big five carpal penas, including the big three metallo types. And here and published in thorax is a study we did comparing what happened when the same respiratory specimens were put through these machines and put through the hospital's diagnostic laboratory. So here in 72 hours, the routine diagnostic lab had got the, the specimen contained a pseudomonas very multi resistant and it had a carbapenem. They still didn't know which carate, but the biofire had the result pseudomonas. So metalloenzyme in two hours and the curetis Univ had it in six hours and also told us that the bug was likely to be resistant to fluoroquinolones. And that somewhere in that respiratory secretion, there was some other bug. It wasn't like to be the pseudomonas with an um big er, er macrolide resistance. So they very rapid results. I'm finding a maternal carbapenamase producer. What can you do? Well, adopt appropriate therapy, er Azonam, avibactam, certainly against Enterobacter. Ri think a Tron is a rather marginal drug against pseudomonas and it's not active against Acinol would be another option. Er, colistin, of course, the classical agent and then things like Fosphomycin Rcin Tigecycline may have some activity probably better as combination agents and UCAS at least only now has great points for them against E coli not pseudomonas in terms of infection control. I don't think there's any big distinction between the carbapenemases and producers of other carbapenemases. You should do your utmost to prevent spread among patients. There's lots of good advice in national guidelines. Um Wilson et al the UK guidelines. If you want to look at them, there may be a reporting requirement to public health authorities, you know the rules locally. What I would say is that there has been less association of maternal carbapenamase producers with epidemic strains than with K PC which has associated. Remember that's a non maternal carb which has associated itself with a globally successful Klebsiella strain sequence type 258 which is dominant in some countries and which has also gone in to hypervirulent veins. So about that, uh so much more association with K PC with really disturbing strains from an infection control point of view than with metallics. But in my final moment, let me touch on one. I won't call it disturbing but intriguing possibility which is that we might be overrating the significance of metal enzymes. They may look worse in vitro than they are in vivo. Now, I stress, I don't say that's the case but it's possible the point to this that to function a metalloenzyme, a metal carbapenemase need zinc and in the body zinc is tightly bound. The concentration is 15 times lower than in routine susceptibility test media. And we know from our EDTA synergy test that these enzymes don't work so well, they don't give resistance if they can't get zinc. Our EDTA was binding zinc up and take it away. So that begs the question. Can these things function properly in vivo? Well, here is a case series study by David Nicolaus group. So very well respected group in the United States. 58 patients who have bacter bacteremias due to MDM producing half of them got active therapy. So this was this was Ceftazidine aact given together with Azonam. So effectively Aztreonam, Aam and 29 who got ineffective, inactive initial empiric therapy, usually Meriem sometimes alone, sometimes in combination. But after 3 to 5 days, once the car a producer was recognized, this was generally switched to active therapy, but it's accepted that in a a bacteremic patient, early active therapy is desirable for good outcomes but actually, no, no significant difference in outcomes. The numbers just bounce about. Does it prove that these enzymes are not so bad in vivo as in vitro, I don't know it points that way. But you could say, well, this arm, there were a lot more urosepsis cases and in this um far more bacteremia origin and far more, forgive me pneumonia origin cases. And we know that sis is associated with better outcomes than with, than the bacteremia associated with a pneumonia. So it's a pointer, I think rather than definitive, there is a final point and that's the metallo enzymes aren't or equal. Now, look at this number KD, the dissociation constant. This isn't a chemistry lecture, but the lower this number is the tighter the enzyme binds the zinc. So the common MDM enzyme is MDM one, it's got a dissociation constant of one micromole. And if we move to low zinc, it's down to 1% of normal activity. But MDM five and MDM seven are now quite common variants. They've got substantially lower dissociation constants. They bind the zinc be more strongly and they're more active at low zinc concentrations. And N DM 15 is rare, but it binds even tighter still and retains even more of its activity traps. Just traps. N DM is evolving to be more efficient at giving l high activity resistance in the zinc start conditions of the body. And if that's really important, then either we're going to have to tweak those detection tests, do them with different EDTA concentrations, or we're going to have to sequence er, the er suspected Carbenin a producers. So, ladies and gentlemen, what I've tried to show is that carbapenamase producers are a growing problem and that the tunnel car pen ma s producers are widespread and that they're dominant as carbapenamase producers in some countries, particularly in South Asia. The common one N DM confers very obvious carbapenem resistance in vitro. Now, depending on what drugs you test. First line, you might be able to make a very good guess that an ice that has got a metallic carbon Penina, if for example, it's susceptible to a three and um avibactam, but resistant to ceftazidine, avibactam, it's an bacteral, that's a very good pointer that it's got a metallo enzyme. But if you're not sure you can go on to do further tests and we talked about Imipen and EDTA synergy, PCR, immunochromatography or biochemical methods, the carer NP with and without EDTA added, you can also detect metal carbon pens producers directly in respiratory secretions using one multiplex er er syndromic panels such as such as the, the Biot fire. And that gives you a very swift result. There are still concerns, doubts I would say about the clinical significance of metol carbapenemase producers in that they may struggle to acquire zinc in infection. But at least for the time being, it would be a very, very brave clinician who simply said, oh, it's only a metalloenzyme. I can ignore it. I think until insignificance is proved, one must treat the er, patient and treat the detection of such an enzyme as very significant. Thank you.