1 00:00:16,420 --> 00:00:17,020 PROFESSOR: OK. 2 00:00:17,020 --> 00:00:19,580 Let's get going here. 3 00:00:19,580 --> 00:00:23,410 So this week I'll be talking about bacteria and viruses. 4 00:00:23,410 --> 00:00:26,380 And these are really significant topics, 5 00:00:26,380 --> 00:00:29,260 because I think it's something that we often 6 00:00:29,260 --> 00:00:32,229 don't think about the magnitude of the problems 7 00:00:32,229 --> 00:00:35,590 and what kind of crises we're approaching 8 00:00:35,590 --> 00:00:37,750 with respect to the therapeutic treatment 9 00:00:37,750 --> 00:00:39,252 of infectious disease. 10 00:00:39,252 --> 00:00:40,960 So what I want to try and get home to you 11 00:00:40,960 --> 00:00:45,400 this week is the variety of different microorganisms 12 00:00:45,400 --> 00:00:48,490 that threaten our health, and just 13 00:00:48,490 --> 00:00:50,440 talk to you about the sorts of issues 14 00:00:50,440 --> 00:00:54,520 that are really prominent in the news concerning resistance 15 00:00:54,520 --> 00:00:56,170 to therapeutic agents. 16 00:00:56,170 --> 00:00:59,000 But in order to do that, we've got to meet some bacteria, 17 00:00:59,000 --> 00:01:01,810 meet some viruses, and understand 18 00:01:01,810 --> 00:01:04,989 that some of their lifestyles, their mechanisms, 19 00:01:04,989 --> 00:01:08,890 so that we can understand what kinds of agents 20 00:01:08,890 --> 00:01:12,790 are used and developed to try to mitigate these diseases, 21 00:01:12,790 --> 00:01:15,190 because it's only through a molecular mechanistic 22 00:01:15,190 --> 00:01:19,300 understanding of the life cycles of viruses and bacteria 23 00:01:19,300 --> 00:01:23,410 that we can understand how many of these therapeutic agents 24 00:01:23,410 --> 00:01:26,450 work and what may be happening in resistance development. 25 00:01:26,450 --> 00:01:32,410 Now I find this particular slide a little daunting, 26 00:01:32,410 --> 00:01:35,770 but I want to point out to you that it concerns the world's 27 00:01:35,770 --> 00:01:37,450 deadliest animals. 28 00:01:37,450 --> 00:01:40,660 So we worry a lot about tigers, and sharks, 29 00:01:40,660 --> 00:01:45,250 and things like that, nasty poisonous snakes, 30 00:01:45,250 --> 00:01:48,550 bites from dogs with rabies, and so on. 31 00:01:48,550 --> 00:01:52,030 I'm going to leave this black bar here, sort of unmentioned. 32 00:01:52,030 --> 00:01:54,760 I don't know what year this is, but if we talk about daunting, 33 00:01:54,760 --> 00:01:56,140 that's pretty serious. 34 00:01:56,140 --> 00:02:00,220 And then the biggest killer on this screen is the mosquito. 35 00:02:00,220 --> 00:02:02,560 But it isn't actually the mosquito, 36 00:02:02,560 --> 00:02:05,500 it's the protozoal microorganisms 37 00:02:05,500 --> 00:02:09,039 that the mosquito carries from one person to another 38 00:02:09,039 --> 00:02:14,950 that really make that such a serious consideration. 39 00:02:14,950 --> 00:02:19,180 But what's not here are all the bacteria and viruses that 40 00:02:19,180 --> 00:02:21,780 actually are far more serious. 41 00:02:21,780 --> 00:02:23,230 And the numbers on the next stage 42 00:02:23,230 --> 00:02:28,100 will show you just quite how shocking these numbers are. 43 00:02:28,100 --> 00:02:31,360 If you're interested in infectious disease 44 00:02:31,360 --> 00:02:36,610 as a field, because I think anyone going towards MD, 45 00:02:36,610 --> 00:02:39,625 MD/PhD infectious diseases, it really 46 00:02:39,625 --> 00:02:42,940 is a critical area that we have to get to grips with. 47 00:02:42,940 --> 00:02:45,680 There are not enough vaccines in the world. 48 00:02:45,680 --> 00:02:49,900 There is not enough treatment with a very microbe 49 00:02:49,900 --> 00:02:53,640 specific anti-infective agents. 50 00:02:53,640 --> 00:02:57,203 So I encourage you to look at the CDC. 51 00:02:57,203 --> 00:02:58,870 There's a few other places where there's 52 00:02:58,870 --> 00:03:03,730 loads of information collated, such as the NIAID, which 53 00:03:03,730 --> 00:03:07,000 is the NIH Center for Infectious Disease, 54 00:03:07,000 --> 00:03:08,710 and the World Health Organization. 55 00:03:08,710 --> 00:03:12,350 So there's lots of places where you can find stuff out. 56 00:03:12,350 --> 00:03:14,770 So what we're going to be talking about in the next three 57 00:03:14,770 --> 00:03:20,710 classes are our smallest enemies, things like bacteria, 58 00:03:20,710 --> 00:03:24,970 fungal infections from things like yeast or Aspergillus, 59 00:03:24,970 --> 00:03:28,780 which would cause candida and aspergillosis. 60 00:03:28,780 --> 00:03:31,370 Protozoal disease we won't mention, 61 00:03:31,370 --> 00:03:33,010 but those are the types of diseases 62 00:03:33,010 --> 00:03:37,540 that are carried by things like ticks, mosquitoes, 63 00:03:37,540 --> 00:03:39,520 tsetse flies. 64 00:03:39,520 --> 00:03:41,990 We think of those as the infectious agent, 65 00:03:41,990 --> 00:03:46,630 but it's really what those organisms carry and cause 66 00:03:46,630 --> 00:03:49,060 the spread of disease that's important there. 67 00:03:49,060 --> 00:03:52,420 And we won't either talk about prion diseases, which 68 00:03:52,420 --> 00:03:55,090 are the diseases that don't involve 69 00:03:55,090 --> 00:03:57,220 an infectious microorganism, but are 70 00:03:57,220 --> 00:04:00,700 believed to be spread from protein to protein 71 00:04:00,700 --> 00:04:05,080 through the nucleation of new prions from existing prions. 72 00:04:05,080 --> 00:04:07,900 What we'll focus on in the first class 73 00:04:07,900 --> 00:04:12,790 is bacteria and in the other two on viruses, 74 00:04:12,790 --> 00:04:15,550 with an eye to looking at antibiotics 75 00:04:15,550 --> 00:04:19,730 and antiviral agents, how they work, where they go wrong. 76 00:04:19,730 --> 00:04:22,120 And this is where the numbers get fairly shocking. 77 00:04:22,120 --> 00:04:25,210 So for example, bacterial infections 78 00:04:25,210 --> 00:04:29,320 of the lower respiratory tract, that's deep in the lungs, 79 00:04:29,320 --> 00:04:31,330 cause 4 million deaths a year. 80 00:04:31,330 --> 00:04:35,530 Think back to the numbers you just saw on that first slide. 81 00:04:35,530 --> 00:04:39,340 These are things like strep pneumoniae, 82 00:04:39,340 --> 00:04:41,140 Klebsiella pneumoniae. 83 00:04:41,140 --> 00:04:42,970 They're called pneumonias because they're 84 00:04:42,970 --> 00:04:46,150 infectious diseases of the lung, but the organisms 85 00:04:46,150 --> 00:04:50,650 that cause them are of the Streptomyces, and Klebsiella, 86 00:04:50,650 --> 00:04:53,950 and Staphylococcus aureus specifically. 87 00:04:53,950 --> 00:04:57,820 But there are others that cause lung infections and lower 88 00:04:57,820 --> 00:04:59,840 respiratory disease. 89 00:04:59,840 --> 00:05:03,580 These are particularly troublesome in areas 90 00:05:03,580 --> 00:05:06,520 where the atmosphere is bad. 91 00:05:06,520 --> 00:05:12,250 In big cities where there's a lot of insult from emissions 92 00:05:12,250 --> 00:05:14,470 and such that make the lungs weaker, 93 00:05:14,470 --> 00:05:16,970 then these sorts of organisms can really 94 00:05:16,970 --> 00:05:21,420 take a hold more readily, so they are more serious. 95 00:05:21,420 --> 00:05:23,510 There are many, many microorganisms 96 00:05:23,510 --> 00:05:25,650 that cause pneumonias. 97 00:05:25,650 --> 00:05:27,500 And sometimes it's a real problem 98 00:05:27,500 --> 00:05:31,370 to track down the precise microorganism, which 99 00:05:31,370 --> 00:05:35,540 makes the issue of treatment really difficult, really 100 00:05:35,540 --> 00:05:36,590 challenging. 101 00:05:36,590 --> 00:05:40,160 So I'm going to talk in a minute about absolute identification 102 00:05:40,160 --> 00:05:44,090 of infectious agents, so we can do better jobs of specifically 103 00:05:44,090 --> 00:05:47,410 targeting the causative agents. 104 00:05:47,410 --> 00:05:50,660 Diarrheal disease-- 2 million deaths. 105 00:05:50,660 --> 00:05:53,510 These are organisms like Campylobacter jejuni 106 00:05:53,510 --> 00:05:55,670 and Salmonella enterica. 107 00:05:55,670 --> 00:06:02,780 We tend to have these crises, because romaine my 108 00:06:02,780 --> 00:06:06,060 is contaminated with infection. 109 00:06:06,060 --> 00:06:09,330 There are very few deaths in the developed world. 110 00:06:09,330 --> 00:06:11,840 We get down to that very quickly, 111 00:06:11,840 --> 00:06:14,780 say stop eating Romaine lettuce until we figure out 112 00:06:14,780 --> 00:06:16,310 what's going on here-- 113 00:06:16,310 --> 00:06:17,730 very, very few. 114 00:06:17,730 --> 00:06:19,970 But once again, in the developing world, 115 00:06:19,970 --> 00:06:21,770 these can run rampant. 116 00:06:21,770 --> 00:06:25,250 And they can grab small children and older people who 117 00:06:25,250 --> 00:06:29,210 are already compromised, already a little bit not quite 118 00:06:29,210 --> 00:06:33,260 with strong immune systems, and people generally 119 00:06:33,260 --> 00:06:36,620 die of dehydration, because these diseases really 120 00:06:36,620 --> 00:06:37,940 hit the GI tract. 121 00:06:37,940 --> 00:06:41,240 It causes leaking us in the GI tract and really, really 122 00:06:41,240 --> 00:06:42,870 serious diarrheal disease. 123 00:06:42,870 --> 00:06:44,620 So those are the bad boys there. 124 00:06:44,620 --> 00:06:47,660 But once again, there are many others. 125 00:06:47,660 --> 00:06:51,980 Tuberculosis is yet another really serious 126 00:06:51,980 --> 00:06:56,600 infectious disease caused by Mycobacterium tuberculosis, 127 00:06:56,600 --> 00:07:02,860 that's the main one of the mycobacteria that is a threat. 128 00:07:02,860 --> 00:07:06,510 It used to be called consumption in the old days, 129 00:07:06,510 --> 00:07:10,640 because people almost looked consumed by the disease. 130 00:07:10,640 --> 00:07:13,550 They would just get thinner and thinner. 131 00:07:13,550 --> 00:07:16,190 Literally it was a wasting disease. 132 00:07:16,190 --> 00:07:19,160 People would be sent up into the mountains of Switzerland 133 00:07:19,160 --> 00:07:21,860 to try to recover from consumption, 134 00:07:21,860 --> 00:07:24,860 to where the air is clearer and cleaner, 135 00:07:24,860 --> 00:07:27,410 and maybe hope that they can recuperate. 136 00:07:27,410 --> 00:07:30,410 But TB-- look at these numbers. 137 00:07:30,410 --> 00:07:34,640 In 2015 there were almost 10 million new cases. 138 00:07:34,640 --> 00:07:38,950 There are about 1.2 million deaths from TB. 139 00:07:38,950 --> 00:07:42,800 A serious situation with TB is that it's often 140 00:07:42,800 --> 00:07:46,880 found co-infecting with the HIV virus, where you just 141 00:07:46,880 --> 00:07:48,200 can't fight the TB. 142 00:07:48,200 --> 00:07:52,430 So eventually, if you're infected with the HIV virus, 143 00:07:52,430 --> 00:07:56,630 it's the TB that gets you due to the weakening caused 144 00:07:56,630 --> 00:07:58,580 by the infection with TB. 145 00:07:58,580 --> 00:08:00,710 So these numbers are shocking in light 146 00:08:00,710 --> 00:08:03,650 of the numbers I showed you on the previous slide, right. 147 00:08:03,650 --> 00:08:08,210 Look at these numbers if you go to snakes and things like that. 148 00:08:08,210 --> 00:08:10,430 They're meaningless numbers compared 149 00:08:10,430 --> 00:08:13,880 to infectious diseases. 150 00:08:13,880 --> 00:08:22,160 So now, and I'm going to talk to you about the origins of this, 151 00:08:22,160 --> 00:08:26,360 many, many infectious agents that we thought we 152 00:08:26,360 --> 00:08:27,680 had conquered-- 153 00:08:27,680 --> 00:08:29,180 we thought we could take care of it. 154 00:08:29,180 --> 00:08:33,004 You just take this course of antibiotics and you're off, 155 00:08:33,004 --> 00:08:34,100 you're set. 156 00:08:34,100 --> 00:08:37,880 But now, because of the rapid mutation rates 157 00:08:37,880 --> 00:08:41,570 in bacteria and viruses, certain pathogens 158 00:08:41,570 --> 00:08:44,420 have completely worked out mechanisms 159 00:08:44,420 --> 00:08:46,520 to escape therapeutic agent. 160 00:08:46,520 --> 00:08:48,680 And I'm going to talk to you about those mechanisms 161 00:08:48,680 --> 00:08:50,690 towards the end of this class. 162 00:08:50,690 --> 00:08:53,780 So basically you can dose a person one day 163 00:08:53,780 --> 00:08:56,750 with a normal dose of an antibiotic agent, 164 00:08:56,750 --> 00:09:00,620 and then 10 months later that normal dose or 10 times 165 00:09:00,620 --> 00:09:03,740 or 100 times that dose stops working. 166 00:09:03,740 --> 00:09:04,940 Why is that? 167 00:09:04,940 --> 00:09:08,870 It's due to resistance acquisition due to rapid cell 168 00:09:08,870 --> 00:09:14,360 division and mistakes made on replication and transcription, 169 00:09:14,360 --> 00:09:17,150 that then may one in a million times 170 00:09:17,150 --> 00:09:20,510 confer an advantage on the microorganism. 171 00:09:20,510 --> 00:09:24,610 All of a sudden the drugs don't work anymore. 172 00:09:24,610 --> 00:09:30,530 The WHO and various community notice boards 173 00:09:30,530 --> 00:09:35,320 call this set of infectious agents the escape pathogens. 174 00:09:35,320 --> 00:09:37,790 It helps us remember which ones these are, 175 00:09:37,790 --> 00:09:41,480 because these are pathogens that escape treatment, 176 00:09:41,480 --> 00:09:43,880 because they've developed resistance 177 00:09:43,880 --> 00:09:46,310 to multiple drug cocktails. 178 00:09:46,310 --> 00:09:49,550 So commonly, when someone has a particular disease 179 00:09:49,550 --> 00:09:52,460 they don't take one drug, they take two or three 180 00:09:52,460 --> 00:09:56,210 to hit lots of pathways at once in the hope 181 00:09:56,210 --> 00:09:58,610 that resistance won't develop fast. 182 00:09:58,610 --> 00:10:01,820 But the escape pathogens have collectively 183 00:10:01,820 --> 00:10:04,910 acquired resistance to several antibiotics, 184 00:10:04,910 --> 00:10:07,460 meaning there's no good treatment. 185 00:10:07,460 --> 00:10:12,440 So the letters of escape stand for Enterococcus faecium, 186 00:10:12,440 --> 00:10:17,030 Staph aureus, Klebsiella pneumoniae, Acinetobacter 187 00:10:17,030 --> 00:10:20,210 baumannii, Pseudomonas aeruginosa, and some 188 00:10:20,210 --> 00:10:23,090 of the Enterobacter species. 189 00:10:23,090 --> 00:10:30,210 Some of these infectious agents are what result from-- 190 00:10:30,210 --> 00:10:32,550 I always say this wrong-- 191 00:10:32,550 --> 00:10:34,560 nosocomial infections. 192 00:10:34,560 --> 00:10:37,680 Does anyone know what those are? 193 00:10:37,680 --> 00:10:41,450 These are infections that people get in hospitals. 194 00:10:41,450 --> 00:10:44,280 So Tom Brady had a knee operation. 195 00:10:44,280 --> 00:10:47,730 He got an infection in his knee that came from the surgery, 196 00:10:47,730 --> 00:10:48,330 right. 197 00:10:48,330 --> 00:10:50,970 These are hospital acquired infections, 198 00:10:50,970 --> 00:10:56,370 because you can't sometimes clear an area enough, 199 00:10:56,370 --> 00:10:58,710 and there's infectious agents around. 200 00:10:58,710 --> 00:11:03,240 So Acinetobacter baumannii was dubbed the Iraqi Bug 201 00:11:03,240 --> 00:11:07,200 for many, many years, because the vets coming back from Iraq 202 00:11:07,200 --> 00:11:10,080 were going to military hospitals, 203 00:11:10,080 --> 00:11:13,230 and these were abundant with cases 204 00:11:13,230 --> 00:11:15,390 of Acinetobacter baumannii. 205 00:11:15,390 --> 00:11:18,900 So that moved on to the escape pathogen list. 206 00:11:18,900 --> 00:11:21,090 So these are things to watch out for. 207 00:11:21,090 --> 00:11:23,880 It's the reason that nosocomial infections-- 208 00:11:23,880 --> 00:11:25,590 I hope I'm saying it right, otherwise 209 00:11:25,590 --> 00:11:28,260 you're going to go off and Google it and realize 210 00:11:28,260 --> 00:11:29,470 I said it wrong. 211 00:11:29,470 --> 00:11:33,060 It's the reason why old school physicians wore bow ties 212 00:11:33,060 --> 00:11:35,640 and not ties. 213 00:11:35,640 --> 00:11:38,490 Can you imagine why? 214 00:11:38,490 --> 00:11:40,800 So if you're wearing a tie, which I seldom 215 00:11:40,800 --> 00:11:44,790 wear to be honest, and you're working over a patient, 216 00:11:44,790 --> 00:11:47,850 the tie can be the thing that carries the infection, 217 00:11:47,850 --> 00:11:50,520 because it gets closer to infected areas. 218 00:11:50,520 --> 00:11:52,140 This is old school stuff. 219 00:11:52,140 --> 00:11:55,230 And so originally the physicians wore bow ties 220 00:11:55,230 --> 00:11:58,300 in order to distinguish themselves as important people, 221 00:11:58,300 --> 00:12:02,460 but not to wear ties that might carry infectious agents. 222 00:12:02,460 --> 00:12:04,530 That's sort of a scary thing. 223 00:12:04,530 --> 00:12:08,460 So with all this said, let me just 224 00:12:08,460 --> 00:12:11,760 lead you in to talk about bacteria antibiotics 225 00:12:11,760 --> 00:12:13,600 and resistance development. 226 00:12:13,600 --> 00:12:20,250 So we often name bacteria somewhat by their shape. 227 00:12:20,250 --> 00:12:25,110 So the long rod shaped ones are cocci. 228 00:12:25,110 --> 00:12:26,580 The round ones are cocci. 229 00:12:26,580 --> 00:12:31,710 The rod ones, whatever they are, come on one of you. 230 00:12:31,710 --> 00:12:34,680 And then what did the rod shape ones? 231 00:12:34,680 --> 00:12:35,990 Bacilli. 232 00:12:35,990 --> 00:12:36,870 I had a blank moment. 233 00:12:36,870 --> 00:12:39,510 So the rod shaped ones are bacilli. 234 00:12:39,510 --> 00:12:41,160 And then there's some others that 235 00:12:41,160 --> 00:12:44,550 have a different morphology like Campylobacter jejuni 236 00:12:44,550 --> 00:12:46,860 that have kind of a corkscrew shape. 237 00:12:46,860 --> 00:12:50,010 And that's thought to be important in their motility, 238 00:12:50,010 --> 00:12:51,990 digging through the mucous layers 239 00:12:51,990 --> 00:12:53,830 in the epithelial layers. 240 00:12:53,830 --> 00:12:56,933 So here I show you several shapes of bacteria. 241 00:12:56,933 --> 00:12:58,350 And I'm just going to, once again, 242 00:12:58,350 --> 00:13:01,950 reinforce what diseases some are associated 243 00:13:01,950 --> 00:13:05,430 with and some other diseases that you might be surprised by. 244 00:13:05,430 --> 00:13:08,180 So yes, we know about salmonella and the E. 245 00:13:08,180 --> 00:13:09,750 coli and food poisoning. 246 00:13:09,750 --> 00:13:12,600 But Helicobacter pylori, which is 247 00:13:12,600 --> 00:13:15,690 one of these flagellated bacteria, 248 00:13:15,690 --> 00:13:18,900 can infect the stomach. 249 00:13:18,900 --> 00:13:21,000 It's often the cause of ulcers. 250 00:13:21,000 --> 00:13:24,120 So it's a causative agent of stomach ulcers, 251 00:13:24,120 --> 00:13:29,250 but that has in turn led to a considerable risk 252 00:13:29,250 --> 00:13:31,960 factor in stomach cancer. 253 00:13:31,960 --> 00:13:34,380 So what we thought was just an infection 254 00:13:34,380 --> 00:13:38,340 causes a constellation of other problems, including cancers. 255 00:13:38,340 --> 00:13:41,880 And more and more microbial agents 256 00:13:41,880 --> 00:13:47,510 are now associated with cancers, in particular the viruses. 257 00:13:47,510 --> 00:13:51,060 Neisseria, these come along with the sexually 258 00:13:51,060 --> 00:13:54,280 transmitted diseases such as gonorrhea. 259 00:13:54,280 --> 00:13:57,135 Neisseria meningitidis is the one that causes meningitis. 260 00:13:57,135 --> 00:14:02,700 It is a very, very often fatal infection of the meninges. 261 00:14:02,700 --> 00:14:06,420 Staph aureus lots of infections around the body, 262 00:14:06,420 --> 00:14:08,580 just gruesome things like cellulitis, 263 00:14:08,580 --> 00:14:11,310 wound infections, toxic shock. 264 00:14:11,310 --> 00:14:13,200 Streptococcal bacteria, I've already 265 00:14:13,200 --> 00:14:16,530 mentioned-- the pneumonias, and then Campylobacter. 266 00:14:16,530 --> 00:14:20,550 And now another complicated factor of infection-- 267 00:14:20,550 --> 00:14:26,490 so I talked to you about stomach ulcers and stomach cancer. 268 00:14:26,490 --> 00:14:30,210 Another thing that seems to be coming along with infections 269 00:14:30,210 --> 00:14:31,890 is autoimmunity. 270 00:14:31,890 --> 00:14:35,550 So in the last section of the class you heard about immunity 271 00:14:35,550 --> 00:14:38,880 and you also heard about tolerance, 272 00:14:38,880 --> 00:14:42,780 that we don't react to things that are ourselves, 273 00:14:42,780 --> 00:14:44,940 otherwise we'd be in deep trouble. 274 00:14:44,940 --> 00:14:47,850 Autoimmunity can suddenly pop up from 275 00:14:47,850 --> 00:14:50,670 certain bacterial infections, because bacteria 276 00:14:50,670 --> 00:14:54,450 tend to cloak themselves with unusual sugar 277 00:14:54,450 --> 00:14:56,850 polymers and other kinds of structures 278 00:14:56,850 --> 00:14:58,980 that the body doesn't really know what to do with. 279 00:14:58,980 --> 00:15:01,500 And in some cases they kind of mimic 280 00:15:01,500 --> 00:15:03,780 things that are in the human body. 281 00:15:03,780 --> 00:15:07,920 So they they are mimetics of normal structures 282 00:15:07,920 --> 00:15:09,330 in the human body. 283 00:15:09,330 --> 00:15:12,210 And the body just doesn't notice them at all. 284 00:15:12,210 --> 00:15:15,300 And then there are incidences where 285 00:15:15,300 --> 00:15:18,420 certain bacterial infections later on 286 00:15:18,420 --> 00:15:20,830 cause autoimmune disease. 287 00:15:20,830 --> 00:15:22,650 So a bacteria may come along. 288 00:15:22,650 --> 00:15:25,680 It may have something that looks kind of like something human, 289 00:15:25,680 --> 00:15:27,240 but not quite. 290 00:15:27,240 --> 00:15:30,150 The human body responds, develops antibodies, 291 00:15:30,150 --> 00:15:35,220 and then they cross talk back to aspects of our physiology. 292 00:15:35,220 --> 00:15:39,260 So Campylobacter jejuni is often a contaminant in poultry. 293 00:15:39,260 --> 00:15:41,240 It's a severe GI infection. 294 00:15:41,240 --> 00:15:44,690 But later on people get diseases such as Guillain-Barre, 295 00:15:44,690 --> 00:15:49,790 which is a neuropathy where the ends of your limbs 296 00:15:49,790 --> 00:15:52,310 become numb and non-functional. 297 00:15:52,310 --> 00:15:55,250 So there was a famous football player, 298 00:15:55,250 --> 00:15:57,110 the one they called "The Refrigerator," who 299 00:15:57,110 --> 00:16:01,160 had a serious case of Guillain-Barre resulting 300 00:16:01,160 --> 00:16:04,070 from very much an infectious disease, which 301 00:16:04,070 --> 00:16:07,810 converted into autoimmunity. 302 00:16:07,810 --> 00:16:10,660 So let's now look at antibiotic targets. 303 00:16:10,660 --> 00:16:13,510 And to look at antibiotic targets, 304 00:16:13,510 --> 00:16:16,300 I think the first clear place to look 305 00:16:16,300 --> 00:16:18,610 is at the bacterial cell wall. 306 00:16:28,610 --> 00:16:32,090 Now when we first started talking about prokaryotes, 307 00:16:32,090 --> 00:16:36,260 things that include bacteria, we talked about the fact 308 00:16:36,260 --> 00:16:38,690 that these single celled organisms 309 00:16:38,690 --> 00:16:44,270 have to have a robust cell wall to prevent osmotic shock. 310 00:16:44,270 --> 00:16:45,770 They have to have some kind of thing 311 00:16:45,770 --> 00:16:49,730 to keep them from taking up too much water 312 00:16:49,730 --> 00:16:53,420 and basically exploding because of osmosis. 313 00:16:53,420 --> 00:16:57,530 Water floods in to balance the salt concentrations. 314 00:16:57,530 --> 00:17:01,260 So they have a complex cell wall, 315 00:17:01,260 --> 00:17:09,500 which is made of a macro molecule called peptidoglycan 316 00:17:09,500 --> 00:17:12,650 And it's usually one word, but I want to just underline 317 00:17:12,650 --> 00:17:16,940 peptidoglycan because it's a fascinating polymer that's 318 00:17:16,940 --> 00:17:21,575 made up of peptides and linear carbohydrate polymers. 319 00:17:22,290 --> 00:17:25,190 So if you look at this typical bacterium, 320 00:17:25,190 --> 00:17:29,910 this is just a cartoon of the peptidoglycan. 321 00:17:29,910 --> 00:17:38,210 So it's a cross-linked polymer, we 322 00:17:38,210 --> 00:17:43,250 in one direction it has repeating carbohydrate units. 323 00:17:43,250 --> 00:17:46,910 I'm not drawing those complex hexose structures there. 324 00:17:46,910 --> 00:17:49,250 I'm just drawing it in cartoon form. 325 00:17:49,250 --> 00:17:53,755 And those are carbohydrates known as NAG and NAN. 326 00:17:57,890 --> 00:18:03,140 NAG is N-Acetyl Glucosamine. 327 00:18:03,140 --> 00:18:04,850 It's a hexose sugar. 328 00:18:04,850 --> 00:18:07,420 NAN is N-AcetylMuramic acid. 329 00:18:07,420 --> 00:18:09,890 It's another modified sugar. 330 00:18:09,890 --> 00:18:12,010 And on the one of those sugars, there 331 00:18:12,010 --> 00:18:19,190 is a reactive site that allows you to basically cross-link 332 00:18:19,190 --> 00:18:21,830 these polymers into a mesh work. 333 00:18:21,830 --> 00:18:26,150 So it's a feat of engineering to build this amazing polymer. 334 00:18:26,150 --> 00:18:29,870 It starts being built on the inside, on the cytoplasm. 335 00:18:29,870 --> 00:18:32,960 And then the components get flipped onto the other side 336 00:18:32,960 --> 00:18:35,660 of the cytoplasm of bacteria. 337 00:18:35,660 --> 00:18:38,120 Then they get polymerized in place 338 00:18:38,120 --> 00:18:41,780 to make this complex mesh work of a polymer 339 00:18:41,780 --> 00:18:46,190 that creates the rigidity of the bacterial cell wall. 340 00:18:46,190 --> 00:18:50,270 It's generically known as a peptidoglycan. 341 00:18:50,270 --> 00:18:53,720 Different bacteria have different peptidoglycans. 342 00:18:53,720 --> 00:18:56,240 There are several modifications that 343 00:18:56,240 --> 00:19:01,430 might be specific to particular bacterial sera 344 00:19:01,430 --> 00:19:03,110 But this is the generic structure, 345 00:19:03,110 --> 00:19:05,600 where you have a polymer that's built of sugars. 346 00:19:05,600 --> 00:19:07,910 You can recognize the sugar structure there 347 00:19:07,910 --> 00:19:11,690 going in one direction and the peptide component that 348 00:19:11,690 --> 00:19:15,350 cross-links across in order to make this mesh. 349 00:19:15,350 --> 00:19:18,350 And bacterial wall have different amounts of this, 350 00:19:18,350 --> 00:19:21,770 but it'll build up to a really strong, rigid mesh 351 00:19:21,770 --> 00:19:25,370 work that's permeable to things, small molecules and water. 352 00:19:25,370 --> 00:19:26,960 There is holes, and so on. 353 00:19:26,960 --> 00:19:29,600 But it creates a mechanical rigidity 354 00:19:29,600 --> 00:19:33,110 so that osmotic shock doesn't occur on the bacteria. 355 00:19:33,110 --> 00:19:34,160 Any questions about that? 356 00:19:34,160 --> 00:19:35,230 Does that makes sense? 357 00:19:35,230 --> 00:19:38,060 So that, in a sense, it's their exoskeleton, 358 00:19:38,060 --> 00:19:40,670 if you want to think about it like that. 359 00:19:40,670 --> 00:19:43,610 So the properties are rigid. 360 00:19:43,610 --> 00:19:46,610 Without it, the bacteria would suffer osmotic shock. 361 00:19:46,610 --> 00:19:52,160 And it's plenty permeable to allow 2-nanometer type 362 00:19:52,160 --> 00:19:57,048 pores in order for nutrients and water to go into the structure. 363 00:19:57,048 --> 00:19:57,715 [VIDEO PLAYBACK] 364 00:19:57,715 --> 00:19:58,965 - --have E. coli growing here. 365 00:19:58,965 --> 00:19:59,712 And it's living. 366 00:19:59,712 --> 00:20:00,920 You can see it start to grow. 367 00:20:00,920 --> 00:20:02,110 Here we add penicillin. 368 00:20:02,110 --> 00:20:03,568 We're going to see these bacteria-- 369 00:20:03,568 --> 00:20:06,440 PROFESSOR: These are bacteria, rod-shaped bacteria. 370 00:20:06,440 --> 00:20:08,495 - There wasn't any microphone on this, so-- 371 00:20:08,495 --> 00:20:10,870 PROFESSOR: And I'm going to ask you to just keep watching 372 00:20:10,870 --> 00:20:11,900 this kind of carefully. 373 00:20:11,900 --> 00:20:13,442 - There goes another one, boop, boop. 374 00:20:13,442 --> 00:20:14,300 [LAUGHTER] 375 00:20:14,300 --> 00:20:17,390 - Poking holes in the cell wall, boom, bacteria is dead. 376 00:20:17,390 --> 00:20:20,750 PROFESSOR: Look at some of the bacteria disappearing. 377 00:20:20,750 --> 00:20:22,160 All right, I guess 378 00:20:22,160 --> 00:20:25,970 [INTERPOSING VOICES] 379 00:20:25,970 --> 00:20:27,370 OK, then we're going to-- 380 00:20:27,370 --> 00:20:29,120 [INTERPOSING VOICES] 381 00:20:29,120 --> 00:20:31,237 So we're going to leave it. 382 00:20:31,237 --> 00:20:31,820 [END PLAYBACK] 383 00:20:31,820 --> 00:20:33,390 Let's go back one. 384 00:20:33,390 --> 00:20:35,630 OK, now what was that? 385 00:20:35,630 --> 00:20:40,430 OK, so I've told you bacteria would suffer osmotic shock 386 00:20:40,430 --> 00:20:42,980 without peptidoglycan. 387 00:20:42,980 --> 00:20:45,440 Those are bacteria that you see popping, 388 00:20:45,440 --> 00:20:49,490 as the person who was talking said, 389 00:20:49,490 --> 00:20:53,880 because the peptidoglycan cannot be made. 390 00:20:53,880 --> 00:20:55,910 There is an antibiotic that's added. 391 00:20:55,910 --> 00:20:59,990 It is penicillin that's added to the bacteria. 392 00:20:59,990 --> 00:21:04,370 And it stops-- as bacteria grow, they have to make a bunch more 393 00:21:04,370 --> 00:21:06,910 peptidoglycan, because if you're doubling, 394 00:21:06,910 --> 00:21:08,930 you've got to make twice as much peptido-- 395 00:21:08,930 --> 00:21:11,420 you've got to double the amount of peptidoglycan. 396 00:21:11,420 --> 00:21:13,880 If you have something that inhibits 397 00:21:13,880 --> 00:21:17,090 the peptidoglycan being made, you 398 00:21:17,090 --> 00:21:20,660 have a bacterium that's trying to stretch out what it has, 399 00:21:20,660 --> 00:21:23,310 it's not resistant to osmotic shock. 400 00:21:23,310 --> 00:21:26,030 And what you saw was the bacteria basically undergoing 401 00:21:26,030 --> 00:21:30,230 cell death via osmotic shock, pretty graphic, pretty visual. 402 00:21:30,230 --> 00:21:36,470 So penicillin was one of the first antibiotics 403 00:21:36,470 --> 00:21:38,660 that was described for the treatment 404 00:21:38,660 --> 00:21:40,850 of bacterial infections. 405 00:21:40,850 --> 00:21:43,980 And we'll go to the timeline of that in a moment. 406 00:21:43,980 --> 00:21:47,660 So when we talk about bacteria, the original definition 407 00:21:47,660 --> 00:21:54,920 of bacteria is in three different subtypes, 408 00:21:54,920 --> 00:22:06,030 gram-negative, gram-positive, and mycobacterial. 409 00:22:12,570 --> 00:22:16,500 This is actually the first way that people 410 00:22:16,500 --> 00:22:19,920 would take a look at your cell-- at the bacterial cells 411 00:22:19,920 --> 00:22:24,240 and diagnose roughly what kind of bacteria they were. 412 00:22:24,240 --> 00:22:27,480 Did they fall-- which of these broad families 413 00:22:27,480 --> 00:22:28,530 did they fall into? 414 00:22:28,530 --> 00:22:30,900 Because it would help in defining how 415 00:22:30,900 --> 00:22:33,550 you would treat the infectious disease. 416 00:22:33,550 --> 00:22:42,450 So I want to show you the difference between the cell 417 00:22:42,450 --> 00:22:47,220 wall of these various types of bacteria. 418 00:22:47,220 --> 00:22:51,030 And the truth is, if you have an infectious disease, 419 00:22:51,030 --> 00:22:55,770 your wish is, if you had to pick one of the three, 420 00:22:55,770 --> 00:22:58,150 that you have a gram-positive disease. 421 00:22:58,150 --> 00:23:00,870 And I'll explain why that is in a moment, 422 00:23:00,870 --> 00:23:05,130 because it's all to do with how drugs can get into the bacteria 423 00:23:05,130 --> 00:23:08,880 to inhibit vital functions in order that they die 424 00:23:08,880 --> 00:23:11,290 and they don't take over your system. 425 00:23:11,290 --> 00:23:14,860 So let's look first at gram-positive bacteria. 426 00:23:14,860 --> 00:23:15,760 They're shown here. 427 00:23:15,760 --> 00:23:18,720 This is a section of a bacterium. 428 00:23:18,720 --> 00:23:21,750 Gram-positive have a single cell wall. 429 00:23:28,430 --> 00:23:35,760 And they also have a thick layer of peptidoglycan. 430 00:23:38,490 --> 00:23:41,610 So they gain rigidity by basically 431 00:23:41,610 --> 00:23:45,750 having an extracellular thick layer of peptidoglycan 432 00:23:45,750 --> 00:23:47,310 coating them. 433 00:23:47,310 --> 00:23:49,080 There is a schematic of it here. 434 00:23:49,080 --> 00:23:51,420 So here would be the inner cell wall. 435 00:23:51,420 --> 00:23:53,760 And here would be the peptidoglycan, 436 00:23:53,760 --> 00:23:59,040 shown in orange and pale, buff-colored circles. 437 00:23:59,040 --> 00:24:02,010 So that would be where their peptidoglycan is. 438 00:24:02,010 --> 00:24:05,580 And then there are some other glyco conjugates that 439 00:24:05,580 --> 00:24:07,060 actually stick out beyond that. 440 00:24:07,060 --> 00:24:10,230 But there is only one cytoplasmic membrane. 441 00:24:10,230 --> 00:24:13,440 That's the standard double bilayer. 442 00:24:13,440 --> 00:24:17,550 And the peptidoglycan is quite thick, relatively, 443 00:24:17,550 --> 00:24:21,550 20 to 80 nanometers across. 444 00:24:21,550 --> 00:24:22,860 So that's how wide it is. 445 00:24:22,860 --> 00:24:25,200 And you can, if you've got a-- if you've 446 00:24:25,200 --> 00:24:27,330 stain a bacterium under a microscope, 447 00:24:27,330 --> 00:24:30,480 you would see that, the thickness of that wall, 448 00:24:30,480 --> 00:24:32,980 but the absence of a double wall. 449 00:24:32,980 --> 00:24:36,420 The gram-negative bacteria have a double wall. 450 00:24:43,920 --> 00:24:52,030 The inner membrane is pretty standard. 451 00:24:52,030 --> 00:24:54,600 It's just typical phospholipids. 452 00:24:54,600 --> 00:24:58,020 It looks like the inner cytoplasmic membrane 453 00:24:58,020 --> 00:25:01,230 of the gram-positive bacteria. 454 00:25:01,230 --> 00:25:05,440 And then it has an outer wall. 455 00:25:05,440 --> 00:25:09,000 So the inner membrane is typical. 456 00:25:09,000 --> 00:25:14,610 And then the outer wall has one leaflet 457 00:25:14,610 --> 00:25:16,320 that looks kind of normal. 458 00:25:16,320 --> 00:25:19,200 And then it has a second leaflet that's sort of decorated, 459 00:25:19,200 --> 00:25:21,180 honestly, like a Christmas tree. 460 00:25:21,180 --> 00:25:23,130 There is all kinds of things sticking out 461 00:25:23,130 --> 00:25:27,150 there that interact with hosts that they infect, and so on. 462 00:25:27,150 --> 00:25:30,180 And the space between the two walls 463 00:25:30,180 --> 00:25:44,280 is called the periplasmic space, because it's between. 464 00:25:44,280 --> 00:25:46,120 It's not the cytoplasm. 465 00:25:46,120 --> 00:25:48,480 It's what's called the periplasm. 466 00:25:48,480 --> 00:25:50,325 Now, what's interesting about these, 467 00:25:50,325 --> 00:25:54,270 the gram-negative bacteria, is they have quite a bit 468 00:25:54,270 --> 00:25:59,692 less peptidoglycan, only about 7 to 8 nanometers. 469 00:26:05,600 --> 00:26:07,250 So that's pretty interesting. 470 00:26:07,250 --> 00:26:11,720 But they sort of gain robustness from that second wall structure 471 00:26:11,720 --> 00:26:14,040 that's coating on the outside. 472 00:26:14,040 --> 00:26:18,260 Now, their challenge with gram-negative bacteria relative 473 00:26:18,260 --> 00:26:22,460 to gram-positive bacteria is any drugs you develop have to make 474 00:26:22,460 --> 00:26:23,780 a pretty-- 475 00:26:23,780 --> 00:26:27,260 if they're targeted at intracellular sites, 476 00:26:27,260 --> 00:26:30,860 they have to get through two walls, not just one wall. 477 00:26:30,860 --> 00:26:33,020 So they are harder to treat. 478 00:26:33,020 --> 00:26:35,300 And they also have a lot of characteristics 479 00:26:35,300 --> 00:26:38,420 that make them more prone to resistance development. 480 00:26:38,420 --> 00:26:42,080 So I want to point out to you, on this electron micrograph, 481 00:26:42,080 --> 00:26:46,400 you can actually see the double wall, the dark band of space 482 00:26:46,400 --> 00:26:49,280 and then another dark band, whereas here you 483 00:26:49,280 --> 00:26:52,160 see a thin single wall, but you see 484 00:26:52,160 --> 00:26:55,080 a lot of junk on the outside. 485 00:26:55,080 --> 00:26:58,640 Is everyone seeing the differences just 486 00:26:58,640 --> 00:26:59,850 to look at them? 487 00:26:59,850 --> 00:27:05,690 OK, so what's this gram thing about? 488 00:27:05,690 --> 00:27:07,310 What does this stand for? 489 00:27:10,190 --> 00:27:29,220 It simply stands for a chemical dye that stains peptidoglycan. 490 00:27:29,220 --> 00:27:33,240 And it was invented or discovered by Professor Gram. 491 00:27:33,240 --> 00:27:34,110 That was his name. 492 00:27:34,110 --> 00:27:36,960 So when someone says you got a gram-positive infection, 493 00:27:36,960 --> 00:27:39,420 gram-negative infection, it's how 494 00:27:39,420 --> 00:27:42,870 those cells look when they've been treated with this stain. 495 00:27:42,870 --> 00:27:48,240 Gram positives show up very positive to the stain 496 00:27:48,240 --> 00:27:50,790 because there is a lot of peptidoglycan 497 00:27:50,790 --> 00:27:55,950 on the outside that absorbs the dye and shows a strong color. 498 00:27:55,950 --> 00:28:01,500 The gram-negatives don't show very well with a Gram stain, 499 00:28:01,500 --> 00:28:05,520 because the peptidoglycan is tucked in the periplasm, 500 00:28:05,520 --> 00:28:07,870 not on the outside of the cell. 501 00:28:07,870 --> 00:28:11,580 So if someone does a quick check on a bacterial streak 502 00:28:11,580 --> 00:28:13,380 or an infection that you have, they 503 00:28:13,380 --> 00:28:15,180 might treat it with the Gram stain 504 00:28:15,180 --> 00:28:18,360 and say gram-positive or gram-negative just 505 00:28:18,360 --> 00:28:23,490 based on that simple color analysis. 506 00:28:23,490 --> 00:28:27,360 And so in one case, the peptidoglycan 507 00:28:27,360 --> 00:28:29,370 is abundant and accessible. 508 00:28:29,370 --> 00:28:33,660 In the other case, it's very, very much thinner and less 509 00:28:33,660 --> 00:28:35,400 accessible to the dyes. 510 00:28:35,400 --> 00:28:37,770 Now, this probably looks like stone-age stuff 511 00:28:37,770 --> 00:28:40,530 to you, because how much can you learn 512 00:28:40,530 --> 00:28:43,020 by these simple colorimetric stains? 513 00:28:43,020 --> 00:28:46,470 We're certainly moving in very, very different directions. 514 00:28:46,470 --> 00:28:51,450 But let me just finish off with the third type of bacteria, 515 00:28:51,450 --> 00:28:58,050 the mycobacteria, which include Mycobacterium tuberculosis. 516 00:28:58,050 --> 00:29:00,920 And they have a different kind of wall, again. 517 00:29:00,920 --> 00:29:02,340 And they're pretty unusual. 518 00:29:02,340 --> 00:29:04,950 And they are really, really hard to treat, 519 00:29:04,950 --> 00:29:08,610 because it's almost impossible to get therapeutic agents 520 00:29:08,610 --> 00:29:10,530 into mycobacteria. 521 00:29:10,530 --> 00:29:14,490 I used to work on a team with Novartis in Singapore. 522 00:29:14,490 --> 00:29:17,070 And they said, doing anything with mycobacteria 523 00:29:17,070 --> 00:29:20,490 was like trying to do biochemistry on a wax candle, 524 00:29:20,490 --> 00:29:21,092 literally. 525 00:29:21,092 --> 00:29:22,800 You just can't work with it, because they 526 00:29:22,800 --> 00:29:25,920 have a thick additional wall that's kind of different again. 527 00:29:25,920 --> 00:29:27,040 Did you have a question? 528 00:29:27,040 --> 00:29:27,540 No. 529 00:29:27,540 --> 00:29:30,360 Sorry, I thought I saw your hand up. 530 00:29:30,360 --> 00:29:33,750 So what they have is a typical cell 531 00:29:33,750 --> 00:29:36,660 wall then some peptidoglycan, but then they 532 00:29:36,660 --> 00:29:41,760 have this thick mycobacterial layer 533 00:29:41,760 --> 00:29:45,720 which comprises what are known as mycolic acids, which 534 00:29:45,720 --> 00:29:50,640 basically add this thick layer of greasy hydrophobic material 535 00:29:50,640 --> 00:29:54,600 on the outside of the mycobacteria that's 536 00:29:54,600 --> 00:29:57,120 pretty impenetrable. 537 00:29:57,120 --> 00:29:59,040 The cell wall is quite different. 538 00:29:59,040 --> 00:30:01,110 It doesn't have an outer coat. 539 00:30:01,110 --> 00:30:05,430 It's like gram-positives in that respect. 540 00:30:05,430 --> 00:30:07,215 But it doesn't stain very strongly. 541 00:30:11,740 --> 00:30:20,010 So it has a weak, what's known as Gram stain. 542 00:30:20,010 --> 00:30:21,510 So sometimes if you've got something 543 00:30:21,510 --> 00:30:24,780 that gives a sort a so-so response to the Gram stain, 544 00:30:24,780 --> 00:30:27,690 you might say, oh, it looks like a mycobacterium 545 00:30:27,690 --> 00:30:29,310 because of what's happening. 546 00:30:29,310 --> 00:30:34,110 Now, mycobacteria TB is a huge threat, 547 00:30:34,110 --> 00:30:39,190 because its treatment, its current treatment-- and it's 548 00:30:39,190 --> 00:30:42,400 the same treatment that's been around for, like, 30 549 00:30:42,400 --> 00:30:45,370 years or something-- is a treatment with four 550 00:30:45,370 --> 00:30:48,280 different antibacterial agents that 551 00:30:48,280 --> 00:30:51,490 hit a bunch of different sites in the lifecycle 552 00:30:51,490 --> 00:30:53,050 of the bacteria. 553 00:30:53,050 --> 00:30:56,780 It includes these compounds shown here 554 00:30:56,780 --> 00:31:03,880 which are isoniazid, rifampicin, ethambutol, and pyrazinamide. 555 00:31:03,880 --> 00:31:07,610 And it's a six-month treatment with those medications, 556 00:31:07,610 --> 00:31:12,430 so handful, four different medications for six months. 557 00:31:12,430 --> 00:31:15,520 So what they were realizing in the developing world 558 00:31:15,520 --> 00:31:17,920 is that there was terrible compliance. 559 00:31:17,920 --> 00:31:20,680 The drugs are cheap, but there was no compliance. 560 00:31:20,680 --> 00:31:23,590 People just were not taking the pills, because they're like, 561 00:31:23,590 --> 00:31:27,490 I'm tired of taking these pills every day for six months. 562 00:31:27,490 --> 00:31:31,570 So what was developed was what's known as the DOTs program. 563 00:31:31,570 --> 00:31:33,300 Has anyone never heard of this? 564 00:31:33,300 --> 00:31:36,590 Is anyone interested in infectious disease? 565 00:31:36,590 --> 00:31:40,810 It was a situation where it was a social system set up 566 00:31:40,810 --> 00:31:42,730 in order to make sure people took 567 00:31:42,730 --> 00:31:47,500 these drugs every day for six months in order to comply. 568 00:31:47,500 --> 00:31:51,400 So social workers would go to the villages in remote areas 569 00:31:51,400 --> 00:31:54,330 and watch people take the medications. 570 00:31:54,330 --> 00:31:56,470 So it's directly observed treatment 571 00:31:56,470 --> 00:31:59,380 to make sure they followed through, because if they had 572 00:31:59,380 --> 00:32:04,630 regular TB, not very resistant TB, you could overcome it, 573 00:32:04,630 --> 00:32:06,970 provided that you took these medications. 574 00:32:06,970 --> 00:32:09,610 But still, it's a hugely debilitating thing 575 00:32:09,610 --> 00:32:11,710 to have to deal with these treatments. 576 00:32:11,710 --> 00:32:13,950 Now, there are two strains of TB. 577 00:32:13,950 --> 00:32:16,360 One is called MDR-TB. 578 00:32:16,360 --> 00:32:20,590 And the other ones called XMDR-TB You'll occasionally 579 00:32:20,590 --> 00:32:25,720 hear of these on TV programs. 580 00:32:25,720 --> 00:32:29,920 MDR is resistant to three of the four medications. 581 00:32:29,920 --> 00:32:34,600 And XMDR, which stands for extremely MultiDrug Resistant, 582 00:32:34,600 --> 00:32:38,180 is resistant to every single one of those medications. 583 00:32:38,180 --> 00:32:40,660 New medications, different mechanisms of action 584 00:32:40,660 --> 00:32:43,072 are sorely needed. 585 00:32:43,072 --> 00:32:44,530 All right, this is just what things 586 00:32:44,530 --> 00:32:46,960 look like with the Gram stains. 587 00:32:46,960 --> 00:32:51,340 So here you see gram-positive Bacillus anthracis. 588 00:32:51,340 --> 00:32:54,187 That's the deep purple rods. 589 00:32:54,187 --> 00:32:56,020 You know that's a gram-positive because it's 590 00:32:56,020 --> 00:32:58,180 a deep purple stain. 591 00:32:58,180 --> 00:33:00,730 The other cells in this picture are white cells. 592 00:33:00,730 --> 00:33:04,330 So you can really pick out the gram-positive. 593 00:33:04,330 --> 00:33:07,510 This is the structure of the chemical dye 594 00:33:07,510 --> 00:33:10,720 that stains peptidoglycan through absorbing 595 00:33:10,720 --> 00:33:13,300 into the peptidoglycan. 596 00:33:13,300 --> 00:33:15,490 It's a very sort of physical interaction 597 00:33:15,490 --> 00:33:17,950 of the dye with the polymer. 598 00:33:17,950 --> 00:33:21,160 And over on this slide, it's a mixture of gram-positive 599 00:33:21,160 --> 00:33:22,540 and gram-negative. 600 00:33:22,540 --> 00:33:28,040 And you can pick up the gram-positive 601 00:33:28,040 --> 00:33:30,740 and differentiate them from the gram-negative, which 602 00:33:30,740 --> 00:33:33,620 just stains sort of kind of weakly pink. 603 00:33:33,620 --> 00:33:37,700 And then mycobacteria, which are formerly gram-positive, 604 00:33:37,700 --> 00:33:41,210 don't stain very well because of that thick mycolic 605 00:33:41,210 --> 00:33:43,400 acid hydrophobic wall. 606 00:33:43,400 --> 00:33:45,170 So what would you do nowadays? 607 00:33:45,170 --> 00:33:48,800 Would you pull out a stain and drop it on bacteria 608 00:33:48,800 --> 00:33:52,680 and get some vague response? 609 00:33:52,680 --> 00:33:57,880 What's open to you now in the 21st century? 610 00:33:57,880 --> 00:34:00,630 You have a tiny sample of a bacterium. 611 00:34:00,630 --> 00:34:01,200 Grow it up. 612 00:34:01,200 --> 00:34:01,950 What would you do? 613 00:34:01,950 --> 00:34:04,740 You could tell exactly what it is. 614 00:34:04,740 --> 00:34:05,680 AUDIENCE: PCR. 615 00:34:05,680 --> 00:34:08,850 PROFESSOR: Yeah, you'd PCR up the genomic DNA 616 00:34:08,850 --> 00:34:12,000 and then go match it, because the thing that we, in addition 617 00:34:12,000 --> 00:34:15,179 to the human genome, there are thousands 618 00:34:15,179 --> 00:34:20,219 of pathogenic bacteria sequences that are completely annotated, 619 00:34:20,219 --> 00:34:21,090 known. 620 00:34:21,090 --> 00:34:24,929 The [INAUDIBLE] has a massive compilation of these sequences. 621 00:34:24,929 --> 00:34:27,840 And you just go and you find out what the bacterium 622 00:34:27,840 --> 00:34:29,710 is based on the sequence. 623 00:34:29,710 --> 00:34:33,340 So now rapid sequencing efforts-- 624 00:34:33,340 --> 00:34:37,739 maybe they're just a few number of key places in a genome 625 00:34:37,739 --> 00:34:41,699 that you would go towards and just do a really fast array 626 00:34:41,699 --> 00:34:46,110 and figure out what's there and within what bacterium it is, 627 00:34:46,110 --> 00:34:49,500 which gives you a much better clue as to how to treat it 628 00:34:49,500 --> 00:34:51,659 than the vague, ambiguous stains. 629 00:34:51,659 --> 00:34:55,710 So even though stains keep going, there is now other ways. 630 00:34:55,710 --> 00:34:58,440 Unfortunately, not everyone has the instrumentation 631 00:34:58,440 --> 00:35:00,480 to do rapid sequencing. 632 00:35:00,480 --> 00:35:02,540 So nowadays, there is a lot, lot, 633 00:35:02,540 --> 00:35:07,410 lot of interest in faster dipstick sorts of tests 634 00:35:07,410 --> 00:35:10,980 that can distinguish between different bacterial strains 635 00:35:10,980 --> 00:35:16,500 by, for example, interrogating that coat of glyco-conjugates 636 00:35:16,500 --> 00:35:18,780 that's on the outside of the bacteria, 637 00:35:18,780 --> 00:35:21,570 dipstick paper tests that can give you 638 00:35:21,570 --> 00:35:23,460 an idea of what organism and what 639 00:35:23,460 --> 00:35:27,960 serotype so you can move forward and do a much more rational 640 00:35:27,960 --> 00:35:31,290 treatment of those organisms. 641 00:35:31,290 --> 00:35:33,210 OK, let's see what's-- yes. 642 00:35:33,210 --> 00:35:38,280 All right, so where did the antibiotics first come from? 643 00:35:38,280 --> 00:35:41,340 Any questions so far? 644 00:35:41,340 --> 00:35:45,355 OK, so where did the first antibiotics come from? 645 00:35:45,355 --> 00:35:48,370 From a couple of accidental discoveries. 646 00:35:48,370 --> 00:35:52,690 Who has heard of the Fleming experiment? 647 00:35:52,690 --> 00:35:56,610 Who knows about that discovery of penicillin? 648 00:35:56,610 --> 00:36:00,470 Yeah, so there was an original observation 649 00:36:00,470 --> 00:36:03,140 that predated that which sort of suggests that Pasteur 650 00:36:03,140 --> 00:36:05,620 was a pretty smart guy, because he contributed 651 00:36:05,620 --> 00:36:07,790 in a lot of different areas. 652 00:36:07,790 --> 00:36:12,830 He discovered that some bacteria tend to release substances 653 00:36:12,830 --> 00:36:15,140 that kill other bacteria. 654 00:36:15,140 --> 00:36:19,250 That was in the 1870s. 655 00:36:19,250 --> 00:36:21,590 Then later on, there was another sort 656 00:36:21,590 --> 00:36:24,440 of spread of antibiotic agents. 657 00:36:24,440 --> 00:36:27,920 And it came with the discovery that we had things 658 00:36:27,920 --> 00:36:30,500 like arsenic derivatives actually 659 00:36:30,500 --> 00:36:36,080 showed some value in treating the organism that 660 00:36:36,080 --> 00:36:37,520 causes syphilis. 661 00:36:37,520 --> 00:36:40,700 So talk about the treatment being-- 662 00:36:40,700 --> 00:36:43,200 the cure being worse than the infection. 663 00:36:43,200 --> 00:36:45,110 People were being treated, seriously, 664 00:36:45,110 --> 00:36:48,980 with these arsenic derivatives in the hope of wiping out 665 00:36:48,980 --> 00:36:51,170 the infectious agent that caused syphilis. 666 00:36:51,170 --> 00:36:53,990 But you know, sometimes it was a mixed bag. 667 00:36:53,990 --> 00:36:58,010 But where things started to get a lot more interesting was that 668 00:36:58,010 --> 00:37:02,720 in 1928, there was this sort of famous historic story 669 00:37:02,720 --> 00:37:08,330 of Fleming discovering that some bacteria seemed 670 00:37:08,330 --> 00:37:15,590 to be inhibited by a particular agent that came from a fungus. 671 00:37:15,590 --> 00:37:18,360 And this was the origin of penicillin. 672 00:37:18,360 --> 00:37:21,590 So he would have a Petri dish where he was growing bacteria. 673 00:37:21,590 --> 00:37:24,380 And he noticed that in some of his samples, 674 00:37:24,380 --> 00:37:26,930 there was inhibition of bacterial growth 675 00:37:26,930 --> 00:37:29,990 due to an exogenous agent that had somehow 676 00:37:29,990 --> 00:37:32,600 contaminated the plates. 677 00:37:32,600 --> 00:37:35,840 So in that story, that was the substance 678 00:37:35,840 --> 00:37:38,600 that was named as penicillin. 679 00:37:38,600 --> 00:37:41,660 The mold from the-- mold is the fungus-- 680 00:37:41,660 --> 00:37:45,200 actually inhibited the growth of staphylococcus bacteria. 681 00:37:45,200 --> 00:37:47,150 And it was called penicillin. 682 00:37:47,150 --> 00:37:50,300 And then a lot more time went by. 683 00:37:50,300 --> 00:37:56,360 But in the 1940s, the active ingredient was discovered. 684 00:37:56,360 --> 00:38:02,120 So 1940s is sort of slap bang about, I would say, 685 00:38:02,120 --> 00:38:06,020 a couple of years into the Second World War. 686 00:38:06,020 --> 00:38:11,150 And they were able to mobilize the production of this agent. 687 00:38:11,150 --> 00:38:12,710 Towards the later end of the war, 688 00:38:12,710 --> 00:38:15,470 people had penicillin available to them. 689 00:38:15,470 --> 00:38:18,830 And it's basically pretty well believed 690 00:38:18,830 --> 00:38:22,460 that, if it wasn't for the antibiotic agents that 691 00:38:22,460 --> 00:38:23,690 emerged-- 692 00:38:23,690 --> 00:38:26,060 you know, the war ended in 1945. 693 00:38:26,060 --> 00:38:28,570 If it wasn't for those agents that emerged, 694 00:38:28,570 --> 00:38:32,120 there would have been way way, way more deaths from the war. 695 00:38:32,120 --> 00:38:35,000 As it was, there were way too many. 696 00:38:35,000 --> 00:38:38,150 So penicillin was the first antibiotic 697 00:38:38,150 --> 00:38:41,180 that was discovered with a discreet mechanism of action. 698 00:38:41,180 --> 00:38:44,880 And it was discovered at a very, very important time. 699 00:38:44,880 --> 00:38:47,030 So that was all great news. 700 00:38:47,030 --> 00:38:49,230 Penicillin was produced widely. 701 00:38:49,230 --> 00:38:51,620 Some of you may be allergic to penicillin. 702 00:38:51,620 --> 00:38:53,750 There are other options nowadays. 703 00:38:53,750 --> 00:38:55,850 But it's the cheapest and most viable 704 00:38:55,850 --> 00:39:00,690 of the first-line antibiotics. 705 00:39:00,690 --> 00:39:01,240 Here we go. 706 00:39:04,216 --> 00:39:06,980 And this thing, this pointer has a mind of its own. 707 00:39:06,980 --> 00:39:08,460 It sort of changed its mind. 708 00:39:08,460 --> 00:39:12,160 But the problem was the bacterial species 709 00:39:12,160 --> 00:39:17,000 started to survive treatment due to development of resistance. 710 00:39:17,000 --> 00:39:19,250 And all of a sudden, something that worked really well 711 00:39:19,250 --> 00:39:20,720 wasn't working anymore. 712 00:39:20,720 --> 00:39:24,880 So let's try and think about peptidoglycan, 713 00:39:24,880 --> 00:39:28,120 what penicillin looks like and what it does, 714 00:39:28,120 --> 00:39:31,090 and how penicillin resistance emerges. 715 00:39:31,090 --> 00:39:33,670 Those are the three things I'm going to cover here. 716 00:39:33,670 --> 00:39:36,190 OK, so what does penicillin do? 717 00:39:36,190 --> 00:39:44,680 Penicillin stops the formation of this big macromolecular 718 00:39:44,680 --> 00:39:48,790 peptidoglycan polymer by stopping the last cross-link, 719 00:39:48,790 --> 00:39:51,010 stopping the chemistry that happens 720 00:39:51,010 --> 00:39:54,970 to join the peptide chains to make a cross-linked polymer. 721 00:39:54,970 --> 00:39:57,640 And anyone who is in the mechanical engineering area 722 00:39:57,640 --> 00:40:00,910 will know that polymers that are just strands 723 00:40:00,910 --> 00:40:02,980 are much weaker than polymers that 724 00:40:02,980 --> 00:40:04,900 are crossed-linked structures which 725 00:40:04,900 --> 00:40:07,790 have tensile strength in both directions. 726 00:40:07,790 --> 00:40:11,160 So the uncross-linked peptidoglycan was weak. 727 00:40:11,160 --> 00:40:14,140 And what penicillin specifically did 728 00:40:14,140 --> 00:40:17,350 was inhibit forming that cross-link. 729 00:40:17,350 --> 00:40:19,300 What does penicillin look like? 730 00:40:19,300 --> 00:40:20,650 Here it is. 731 00:40:20,650 --> 00:40:22,150 It's a cool structure. 732 00:40:22,150 --> 00:40:26,830 It's what's known as a natural product, five ring, four ring, 733 00:40:26,830 --> 00:40:28,600 an interesting structure. 734 00:40:28,600 --> 00:40:32,050 And what it would do is it would interact with the enzyme 735 00:40:32,050 --> 00:40:34,470 the cross-linked the peptidoglycan 736 00:40:34,470 --> 00:40:37,240 and basically stop it dead in its tracks. 737 00:40:37,240 --> 00:40:39,940 What did the bacteria do? 738 00:40:39,940 --> 00:40:44,210 The key part of this structure is this four-membered ring 739 00:40:44,210 --> 00:40:47,210 within amide bond in it. 740 00:40:47,210 --> 00:40:51,980 The bacteria evolved an enzyme to chop it open basically 741 00:40:51,980 --> 00:40:53,900 making it completely inactive. 742 00:40:53,900 --> 00:40:58,010 So beta lactamase was evolved in the bacterial populations. 743 00:40:58,010 --> 00:41:01,040 It was probably derived from some other enzyme that 744 00:41:01,040 --> 00:41:03,440 did some useful function, but not 745 00:41:03,440 --> 00:41:05,900 targeted to the penicillins. 746 00:41:05,900 --> 00:41:09,140 But the bacteria started to survive 747 00:41:09,140 --> 00:41:12,230 because they made a ton of an enzyme called beta lactamase. 748 00:41:12,230 --> 00:41:14,780 And then it completely stopped working. 749 00:41:14,780 --> 00:41:18,350 So the chemists came up with other options, 750 00:41:18,350 --> 00:41:21,440 because they said, well, you know, if that doesn't work, 751 00:41:21,440 --> 00:41:24,950 we've got other antibiotics in our arsenal. 752 00:41:24,950 --> 00:41:26,840 And there is a compound that was used 753 00:41:26,840 --> 00:41:31,400 for years as a last line of resort antibiotic known 754 00:41:31,400 --> 00:41:33,020 as vancomycin. 755 00:41:33,020 --> 00:41:38,330 It was very, very important, so very serious infections, 756 00:41:38,330 --> 00:41:41,060 and really preserved for that use. 757 00:41:41,060 --> 00:41:45,710 And they thought that vancomycin might be a drug that 758 00:41:45,710 --> 00:41:47,450 just couldn't be defeated. 759 00:41:47,450 --> 00:41:50,360 This big molecule here is vancomycin. 760 00:41:50,360 --> 00:41:54,800 This little piece of peptide is actually the peptide 761 00:41:54,800 --> 00:41:56,350 that's in that cross-link. 762 00:41:56,350 --> 00:41:59,300 And vancomycin basically, like a glove, 763 00:41:59,300 --> 00:42:03,860 sat on that piece of peptide and stopped it being cross-linked. 764 00:42:03,860 --> 00:42:05,660 And what did the bacteria do? 765 00:42:05,660 --> 00:42:10,010 They evolved a set of enzymes to completely change 766 00:42:10,010 --> 00:42:12,400 that little piece of peptide into something 767 00:42:12,400 --> 00:42:16,130 that bound more poorly, giving you resistance 768 00:42:16,130 --> 00:42:18,030 to vancomycin as well. 769 00:42:18,030 --> 00:42:21,710 So when there is one drug involved, 770 00:42:21,710 --> 00:42:24,390 it's pretty easy to get resistance quite quickly. 771 00:42:24,390 --> 00:42:29,270 You just mutate one enzyme and you get a resistant strain. 772 00:42:29,270 --> 00:42:33,200 And the enzyme that can beat the antibiotic will win. 773 00:42:36,620 --> 00:42:40,940 If you've got a compound that takes five different enzymes 774 00:42:40,940 --> 00:42:46,520 or an antibiotic that has a very complex mechanism of action, 775 00:42:46,520 --> 00:42:50,660 you might say, well, this is never going to be defeated. 776 00:42:50,660 --> 00:42:54,200 It took five additional enzymes to evolve 777 00:42:54,200 --> 00:42:57,500 to make the peptidoglycan a different structure. 778 00:42:57,500 --> 00:43:00,140 And it's not that within every bacterium, 779 00:43:00,140 --> 00:43:02,660 you mutate five different enzymes 780 00:43:02,660 --> 00:43:04,700 and get them all working as a team. 781 00:43:04,700 --> 00:43:07,850 What was happening in these infections 782 00:43:07,850 --> 00:43:10,760 is that a plasmid with the set of enzymes 783 00:43:10,760 --> 00:43:13,880 was being passed around amongst bacteria. 784 00:43:13,880 --> 00:43:18,170 So a new bacterium could acquire resistance to this compound 785 00:43:18,170 --> 00:43:21,080 without evolving a whole bunch of new enzymes, 786 00:43:21,080 --> 00:43:24,350 but rather by lateral transfer of plasmids 787 00:43:24,350 --> 00:43:28,700 encoding the genes that it took to make the vancomycin 788 00:43:28,700 --> 00:43:30,950 inactive. 789 00:43:30,950 --> 00:43:34,280 All right, so let me just tell you a few of the targets. 790 00:43:34,280 --> 00:43:36,440 And then there is one movie I want to show you 791 00:43:36,440 --> 00:43:37,710 that's kind of cool. 792 00:43:37,710 --> 00:43:42,950 So currently, when we inhibit bacteria with antibiotics, 793 00:43:42,950 --> 00:43:45,020 there are a number of essential processes 794 00:43:45,020 --> 00:43:48,240 that are targeted with common antibiotics. 795 00:43:48,240 --> 00:43:50,480 So this would be a typical bacterium. 796 00:43:50,480 --> 00:43:59,900 One target of action is DNA synthesis and DNA polymerase. 797 00:43:59,900 --> 00:44:02,510 And the enzyme that is targeted is 798 00:44:02,510 --> 00:44:05,690 one we've talked about, topoisomerase. 799 00:44:05,690 --> 00:44:09,050 And that is inhibited by the fluoroquinolones 800 00:44:09,050 --> 00:44:13,700 such as ciprofloxacin that actually targets specifically 801 00:44:13,700 --> 00:44:15,050 the bacterial polymerase. 802 00:44:15,050 --> 00:44:18,830 So that's one way, inhibit DNA replication, 803 00:44:18,830 --> 00:44:20,690 bacteria can't divide. 804 00:44:20,690 --> 00:44:25,130 Another set of antibiotics are those 805 00:44:25,130 --> 00:44:28,230 that inhibit protein synthesis. 806 00:44:28,230 --> 00:44:30,770 So in particular, you know the tunnel 807 00:44:30,770 --> 00:44:34,010 that comes out of the ribosome where the growing polypeptide 808 00:44:34,010 --> 00:44:38,240 chain emerges after reading the messenger RNA 809 00:44:38,240 --> 00:44:40,820 and translating the messenger into protein? 810 00:44:40,820 --> 00:44:43,780 There are antibiotics to basically stick in that tunnel 811 00:44:43,780 --> 00:44:45,830 and stop protein synthesis. 812 00:44:45,830 --> 00:44:48,610 And those are things like the aminoglycosides. 813 00:44:48,610 --> 00:44:50,960 And they block exit from the ribosome. 814 00:44:50,960 --> 00:44:53,600 But you could imagine that mutating. 815 00:44:53,600 --> 00:44:56,330 There are the ones that inhibit cell wall biosynthesis 816 00:44:56,330 --> 00:44:59,540 that I've already talked to about, the penicillins, 817 00:44:59,540 --> 00:45:01,680 the vancoymcin. 818 00:45:01,680 --> 00:45:06,820 And then there are others that inhibit folate synthesis. 819 00:45:06,820 --> 00:45:09,430 And then there is a lot of synthetic drugs, 820 00:45:09,430 --> 00:45:11,840 but also a lot of natural product drugs. 821 00:45:11,840 --> 00:45:15,040 So both nature and chemistry have teamed up 822 00:45:15,040 --> 00:45:18,700 to inhibit all of these essential steps. 823 00:45:18,700 --> 00:45:22,610 OK, so how do you test for antibiotic resistance? 824 00:45:22,610 --> 00:45:26,660 You use plates where you're growing particular strains 825 00:45:26,660 --> 00:45:29,060 of bacteria on a plate. 826 00:45:29,060 --> 00:45:32,090 This would be a colony. 827 00:45:32,090 --> 00:45:34,130 And it's growing outwards. 828 00:45:34,130 --> 00:45:37,160 Where there is a colony but there is no growth around it, 829 00:45:37,160 --> 00:45:39,770 it means there is something in that plate that 830 00:45:39,770 --> 00:45:42,300 is inhibiting bacterial growth. 831 00:45:42,300 --> 00:45:44,900 So these are very clear types of ways 832 00:45:44,900 --> 00:45:47,450 that people check to see if bacteria 833 00:45:47,450 --> 00:45:49,670 have become resistant to drugs. 834 00:45:49,670 --> 00:45:51,860 You would look for that zone of inhibition. 835 00:45:51,860 --> 00:45:55,010 Does it disappear with some of the resistant strains, 836 00:45:55,010 --> 00:45:56,870 for example? 837 00:45:56,870 --> 00:45:59,510 And these get pretty sophisticated 838 00:45:59,510 --> 00:46:03,590 now where you can test a bunch of antibiotics in one 839 00:46:03,590 --> 00:46:06,440 go, where each of these colored dots 840 00:46:06,440 --> 00:46:08,900 represents an area where there is treatment 841 00:46:08,900 --> 00:46:11,720 with one antibiotic or another. 842 00:46:11,720 --> 00:46:13,320 So what's the problem? 843 00:46:13,320 --> 00:46:15,710 The problem is this graph, that as soon 844 00:46:15,710 --> 00:46:19,790 as an antibiotic is introduced, just a few years go by. 845 00:46:19,790 --> 00:46:22,490 And there is resistance to that antibiotic. 846 00:46:22,490 --> 00:46:27,230 So resistance basically is the gradual acquisition 847 00:46:27,230 --> 00:46:30,200 of machinery to somehow inactivate 848 00:46:30,200 --> 00:46:31,710 the antibiotic treatment. 849 00:46:31,710 --> 00:46:34,400 So if you take a look, here on the top 850 00:46:34,400 --> 00:46:36,610 is where the drug is introduced. 851 00:46:36,610 --> 00:46:39,450 And on the bottom is when resistance was developed. 852 00:46:39,450 --> 00:46:41,730 So let's go to something we're familiar. 853 00:46:41,730 --> 00:46:45,440 Here is penicillin, people introduced about 1940 854 00:46:45,440 --> 00:46:47,180 to the general population. 855 00:46:47,180 --> 00:46:51,590 By about '47, there was resistance to penicillin. 856 00:46:51,590 --> 00:46:55,880 And you can see, this is really just a really serious sort 857 00:46:55,880 --> 00:46:57,320 of series of events. 858 00:46:57,320 --> 00:47:01,500 So what I want to show you was resistance in action. 859 00:47:01,500 --> 00:47:05,360 And that'll be the last thing I talk about today, 860 00:47:05,360 --> 00:47:09,620 because I just want to give you a feel for what 861 00:47:09,620 --> 00:47:11,165 does resistance look like. 862 00:47:14,100 --> 00:47:15,780 So this was an experiment that was 863 00:47:15,780 --> 00:47:22,980 done at Harvard on just a visualization of resistance 864 00:47:22,980 --> 00:47:24,690 development. 865 00:47:24,690 --> 00:47:27,780 I think what's so fascinating is you could then 866 00:47:27,780 --> 00:47:33,030 go back to the plate and pluck the first pioneers who 867 00:47:33,030 --> 00:47:35,820 crossed that line and find out what that was. 868 00:47:35,820 --> 00:47:39,780 What was that mutation that let the population expand, 869 00:47:39,780 --> 00:47:40,300 and so on? 870 00:47:40,300 --> 00:47:44,460 So you could really map out the entire evolution of very, very 871 00:47:44,460 --> 00:47:46,090 strong resistance. 872 00:47:46,090 --> 00:47:47,820 So in the next class, I'll talk to you 873 00:47:47,820 --> 00:47:49,510 about resistance mechanisms. 874 00:47:49,510 --> 00:47:53,460 And then we'll talk about viruses and resistance 875 00:47:53,460 --> 00:47:55,790 to antivirals.