When An­tibi­otic Re­sis­tance in vitro Does Not Tell You...

...What You Need To Know

by Terry Roe­mer

In­creas­ingly, it is be­com­ing ap­par­ent that there is a deep dif­fer­ence be­tween what hap­pens in vivo and in vitro. The idea that one can com­fort­ably ex­trap­o­late from the bench to, say, the bed­side, is re­ced­ing rapidly. Liv­ing things, not even the small­est ones, are no agar plates, how­ever con­ve­nient and in­ex­pen­sive these may be. Each field of Bi­ol­ogy has to learn this les­son, not the least that of an­tibi­otic re­search. Gone are the days when a lab­o­ra­tory re­sult alone was thought to re­veal what goes on in a per­son sick with a mi­cro­bial in­fec­tion. Where do such dis­tinc­tions show up and why do they mat­ter?

Fig­ure 1. Source

Re­cently, Dan An­der­s­son and col­leagues from Up­p­sala Uni­ver­sity Swe­den, pub­lished a truly unique and im­por­tant study on the many mech­a­nisms of mecil­li­nam (aka amdinocillin) re­sis­tance in Es­cherichia coli. Al­though sev­eral such stud­ies have been per­formed in the past, what sep­a­rates this work from those pre­vi­ously pub­lished is in pro­vid­ing an un­der­stand­ing of the ge­netic and en­vi­ron­men­tal ba­sis of an im­por­tant drug re­sis­tance para­dox of mecil­li­nam, namely why is re­sis­tance to this β‑lactam an­tibi­otic so read­ily ob­served in the lab­o­ra­tory (i.e. on a petri dish) but rarely iden­ti­fied in a clin­i­cal set­ting where this drug is widely used to treat E. coli uri­nary tract in­fec­tions?

In­deed, re­sis­tance mu­tants arise in vitro at fre­quen­cies of 8x10^–8 to 2x10^–5 per cell, de­pend­ing on the drug con­cen­tra­tion used dur­ing se­lec­tion, but at much lower rates in clin­i­cal iso­lates. The lab­o­ra­tory mu­tants in­volve many dis­tinct func­tions and as many as 38 known genes, rang­ing from the drug tar­get (pbpB) to yet other as­pects of cell wall syn­the­sis, cell di­vi­sion, res­pi­ra­tion, ri­bo­somes, tRNA syn­thetases, as well as the ppGpp-me­di­ated strin­gent re­sponse path­way. Fur­ther­more, based on whole genome se­quenc­ing and pre­cise al­lelic re­place­ment, each of these mu­ta­tions has been rig­or­ously demon­strated to be the cause for the ob­served drug re­sis­tance. All this di­ver­sity is quite in­ter­est­ing and the sub­ject of much re­search, but it is not the point here. What is star­tling is that the rare mecil­li­nam re­sis­tant E. coli clin­i­cal iso­lates are sur­pris­ingly dif­fer­ent: all iso­lates carry only a sin­gle re-oc­cur­ring class of mu­ta­tions, the loss of func­tion of cysB, a gene in­volved in cys­teine biosyn­the­sis. Just how does de­ple­tion of cys­teine lev­els con­fer mecil­li­nam re­sis­tance? This is not known, but the dif­fer­ent drug re­sis­tance spec­tra un­der two growth con­di­tions leads to in­sights into the para­dox­i­cal rate of drug re­sis­tance ob­served in vitro and in vivo.

Fig­ure 2: Pu­ta­tive Amd re­sis­tance-en­cod­ing genes in clin­i­cal strains. Genes marked by gray shad­ing have a mu­ta­tion that is not present in any of the ref­er­ence genomes. Spe­cific mu­ta­tions are listed in Ta­ble 4 of the pa­per. Source

The An­der­s­son lab went on to demon­strate that whereas mu­tants se­lected in vitro all share sim­i­lar fit­ness costs (again, as­sayed in an in vitro con­di­tion), the cysB mu­ta­tions do not in­cur a fit­ness cost when the strains are grown in a more rel­e­vant urine-rich medium (btw, the urine was sup­plied by one of the in­ves­ti­ga­tors). These ex­per­i­ments demon­strate that among the broad set of mu­ta­tions that can con­fer mecil­li­nam re­sis­tance un­der the stan­dard way of de­ter­min­ing drug re­sis­tance, only the rare cysB mu­tants pos­sess suf­fi­cient fit­ness to per­sist in the set­ting of an in­fec­tion. How­ever, even these mu­tants are rarely found in an in­fected blad­der, prob­a­bly be­cause mecil­li­nam reaches lev­els  so high as to be in­hibitory even to these mu­tants. In sim­ple terms, a high growth rate of the pathogen is re­quired in this en­vi­ron­ment, if it is to counter its con­stant ex­pul­sion from the uri­nary tract.

Fig­ure 3: Box plot of fit­ness of lab­o­ra­tory-se­lected E. coli iso­lates (LI) and clin­i­cal iso­lates (CI) in MH medium and urine, show­ing (from top to bot­tom) the max­i­mum fit­ness value, the up­per quar­tile, the me­dian, the lower quar­tile, and the min­i­mum fit­ness value. A sin­gle out­lier is shown as a cir­cle. Fit­ness was mea­sured as the rel­a­tive growth rate, where the par­ent strain was set to 1.0. Source

In a gen­eral sense, hav­ing a pathogen be­come re­sis­tant less of­ten in a per­son than in the lab is good news. The bad news is that to as­sess the sig­nif­i­cance of re­sis­tance aris­ing re­quires more com­pli­cated and ex­pen­sive tech­nolo­gies. How­ever, the demon­stra­tion of the im­por­tance of the en­vi­ron­ment in drug re­sis­tance is crit­i­cal to all of us in­volved in early an­tibac­te­r­ial dis­cov­ery. So of­ten we lower the pri­or­ity of new leads based on tra­di­tional and an­ti­quated mea­sures of re­sis­tance. I have the un­com­fort­able feel­ing that a po­ten­tially large num­ber of use­ful drugs sit in bot­tles on a shelf and are not fur­ther tested be­cause they re­sulted in a "high rate" of re­sis­tance. Clearly, we need to think more deeply about the is­sues, es­pe­cially re­gard­ing the rel­e­vance of drug re­sis­tance in the in­fec­tious set­ting. The work of An­der­s­son and col­leagues em­pha­sizes the im­por­tance of a higher or­der un­der­stand­ing of an­tibi­otic drug re­sis­tance and serves as a frame­work for eval­u­at­ing re­sis­tance in a more mean­ing­ful con­text. It em­pha­sizes the need for new method­olo­gies to mea­sure an­tibi­otic drug re­sis­tance in an­i­mal mod­els of in­fec­tion that cap­ture 1) phys­i­ol­ogy of the pathogen, es­pe­cially its level of fit­ness, 2) drug ex­po­sure lev­els, and 3) host re­sponse mech­a­nisms such as the in­nate im­mune sys­tem. Alas, these are all ig­nored by tra­di­tional in vitro drug re­sis­tance as­says. With­out re­vis­ing our think­ing, we risk ig­nor­ing or de­pri­or­i­tiz­ing po­ten­tially novel ther­a­peu­tic an­timi­cro­bial agents based on faulty as­sump­tions of their propen­sity for drug re­sis­tance. This is some­thing we can­not af­ford to do in our search for novel agents to com­bat the an­tibi­otic re­sis­tance epi­demic.

Ref­er­ence

Thulin E, Sundqvist M, An­der­s­son DI. 2015. Amdinocillin (Mecil­li­nam) Re­sis­tance Mu­ta­tions in Clin­i­cal Iso­lates and Lab­o­ra­tory-Se­lected Mu­tants of Es­cherichia coli. An­timi­crob Agents Chemother, 59, 1718–1727. PMID 25583718

 

Terry is a Dis­tin­guished Sci­en­tist at Merck & Co.