Title: Antibiotic resistant characteristics from 16S rRNA
Author: Casey R. Richardson
Affiliation: Morton Bioinformatics, Morton, Texas, United States of America Abstract Background: Microbiota have evoved to accimate themseves to many environments. !"manity is become ever increasin#y medicated and many of those medications are antibiotics. Sady, Microbiota are ada$tin# to medication and %ith each $assin# #eneration they become more diffic"t to s"bd"e. The 16S sma s"b"nit of bacteria ribosoma rRNA $rovides a %eath of information for cassifyin# the s$ecies eve taxonomy of bacteria. Methodology/Principal Findings: &x$eriments %ere coected "tii'in# broad and narro% s$ectr"m antibiotics, %hich act $rimariy on (NA. )n each ex$eriment a staticay si#nificant, "ni*"e and $redictabe $attern of se*"entia and thermodynamic stabiity or instabiity %as fo"nd to correate to antibiotic resistance. Conclusions/Significance: Cassification of antibiotic resistance is $ossibe for some s$ecies and antibiotic combinations "sin# the 16S rRNA se*"entia and thermodynamic $ro$erties. Introduction &xtremo$hies thrive in "ncommony harsh environments. +or exam$e, thermo$hies ive in tem$erat"res ran#in# from ,- to 1..C /10 /.0 /10, hao$hies d%e in environments %ith hi#h sat content /,0, and most im$ortanty antibiotic resistant ife forms %hich fo"rish des$ite antibiotics. Antibiotics resistance is an im$ortant area of st"dy beca"se many diseases are deveo$in# resistance to antibiotics. ) hy$othesi'e that 2ro3aryotic extremo$hies can characteri'e by their 16S rRNA se*"entia characteristics. +eat"res ) have examined to 4"stify this hy$othesis are n"ceotide and din"ceotide fre*"encies, adenosine and "raci 5A6U7 and #"anine and cytosine 586C7 content, ad4"sted base $airi# $ro$ensity 2b, ad4"sted base $air distance d(, ad4"sted Shannon entro$y d9, and minim"m free ener#y 5mfe7 /-0. Secondary str"ct"re and 86C content have been sho%n to characteri'e the ivin# environment of bacteria /60. 16S rRNA have aso been sho%n to $ay a $art in a bacteria:s fitness /;0. <ne recent st"dy has been $erformed on the h"man #"t:s microbiota ada$tation to Ci$rofoxacin 5c$7, a synthetic antibiotic "sed to treat bacteria infections /=0. Another st"dy has been $erformed on the s%ine #"t:s microbiota in the $resence of AS2.-> 5chortetracycine, s"fametha'ine, and $eniciin7 and Carbadox /?0. A third st"dy %as $erformed on the microbiota of mice in the $resence of Am$iciin, @ancomycin and an antibiotic coc3tai com$osed of Metronida'oe, Neomycin, and @ancomycin 5MN@7 /1>0. Neomycin is an amino#ycosides b"t the other antibiotics in the st"dies are not. Amino#ycosides are 3no%n to have a 16S resistant $ath%ay conferred by methytransferases /11A1,0. St"dy of antibiotic resistant bacteria has foc"sed on atera #ene transfer, %hich confers antibiotic resistance b"t most have not foc"sed on #enera or s$eciesAeve determinants observabe from 16S rRNA /1-A1?0. ) conc"de that base $airin# and din"ceotide fre*"encies in the 16S rRNA characteri'e s$ecies of bacteria that are abe to %ithstand s$ecific antibiotics. This $a$er ex$ains a method of $redictin# broadAs$ectr"m antibiotic resistance to he$ faciitate a transition to tar#eted 5even if remainin# broadAs$ectr"m7 antibiotic thera$y /.>0. Materials and Methods rRNA data %as $rovided by the S)B@A database. 2yrose*encin# data %as obtained from NCB) SRA and by direct contact %ith a"thors. &ach read %as c"stered into an o$erationa taxonomic "nit 5<TU7 at a $ercent id of ?;C "sin# US&ARC!. Beca"se $yrose*"encin# data res"ts in reads of reative ab"ndance, each sam$e:s r"n %as rarefied by dividin# by the tota n"mber of reads for each individ"a sam$e. 2ercenta#e of re$resentation ao%s com$arison bet%een sam$es of "ne*"a tota ab"ndance. &ach <TU %as ma$$ed to the nearest S)B@A f" en#th 16S se*"ence /.10 /..0. +or com$arison $"r$oses, each <TU of the 2NAS data %as aso ma$$ed to the f" 8reen#enes database and the res"ts %ere e*"ivoca. @ienneaRNA %as "sed to coect thermodynamic $ro$erties of 16S rRNA. #enrnastats.$ and #enRandomRNA.$ %ritten by Staney N8 D%an# Boon# %ere modified to $rovide a the characteristics and random ne#ative rRNA to test the m"ti$e inear re#ression. /.10 M"ti$e inear re#ression %as $erformed %ith the R $ro#rammin# an#"a#e. 2earson:s correation coefficients, binomia distrib"tions and t distrib"tions %ere cac"ated "sin# Microsoft &xce. esults and !iscussion ) examined the thermodynamic stabiity of 1.;,-., #enes from 8reen#enes database and fo"nd that 16S rRNA co"d be "sed to cassify extremo$hies based on their maxim"m free ener#y and normai'ed maxim"m free ener#y. /..0 Thermo$ies have hi#hy stabe rRNA thermodynamic $ro$erties. /.,,.-0 ) add from my observation that hao$hies aso have a hi#hy stabe secondary str"ct"re and $hyto$asma have a very %ea3 secondary str"ct"re. 2hyto$asma have no ce %a and m"st ada$t to the vario"s sit"ations they are $resented %ithE this abiity is refected in their "nstabe rRNA. Conc"din# that secondary str"ct"re co"d be "sed the cassify extremo$hies, ) anay'ed the $yrose*encin# data from the h"man #"t:s microbiota ta3en in the $resence or absence of c$. Ci$rofoxacin is a broadAs$ectr"m *"inoones that inhibits se$aration of bacteria (NA. )n F)ncom$ete Recovery and )ndivid"ai'ed Res$onses of the !"man (ista 8"t Microbiota to Re$eated Antibiotic 2ert"rbationG 52NAS7, 1> sam$es %ere ac*"ired before, d"rin#, or after c$ administration and = sam$es %ere ta3en d"rin# the re#iment of c$. /=0 My anaysis foc"ses on the tota sam$es ta3en and the sam$es ta3en d"rin# c$. Both datasets %ere anay'ed as a %hoe "sin# 2earson:s correation coefficient %ith a c"t off va"e of $ H .>-. Tabe 1 sho%s 2NAS intrinsic 16S RNA thermodynamic characteristics. Three casses %ere chosen to re$resent the bios$here of the h"man #"t %ith and %itho"t the seective $ress"re of an antibiotic. Tota ex$ression is the s"m of a the rarefied se*"ences from 16S reads for a individ"as invoved in the ex$eriment. ) considered the coective #ro"$ of $atients as one bios$here "nder e*"a seective $ress"re from a sin#e antibiotic. Tota resistance is the s"m of a the rarefied of an <TU:s 16S reads for a sam$es ta3en d"rin# antibiotics for a individ"as. Avera#e $ercent resistance is the 16S rarefied reads ta3en d"rin# antibiotics divided by tota amo"nt of rarefied reads avera#ed for the three individ"as. +inay, a sim$e $ercent resistance %as cac"ated dividin# the rarefied tota ex$ression and tota resistance. The $ercent resistance casses sho% %hich bacteria thrived "nder the seective $ress"re of antibiotics. A bios$here a$$roach to the h"man #"t is made $ossibe by a o% 5.1;7 standard deviation bet%een the s"b4ects in the cate#ory of $ercent resistance. This is beca"se c$ aone is actin# on the bacteria %ith no assistance from the individ"a:s imm"ne system. &nvironmenta factors beyond the medication and bacteria ada$tabiity i3e code$endency and ocation are aso 3ey to the s"rviva of bacteria. These factors are re$resented in the .1; standard deviation bet%een individ"as. Tabe 1 sho%s $ercent resistance hi#hy correates %ith Shannon enthro$y 597, a meas"rement of the str"ct"ra dissimiarity of a RNA. Aso the ad4"sted base $air distance 5(7 has a o% $ va"e 50.4090247. 2b is the n"mber of base $airs observed in the secondary str"ct"re and mfe is 8ibbs free ener#y. )n Tabe . , the tota resistance and tota ex$ression ro%s sho% that s$ecies %ith stron#er secondary str"ct"res are abe to be #eneray more ex$ressed before, d"rin#, and after c$. The $ercent resistance ro% sho%s the im$ortance of ada$tation in a s$ecies: abiity to fo"rish. <r#anisms are characteri'ed by maxim"m $resence in the #"t d"rin# a re#iment of c$ by havin# a oca maxim"m to diversity %hie maintainin# an overa minim"m secondary str"ct"re. Tabe 1 sho%s the 2earson:s Correation coefficient and $ va"e of 16S rRNA se*"ence co"nts for a combination of A6U 4"xta$osed %ith 86C. A hi#h concentration of 86C, the more stabe #enetic str"ct"re, eads to ess overa ex$ression b"t a hi#her abiity to resist the effects of c$. A6U, the ess stabe n"ceotides, correate %ith #reater abiity to resist c$ and hi#her overa ex$ression b"t a esser $ercent of resistance. The trend is ma#nified ony %hen the $ercenta#e re$resentation of "raci and #"anine do"bes. +"rther st"dyin# the effects of antibiotics on bacteria based on the se*"entia and thermodynamic characteristics of 16S rRNA, ) anay'ed $yrose*encin# data from FAntibiotics in +eed )nd"ce 2ro$ha#es in S%ine +eca MicrobiomesG 5mBio7. /?0 )n mBio, fo"r #ro"$s of $i#s %ere either nonAmedicated, #iven AS2.->, or a o% or hi#h dose of Carbadox. +o"r s%ine %ere #iven nonAmedicated feed and meas"red - times over the co"rse of ;> days. Six s%ine %ere #iven AS2.-> for 1, days and meas"red t%ice d"rin# treatment. +o"r s%ine %ere #iven a s"bthera$e"tic dose of Carbadox and %ere meas"red once at 1, days. Six s%ine %ere #iven a thera$e"tic dose of Carbadox for .= days and meas"red t%ice. Three cate#ories simiar to those "sed for the 2NAS anaysis %ere seected. Tota resistance %as recorded as the amo"nt of rarified ab"ndances d"rin# AS2.->, s"bthera$e"tic Carbadox or thera$e"tic Carbadox. NonAmedicated ex$ression %as recorded as the tota and avera#e nonAmedicated rarefied ab"ndances. )n mBio there %as ony one $retreatment sam$e and no sam$es ta3en foo%in# the cessation of treatment, so avera#e $ercent resistance %as recorded as the avera#e across each s%ine ex$ression d"rin# AS2.-> or Carbadox divided by the avera#e nonAmedicated amo"nt. Simiar to the c$ anaysis, there %as a o% standard deviation 5.1-7 bet%een the medicated individ"as: ex$ression. NonAmedicated individ"as had neary a do"be standard deviation 5..?7 sho%in# that %itho"t the seective $ress"re of antibiotics, a biome ada$ts to the environment of the individ"a. Tabe , sho%s the $robabiities of correations from rarefied ab"ndances for the three seected cate#ories. Avera#e $ercent resistance is notaby staticay %ea3er in the mBio anaysis, b"t tota resistance and nonA medicated ex$ression have statisticay si#nificant res"ts. )n Tabe ,, the 2b and M+& co"mns sho% a o% $ va"e for a tota resistance and nonAmedicated cate#ories. 2b res$onds simiary to the c$ ex$eriment b"t rRNA M+& $ays a different and si#nificant roe in an or#anism:s abiity to resist and thrive in AS2.-> and Carbadox. Tabe - sho%s that M+& has a stron# ne#ative correation to ex$ression d"rin# an antibiotic and ex$ression in a nonAmedicated settin#. A ne#ativey correated M+& means that "ni3e the c$ ex$eriment, a thermodynamicay stabe rRNA is seected by the AS2.-> and Carbadox. Tabe 6 sho%s that correations and $ va"es from A6U and 86C co"nts are stron#y inverted from the c$ ex$eriment. Stabe din"ceotide 88 is seected by AS2.-> and Carbadox %hie thermodynamicay oose din"ceotide UA has a staticay si#nificant reationshi$ to avera#e $ercent resistance. Beca"se 2NAS st"dies and mBio st"dies $rovide a "ni*"e vie% of thermodynamics of 16S rRNA d"rin# antibiotics, a third $a$er:s data %as anay'ed. )n IC):s F@ancomycinAresistant &nterococc"s domination of intestina microbiota is enabed by antibiotic treatment in mice and $recedes boodstream invasion in h"mansG mice ie"m, ie"m %a, cec"m, and feces %ere anay'ed in the $resence of Am$iciin, @ancomycin, and an antibiotic coc3tai containin# Metronida'oe, Neomycin, and @ancomycin 5MN@7. /1>0 )e"m and cec"m %as extracted from the mo"se ma3in# each ex$eriment termina. Cate#ories %ere created simiar to the mBio $a$er. An avera#e tota ex$ression meas"red the avera#e rarefied ex$ression of 16S reads in the "ntreated mice, combinin# ie"m, ie"m %a and cec"m data into one meas"rement. Three tota resistance cate#ories contained the s"m of a rarefied reads ta3en in the $resence of Am$icin, MN@, and @ancomycin. An avera#e $ercent resistance cate#ory %as aso created %ith the avera#e ex$ression d"rin# an antibiotic divided by the avera#e "ntreated ex$ression. Simiar to the $revio"s ex$eriments, there %as a o% standard deviation bet%een the s$ecimen on medication 5.167. Tabe ; sho%s the thermodynamic characteristics of IC) mice data. )t is im$ortant to remember that @ancomycin is imited to #ram $ositive bacteria and Am$iciin has a narro% abiity to affect #ram ne#ative bacteria. Simiar to mBio:s ex$eriment %ith Carbadox and AS2.->, 2b and M+& $ay an im$ortant roe in characteri'in# the abiity of a s$ecies to resist the effects of an antibiotic. Stabiity a#ain is favored for resistin# a three antibiotics. Uni3e mBio:s res"ts, base $airin# and minim"m free ener#y are abe to characteri'e the $ercent resistance cate#ory. Tabe = sho%s that A6U sti $ay an im$ortant roe in characteri'in# an or#anism:s abiity to resist antibiotics, b"t 86C have a ne#i#ibe effect on both. The reativey %ea3 dimer UC meas"red as a $ercent $ositivey correates to tota resistance and avera#e $ercent resistance. The very stabe dimer 88 meas"red as a $ercent si#nificanty ne#ativey correates to avera#e $ercent resistance and tota resistance. IC):s data aso contained a st"dy on the intestina microbiota of ao#eneic hemato$oietic stem ce trans$antation 5aoA!SCT7. These $atients had an "nderyin# cancer that had been treated by chemothera$y. As the a"thors $ointed o"t in IC), it is diffic"t to dra% conc"sions from this data beca"se of the m"tifod eve of contrib"tin# factors to the $atient:s microbiota antibiotics. &ach medication seects for a different set of criteria based on the method of action and most $atients %ere on m"ti$e antibiotics for different d"rations at different dosa#es. This %as sho%n in the standard deviation of the sam$es ta3en d"rin# medicine bein# si#nificanty ar#er than the 2NAS and mBio 51.>?7. The $atients %ith the east dr"# chan#es had a o%er standard deviation amon# their sam$es. Tabe ? sho%s that bacteria %ere seected from either s$ectr"m of the thermodynamic s$ectr"m, simiar to both 2NAS thermodynamicay instabe and mBio thermodynamicay stron#. The %ea3est din"ceotide UU $ositivey correates to s"rviva of a bacteri"m in some $atients b"t ne#ativey correates to other $atients. To f"rther investi#ate the reationshi$ of antibacteria resistance and ex$ression, -, feat"res %ere recorded and in$"t into a m"ti$e inear re#ression 5MBR7. Binear re#ression is a method of $redictin# an o"tcome of a de$endant variabe #iven a sin#e or m"ti$e ex$anatory variabes. &x$anatory variabes are essentiay feat"res "sed to describe a mathematica ine that re$resents the de$endent variabes. Coefficients are modifiers of ex$anatory variabes deveo$ed that match in$"t data to a de$endent 5or trainin#7 variabe. Mono and din"ceotide fre*"encies as %e as , thermodynamic stabiity *"antities and a n"ceotide and thermodynamic $redicators %ere normai'ed to se*"ence en#th. Three inear modes %ere deveo$ed based on the 2NAS, mBio, and IC) data. Jhen the inear mode %as as3ed to $redict the sim$e $ercent of resistance of 2NAS, it %as acc"rate to .>C of the act"a resistance in ;=C of the rRNA. )t %as acc"rate to 1>C of act"a resistance for -;C of a res$onses. The binomia distrib"tion of 1>C 51K1>7 and .>C 51K.7 acc"racy for .=.; sam$es is >. Tabe 1> sho%s that the abiity to $redict resistance %as #enerai'ed across the famiies of the bacteria 3in#dom. The ar#e +irmic"tes and Bacteroidetes famiies both $erformed %e, as evidenced by the avera#e acc"racy of 1,C. This sho%s that the h"man #"t:s microbiota:s abiity to resist antibiotics can be *"antified by rRNA se*"entia and thermodynamic characteristics. To increase the s$ecificity of the inear mode, the rRNA of the 2NAS sam$es %ere sh"ffed "sin# a sim$e monon"cide sh"ffe and added to the trainin# set. The random se*"ences %ere identified by ne#ative n"mber or n"mbers %ith a $ercent ess than .>1 in =1C of the time. ??C of a sh"ffed se*"ences $ro$ery identified as havin# a $ercent resistance of ess than ... This mar#inay decreased the acc"racy of the MBR to ;-C. A se$arate m"ti$e inear re#ression mode %as b"it based on the mBio data %ith the same feat"res as the 2NAS $a$er. The $redicted variabe had to be atered to fit the data beca"se there %as no $recedin# or contin"in# data after treatment from the ex$eriment. )nstead a variabe %as seected simiar to $ercent resistance 5the avera#e amo"nt of resistance divided by the s"m of the avera#e nonAmedicated and avera#e medicated sam$es7. This n"mber is "sef" beca"se i3e $ercent resistance it tes %hat resistance to ex$ect #iven an initia amo"nt. The s"bAthera$e"tic Carbox MBR $erformed simiar to the c$ ex$eriment yieded an acc"racy of .>C for ;=C of the data. The thera$e"tic Carbox and AS2.-> had a o%er acc"racy of .>C for -6C and -,C, res$ectivey, of the sam$es. Tabe 11 sho%s that the abiity to $redict the AS2.->, s"b thera$e"tic and thera$e"tic Carbox. +irmac"tes $erformed %ith an avera#e acc"racy of .-C, 16C and .-C for AS2.->, S"bthera$e"tic and Thera$e"tic Carbox, res$ectivey. Bacteroidetes i3e%ise $erformed %ith an avera#e acc"racy of .1C, 1-C and .1C. Jhen monon"cide sh"ffed reads %ere added to increase s$ecificity, the avera#e acc"racy increased in AS2.-> to 6.C. S"bthera$e"tic Carbox i3e%ise increased to ;6C and thera$e"tic Carbox increased to 61C. Randomy sh"ffed se*"ences %ere correcty identified by the modes as havin# a $ercent resistance of ess than .>1 =.C of the time. A third MBR %as created for the IC) data b"t it did not $erform as acc"ratey as the 2NAS data or mBio data, b"t the res"ts %ere sti statistcay si#nificant based on the Binomia distrib"tion 5see Tabe 1.7. )n the Am$iciin, @ancomycin and MN@ ex$eriment the MBR %as acc"rate to .>C of the act"a resistance in 1=C, ,-C and 1;C of the rRNA. <ne ex$anation for the o%er acc"racy is the o%er de$th. There %ere si#nificanty fe%er +irmac"tes 5-617 and Bacteroidetes 5-7 in the IC) data %hen com$ared %ith the 2NAS 5..1-, ,;=7. Aso the medications "sed in the IC) ex$eriment had a narro%er s$ectr"m of inf"ence. Am$iciin ony effects some #ram ne#atives %hie @ancomycin affects ony #ram $ositives, b"t the MN@ coc3tai sho"d effect both #ram $ositive and ne#ative bacteria. 16S rRNA se*"entia characteristics extend beyond taxonomy and are abe to #"ide "s in a s$eciesAbased anaysis of antibiotic resistance. 16S rRNA %o"d not $rovide information on strainAbased resistance beca"se t%o strains share the same 16S rRNA. The MBRs %ere "sed to ma3e $redictions concernin# the $ercent resistance from 1. famiies fo"nd in the #"t from the S)B@A database. A contentio"s of each MBR %as made and 1;. s$ecies %ere fo"nd to have a hi#h $robabiity of antibiotic resistance. The res"ts inc"ded &"bacteri"m brachy, a chest infection %hich re*"ires m"ti$e days of $eniciin /.60 and Acanthamoeba, %hich can ca"se bindness %hen $resent in the eye and is imm"ne to many contact disinfectants /.;0. Aso inc"ded %as 2revotea timonensis, %hich sho%ed imm"nity to $eniciin in a st"dy /.=0. Additionay Stre$tococc"s, the ca"se of stre$ throat, %as isoated. An "nc"t"red +"sobacteri"m %as aso identified as $otentiay antibiotic resistant. Some +"sobacteri"m infections are caed BemierreLs syndrome, a com$ication of stre$ throat in %hich the esions ca"sed by stre$ throat become infected, "timatey ca"sin# severe $ne"monia /.?0 The Antibiotic Resistance 8enes (atabase 5AR8B7 contains a ist of #en"s and s$ecies 3no%n to have antibiotic resistance. 8enera, %hich %ere $redicted to have at east 1>C $ercent resistant in c$, AS2.-> and Carbadox ex$eriments %ere cross correated %ith the AR8B. .1 8enera %ere vaidated by AR(B and are sho%n in Tabe 11. Most 8enera %ere resistant to tetracycine. M"tations in the 16S re#ion are 3no%n to ca"se resistance to tetracycine. ? of the .1 %ere aso resistant to bacitracin, an in#redient in Neos$orin /1>0 /110. 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Bioinformatics .1M 11.1A111>. 6. 8atier NB, I. R. 51??;7 Reationshi$s Bet%een 8enomic 86C Content, RNA Secondary Str"ct"res, and <$tima 8ro%th Tem$erat"re in 2ro3aryotes. I<URNAB <+ M<B&CUBAR &@<BUT)<N ,,M 61.A616. ;. B"ra3ovs3y (&, 2ro3horova )@, Ser#iev 2@, Mion 2, Ser#eeva <@, et a. 5.>1.7 )m$act of methyations of m.8?66Km-C?6; in 16S rRNA on bacteria fitness and transation initiation. N"ceic Acids Res. =. (ethefsen B, Reman (A 5.>117 )ncom$ete recovery and individ"ai'ed res$onses of the h"man dista #"t microbiota to re$eated antibiotic $ert"rbation. 2roc Nat Acad Sci U S A 1>= S"$$ 1M ,--,A,-61. ?. Aen !D, Booft T, Bayes (<, !"m$hrey S, Bevine UO, et a. 5.>117 Antibiotics in feed ind"ce $ro$ha#es in s%ine feca microbiomes. MBio .. 1>. Ubeda C, Ta"r O, Ien* RR, &*"inda MI, Son T, et a. 5.>1>7 @ancomycinAresistant &nterococc"s domination of intestina microbiota is enabed by antibiotic treatment in mice and $recedes boodstream invasion in h"mans. I Cin )nvest 1.>M ,11.A,1,1. 11. (e Stasio &A, Moa'ed (, Noer !+, (ahber# A& 51?=?7 M"tations in 16S ribosoma RNA disr"$t antibioticAARNA interactions. &MB< I =M 1.11A1.16. 1.. Becerc* R, Co"rvain 2 51??17 Bacteria resistance to macroide, incosamide, and stre$to#ramin antibiotics by tar#et modification. Antimicrob A#ents Chemother 1-M 1.6;A1.;.. 11. B"sh D, 2"cci MI 5.>117 Ne% antimicrobia a#ents on the hori'on. Biochem 2harmaco =.M 1-.=A 1-1?. 1,. Jachino I, Ara3a%a O 5.>1.7 &xo#eno"sy ac*"ired 16S rRNA methytransferases fo"nd in amino#ycosideAresistant $atho#enic 8ramAne#ative bacteriaM An "$date. (r"# Resistance U$dates. 1-. To%nsend I2, Bohn T, Niesen DM 5.>1.7 Assessin# the $robabiity of detection of hori'onta #ene transfer events in bacteria $o$"ations. +ront Microbio 1M .;. 16. Oamane D, Jachino I, S"'"3i S, Shibata N, Dato !, et a. 5.>>;7 16S rRNA methyaseA$rod"cin#, #ramAne#ative $atho#ens, Ia$an. &mer# )nfect (is 11M 6,.A6,6. 1;. Dristiansson &, +ic3 I, Ian'on A, 8rabic R, R"t#ersson C, et a. 5.>117 2yrose*"encin# of antibioticA contaminated river sediments reveas hi#h eves of resistance and #ene transfer eements. 2BoS <ne 6M e1;>1=. 1=. Stecher B, (en'er R, Maier B, Bernet +, Sanders MI, et a. 5.>1.7 8"t infammation can boost hori'onta #ene transfer bet%een $atho#enic and commensa &nterobacteriaceae. 2roceedin#s of the Nationa Academy of Sciences 1>?M 1.6?A1.;,. 1?. +an#Ayo" O, (an O, Iin#Aye 2, Chon# C, NhiA*ian# 9, et a. 5.>1>7 !i#h $revaence of $asmidA mediated 16S rRNA methyase #ene rmtB amon# &scherichia coi cinica isoates from a Chinese teachin# hos$ita. BMC )nfect (is 1>. .>. +abrice A, (idier R 5.>>?7 &x$orin# microbia diversity "sin# 16S rRNA hi#hAthro"#h$"t methods. I Com$"t Sci Syst Bio .M ;,A?.. .1. 2r"esse &, 9"ast C, Dnitte D, +"chs BM, B"d%i# J, et a. 5.>>;7 S)B@AM a com$rehensive onine reso"rce for *"aity chec3ed and ai#ned ribosoma RNA se*"ence data com$atibe %ith ARB. N"ceic Acids Res 1-M ;1==A;1?6. ... (eSantis TN, !"#enhot' 2, Barsen N, Ro4as M, Brodie &B, et a. 5.>>67 8reen#enes, a chimeraA chec3ed 16S rRNA #ene database and %or3bench com$atibe %ith ARB. A$$ &nviron Microbio ;.M ->6?A->;.. .1. Boon# SN8D, Mishra SD 5.>>;7 Uni*"e fodin# of $rec"rsor microRNAsM 9"antitative evidence and im$ications for de novo identification. Rna 11M 1;>A1=;. .,. 2ace B, Cam$be BB 51?6;7 Correation of maxima #ro%th tem$erat"re and ribosome heat stabiity. 2roc Nat Acad Sci U S A -;M 111>. .-. Noer !+, Joese CR 51?=17 Secondary str"ct"re of 16S ribosoma RNA. Science .1.M ,>1A,11. .6. Rochford IC 51?=>7 2e"ro$"monary infection associated %ith &"bacteri"m brachy, a ne% s$ecies of &"bacteri"m. I Cin Microbio 1.M ;..A;.1. .;. MarcianoACabra +, Cabra 8 5.>>17 Acanthamoeba s$$. as a#ents of disease in h"mans. Cin Microbio Rev 16M .;1A1>;. .=. 8a'"nova <<, Ba"nay T, Rao"t (, Ro"x @ 5.>>;7 2revotea timonensis s$. nov., isoated from a h"man breast abscess. )nt I Syst &vo Microbio -;M ==1A==6. .?. Che"n# JO, Beas I 5.>>;7 +"sobacteri"mM e"sive ca"se of ifeAthreatenin# se$tic thromboemboism. Can +am 2hysician -1M 1,-1A1,-1. 1>. Ross I), &ady &A, Cove I!, C"niffe JI 51??=7 16S rRNA m"tation associated %ith tetracycine resistance in a #ramA$ositive bacteri"m. Antimicrob A#ents Chemother ,.M 1;>.A1;>-. 11. Bi" B, 2o$ M 5.>>?7 AR(BAAAntibiotic Resistance 8enes (atabase. N"ceic Acids Res 1;M (,,1A ,,;. Table 1. PNAS probabilities of correlations to thermodynamic metrics. Pb Npb Mfe Nmfe Q NQ N Total resistance 0.00040! ".4#$%0# 0.1!"1&' 0.0004#& 0.1'929 0.242'9! 0.1"!!4 0.2222&' A(era)e percent resistance 0.000&91 4.&2$%0# 0.00011# 1.49$%0' 1.9!$%0' 4.&1$%0# !.99$%0' 2$%0' Total e*pression 0.00019# 1.0"$%0' 0.41!44' 0.02#!#! 0.4'!&4# 0."!00&# 0.409024 0.41'01!
Table 2. PNAS correlations and p (al+e of correlations to normali,ed n+mber of base pairs obser(ed in the secondary str+ct+re Npb Pearson-s r P (al+e Total resistance >.>;-6.? 1.,&A>- A(era)e percent resistance A>.>?.1 1.--&A>; $*pression >.>?>1., ..?1&A>; Tabe 1. 2NAS 2earson:s correation and $ va"e for tota amo"nt of A6U, 86C, UU and 88 A./ 0.1 // 00 Pearson2s r p (al+e Pearso n2s r p (al+e Pearson2s r p (al+e Pearson2s r p (al+e Total resistance 0.10'"49 ".''$%09 % 0.04'! 9 0.00'941 0.1414'2 "."'$%1' %0.0#0&' %0.0#0&' A(era)e percent resistance %0.0"0&1 0.04#1## % 0.0121 4 0.2'291& %0.0#019 0.0004!1 0.021&9 0.021&9 Total e*pression 0.0!!'04 '.9$%0& % 0.022' " 0.10!4&& 0.11"0'# 2.'&$%10 %0.04411 %0.04411
Table 4. m3io probabilities of correlation of thermodynamics to ab+ndance cate)ories. Pb Npb Mfe Nmfe Q NQ Total resistance asp2'0 &.41$%0! 0.000'9# 1.0'$%10 !.1'$%0& 0 . 0 # 1 0 # 0.2"00'9 0.02#0 '" s+bther !.'#$%0& 0.0002#1 1.1"$%0! #.2!$%0# 0 . " ' & & 0 4 0."#!&&" 0.2224 1# Ther 0.001&& 0.00&1#' 2.0!$%0& 4.1&$%0& 0 . 4 ! & 2 4 1 0."49''! 0."449 " A(era)e percent resistance asp2'0 0.2401 0.4&&2!2 0."'9#!4 0.1"#4&1 0 . 4 & 1 2 0.42414! 0.4902 "! & ' s+bther 0.21'492 0.2"'"# 0.00! 0.00291# 0 . 4 & 0 " 9 0.41!"&# 0."!'! 4& Ther 0.0!'0#2 0."4#001 0.1"0'" 0.4&##!' 0 . 2 1 # 2 " ! 0.""&'0' 0.1"'4 '4 Non%medicated e*pression nonmed #.1&$%0! 4.04$%0# 2.4#$%09 1.!&$%0# 0 . " 4 " 2 ! 2 0."91'42 0.21!' 99 nonmeda(era)e &.19$%09 1.#9$%0# 4.2$%10 1.1'$%0# 0 . 2 1 2 ! ! 9 0.4&"4"1 0.10!2 "! Table '. m3io M4$ characteri,es antibiotic adaptation and o(erall e*pression. M4$ Pearson-s r p (al+e Total resistance asp2'0 %0.12"#4 1.0'$%10 S+bther %0.10!91 1.1"$%0! Ther %0.09!#4 2.0!$%0& A(era)e percent resistance asp2'0 0.00&019 0."'9#!4 S+bther 0.04&0"2 0.00! Ther %0.0219' 0.1"0'" Total non% medicated Nonmed a(era)e %0.1194# 4.2$%10 e*pression Table #. m3io Pearson2s correlations and p (al+es for total amo+nt of A6U, 86C, UU and 88 A./ 0.1 /A 00 Pearson2s r p (al+e Pearson2s r p (al+e Pearson2s r p (al+e Pearson2s Total resistance asp2'0 %0.002'4 0.44!"4 0.11#0'' 1.2'$%09 %0.00!12 0.""! !'2 0.100"!4 s+bther 0.00"91# 0.420'#1 0.09"1"9 !.!4$%0& 0.01'192 0.21! "#9 0.092&4" ther %0.0""04 0.04'"22 0.0!42 &.!!$%0# %0.0"1'1 0.0'" "0# 0.104"1' A(era)e percent resistance asp2'0 0.0240'! 0.109029 %0.001&" 0.4#4#' # 0.0"2#!! 0.04& 0!# %0.01#&# s+bther 0.044442 0.011419 %0.0''1# 0.002"' & 0.0!091! 1.#&$% 0' %0.0#0' ther 0.0"'101 0.0"#1"! 0.014'1& 0.22!#! & 0.0"944! 0.021 #!' 0.002101 Total non%medicated e*pression Nonmed a() %0.00149 0.4#9#&4 0.1090## 1.0&$%0! %0.01!9' 0.1## 022 0.09!'!4 Table &. 516 Pearson2s correlations and p (al+es of n+mber of base pairs obser(ed in secondary str+ct+re of Pb and M4$ Pb M4$ Pearson-s r p (al+e Pearson-s r p (al+e Total resistance Ampicillin 0.1"20!' 0.00024' %0.0!994 0.00!9"' 7ancomycin 0.09110# 0.00!219 %0.0!2# 0.014!42 M7N 0.120199 0.000 %0.0!9!" 0.009009 A(era)e percent resistance Ampicillin 0.14 4.!1$%0' %0.124"4 0.000'19 7ancomycin 0.1&4&91 1.!4$%0# %0.1!2'' #.'&$%0& M7N 0.1&"&!1 2.1$%0# %0.19'21 1.11$%0& Total non%medicated e*pression +ntreated a(era)e 0.0#1!1# 0.0'19!" %0.02#&# 0.2409'# Table !. 516 Pearson2s correlations and p (al+es for total amo+nt of A6U, 86C, UU and 88 A./ 0.1 /1 00 Pearson2s r p (al+e Pe ars on2 s r p (al+e Pearson2s r p (al+e Pearson2s Total resistance Ampicillin 0.09""!2 0.00#9#2 0.0 "4 #' " 0.1!11!4 0.0&1!#4 0.029 "2" %0.0#&92 7ancomycin 0.0'40'' 0.0&&'91 0.0 "! 4# & 0.1''9#4 0.100!## 0.00" 9"9 %0.004#2 M7N 0.0!2"0& 0.01'1"& 0.0 4" 4" 2 0.12#&#& 0.09"491 0.00# 90# %0.0"991 A(era)e percent resistance Ampicillin 0.12024 0.000&'9 0.0 "' 0# # 0.1&!""! 0.090'&! 0.00! '"9 %0.119&9 7ancomycin 0.11"!'! 0.001"4" 0.0 '" "4 ' 0.0!0"42 0.141&"2 9.0!$% 0' %0.0!!&& M7N 0.11'#4" 0.00114! 0.0 4" #" ! 0.12'#41 0.14#904 '.19$% 0' %0.1002# Total non%medicated e*pression +ntreated a(era)e 0.0'4&# 0.0&4929 0.0 "0 !& 9 0.20!'09 0.00"14# 0.4#& 0# %0.0""'' Table 9. 516 allo%8S1T Pearson2s correlations and p (al+es for total amo+nt of // // Patient A(era)e $*pression Pearson-s r p (al+e A %0.1'2"2 0.014!1& 3 0.140"'! 0.022#2' 1 0.11&2#1 0.04&4" %0.1'141 0.01'"1# $ 0.12#492 0.0"'&09 Table 10. PNAS acc+racy of predictions by families Ta*onomy Name Total n+mber of r9NA 10: acc+racy 20: acc+racy A(era)e acc+racy A(era)e error 3inomial 10: 3inomial 20: ;in)dom 3acteria 2!2& 142' 219! 0.1"!""# 0.&1'4'& 0 0 4amily 94" 1 1 1 0.02#1'1 0.02#"0# 0." 0.' Teneric+tes 2" 12 19 0.10424 0.2492' 0.01420!0&' 0.0010'# 1yanobacteria # 4 # 0.09'!9" 0.''2499 0.0'9'"' 0.01'#2' 3acteroidetes 4&! 22! "#0 0.142242 2.!!!""2 1.9#""&$%1# '.#2$%"0 Syner)istetes 1 1 1 0.04&!1" 0.011"'# 0." 0.' 4irmic+tes 22"' 11"2 1&4' 0.1"!!#2 0.2'19"1 0 0 4+sobacteria 1 1 0.149& 0.04"## 0.& 0.' Proteobacteria 29 2" 2# 0.0!#2!4 0.1&'&1! '.2#12'$%0! #.!1$%0# 1andidatedi(isionTM& 2 0.20"21" 0.0#!#"# 0.49 0.2' Actinobacteria "& 19 2# 0.1"&904 0.044192 0.00""44&9& 0.00#221 <entisphaerae " " 0.1"4#'! 0.0##!'4 0."4" 0.12' 7err+comicrobia 10 4 9 0.1090&' #.2&09!& 0.200120949 0.009&## Acidobacteria 1 1 1 0.02'9'# 0.000!44 0." 0.' Table 11. m3io acc+racy of predictions by families ASP 2'0 S+bTher Ther Ta*onomy Name Total n+mber of r9NA A(era)e acc+racy 3inomial 10: 3inomial 20: A(e ra) e acc +ra cy 3inomial 10: 3inomial 20: A(era)e acc+racy ;in)dom 3acteria 24!1 0.24!'#4 0.01#40! 2.02$%0# 0.1 #!' 2" 0 0 0.2"24"& 4amily 94" 2 0.4'!"4# 0.49 0.' 0.1 !42 0& 0.49 0.' 0.''4129 Teneric+te s &" 0.2"&&29 0.09999" 0.0924"9 0.2 !0! 2" 0.00"4&' 0.0201"& 0."'!191 1yanobact eria 41 0."1&204 0.110&# 0.1112# 0." &'2 !! 0.12#1' 0.04#!&4 0."9'!'' Planctomy cetes " 0.140!&! 0.441 0.12' 0.0 '"" '& 0.02& 0.12' 0.14"149 Syner)iste tes 1 0.0!2'12 0." 0.' 0.0 #12 '2 0." 0.' 0.0190&4 4irmic+tes 1#0' 0.2''""9 0.01"!9! 0.000111 0.1 #21 9' 0 0 0.22&4# $l+simicro bia 4 0."0!!99 0.2401 0.2' 0.4 "'0 #2 0.2401 0."&' 0.1#90'1 4+sobacte ria 2 0.2"11! 0.49 0.2' 0.1 '&9 0.42 0.' 0.209# 3acteroid etes #'4 0.2"09'& 0.01"#04 0.001"#1 0.1 '44 '4 2.!"$%4' !.21$%'4 0.21"9!& Proteobac teria 19 0.192"04 0.191#"9 0.0'1&' 0.2 4#9 " 0.1'2'29 0.0221&9 0."9'2## 1andidate di(isionT M& 2 0.141##! 0.49 0.2' 0.0 41" '# 0.09 0.2' 0.0"#&#" 4ibrobact eres ' 0.192'&2 0.1#!0& 0."12' 0.1 "92 !" 0."0!& 0.1'#2' 0."#"!0& Actinobac teria 4 0.20##22 0.411# 0."&' 0.1 9!# 0.411# 0.2' 0.21#999 09 <entispha erae 20 0.2"110" 0.191#"9 0.1201"4 0.1 1## &4 0.00101! 0.0010!& 0.2209"9 7err+comi crobia 4 0.242"14 0.2401 0.2' 0." 2"# &1 0.2401 0.2' 0.4!4 Spirochae tes 42 0.2#2'"# 0.119#"9 0.11#!2" 0.1 9!# &4 0.0#&!'1 0.00'!02 0.2"&9!4 Tabe 1.. IC) acc"racy of $redictions by famiies AM2 @ancomyc in MN@ Taxonomy Name Tota n"mber of rRNA Avera#e acc"racy Binomia 1>C Binomia .>C Ave ra# e acc "ra cy Binomia 1>C Binomia .>C Avera#e acc"racy Din#dom Bacteria 661 >.1>161 1.-;&A>6 ..?6&A>= >.. =,? 1; =.>;&A>- >.>>.6-= >.->,>- +amiy @err"comi crobia . >.6>1;.1 >.,. >.- >., .11 -? >.,? >.- >.?.11-? Cyanobact eria - >.1=>1=. >.16=>; >.>11.- >.1 11; 16 >.16=>; >.11.- >.1>;=6? 2roteobact eria .; >.,.,?-= >.>>,.16 >.>>6616 >.1 -;, 1; >.>1=?>6 >.>1,?. >.6,-.1= Chorofe xi 1 >.6.1?.- >.; >.- >.1 1>. ? >.; >.- >.=6?;1 +irmic"tes -61 >..==11. >.>>>.? -.11&A>- >.. 6;. ;= >.>>;--6 >.>1?1>6 >.,==6, Acidobact 1 >..11;6, >.; >.- >.. >.; >.- >..61>;1 eria 61> ;1 Actinobac teria -6 >.1=1.-; >.>>1,.1 ..6.&A>- >., 1,1 >. ..6.&A>- =.>6&A>- >.61161- Bacteroid etes - >.1;1>,1 >.11.1 >.11.- >.1 1-- ?; >.16>1- >.>11.- >.11,;-1 Nitros$ira e 1 >.?>11>. >.; >.- >., 6?; =; >.; >.- >.,6?;=; Teneric"te s . >..6>->1 >.,? >.- >.. ,.6 -1 >.,? >.- >..,.6-1 Tabe 11. Seect #enera from $redictions ma$$ed to AR(B %ith seect dr"# resistances 8en"s tetracycine bacitracin choram$henico incosamide macroide stre$tomycin vancomycin +ine#odia x &"bacteri"m x x x x Bactococc"s x x x x Stre$tococc"s x x x x x +aecaibacteri"m x R"minococcaceae x 2revote x Acidaminococc"s x x Anaerotr"nc"s x Seenomonas x x Anaerococc"s x x &nterococc"s x x x x x x x Anaerosti$es x x Bactobaci"s x x x x x 2ediococc"s x x Co$rococc"s x x Costridi"m x x x x x x x R"minococc"s x (orea x x B"tyrivibrio x 8ran"icatea x Tota co"nt 1= ? 6 6 - - -