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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.
Ackno"ledg#ents
) than3 Bes (ethefsen for $rovidin# his 16S rRNA data and for he$ %ith conce$t"ai'in# the com$arison
of sam$es. ) aso %o"d i3e to than3 !eather D. Aen for $rovidin# her data from mBio and Cares
Ubeda for $rovidin# data from his IC) ex$eriments. +inay, ) than3 the NCB) SRA for hostin# said data.
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antibioticAARNA interactions. &MB< I =M 1.11A1.16.
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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&#2 %0.0!9!" 0.009009
A(era)e percent
resistance
Ampicillin 0.14&#11 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&#1
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 - - -

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