Research ArticleIn Vitro Activity of 120573-Lactams in Combination with120573-Lactamase Inhibitors againstMycobacterium tuberculosisClinical Isolates

Fu Li 1 Li Wan2 Tongyang Xiao 1 Haican Liu 1 Yi Jiang1

Xiuqin Zhao 1 Ruibai Wang 1 and KanglinWan 1

1State Key Laboratory for Infectious Diseases Prevention and Control Collaborative Innovation Center for Diagnosis andTreatment of Infectious Diseases National Institute for Communicable Disease Control and Prevention Chinese Center forDisease Control and Prevention Beijing 102206 China2Department of Physiology Xiangya School of Medicine Central South University Changsha Hunan 410078 China

Correspondence should be addressed to Ruibai Wang wangruibaiicdccn and Kanglin Wan wankanglinicdccn

Received 19 February 2018 Accepted 4 June 2018 Published 2 July 2018

Academic Editor Isabel Portugal

Copyright copy 2018 Fu Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives Evaluating the activity of nineteen120573-lactams in combinationwith different120573-lactamase inhibitors to determine themostpotent combination againstMycobacterium tuberculosis (MTB) in vitroMethods Drug activity was examined by drug susceptibilitytest with 122 clinical isolates from China Mutations of blaC and drug targets ldtMt1 ldtMt2 dacB2 and crfA were analyzed bynucleotide sequencing Results Tebipenem (TBM) in combination with clavulanate (CLA) exhibited the highest anti-TB activityTheMIC of 120573-lactam antibiotics was reduced most evidently in the presence of CLA compared to avibactam (AVI) and sulbactam(SUB) Eight polymorphism sites were identified in blaC which were not associated with 120573-lactams resistance Interestinglyone strain carrying G514A mutation in blaC was highly susceptible to 120573-lactams regardless of the presence of inhibitors Thetranspeptidase encoding genes ldtMt1 ldtMt2 and dacB2 harboured threemutations twomutations and onemutation respectivelybut no correlation was found between these mutations and drug resistance Conclusion The activity of 120573-lactams against MTB anddifferent synergetic effect of 120573-lactamase inhibitors were indicated TBMCLA exhibited the most activity and has a great prospectin developing novel anti-TB regimen however further clinical research is warranted Moreover the resistance to the 120573-lactamantibiotics might not be conferred by single target mutation in MTB and requires further studies

1 Introduction

Tuberculosis (TB) is a great threat to the global public healththat caused an estimated 13 million deaths in 2016 [1] Drugresistance has been a major obstacle in the progress of elim-inating TB especially due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) MTBworldwide [2ndash4] With the rising incidence of drug-resistantTB the traditional first-line and second-line anti-TB drugsfail to fulfill the current needs of TB treatments In suchcases developing novel drugs or drug regimens is urgent andnecessary for effective control of TB Although significantprogress has been made in identification of several newanti-TB compounds such as bedaquiline delamanid and

pretomanid which are in phase II or III trials [5 6] longduration and high expenses lead to slow development ofnew drugs Repurposing the currently used antibiotics maycontribute to the development of novel anti-TB regimens120573-lactams disrupt the cell wall biosynthesis of bacteria

by preventing the formation of peptide cross-links betweenthe adjacent polysaccharide chains in the peptidoglycan layer[7] In Mycobacterium tuberculosis (MTB) two kinds oftranspeptidase the LD-transpeptidase (mainly encoded byldtMt1 and ldtMt2) and the DD-transpeptidase (encoded bydacB2) catalyze the cell wall biosynthesis [8 9] The LD-transpeptidase which is responsible for the formation of over80 transpeptide linkages (3997888rarr3 linkages) in MTB is thetarget of carbapenems [10ndash12] The DD-transpeptidase that

HindawiBioMed Research InternationalVolume 2018 Article ID 3579832 8 pageshttpsdoiorg10115520183579832

2 BioMed Research International

mainly catalyzes the formation of 4997888rarr3 linkages is a kind ofpenicillin-binding proteins (PBPs) which can be inhibited bymeropenem (MEM) [13]

The resistance to 120573-lactam antibiotics encompassesthe activity of 120573-lactamases Thousands of different 120573-lactamases have been identified in various environmentaland pathogenic species of bacteria [14] MTB is intrinsicallyresistant to 120573-lactams due to the expression of endogenous120573-lactamase BlaC active against a broad spectrum of 120573-lactams CLA AVI and SUB are 120573-lactamase inhibitorsthat inhibit the hydrolysis of 120573-lactams Recent studieshave reported that a combination of the 120573-lactams and 120573-lactamase inhibitors is effective against MTB in vitro andin vivo [15ndash17] The efficacy safety and tolerability of car-bapenems such as MEM imipenem and ertapenem (ETP)against MTB have been evaluated in several clinical studies[18] Amoxicillinclavulanate (AMXCLA) combination hasbeen evaluated to be efficient against MTB in vitro and isthus categorized into Group 5 anti-TB drugs by WHO [1920] This suggests that combinations of 120573-lactams and 120573-lactamase inhibitors are prospective candidates in developingmore effective treatment against TB

In this study to determine the most active and applicablecombination of 120573-lactam and 120573-lactamase inhibitors the invitro anti-TB activity of nineteen 120573-lactams belonging to dif-ferent subclasses either alone or in combinationwith different120573-lactamase inhibitors was tested using drug susceptibilitytests A total of 122MTB clinical isolates collected fromChinawere used Mutations in blaC and three main 120573-lactamstargets ldtMt1 ldtMt2 and dacB2 were analyzed to determinethe relationship between drug target mutations and 120573-lactamresistance In addition T184A mutation in an unannotatedgene crfA reported to confer carbapenem resistance inMTBwas also detected

2 Materials and Methods

21 Strains A total of 122 identified clinical MTB isolatesincluding 47 susceptible (S) 64 MDR and 11 XDR isolatesused in this study were collected from seven provincial TBhospitals of China in Beijing Anhui FujianGuizhouHenanXinjiang and Sichuan All the strains were preserved in thestrain bank of National Institute for Communicable DiseaseControl and Prevention Chinese Center for Disease Controland Prevention

22 120573-Lactams and 120573-Lactamase Inhibitors MEM was pur-chased from Sigma-Aldrich Co (St Louis MO USA) Tebi-penem doripenem biapenem ertapenem amoxicillinampicillin methicillin oxacillin cloxacillin nafcillin diclox-acillin piperacillin cephalexin cefuroxime cefixime cef-pirome aztreonam and moxalactam and three 120573-lactamaseinhibitors CLA AVI and SUB used in this study werepurchased from MCE Co (Monmouth Junction NJ USA)(Table 1)

23 Drug Susceptibility Test Drug susceptibility test wasperformed by broth microdilution method using 96-wellmicroplate Briefly a loop ofMTB culture in the late log phase

Table 1The 120573-lactams and 120573-lactamase inhibitors for drug suscep-tibility tests

Classification Agent GenerationCarbapenem Tebipenem

MeropenemDoripenemBiapenemErtapenem

Penicillin AmoxicillinAmpicillinMethicillinOxacillinCloxacillinNafcillin

DicloxacillinPiperacillin

Cephalosporin Cephalexin First generationCefuroxime Second generationCefixime Third generationCefpirome Fourth generation

Monobactam AztreonamOxacephem Moxalactam120573-lactamase inhibitor Clavulanate

AvibactamSulbactam

was fully ground and adjusted with saline to a cell densityof 3 times 108 cellsmL (1 McFarland standard) The suspensionwas diluted 1 20 with 7H9 broth (Difco Detroit MI USA)containing 10 OADC (BD Franklin Lakes NJ USA)Six groups of serial twofold drug dilutions were preparedin the 7H9 broth containing either five different constantconcentrations of each 120573-lactamase inhibitor or no inhibitorat allThe final reactionmix contained 100 120583l of drug solutionand equal volume of bacterial suspension in each well Theplates were sealed and incubated at 37∘C for 7 days Then theindicator (20 120583l Alamar Blue mixed with 50 120583l 5 Tween-80) was added to the drug-containing group when the drug-free control showed color change (blue to pink) The lowestdrug concentration that inhibited the strain growth andprevented color change was recorded as the MIC value Drugsusceptibility tests for each strain were repeated twice

The final concentrations of tebipenem ranged from 0125to 16 120583gml and for the other carbapenems 025 to 32120583gml concentrations were tested in drug susceptibility testsAmpicillin (AMP) and cefuroxime (CXM) were used atconcentrations ranging from 05 to 64 120583gml and the otherchemicals were tested at concentrations ranging from 1 to 128120583gml The final concentration of each 120573-lactamase inhibitorwas 0625120583gml 125120583gml 25120583gml 5120583gml and 10120583gmlrespectively

24 Polymerase Chain Reaction (PCR) and Sequencing Ge-nomic DNA was isolated from mycobacterial cultures byboiling method and was used for amplification of blaC

BioMed Research International 3

Table 2 Primers and conditions used for amplification and sequencing

Primers Sequence (51015840-31015840) PCR conditions Product length (bp)Denaturationa (s) Annealing (∘C s) Elongationb (s)

BlaC-F ATGCGCAACAGAGGATTCGGTC 30 61 30 65 924BlaC-R CTATGCAAGCACACCGGCAACGLdtMt1-F ATGCGTCGAGTGGTTCGTTATC 30 58 30 55 756LdtMt1-R CTAGCCGACCACCTCAATGGLdtMt2-F ATGCCAAAGGTGGGGATTGC 30 59 30 90 1227LdtMt2-R TTACGCCTTGGCGTTACCGGCDacB2-F ACCAGCAACTGCTGGATTTC 30 60 30 85 1196DacB2-R CGTTGATGACCAACGTCTTCCrfA-F ACCCGGCTCACAGAGAATCG 30 60 30 40 457CrfA-R TATCACCGGTAGGCCATGCNote a denaturation temperature was 94∘C b elongation temperature was 72∘C All PCRs were performed for 31 cycles and each reaction was initialized bydenaturation at 94∘C for 10 min and terminated with a final elongation step at 72∘C for 10 min

ldtMt1 ldtMt2 dacB2 and crfA by polymerase chain reaction(PCR) The primer sets and conditions for amplificationare shown in Table 2 The PCR products were sequenced(TSINGKE Biological Technology Beijing China) and thegene polymorphisms were identified by aligning with thereference strainH37Rv (GenBank accession noNC 000962)using MEGA 70 software

3 Results

31 Determination of Inhibitorsrsquo Concentrations The effectiveconcentration of each 120573-lactamase inhibitor was determinedby drug susceptibility tests against six MTB clinical isolateswith nineteen 120573-lactams either alone or in combination withfive different concentrations of 120573-lactamase inhibitors Theresults showed that the MICs did not change when 5 120583gmlconcentration of inhibitors was used (Table 3) Thus wedecided to use inhibitors at a fixed final concentration of5 120583gml in the subsequent experiments All the carbapen-ems AMX ampicillin (AMP) and cefuroxime (CXM) wereselected to perform further tests because the other 120573-lactamsshowed no activity against MTB in the initial screening

32 MIC Profiles of 120573-Lactams against Clinical Isolates TheMIC profiles of the clinical isolates are shown in Table 4 Inthe case of carbapenems TBM and biapenem (BIA) showedbetter activity with MIC90 of 16 120583gml when they were usedalone The MICs of carbapenems were greatly reduced (4-8 folds) except for ETP (1-4 folds) in the presence of 120573-lactamase inhibitors TBM in combination with CLA showedthemost inhibitory activity withMIC50 andMIC90 of 1 120583gmland 2 120583gml respectively followed by BIA (2 120583gml and 4120583gml) and doripenem (DOR) (2 120583gml and 8 120583gml) MEMin combination with CLA also exhibited good activity withMIC50 and MIC90 of 2 120583gml and 8 120583gml respectively butit was lower than that of TBM and BIA ETP in combinationwith any of the inhibitors showed the least activitywithMIC90of gt16 120583gml In the case of penicillins and cephalosporinsAMXCLA showed highest activity against MTB with MIC50and MIC90 of 2 120583gml and 8 120583gml respectively followed

by AMPCLA (4 120583gml and 16 120583gml) CXM alone or incombination with any of the inhibitors showed no anti-TBactivity even when the highest drug concentrationwas tested

Comparison of the synergistic effect of the three different120573-lactamase inhibitors shows that CLA exhibited the mostpotent activity in inhibiting the 120573-lactamase Overall theMICs of 120573-lactams were reduced most prominently (4-32folds) in the presence of CLA followed by AVI (4-8 folds)However the least activity was observed in case of SUB asMIC50 and MIC90 of all the tested drug combinations withSUB showed lesser reduction (2-8 folds) than that in thepresence of CLA and AVI especially when used in combi-nation with TBM BIA and AMP

33 Relationship between Drug Target Mutation and Resis-tance Since the 120573-lactamase BlaC LD-transpeptidase andDD-transpeptidase are potential targets of 120573-lactams theencoding genes including blaC ldtMt1 ldtMt2 and dacB2were analyzed to illustrate the role of drug target mutationsin the emergence of the 120573-lactams resistance in MTB Inaddition mutation at position T184A in an unannotatedprotein encoding gene crfA which was earlier reported toconfer carbapenem resistance inMTB [21] was also analyzedMutations in the five genes are shown in Table 5 and theMICprofiles of the mutant strains are listed in the supplementarymaterials As observed 164 (20122) of clinical isolatescarried 8 mutation sites in blaC The silent mutation C786T(11 isolates) was the most predominant mutation Threenonsynonymousmutations T333G T492A andG514A wereobserved in blaC while none of these mutations was relatedto 120573-lactam resistance Interestingly one strain GZ10116which carried G514A mutation was highly susceptible to 120573-lactams irrespective of the presence of inhibitors

There were 1 1 and 2 strains harbouring A111G G448Aand C659T mutations respectively on ldtMt1 1 strain and 4strains respectively harboured C594T and A776G mutationin ldtMt2 Only one strain which harboured A776Gmutationin ldtMt2 was resistant to all the combinations of 120573-lactamsand inhibitors However we did not find any mutationassociated with the 120573-lactam resistance in 1198971198891199051198721199051 and ldtMt2

4 BioMed Research International

Table3MIC

profi

leso

f120573-la

ctam

salone

orin

combinatio

nwith

inhibitorsagainstsixMTB

isolates

Inhibitors

Con

centratio

nMIC

range(120583gml)

TBM

MEM

DOR

BIA

ETP

AMX

AMP

MET

OXA

CLO

NAF

DIC

PIP

LEX

CXM

CFM

CPO

ATM

MOX

Clavulanate

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625lt1-2

4-8

8-16

8-16

16-32

16-32

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

64-gt128

32-gt64gt128

64-gt128gt128gt128

125

lt1

44-8

4-8

16-32

8-32

16-64

64-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

16-64gt128

32-gt128gt128gt128

25

lt1

2-4

4-8

2-4

8-32

4-32

4-64

32-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1

2 -4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

10lt1

2-4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

Avibactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

4-8

8-16

8-16

8-16

16-gt32

64-128

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

3 2-gt128

32-gt64gt128

32-gt128gt128gt128

125

2-4

8-16

4-16

8-16

16-32

32-128

16-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

lt1-4

8-16

4-8

4-8

8-32

32-64

8-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1-2

4-16

2-4

2-4

8-16

8-32

8-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

10lt1-2

4-16

2-4

2-4

8-16

8-32

4-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

3 2-gt128

8-32gt128

32-gt128gt128gt128

Sulbactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

8-16

16-32

8-16

8-16

32-gt32

64-gt128

32-64

128-gt128

128-gt128gt128gt128gt128gt128

32-gt128

32-gt64gt128

32-gt128gt128gt128

125

4-16

16-32

8-16

4-16

16-32

64-gt128

16-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

4-8

16-32

4-16

4-8

16-32

32-gt128

8-64

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

52-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

102-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

64-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

NoteTB

Mtebipenem

MEM

merop

enem

DOR

dorip

enem

BIAb

iapenemE

TPertapenem

AMX

amoxicillinA

MPam

picillin

MET

methicillin

OXAo

xacillin

CLOcloxacillin

NAFnafcillinD

IC

diclo

xacillin

PIPpiperacillin

LEX

ceph

alexinC

XMcefuroxim

eCF

McefiximeCP

OcefpiromeAT

Maztreon

amM

OX

moxalactam

BioMed Research International 5

Table 4 MICs of 120573-lactams alone or in combination with a fixedconcentration of 5 120583gml of the inhibitor against 122 MTB isolates

Antibiotic Inhibitor MIC range(120583gml)

MIC50(120583gml)

MIC90(120583gml)

TBM None 05-gt16 8 16CLA 0125-gt16 1 2AVI 0125-gt16 1 2SUB 05-gt16 2 4

BIA None 2-gt32 8 16CLA 025-gt32 2 4AVI 05-gt32 2 4SUB 05-gt32 4 8

DOR None 2-gt32 16 16CLA 05-gt32 2 8AVI 05-gt32 4 8SUB 05-gt32 4 16

MEM None 4-gt32 gt32 gt32CLA 025-gt32 2 8AVI 1-gt32 4 16SUB 4-gt32 8 16

ETP None 8-gt32 gt32 gt32CLA 2-gt32 8 16AVI 4-gt32 8 16SUB 4-gt32 16 32

AMX None 8-gt64 gt64 gt64CLA lt05-32 2 8AVI lt05-64 8 16SUB 2-gt64 8 32

AMP None 32-gt64 64 gt64CLA 05-gt64 4 16AVI 05-gt64 8 16SUB 05-gt64 16 32

CXM None 16-gt64 gt64 gt64CLA 1-gt64 32 gt64AVI 2-gt64 32 gt64SUB 4-gt64 64 gt64

Note MIC5090 MICs that inhibit 50 and 90 of the isolates respectively

Only one mutation T659A was found on dacB2 in fourstrains but it was also not associated with the 120573-lactamresistance In addition the results showed that no mutationwas detected in crfA among all the strains in our study Wealso did not find any strain harbouring mutations in the blaCand transpeptidase genes simultaneously

4 Discussion

Novel drugs or regimens are urgently needed due to the risein the prevalence of multidrug-resistant TB worldwide 120573-lactams are widely used and well-tolerated antibiotics usedfor controlling clinical Gram-positive and Gram-negative

Table 5 Mutations on blaC ldtMt1 ldtMt2 and dacB2 genes

Gene Substitution Amino acid change Number of mutantsblaC G183A Leu61Leu 1

T333G Ser111Arg 3C354T Ser118Ser 1G486C Ala162Ala 1T492A Phe164Leu 1G514A Gly172Ser 1C610T Leu204Leu 1C786T Ile262Ile 11

ldtMt1 A111G Pro37Pro 1G448A Ala150Thr 1C659T Ala220Val 2

ldtMt2 C594T Gly198Gly 1A776G Asp259Gly 4

dacB2 T659A Leu220Gln 4

bacterial infections [22] Recently the combination of 120573-lactamase inhibitor and 120573-lactam was shown to be effectivein restoring the potency of 120573-lactam against MTB [15ndash17] In the present study nineteen commercially available120573-lactams and three 120573-lactamase inhibitors were screenedin vitro to identify the most potent combination againstMTB The results show that carbapenems except for ETPexhibited excellent activity against MTB clinical strains Thismay be due to the inactivation of multiple enzymes bycarbapenems whereas the other120573-lactams did not inhibit theLD-transpeptidase in MTB [10] The activity of AMX wasalso improved notably in the presence of clavulanate Whilethe efficacy of AMXCLA against clinical TB remains con-troversial carbapenems may serve as an optimal alternativein developing novel anti-TB regimens The results showedthat all the tested cephalosporins in our study were inactiveagainst MTB Dincer et al [23] reported that cefazolin incombinationwithCLAwas active againstH37RaThismay bedue to the different strain tested and different methodologyused Additionally the concentration of CLA that is 32120583gml was much higher than the concentration (5 120583gml)used in our test This suggests that a more universal drugsusceptibility testing protocol for determining the MIC of 120573-lactams against MTB is needed in the future

Carbapenems displayed a variable activity in this studyTBM in combination with CLA exhibited the highest activityagainstMTB followed byBIA andDORMIC90 of TBMCLAwas 2 120583gml which can be easily obtained in plasma withthe administration of a single dose of 200 mg TBM pivoxilgranules [24] Comparable activity to that of TBMCLAwas observed in case of BIACLA The maximum plasmaconcentration (119862max) of BIA after a single intravenous dosewas 174 120583gml [25] which exceeded MIC90 (2 120583gml) ofBIACLA Both of BIA and TBM are stable against renaldehydropeptidase-I and are also safe for central nervoussystem since they do not exhibit competitive binding tothe 120574-aminobutyric acid (GABA) receptors [26] HoweverBIA requires intravenous administration that is not easilycomplied with by the patients MEMCLA was previously

6 BioMed Research International

reported to be effective against MDR-TBThe 119862max and AUCvalues of MEMwere 26 120583gml and 272ndash324 120583gsdothml respec-tively after being administered by an intravenous infusionof 500 mg of MEM [27] In our results also MEMCLAexhibited potent activity (MIC90 8 120583gml) againstMTB but itwas weaker than that of TBMCLA which is consistent withprevious study [28] This may be explained by lower 119870m andless breakdown of the covalent tebipenem-BlaC adduct incontrast to that of MEM thus suggesting a higher activity ofTBM against MTB [17 29] Oral administration of the TBMpivoxil had a better protective effect than meropenem in allthe tried sepsis models due to its greater tissue distribution[30] Hence as an oral carbapenem TBM may be a betteroption in developing new anti-TB regimen

MTB harbours a chromosomally encoded 120573-lactamasewhich is selectively susceptible to 120573-lactamase inhibitorsIn this study varied synergistic effects of three different 120573-lactamase inhibitors were observed Consistent with previousstudy [28] the greatest reduction in MIC was achieved byCLA in combinationwith120573-lactams especially inAMXCLA(32-fold in MIC50) AVI a newly developed 120573-lactamaseinhibitor combiningwith ceftazidime exhibited good activityagainst Enterobacteriaceae and Pseudomonas aeruginosa [31]However a combination of AVI with 120573-lactams displayed alesser reduction in MIC of 120573-lactams compared to CLA Arecent report has demonstrated that the efficiency of AVIwas lower than that of CLA due to its bulky rings that mayinfluence its binding to BlaC [32] The unstable covalentadduct of SUB and BlaC may also explain its lesser inhibitingactivity than that of CLA [33] Thus CLA seems to be themost suitable in developing anti-TB regimens encompassing120573-lactams In addition the concentration of 5 120583gml of CLAused in the current studywas lower than the119862max of 59120583gmlachieved after a 250 mg three times a day dosage suggestingthat it can be easily achieved in clinical use [34]

For investigating the relationship between mutations inthe 120573-lactam targets and the drug susceptibility of MTB clin-ical isolates blaC ldtMt1 ldtMt2 and dacB2 in 122MTB clinicalisolates were sequenced and analyzed BlaC hydrolyzes the120573-lactam ring which is essential for the activity of 120573-lactamantibiotics resulting in the 120573-lactams resistance in MTBEight differentmutationswere observed in blaC However wedid not find any association between these mutations and 120573-lactam resistance Interestingly one of the strains harbouringG514A (Gly172Ser) mutation was highly susceptible to 120573-lactam antibiotics in either the presence or the absence ofthe inhibitors Zhang et al [35] have reported that the S111Rmutation on BlaC may be associated with an increased sus-ceptibility of MTB Considering that the mutation Gly172Sermay affect the interaction of BlaC and 120573-lactams [36] wemay speculate that this amino change leads to a conformationchange in BlaC which influences its activity

Mutation on target gene often confers drug resistanceto bacteria Four clinical MTB strains were found to carrymutation A776G in ldtMt2 and two of them were susceptibleto 120573-lactams Similarly among the dacB2 mutants oneexhibited resistance while the other exhibited susceptibilityto 120573-lactams However we did not find any correlationbetween these mutations and the 120573-lactams resistance In

this study we only tested four potential targets and noneof the strains harboured mutations simultaneously on allthe four genes It is reported that the 120573-lactam antibioticshave to bind to at least three of the four identified PBPsto be effective [37] Considering the multiple targets of 120573-lactams we may speculate that the drug resistance may notbe conferred by mutation in a single target Further studiesare required to demonstrate the role of drug target mutationsin the development of 120573-lactam resistance inMTB in order toconsider the inclusion of 120573-lactams in the anti-TB treatmentregimens

5 Conclusions

In summary varied activities of carbapenems penicillinsand cephalosporins alone or in combination with 120573-lactamase inhibitors against MTB were observed TBM incombination with CLA showed the most remarkable activityand has a good prospect in developing novel anti-TB regi-mens Further studies are warranted to investigate the efficacyof TBMCLA in the clinical trials Additionally resistance tothe 120573-lactamsmay not be conferred by single target mutationin MTB and thus requires further investigation

Data Availability

The datasets used to support the findings of this study areavailable upon request from the corresponding author

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was financially supported by the Key project of theState Key Laboratory for Infectious Disease Prevention andControl (2014SKLID104) and the project of the National KeyPrograms of Mega Infectious Diseases (2013ZX10003002-001)

Supplementary Materials

The details of mutations and the MIC profile of each mutantin this study are listed in the supplementary material file(Supplementary Materials)

References

[1] World Health Organization Global tuberculosis report WHODocument World Health Organization 2017

[2] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[3] T Cohen H E Jenkins C Lu M McLaughlin K Floyd andM Zignol ldquoOn the spread and control of MDR-TB epidemicsAn examination of trends in anti-tuberculosis drug resistance

BioMed Research International 3

Table 2 Primers and conditions used for amplification and sequencing

Primers Sequence (51015840-31015840) PCR conditions Product length (bp)Denaturationa (s) Annealing (∘C s) Elongationb (s)

BlaC-F ATGCGCAACAGAGGATTCGGTC 30 61 30 65 924BlaC-R CTATGCAAGCACACCGGCAACGLdtMt1-F ATGCGTCGAGTGGTTCGTTATC 30 58 30 55 756LdtMt1-R CTAGCCGACCACCTCAATGGLdtMt2-F ATGCCAAAGGTGGGGATTGC 30 59 30 90 1227LdtMt2-R TTACGCCTTGGCGTTACCGGCDacB2-F ACCAGCAACTGCTGGATTTC 30 60 30 85 1196DacB2-R CGTTGATGACCAACGTCTTCCrfA-F ACCCGGCTCACAGAGAATCG 30 60 30 40 457CrfA-R TATCACCGGTAGGCCATGCNote a denaturation temperature was 94∘C b elongation temperature was 72∘C All PCRs were performed for 31 cycles and each reaction was initialized bydenaturation at 94∘C for 10 min and terminated with a final elongation step at 72∘C for 10 min

ldtMt1 ldtMt2 dacB2 and crfA by polymerase chain reaction(PCR) The primer sets and conditions for amplificationare shown in Table 2 The PCR products were sequenced(TSINGKE Biological Technology Beijing China) and thegene polymorphisms were identified by aligning with thereference strainH37Rv (GenBank accession noNC 000962)using MEGA 70 software

3 Results

31 Determination of Inhibitorsrsquo Concentrations The effectiveconcentration of each 120573-lactamase inhibitor was determinedby drug susceptibility tests against six MTB clinical isolateswith nineteen 120573-lactams either alone or in combination withfive different concentrations of 120573-lactamase inhibitors Theresults showed that the MICs did not change when 5 120583gmlconcentration of inhibitors was used (Table 3) Thus wedecided to use inhibitors at a fixed final concentration of5 120583gml in the subsequent experiments All the carbapen-ems AMX ampicillin (AMP) and cefuroxime (CXM) wereselected to perform further tests because the other 120573-lactamsshowed no activity against MTB in the initial screening

32 MIC Profiles of 120573-Lactams against Clinical Isolates TheMIC profiles of the clinical isolates are shown in Table 4 Inthe case of carbapenems TBM and biapenem (BIA) showedbetter activity with MIC90 of 16 120583gml when they were usedalone The MICs of carbapenems were greatly reduced (4-8 folds) except for ETP (1-4 folds) in the presence of 120573-lactamase inhibitors TBM in combination with CLA showedthemost inhibitory activity withMIC50 andMIC90 of 1 120583gmland 2 120583gml respectively followed by BIA (2 120583gml and 4120583gml) and doripenem (DOR) (2 120583gml and 8 120583gml) MEMin combination with CLA also exhibited good activity withMIC50 and MIC90 of 2 120583gml and 8 120583gml respectively butit was lower than that of TBM and BIA ETP in combinationwith any of the inhibitors showed the least activitywithMIC90of gt16 120583gml In the case of penicillins and cephalosporinsAMXCLA showed highest activity against MTB with MIC50and MIC90 of 2 120583gml and 8 120583gml respectively followed

by AMPCLA (4 120583gml and 16 120583gml) CXM alone or incombination with any of the inhibitors showed no anti-TBactivity even when the highest drug concentrationwas tested

Comparison of the synergistic effect of the three different120573-lactamase inhibitors shows that CLA exhibited the mostpotent activity in inhibiting the 120573-lactamase Overall theMICs of 120573-lactams were reduced most prominently (4-32folds) in the presence of CLA followed by AVI (4-8 folds)However the least activity was observed in case of SUB asMIC50 and MIC90 of all the tested drug combinations withSUB showed lesser reduction (2-8 folds) than that in thepresence of CLA and AVI especially when used in combi-nation with TBM BIA and AMP

33 Relationship between Drug Target Mutation and Resis-tance Since the 120573-lactamase BlaC LD-transpeptidase andDD-transpeptidase are potential targets of 120573-lactams theencoding genes including blaC ldtMt1 ldtMt2 and dacB2were analyzed to illustrate the role of drug target mutationsin the emergence of the 120573-lactams resistance in MTB Inaddition mutation at position T184A in an unannotatedprotein encoding gene crfA which was earlier reported toconfer carbapenem resistance inMTB [21] was also analyzedMutations in the five genes are shown in Table 5 and theMICprofiles of the mutant strains are listed in the supplementarymaterials As observed 164 (20122) of clinical isolatescarried 8 mutation sites in blaC The silent mutation C786T(11 isolates) was the most predominant mutation Threenonsynonymousmutations T333G T492A andG514A wereobserved in blaC while none of these mutations was relatedto 120573-lactam resistance Interestingly one strain GZ10116which carried G514A mutation was highly susceptible to 120573-lactams irrespective of the presence of inhibitors

There were 1 1 and 2 strains harbouring A111G G448Aand C659T mutations respectively on ldtMt1 1 strain and 4strains respectively harboured C594T and A776G mutationin ldtMt2 Only one strain which harboured A776Gmutationin ldtMt2 was resistant to all the combinations of 120573-lactamsand inhibitors However we did not find any mutationassociated with the 120573-lactam resistance in 1198971198891199051198721199051 and ldtMt2

4 BioMed Research International

Table3MIC

profi

leso

f120573-la

ctam

salone

orin

combinatio

nwith

inhibitorsagainstsixMTB

isolates

Inhibitors

Con

centratio

nMIC

range(120583gml)

TBM

MEM

DOR

BIA

ETP

AMX

AMP

MET

OXA

CLO

NAF

DIC

PIP

LEX

CXM

CFM

CPO

ATM

MOX

Clavulanate

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625lt1-2

4-8

8-16

8-16

16-32

16-32

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

64-gt128

32-gt64gt128

64-gt128gt128gt128

125

lt1

44-8

4-8

16-32

8-32

16-64

64-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

16-64gt128

32-gt128gt128gt128

25

lt1

2-4

4-8

2-4

8-32

4-32

4-64

32-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1

2 -4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

10lt1

2-4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

Avibactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

4-8

8-16

8-16

8-16

16-gt32

64-128

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

3 2-gt128

32-gt64gt128

32-gt128gt128gt128

125

2-4

8-16

4-16

8-16

16-32

32-128

16-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

lt1-4

8-16

4-8

4-8

8-32

32-64

8-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1-2

4-16

2-4

2-4

8-16

8-32

8-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

10lt1-2

4-16

2-4

2-4

8-16

8-32

4-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

3 2-gt128

8-32gt128

32-gt128gt128gt128

Sulbactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

8-16

16-32

8-16

8-16

32-gt32

64-gt128

32-64

128-gt128

128-gt128gt128gt128gt128gt128

32-gt128

32-gt64gt128

32-gt128gt128gt128

125

4-16

16-32

8-16

4-16

16-32

64-gt128

16-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

4-8

16-32

4-16

4-8

16-32

32-gt128

8-64

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

52-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

102-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

64-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

NoteTB

Mtebipenem

MEM

merop

enem

DOR

dorip

enem

BIAb

iapenemE

TPertapenem

AMX

amoxicillinA

MPam

picillin

MET

methicillin

OXAo

xacillin

CLOcloxacillin

NAFnafcillinD

IC

diclo

xacillin

PIPpiperacillin

LEX

ceph

alexinC

XMcefuroxim

eCF

McefiximeCP

OcefpiromeAT

Maztreon

amM

OX

moxalactam

BioMed Research International 5

Table 4 MICs of 120573-lactams alone or in combination with a fixedconcentration of 5 120583gml of the inhibitor against 122 MTB isolates

Antibiotic Inhibitor MIC range(120583gml)

MIC50(120583gml)

MIC90(120583gml)

TBM None 05-gt16 8 16CLA 0125-gt16 1 2AVI 0125-gt16 1 2SUB 05-gt16 2 4

BIA None 2-gt32 8 16CLA 025-gt32 2 4AVI 05-gt32 2 4SUB 05-gt32 4 8

DOR None 2-gt32 16 16CLA 05-gt32 2 8AVI 05-gt32 4 8SUB 05-gt32 4 16

MEM None 4-gt32 gt32 gt32CLA 025-gt32 2 8AVI 1-gt32 4 16SUB 4-gt32 8 16

ETP None 8-gt32 gt32 gt32CLA 2-gt32 8 16AVI 4-gt32 8 16SUB 4-gt32 16 32

AMX None 8-gt64 gt64 gt64CLA lt05-32 2 8AVI lt05-64 8 16SUB 2-gt64 8 32

AMP None 32-gt64 64 gt64CLA 05-gt64 4 16AVI 05-gt64 8 16SUB 05-gt64 16 32

CXM None 16-gt64 gt64 gt64CLA 1-gt64 32 gt64AVI 2-gt64 32 gt64SUB 4-gt64 64 gt64

Note MIC5090 MICs that inhibit 50 and 90 of the isolates respectively

Only one mutation T659A was found on dacB2 in fourstrains but it was also not associated with the 120573-lactamresistance In addition the results showed that no mutationwas detected in crfA among all the strains in our study Wealso did not find any strain harbouring mutations in the blaCand transpeptidase genes simultaneously

4 Discussion

Novel drugs or regimens are urgently needed due to the risein the prevalence of multidrug-resistant TB worldwide 120573-lactams are widely used and well-tolerated antibiotics usedfor controlling clinical Gram-positive and Gram-negative

Table 5 Mutations on blaC ldtMt1 ldtMt2 and dacB2 genes

Gene Substitution Amino acid change Number of mutantsblaC G183A Leu61Leu 1

T333G Ser111Arg 3C354T Ser118Ser 1G486C Ala162Ala 1T492A Phe164Leu 1G514A Gly172Ser 1C610T Leu204Leu 1C786T Ile262Ile 11

ldtMt1 A111G Pro37Pro 1G448A Ala150Thr 1C659T Ala220Val 2

ldtMt2 C594T Gly198Gly 1A776G Asp259Gly 4

dacB2 T659A Leu220Gln 4

bacterial infections [22] Recently the combination of 120573-lactamase inhibitor and 120573-lactam was shown to be effectivein restoring the potency of 120573-lactam against MTB [15ndash17] In the present study nineteen commercially available120573-lactams and three 120573-lactamase inhibitors were screenedin vitro to identify the most potent combination againstMTB The results show that carbapenems except for ETPexhibited excellent activity against MTB clinical strains Thismay be due to the inactivation of multiple enzymes bycarbapenems whereas the other120573-lactams did not inhibit theLD-transpeptidase in MTB [10] The activity of AMX wasalso improved notably in the presence of clavulanate Whilethe efficacy of AMXCLA against clinical TB remains con-troversial carbapenems may serve as an optimal alternativein developing novel anti-TB regimens The results showedthat all the tested cephalosporins in our study were inactiveagainst MTB Dincer et al [23] reported that cefazolin incombinationwithCLAwas active againstH37RaThismay bedue to the different strain tested and different methodologyused Additionally the concentration of CLA that is 32120583gml was much higher than the concentration (5 120583gml)used in our test This suggests that a more universal drugsusceptibility testing protocol for determining the MIC of 120573-lactams against MTB is needed in the future

Carbapenems displayed a variable activity in this studyTBM in combination with CLA exhibited the highest activityagainstMTB followed byBIA andDORMIC90 of TBMCLAwas 2 120583gml which can be easily obtained in plasma withthe administration of a single dose of 200 mg TBM pivoxilgranules [24] Comparable activity to that of TBMCLAwas observed in case of BIACLA The maximum plasmaconcentration (119862max) of BIA after a single intravenous dosewas 174 120583gml [25] which exceeded MIC90 (2 120583gml) ofBIACLA Both of BIA and TBM are stable against renaldehydropeptidase-I and are also safe for central nervoussystem since they do not exhibit competitive binding tothe 120574-aminobutyric acid (GABA) receptors [26] HoweverBIA requires intravenous administration that is not easilycomplied with by the patients MEMCLA was previously

6 BioMed Research International

reported to be effective against MDR-TBThe 119862max and AUCvalues of MEMwere 26 120583gml and 272ndash324 120583gsdothml respec-tively after being administered by an intravenous infusionof 500 mg of MEM [27] In our results also MEMCLAexhibited potent activity (MIC90 8 120583gml) againstMTB but itwas weaker than that of TBMCLA which is consistent withprevious study [28] This may be explained by lower 119870m andless breakdown of the covalent tebipenem-BlaC adduct incontrast to that of MEM thus suggesting a higher activity ofTBM against MTB [17 29] Oral administration of the TBMpivoxil had a better protective effect than meropenem in allthe tried sepsis models due to its greater tissue distribution[30] Hence as an oral carbapenem TBM may be a betteroption in developing new anti-TB regimen

MTB harbours a chromosomally encoded 120573-lactamasewhich is selectively susceptible to 120573-lactamase inhibitorsIn this study varied synergistic effects of three different 120573-lactamase inhibitors were observed Consistent with previousstudy [28] the greatest reduction in MIC was achieved byCLA in combinationwith120573-lactams especially inAMXCLA(32-fold in MIC50) AVI a newly developed 120573-lactamaseinhibitor combiningwith ceftazidime exhibited good activityagainst Enterobacteriaceae and Pseudomonas aeruginosa [31]However a combination of AVI with 120573-lactams displayed alesser reduction in MIC of 120573-lactams compared to CLA Arecent report has demonstrated that the efficiency of AVIwas lower than that of CLA due to its bulky rings that mayinfluence its binding to BlaC [32] The unstable covalentadduct of SUB and BlaC may also explain its lesser inhibitingactivity than that of CLA [33] Thus CLA seems to be themost suitable in developing anti-TB regimens encompassing120573-lactams In addition the concentration of 5 120583gml of CLAused in the current studywas lower than the119862max of 59120583gmlachieved after a 250 mg three times a day dosage suggestingthat it can be easily achieved in clinical use [34]

For investigating the relationship between mutations inthe 120573-lactam targets and the drug susceptibility of MTB clin-ical isolates blaC ldtMt1 ldtMt2 and dacB2 in 122MTB clinicalisolates were sequenced and analyzed BlaC hydrolyzes the120573-lactam ring which is essential for the activity of 120573-lactamantibiotics resulting in the 120573-lactams resistance in MTBEight differentmutationswere observed in blaC However wedid not find any association between these mutations and 120573-lactam resistance Interestingly one of the strains harbouringG514A (Gly172Ser) mutation was highly susceptible to 120573-lactam antibiotics in either the presence or the absence ofthe inhibitors Zhang et al [35] have reported that the S111Rmutation on BlaC may be associated with an increased sus-ceptibility of MTB Considering that the mutation Gly172Sermay affect the interaction of BlaC and 120573-lactams [36] wemay speculate that this amino change leads to a conformationchange in BlaC which influences its activity

Mutation on target gene often confers drug resistanceto bacteria Four clinical MTB strains were found to carrymutation A776G in ldtMt2 and two of them were susceptibleto 120573-lactams Similarly among the dacB2 mutants oneexhibited resistance while the other exhibited susceptibilityto 120573-lactams However we did not find any correlationbetween these mutations and the 120573-lactams resistance In

this study we only tested four potential targets and noneof the strains harboured mutations simultaneously on allthe four genes It is reported that the 120573-lactam antibioticshave to bind to at least three of the four identified PBPsto be effective [37] Considering the multiple targets of 120573-lactams we may speculate that the drug resistance may notbe conferred by mutation in a single target Further studiesare required to demonstrate the role of drug target mutationsin the development of 120573-lactam resistance inMTB in order toconsider the inclusion of 120573-lactams in the anti-TB treatmentregimens

5 Conclusions

In summary varied activities of carbapenems penicillinsand cephalosporins alone or in combination with 120573-lactamase inhibitors against MTB were observed TBM incombination with CLA showed the most remarkable activityand has a good prospect in developing novel anti-TB regi-mens Further studies are warranted to investigate the efficacyof TBMCLA in the clinical trials Additionally resistance tothe 120573-lactamsmay not be conferred by single target mutationin MTB and thus requires further investigation

Data Availability

The datasets used to support the findings of this study areavailable upon request from the corresponding author

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was financially supported by the Key project of theState Key Laboratory for Infectious Disease Prevention andControl (2014SKLID104) and the project of the National KeyPrograms of Mega Infectious Diseases (2013ZX10003002-001)

Supplementary Materials

The details of mutations and the MIC profile of each mutantin this study are listed in the supplementary material file(Supplementary Materials)

References

[1] World Health Organization Global tuberculosis report WHODocument World Health Organization 2017

[2] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[3] T Cohen H E Jenkins C Lu M McLaughlin K Floyd andM Zignol ldquoOn the spread and control of MDR-TB epidemicsAn examination of trends in anti-tuberculosis drug resistance

4 BioMed Research International

Table3MIC

profi

leso

f120573-la

ctam

salone

orin

combinatio

nwith

inhibitorsagainstsixMTB

isolates

Inhibitors

Con

centratio

nMIC

range(120583gml)

TBM

MEM

DOR

BIA

ETP

AMX

AMP

MET

OXA

CLO

NAF

DIC

PIP

LEX

CXM

CFM

CPO

ATM

MOX

Clavulanate

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625lt1-2

4-8

8-16

8-16

16-32

16-32

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

64-gt128

32-gt64gt128

64-gt128gt128gt128

125

lt1

44-8

4-8

16-32

8-32

16-64

64-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

16-64gt128

32-gt128gt128gt128

25

lt1

2-4

4-8

2-4

8-32

4-32

4-64

32-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1

2 -4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

10lt1

2-4

2-4

1-28-16

4-8lt1-3

216-gt128

32-gt128

64-gt128

64-gt128gt128

64-gt128

16-gt128

4-16gt128

16-gt128gt128gt128

Avibactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

4-8

8-16

8-16

8-16

16-gt32

64-128

32-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

3 2-gt128

32-gt64gt128

32-gt128gt128gt128

125

2-4

8-16

4-16

8-16

16-32

32-128

16-64

128-gt128

128-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

lt1-4

8-16

4-8

4-8

8-32

32-64

8-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

5lt1-2

4-16

2-4

2-4

8-16

8-32

8-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

10lt1-2

4-16

2-4

2-4

8-16

8-32

4-32

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

3 2-gt128

8-32gt128

32-gt128gt128gt128

Sulbactam

08-16

16-32

8-16

8-16

32-gt32

64-gt128gt64gt128

128-gt128gt128gt128gt128gt128

64-gt128gt64gt128

64-gt128gt128gt128

0625

8-16

16-32

8-16

8-16

32-gt32

64-gt128

32-64

128-gt128

128-gt128gt128gt128gt128gt128

32-gt128

32-gt64gt128

32-gt128gt128gt128

125

4-16

16-32

8-16

4-16

16-32

64-gt128

16-64

64-gt128

64-gt128

128-gt128gt128gt128gt128

32-gt128

32-64gt128

32-gt128gt128gt128

25

4-8

16-32

4-16

4-8

16-32

32-gt128

8-64

32-gt128

64-gt128

128-gt128

64-gt128gt128gt128

32-gt128

16-32gt128

32-gt128gt128gt128

52-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

128-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

102-8

8-32

4-8

2-8

8-32

16-gt64

4-64

16-gt128

32-gt128

64-gt128

64-gt128gt128gt128

32-gt128

8-32gt128

32-gt128gt128gt128

NoteTB

Mtebipenem

MEM

merop

enem

DOR

dorip

enem

BIAb

iapenemE

TPertapenem

AMX

amoxicillinA

MPam

picillin

MET

methicillin

OXAo

xacillin

CLOcloxacillin

NAFnafcillinD

IC

diclo

xacillin

PIPpiperacillin

LEX

ceph

alexinC

XMcefuroxim

eCF

McefiximeCP

OcefpiromeAT

Maztreon

amM

OX

moxalactam

BioMed Research International 5

Table 4 MICs of 120573-lactams alone or in combination with a fixedconcentration of 5 120583gml of the inhibitor against 122 MTB isolates

Antibiotic Inhibitor MIC range(120583gml)

MIC50(120583gml)

MIC90(120583gml)

TBM None 05-gt16 8 16CLA 0125-gt16 1 2AVI 0125-gt16 1 2SUB 05-gt16 2 4

BIA None 2-gt32 8 16CLA 025-gt32 2 4AVI 05-gt32 2 4SUB 05-gt32 4 8

DOR None 2-gt32 16 16CLA 05-gt32 2 8AVI 05-gt32 4 8SUB 05-gt32 4 16

MEM None 4-gt32 gt32 gt32CLA 025-gt32 2 8AVI 1-gt32 4 16SUB 4-gt32 8 16

ETP None 8-gt32 gt32 gt32CLA 2-gt32 8 16AVI 4-gt32 8 16SUB 4-gt32 16 32

AMX None 8-gt64 gt64 gt64CLA lt05-32 2 8AVI lt05-64 8 16SUB 2-gt64 8 32

AMP None 32-gt64 64 gt64CLA 05-gt64 4 16AVI 05-gt64 8 16SUB 05-gt64 16 32

CXM None 16-gt64 gt64 gt64CLA 1-gt64 32 gt64AVI 2-gt64 32 gt64SUB 4-gt64 64 gt64

Note MIC5090 MICs that inhibit 50 and 90 of the isolates respectively

Only one mutation T659A was found on dacB2 in fourstrains but it was also not associated with the 120573-lactamresistance In addition the results showed that no mutationwas detected in crfA among all the strains in our study Wealso did not find any strain harbouring mutations in the blaCand transpeptidase genes simultaneously

4 Discussion

Novel drugs or regimens are urgently needed due to the risein the prevalence of multidrug-resistant TB worldwide 120573-lactams are widely used and well-tolerated antibiotics usedfor controlling clinical Gram-positive and Gram-negative

Table 5 Mutations on blaC ldtMt1 ldtMt2 and dacB2 genes

Gene Substitution Amino acid change Number of mutantsblaC G183A Leu61Leu 1

T333G Ser111Arg 3C354T Ser118Ser 1G486C Ala162Ala 1T492A Phe164Leu 1G514A Gly172Ser 1C610T Leu204Leu 1C786T Ile262Ile 11

ldtMt1 A111G Pro37Pro 1G448A Ala150Thr 1C659T Ala220Val 2

ldtMt2 C594T Gly198Gly 1A776G Asp259Gly 4

dacB2 T659A Leu220Gln 4

bacterial infections [22] Recently the combination of 120573-lactamase inhibitor and 120573-lactam was shown to be effectivein restoring the potency of 120573-lactam against MTB [15ndash17] In the present study nineteen commercially available120573-lactams and three 120573-lactamase inhibitors were screenedin vitro to identify the most potent combination againstMTB The results show that carbapenems except for ETPexhibited excellent activity against MTB clinical strains Thismay be due to the inactivation of multiple enzymes bycarbapenems whereas the other120573-lactams did not inhibit theLD-transpeptidase in MTB [10] The activity of AMX wasalso improved notably in the presence of clavulanate Whilethe efficacy of AMXCLA against clinical TB remains con-troversial carbapenems may serve as an optimal alternativein developing novel anti-TB regimens The results showedthat all the tested cephalosporins in our study were inactiveagainst MTB Dincer et al [23] reported that cefazolin incombinationwithCLAwas active againstH37RaThismay bedue to the different strain tested and different methodologyused Additionally the concentration of CLA that is 32120583gml was much higher than the concentration (5 120583gml)used in our test This suggests that a more universal drugsusceptibility testing protocol for determining the MIC of 120573-lactams against MTB is needed in the future

Carbapenems displayed a variable activity in this studyTBM in combination with CLA exhibited the highest activityagainstMTB followed byBIA andDORMIC90 of TBMCLAwas 2 120583gml which can be easily obtained in plasma withthe administration of a single dose of 200 mg TBM pivoxilgranules [24] Comparable activity to that of TBMCLAwas observed in case of BIACLA The maximum plasmaconcentration (119862max) of BIA after a single intravenous dosewas 174 120583gml [25] which exceeded MIC90 (2 120583gml) ofBIACLA Both of BIA and TBM are stable against renaldehydropeptidase-I and are also safe for central nervoussystem since they do not exhibit competitive binding tothe 120574-aminobutyric acid (GABA) receptors [26] HoweverBIA requires intravenous administration that is not easilycomplied with by the patients MEMCLA was previously

6 BioMed Research International

reported to be effective against MDR-TBThe 119862max and AUCvalues of MEMwere 26 120583gml and 272ndash324 120583gsdothml respec-tively after being administered by an intravenous infusionof 500 mg of MEM [27] In our results also MEMCLAexhibited potent activity (MIC90 8 120583gml) againstMTB but itwas weaker than that of TBMCLA which is consistent withprevious study [28] This may be explained by lower 119870m andless breakdown of the covalent tebipenem-BlaC adduct incontrast to that of MEM thus suggesting a higher activity ofTBM against MTB [17 29] Oral administration of the TBMpivoxil had a better protective effect than meropenem in allthe tried sepsis models due to its greater tissue distribution[30] Hence as an oral carbapenem TBM may be a betteroption in developing new anti-TB regimen

MTB harbours a chromosomally encoded 120573-lactamasewhich is selectively susceptible to 120573-lactamase inhibitorsIn this study varied synergistic effects of three different 120573-lactamase inhibitors were observed Consistent with previousstudy [28] the greatest reduction in MIC was achieved byCLA in combinationwith120573-lactams especially inAMXCLA(32-fold in MIC50) AVI a newly developed 120573-lactamaseinhibitor combiningwith ceftazidime exhibited good activityagainst Enterobacteriaceae and Pseudomonas aeruginosa [31]However a combination of AVI with 120573-lactams displayed alesser reduction in MIC of 120573-lactams compared to CLA Arecent report has demonstrated that the efficiency of AVIwas lower than that of CLA due to its bulky rings that mayinfluence its binding to BlaC [32] The unstable covalentadduct of SUB and BlaC may also explain its lesser inhibitingactivity than that of CLA [33] Thus CLA seems to be themost suitable in developing anti-TB regimens encompassing120573-lactams In addition the concentration of 5 120583gml of CLAused in the current studywas lower than the119862max of 59120583gmlachieved after a 250 mg three times a day dosage suggestingthat it can be easily achieved in clinical use [34]

For investigating the relationship between mutations inthe 120573-lactam targets and the drug susceptibility of MTB clin-ical isolates blaC ldtMt1 ldtMt2 and dacB2 in 122MTB clinicalisolates were sequenced and analyzed BlaC hydrolyzes the120573-lactam ring which is essential for the activity of 120573-lactamantibiotics resulting in the 120573-lactams resistance in MTBEight differentmutationswere observed in blaC However wedid not find any association between these mutations and 120573-lactam resistance Interestingly one of the strains harbouringG514A (Gly172Ser) mutation was highly susceptible to 120573-lactam antibiotics in either the presence or the absence ofthe inhibitors Zhang et al [35] have reported that the S111Rmutation on BlaC may be associated with an increased sus-ceptibility of MTB Considering that the mutation Gly172Sermay affect the interaction of BlaC and 120573-lactams [36] wemay speculate that this amino change leads to a conformationchange in BlaC which influences its activity

Mutation on target gene often confers drug resistanceto bacteria Four clinical MTB strains were found to carrymutation A776G in ldtMt2 and two of them were susceptibleto 120573-lactams Similarly among the dacB2 mutants oneexhibited resistance while the other exhibited susceptibilityto 120573-lactams However we did not find any correlationbetween these mutations and the 120573-lactams resistance In

this study we only tested four potential targets and noneof the strains harboured mutations simultaneously on allthe four genes It is reported that the 120573-lactam antibioticshave to bind to at least three of the four identified PBPsto be effective [37] Considering the multiple targets of 120573-lactams we may speculate that the drug resistance may notbe conferred by mutation in a single target Further studiesare required to demonstrate the role of drug target mutationsin the development of 120573-lactam resistance inMTB in order toconsider the inclusion of 120573-lactams in the anti-TB treatmentregimens

5 Conclusions

In summary varied activities of carbapenems penicillinsand cephalosporins alone or in combination with 120573-lactamase inhibitors against MTB were observed TBM incombination with CLA showed the most remarkable activityand has a good prospect in developing novel anti-TB regi-mens Further studies are warranted to investigate the efficacyof TBMCLA in the clinical trials Additionally resistance tothe 120573-lactamsmay not be conferred by single target mutationin MTB and thus requires further investigation

Data Availability

The datasets used to support the findings of this study areavailable upon request from the corresponding author

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was financially supported by the Key project of theState Key Laboratory for Infectious Disease Prevention andControl (2014SKLID104) and the project of the National KeyPrograms of Mega Infectious Diseases (2013ZX10003002-001)

Supplementary Materials

The details of mutations and the MIC profile of each mutantin this study are listed in the supplementary material file(Supplementary Materials)

References

[1] World Health Organization Global tuberculosis report WHODocument World Health Organization 2017

[2] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[3] T Cohen H E Jenkins C Lu M McLaughlin K Floyd andM Zignol ldquoOn the spread and control of MDR-TB epidemicsAn examination of trends in anti-tuberculosis drug resistance

BioMed Research International 5

Table 4 MICs of 120573-lactams alone or in combination with a fixedconcentration of 5 120583gml of the inhibitor against 122 MTB isolates

Antibiotic Inhibitor MIC range(120583gml)

MIC50(120583gml)

MIC90(120583gml)

TBM None 05-gt16 8 16CLA 0125-gt16 1 2AVI 0125-gt16 1 2SUB 05-gt16 2 4

BIA None 2-gt32 8 16CLA 025-gt32 2 4AVI 05-gt32 2 4SUB 05-gt32 4 8

DOR None 2-gt32 16 16CLA 05-gt32 2 8AVI 05-gt32 4 8SUB 05-gt32 4 16

MEM None 4-gt32 gt32 gt32CLA 025-gt32 2 8AVI 1-gt32 4 16SUB 4-gt32 8 16

ETP None 8-gt32 gt32 gt32CLA 2-gt32 8 16AVI 4-gt32 8 16SUB 4-gt32 16 32

AMX None 8-gt64 gt64 gt64CLA lt05-32 2 8AVI lt05-64 8 16SUB 2-gt64 8 32

AMP None 32-gt64 64 gt64CLA 05-gt64 4 16AVI 05-gt64 8 16SUB 05-gt64 16 32

CXM None 16-gt64 gt64 gt64CLA 1-gt64 32 gt64AVI 2-gt64 32 gt64SUB 4-gt64 64 gt64

Note MIC5090 MICs that inhibit 50 and 90 of the isolates respectively

Only one mutation T659A was found on dacB2 in fourstrains but it was also not associated with the 120573-lactamresistance In addition the results showed that no mutationwas detected in crfA among all the strains in our study Wealso did not find any strain harbouring mutations in the blaCand transpeptidase genes simultaneously

4 Discussion

Novel drugs or regimens are urgently needed due to the risein the prevalence of multidrug-resistant TB worldwide 120573-lactams are widely used and well-tolerated antibiotics usedfor controlling clinical Gram-positive and Gram-negative

Table 5 Mutations on blaC ldtMt1 ldtMt2 and dacB2 genes

Gene Substitution Amino acid change Number of mutantsblaC G183A Leu61Leu 1

T333G Ser111Arg 3C354T Ser118Ser 1G486C Ala162Ala 1T492A Phe164Leu 1G514A Gly172Ser 1C610T Leu204Leu 1C786T Ile262Ile 11

ldtMt1 A111G Pro37Pro 1G448A Ala150Thr 1C659T Ala220Val 2

ldtMt2 C594T Gly198Gly 1A776G Asp259Gly 4

dacB2 T659A Leu220Gln 4

bacterial infections [22] Recently the combination of 120573-lactamase inhibitor and 120573-lactam was shown to be effectivein restoring the potency of 120573-lactam against MTB [15ndash17] In the present study nineteen commercially available120573-lactams and three 120573-lactamase inhibitors were screenedin vitro to identify the most potent combination againstMTB The results show that carbapenems except for ETPexhibited excellent activity against MTB clinical strains Thismay be due to the inactivation of multiple enzymes bycarbapenems whereas the other120573-lactams did not inhibit theLD-transpeptidase in MTB [10] The activity of AMX wasalso improved notably in the presence of clavulanate Whilethe efficacy of AMXCLA against clinical TB remains con-troversial carbapenems may serve as an optimal alternativein developing novel anti-TB regimens The results showedthat all the tested cephalosporins in our study were inactiveagainst MTB Dincer et al [23] reported that cefazolin incombinationwithCLAwas active againstH37RaThismay bedue to the different strain tested and different methodologyused Additionally the concentration of CLA that is 32120583gml was much higher than the concentration (5 120583gml)used in our test This suggests that a more universal drugsusceptibility testing protocol for determining the MIC of 120573-lactams against MTB is needed in the future

Carbapenems displayed a variable activity in this studyTBM in combination with CLA exhibited the highest activityagainstMTB followed byBIA andDORMIC90 of TBMCLAwas 2 120583gml which can be easily obtained in plasma withthe administration of a single dose of 200 mg TBM pivoxilgranules [24] Comparable activity to that of TBMCLAwas observed in case of BIACLA The maximum plasmaconcentration (119862max) of BIA after a single intravenous dosewas 174 120583gml [25] which exceeded MIC90 (2 120583gml) ofBIACLA Both of BIA and TBM are stable against renaldehydropeptidase-I and are also safe for central nervoussystem since they do not exhibit competitive binding tothe 120574-aminobutyric acid (GABA) receptors [26] HoweverBIA requires intravenous administration that is not easilycomplied with by the patients MEMCLA was previously

6 BioMed Research International

reported to be effective against MDR-TBThe 119862max and AUCvalues of MEMwere 26 120583gml and 272ndash324 120583gsdothml respec-tively after being administered by an intravenous infusionof 500 mg of MEM [27] In our results also MEMCLAexhibited potent activity (MIC90 8 120583gml) againstMTB but itwas weaker than that of TBMCLA which is consistent withprevious study [28] This may be explained by lower 119870m andless breakdown of the covalent tebipenem-BlaC adduct incontrast to that of MEM thus suggesting a higher activity ofTBM against MTB [17 29] Oral administration of the TBMpivoxil had a better protective effect than meropenem in allthe tried sepsis models due to its greater tissue distribution[30] Hence as an oral carbapenem TBM may be a betteroption in developing new anti-TB regimen

MTB harbours a chromosomally encoded 120573-lactamasewhich is selectively susceptible to 120573-lactamase inhibitorsIn this study varied synergistic effects of three different 120573-lactamase inhibitors were observed Consistent with previousstudy [28] the greatest reduction in MIC was achieved byCLA in combinationwith120573-lactams especially inAMXCLA(32-fold in MIC50) AVI a newly developed 120573-lactamaseinhibitor combiningwith ceftazidime exhibited good activityagainst Enterobacteriaceae and Pseudomonas aeruginosa [31]However a combination of AVI with 120573-lactams displayed alesser reduction in MIC of 120573-lactams compared to CLA Arecent report has demonstrated that the efficiency of AVIwas lower than that of CLA due to its bulky rings that mayinfluence its binding to BlaC [32] The unstable covalentadduct of SUB and BlaC may also explain its lesser inhibitingactivity than that of CLA [33] Thus CLA seems to be themost suitable in developing anti-TB regimens encompassing120573-lactams In addition the concentration of 5 120583gml of CLAused in the current studywas lower than the119862max of 59120583gmlachieved after a 250 mg three times a day dosage suggestingthat it can be easily achieved in clinical use [34]

For investigating the relationship between mutations inthe 120573-lactam targets and the drug susceptibility of MTB clin-ical isolates blaC ldtMt1 ldtMt2 and dacB2 in 122MTB clinicalisolates were sequenced and analyzed BlaC hydrolyzes the120573-lactam ring which is essential for the activity of 120573-lactamantibiotics resulting in the 120573-lactams resistance in MTBEight differentmutationswere observed in blaC However wedid not find any association between these mutations and 120573-lactam resistance Interestingly one of the strains harbouringG514A (Gly172Ser) mutation was highly susceptible to 120573-lactam antibiotics in either the presence or the absence ofthe inhibitors Zhang et al [35] have reported that the S111Rmutation on BlaC may be associated with an increased sus-ceptibility of MTB Considering that the mutation Gly172Sermay affect the interaction of BlaC and 120573-lactams [36] wemay speculate that this amino change leads to a conformationchange in BlaC which influences its activity

Mutation on target gene often confers drug resistanceto bacteria Four clinical MTB strains were found to carrymutation A776G in ldtMt2 and two of them were susceptibleto 120573-lactams Similarly among the dacB2 mutants oneexhibited resistance while the other exhibited susceptibilityto 120573-lactams However we did not find any correlationbetween these mutations and the 120573-lactams resistance In

this study we only tested four potential targets and noneof the strains harboured mutations simultaneously on allthe four genes It is reported that the 120573-lactam antibioticshave to bind to at least three of the four identified PBPsto be effective [37] Considering the multiple targets of 120573-lactams we may speculate that the drug resistance may notbe conferred by mutation in a single target Further studiesare required to demonstrate the role of drug target mutationsin the development of 120573-lactam resistance inMTB in order toconsider the inclusion of 120573-lactams in the anti-TB treatmentregimens

5 Conclusions

In summary varied activities of carbapenems penicillinsand cephalosporins alone or in combination with 120573-lactamase inhibitors against MTB were observed TBM incombination with CLA showed the most remarkable activityand has a good prospect in developing novel anti-TB regi-mens Further studies are warranted to investigate the efficacyof TBMCLA in the clinical trials Additionally resistance tothe 120573-lactamsmay not be conferred by single target mutationin MTB and thus requires further investigation

Data Availability

The datasets used to support the findings of this study areavailable upon request from the corresponding author

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was financially supported by the Key project of theState Key Laboratory for Infectious Disease Prevention andControl (2014SKLID104) and the project of the National KeyPrograms of Mega Infectious Diseases (2013ZX10003002-001)

Supplementary Materials

The details of mutations and the MIC profile of each mutantin this study are listed in the supplementary material file(Supplementary Materials)

References

[1] World Health Organization Global tuberculosis report WHODocument World Health Organization 2017

[2] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[3] T Cohen H E Jenkins C Lu M McLaughlin K Floyd andM Zignol ldquoOn the spread and control of MDR-TB epidemicsAn examination of trends in anti-tuberculosis drug resistance

6 BioMed Research International

reported to be effective against MDR-TBThe 119862max and AUCvalues of MEMwere 26 120583gml and 272ndash324 120583gsdothml respec-tively after being administered by an intravenous infusionof 500 mg of MEM [27] In our results also MEMCLAexhibited potent activity (MIC90 8 120583gml) againstMTB but itwas weaker than that of TBMCLA which is consistent withprevious study [28] This may be explained by lower 119870m andless breakdown of the covalent tebipenem-BlaC adduct incontrast to that of MEM thus suggesting a higher activity ofTBM against MTB [17 29] Oral administration of the TBMpivoxil had a better protective effect than meropenem in allthe tried sepsis models due to its greater tissue distribution[30] Hence as an oral carbapenem TBM may be a betteroption in developing new anti-TB regimen

MTB harbours a chromosomally encoded 120573-lactamasewhich is selectively susceptible to 120573-lactamase inhibitorsIn this study varied synergistic effects of three different 120573-lactamase inhibitors were observed Consistent with previousstudy [28] the greatest reduction in MIC was achieved byCLA in combinationwith120573-lactams especially inAMXCLA(32-fold in MIC50) AVI a newly developed 120573-lactamaseinhibitor combiningwith ceftazidime exhibited good activityagainst Enterobacteriaceae and Pseudomonas aeruginosa [31]However a combination of AVI with 120573-lactams displayed alesser reduction in MIC of 120573-lactams compared to CLA Arecent report has demonstrated that the efficiency of AVIwas lower than that of CLA due to its bulky rings that mayinfluence its binding to BlaC [32] The unstable covalentadduct of SUB and BlaC may also explain its lesser inhibitingactivity than that of CLA [33] Thus CLA seems to be themost suitable in developing anti-TB regimens encompassing120573-lactams In addition the concentration of 5 120583gml of CLAused in the current studywas lower than the119862max of 59120583gmlachieved after a 250 mg three times a day dosage suggestingthat it can be easily achieved in clinical use [34]

For investigating the relationship between mutations inthe 120573-lactam targets and the drug susceptibility of MTB clin-ical isolates blaC ldtMt1 ldtMt2 and dacB2 in 122MTB clinicalisolates were sequenced and analyzed BlaC hydrolyzes the120573-lactam ring which is essential for the activity of 120573-lactamantibiotics resulting in the 120573-lactams resistance in MTBEight differentmutationswere observed in blaC However wedid not find any association between these mutations and 120573-lactam resistance Interestingly one of the strains harbouringG514A (Gly172Ser) mutation was highly susceptible to 120573-lactam antibiotics in either the presence or the absence ofthe inhibitors Zhang et al [35] have reported that the S111Rmutation on BlaC may be associated with an increased sus-ceptibility of MTB Considering that the mutation Gly172Sermay affect the interaction of BlaC and 120573-lactams [36] wemay speculate that this amino change leads to a conformationchange in BlaC which influences its activity

Mutation on target gene often confers drug resistanceto bacteria Four clinical MTB strains were found to carrymutation A776G in ldtMt2 and two of them were susceptibleto 120573-lactams Similarly among the dacB2 mutants oneexhibited resistance while the other exhibited susceptibilityto 120573-lactams However we did not find any correlationbetween these mutations and the 120573-lactams resistance In

this study we only tested four potential targets and noneof the strains harboured mutations simultaneously on allthe four genes It is reported that the 120573-lactam antibioticshave to bind to at least three of the four identified PBPsto be effective [37] Considering the multiple targets of 120573-lactams we may speculate that the drug resistance may notbe conferred by mutation in a single target Further studiesare required to demonstrate the role of drug target mutationsin the development of 120573-lactam resistance inMTB in order toconsider the inclusion of 120573-lactams in the anti-TB treatmentregimens

5 Conclusions

In summary varied activities of carbapenems penicillinsand cephalosporins alone or in combination with 120573-lactamase inhibitors against MTB were observed TBM incombination with CLA showed the most remarkable activityand has a good prospect in developing novel anti-TB regi-mens Further studies are warranted to investigate the efficacyof TBMCLA in the clinical trials Additionally resistance tothe 120573-lactamsmay not be conferred by single target mutationin MTB and thus requires further investigation

Data Availability

The datasets used to support the findings of this study areavailable upon request from the corresponding author

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this manuscript

Acknowledgments

This work was financially supported by the Key project of theState Key Laboratory for Infectious Disease Prevention andControl (2014SKLID104) and the project of the National KeyPrograms of Mega Infectious Diseases (2013ZX10003002-001)

Supplementary Materials

The details of mutations and the MIC profile of each mutantin this study are listed in the supplementary material file(Supplementary Materials)

References

[1] World Health Organization Global tuberculosis report WHODocument World Health Organization 2017

[2] L Wang H Zhang Y Ruan et al ldquoTuberculosis prevalencein China 1990ndash2010 a longitudinal analysis of national surveydatardquoThe Lancet vol 383 no 9934 pp 2057ndash2064 2014

[3] T Cohen H E Jenkins C Lu M McLaughlin K Floyd andM Zignol ldquoOn the spread and control of MDR-TB epidemicsAn examination of trends in anti-tuberculosis drug resistance

BioMed Research International 7

surveillance datardquo Drug Resistance Updates vol 17 no 4-6 pp105ndash123 2014

[4] K Dheda T Gumbo N R Gandhi et al ldquoGlobal control oftuberculosis from extensively drug-resistant to untreatabletuberculosisrdquoThe Lancet Respiratory Medicine vol 2 no 4 pp321ndash338 2014

[5] S Tiberi R Buchanan J A Caminero et al ldquoThe challenge ofthe new tuberculosis drugsrdquo La Presse Medicale vol 46 no 2pp e41ndashe51 2017

[6] A Zumla J Chakaya R Centis et al ldquoTuberculosis treatmentand management-an update on treatment regimens trials newdrugs and adjunct therapiesrdquoThe Lancet Respiratory Medicinevol 3 no 3 pp 220ndash234 2015

[7] M I Page ldquoThe Reactivity of 120573-Lactams the Mechanism ofCatalysis and the Inhibition of 120573-LactamasesrdquoCurrent Pharma-ceutical Design vol 5 no 11 pp 895ndash913 1999

[8] J-L Mainardi R Villet T D Bugg C Mayer and M ArthurldquoEvolution of peptidoglycan biosynthesis under the selectivepressure of antibiotics in Gram-positive bacteriardquo FEMSMicro-biology Reviews vol 32 no 2 pp 386ndash408 2008

[9] M LavollayMArthurM Fourgeaud et al ldquoThe peptidoglycanof stationary-phaseMycobacterium tuberculosis predominantlycontains cross-links generated by LD-transpeptidationrdquo Jour-nal of Bacteriology vol 190 no 12 pp 4360ndash4366 2008

[10] W-J Li D-F Li Y-LHu X-E Zhang L-J Bi andD-CWangldquoCrystal structure of LD-transpeptidase LdtMt2 in complexwithmeropenem reveals themechanism of carbapenem againstMycobacterium tuberculosisrdquo Cell Research vol 23 no 5 pp728ndash731 2013

[11] J F Fisher and S Mobashery ldquobeta-Lactam Resistance Mecha-nisms Gram-Positive Bacteria and Mycobacterium tuberculo-sisrdquo Cold Spring Harbor Perspectives in Medicine vol 6 no 52016

[12] V Dubee S Triboulet J-L Mainardi et al ldquoInactivationof Mycobacterium tuberculosis LD-transpeptidase LdtMt1 bycarbapenems and cephalosporinsrdquo Antimicrobial Agents andChemotherapy vol 56 no 8 pp 4189ndash4195 2012

[13] P Kumar K Arora J R Lloyd et al ldquoMeropenem inhibitsDD-carboxypeptidase activity in Mycobacterium tuberculosisrdquoMolecular Microbiology vol 86 no 2 pp 367ndash381 2012

[14] C Brandt S D Braun C Stein et al ldquoIn silico serine 120573-lactamases analysis reveals a huge potential resistome in envi-ronmental and pathogenic speciesrdquo Scientific Reports vol 7 p43232 2017

[15] P Kumar A Kaushik E P Lloyd et al ldquoNon-classical transpep-tidases yield insight into new antibacterialsrdquo Nature ChemicalBiology vol 13 no 1 pp 54ndash61 2017

[16] A Kaushik N C Ammerman R Tasneen et al ldquoIn vitroand in vivo activity of biapenem against drug-susceptibleand rifampicin-resistantMycobacterium tuberculosisrdquo Journal ofAntimicrobial Chemotherapy vol 72 no 8 pp 2320ndash2325 2017

[17] J-E Hugonnet L W Tremblay H I Boshoff C E Barry IIIand J S Blanchard ldquoMeropenem-clavulanate is effective againstextensively drug-resistantMycobacterium tuberculosisrdquo Sciencevol 323 no 5918 pp 1215ndash1218 2009

[18] G Sotgiu L DrsquoAmbrosio R Centis et al ldquoCarbapenems totreat multidrug and extensively drug-resistant tuberculosis Asystematic reviewrdquo International Journal of Molecular Sciencesvol 17 no 3 article no 373 2016

[19] H F Chambers T Kocagoz T Sipit J Turner and P CHopewell ldquoActivity of amoxicillinclavulanate in patients with

tuberculosisrdquoClinical Infectious Diseases vol 26 no 4 pp 874ndash879 1998

[20] P R Donald F A Sirgel A Venter et al ldquoEarly bactericidalactivity of amoxicillin in combination with clavulanic acid inpatients with sputum smear-positive pulmonary tuberculosisrdquoInfectious Diseases vol 33 no 6 pp 466ndash469 2001

[21] P Kumar A Kaushik D T Bell et al ldquoMutation in an unanno-tated protein confers carbapenem resistance inMycobacteriumtuberculosisrdquo Antimicrobial Agents and Chemotherapy vol 61no 3 Article ID e02234 2017

[22] O-A Abraham V-M Rosa S-S M Antonio G Claudia andM-E M del Rosario ldquoReview of antibiotic and non-antibioticproperties of beta-lactam moleculesrdquo Anti-Inflammatory ampAnti-Allergy Agents in Medicinal Chemistry vol 15 no 1 pp 3ndash14 2016

[23] I Dincer A Ergin and T Kocagoz ldquoThe vitro efficacy of 120573-lactam and 120573-lactamase inhibitors against multidrug resistantclinical strains of Mycobacterium tuberculosisrdquo InternationalJournal of Antimicrobial Agents vol 23 no 4 pp 408ndash411 2004

[24] M Nakashima J Morita and K Aizawa ldquoPharmakokinet-ics and safety of tebipenem pivoxil fidne granules an oralcarbapenem antibiotic in healthy male volunteersrdquo JapaneseJournal of Chemotherapy vol 57 no 1 pp 90ndash94 2009

[25] C M Perry and T Ibbotson ldquoBiapenemrdquo Drugs vol 62 no 15pp 2221ndash2234 2002

[26] A D Miller A M Ball P B Bookstaver E K Dornblaser andC L Bennett ldquoEpileptogenic potential of carbapenem agentsMechanism of action seizure rates and clinical considerationsrdquoPharmacotherapy vol 31 no 4 pp 408ndash423 2011

[27] G G Zhanel R Wiebe L Dilay et al ldquoComparative review ofthe carbapenemsrdquo Drugs vol 67 no 7 pp 1027ndash1052 2007

[28] YHorita SMaeda Y Kazumi andNDoi ldquoIn vitro susceptibil-ity ofMycobacterium tuberculosis isolates to an oral carbapenemalone or in combination with 120573-Lactamase inhibitorsrdquo Antimi-crobial Agents and Chemotherapy vol 58 no 11 pp 7010ndash70142014

[29] S Hazra H Xu and J S Blanchard ldquoTebipenem a newcarbapenem antibiotic is a slow substrate that inhibits the 120573-lactamase fromMycobacterium tuberculosisrdquo Biochemistry vol53 no 22 pp 3671ndash3678 2014

[30] R Kobayashi M Konomi K Hasegawa M Morozumi KSunakawa and K Ubukata ldquoIn vitro activity of tebipenema new oral carbapenem antibiotic against penicillin-nonsus-ceptible Streptococcus pneumoniaerdquo Antimicrobial Agents andChemotherapy vol 49 no 3 pp 889ndash894 2005

[31] C Pitart F Marco T A Keating W W Nichols and J VilaldquoActivity of ceftazidime-avibactam against fluoroquinolone-resistant Enterobacteriaceae and Pseudomonas aeruginosardquoAntimicrobial Agents and Chemotherapy vol 59 no 6 pp3059ndash3065 2015

[32] H Xu S Hazra and J S Blanchard ldquoNXL104 irreversiblyinhibits the 120573-lactamase from Mycobacterium tuberculosisrdquoBiochemistry vol 51 no 22 pp 4551ndash4557 2012

[33] J-E Hugonnet and J S Blanchard ldquoIrreversible inhibition ofthe Mycobacterium tuberculosis 120573-lactamase by clavulanaterdquoBiochemistry vol 46 no 43 pp 11998ndash12004 2007

[34] A R White C Kaye J Poupard R Pypstra G Woodnuttand B Wynne ldquoAugmentin (amoxicillinclavulanate) in thetreatment of community-acquired respiratory tract infection areview of the continuing development of an antimicrobialagentrdquo Journal of Antimicrobial Chemotherapy vol 53 supple-ment 1 pp i3ndashi20 2004

8 BioMed Research International

[35] D Zhang Y Wang J Lu and Y Pang ldquoIn vitro activity ofbeta-lactams in combination with beta-lactamase inhibitorsagainst multidrug-resistant Mycobacterium tuberculosis iso-latesrdquo Antimicrob Agents Chemother vol 60 no 1 pp 393ndash3992015

[36] L W Tremblay F Fan and J S Blanchard ldquoBiochemical andstructural characterization of Mycobacterium tuberculosis 120573-lactamase with the carbapenems ertapenem and doripenemrdquoBiochemistry vol 49 no 17 pp 3766ndash3773 2010

[37] H F Chambers D Moreau D Yajko et al ldquoCan penicillinsand other beta-lactam antibiotics be used to treat tuberculosisrdquoAntimicrobial Agents and Chemotherapy vol 39 no 12 pp2620ndash2624 1995

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

[35] D Zhang Y Wang J Lu and Y Pang ldquoIn vitro activity ofbeta-lactams in combination with beta-lactamase inhibitorsagainst multidrug-resistant Mycobacterium tuberculosis iso-latesrdquo Antimicrob Agents Chemother vol 60 no 1 pp 393ndash3992015

[36] L W Tremblay F Fan and J S Blanchard ldquoBiochemical andstructural characterization of Mycobacterium tuberculosis 120573-lactamase with the carbapenems ertapenem and doripenemrdquoBiochemistry vol 49 no 17 pp 3766ndash3773 2010

[37] H F Chambers D Moreau D Yajko et al ldquoCan penicillinsand other beta-lactam antibiotics be used to treat tuberculosisrdquoAntimicrobial Agents and Chemotherapy vol 39 no 12 pp2620ndash2624 1995

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom