[ Translating Basic Research Into Clinical Practice ]

Antimicrobial Resistance and RespiratoryInfections

Allison K. Guitor, BSc; and Gerard D. Wright, PhD

ABBREVIATIONS: MDR = mulAFFILIATIONS: From the DepSciences, Michael G. DeGrResearch, McMaster UniversitFUNDING/SUPPORT: This worof Health Research; the NatuCouncil of Canada; and a Can

1202 Translating Basic Res

Since their introduction into health care and clinical practice in the early 20th century, antibiotics

have revolutionized medicine. Alarmingly, these drugs are increasingly threatened by bacteria

that have developed a broad diversity of resistance mechanisms. Antibiotic resistance can be

transferred between bacteria, often on mobile genetic elements; be acquired from the environ-

ment;orarise throughmutationbecauseof selectivepressuresof thedrugs themselves.Thereare

various strategies to resistance, including active efflux of the drug from the bacterial cell, reduced

permeability of the cell envelope, alteration of the drug’s target within the bacterial cell, and

modification ordestructionof theantibiotic.Streptococcus pneumoniae,Haemophilus influenzae,

Pseudomonas aeruginosa, and Mycobacterium tuberculosis frequently are implicated in respira-

tory infections, often manifesting with reduced susceptibility to multiple classes of antibiotics.

Somemechanismsof resistance, suchas the b-lactamases that confer resistance topenicillinsand

related drugs, have been well characterized and are widespread in clinical isolates. Other newly

identified determinants, including the colistin resistance gene mcr-1, are spreading rapidly

worldwide and threaten last-resort treatments of multidrug-resistant organisms. Various ap-

proaches to detecting antibiotic resistance provide surveys of the determinants that are available

for transfer into pathogenic bacteria. Together withmolecular characterization of newly identified

mechanisms, this surveillance can target drug discovery efforts and increase antibiotic steward-

ship. A greater understanding of themechanisms of antibiotic resistance in respiratory pathogens

combined with rapid diagnostics ultimately will reduce treatment failure due to inappropriate

antibiotic use and prevent further spread of resistance. CHEST 2018; 154(5):1202-1212

KEY WORDS: antibiotics; infectious disease; microbiology; molecular biology; resistance

Antibiotics are essential for the control ofinfections in the upper and lower respiratorytracts. The rise of antibiotic resistance is amajor concern to airways clinical practicebecause it can lead to increased mortality,longer hospital stays, and clinical failure.1

The reasons for this crisis in antibiotics areprimarily twofold. First is the inexorablechallenge of microbial evolution that has

tidrug resistant; rRNA = ribosomal RNAartment of Biochemistry and Biomedicaloote Institute for Infectious Diseasey, Hamilton, ON, Canada.k was funded by the Canadian Institutesral Sciences and Engineering Researchada Research Chair in Biochemistry.

CORRESPOND

Biochemistryfor InfectiousW, Hamilton,Copyright � 2Elsevier Inc. ADOI: https://d

earch Into Clinical Practice

decreased sensitivity to all classes ofantibiotics in all important pathogens.Second is the absence of new drugdevelopment. The former is a measure of theintrinsic biology of microorganisms, theirability to adapt to the threat of antibiotics bya variety of mechanisms, and their exposureto the selective pressure of drugs. The latterreflects the economic reality of the return on

ENCE TO: Gerard D. Wright, PhD, Department ofand Biomedical Sciences, Michael G. DeGroote InstituteDisease Research, McMaster University, 1280 Main StON, Canada, L8S 4K1; e-mail: wrightge@mcmaster.ca018 American College of Chest Physicians. Published byll rights reserved.oi.org/10.1016/j.chest.2018.06.019

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investment expected from the development of drugs thatactually effect a cure for acute conditions (in contrast tothat for chronic disease for which drug use may occurover a lifetime). Contributing to the problem is a lack ofinnovation in new antibiotic discovery because ofscientific challenges that reflect the difficulty inidentifying new, safe, and effective drugs. The end resultis that new antibiotic discovery and development are notkeeping pace with the clinical need.

Lower respiratory tract infections are the leading causeof death due to infectious disease globally.2 Of greaterconcern are the populations at risk of acquiringrespiratory infections—namely, the elderly, theimmunocompromised, and children.1,3-5 Changes inimmune function and structural aspects in therespiratory tract, as well as comorbidities, make treatingthe aging population more challenging and choosingappropriate antibiotics paramount.4 With the rise inantibiotic resistance, resolving respiratory illnesses in allpopulations, including the elderly, will continue to be aglobal health-care concern. Understanding themechanisms of antibiotic resistance, their origins, andtheir distribution, therefore, becomes criticallyimportant in the short- and medium-term managementof airways infections. In this review, we discuss thevarious mechanisms of resistance to key classes ofantibiotics in airways pathogens, their selection, andtheir evolution.

Resistance in Respiratory PathogensIn early 2017, theWorldHealthOrganization released a listof priority pathogens for which research and developmentinto antibiotic therapies are needed.6 This list includespathogens that often are implicated in respiratoryinfections, such as carbapenem-resistant Acinetobacterbaumannii; carbapenem-resistant Pseudomonasaeruginosa; carbapenem- and third-generation-cephalosporin-resistant Enterobacteriaceae, includingKlebsiella pneumoniae; Staphylococcus aureus (methicillinresistant, vancomycin intermediate, and vancomycinresistant); penicillin-nonsusceptible Streptococcuspneumoniae; and ampicillin-resistant Haemophilusinfluenzae.6,7 Not featured on this list is the respiratorypathogenMycobacterium tuberculosis because it wasrecognized previously as a global health priority because ofmultidrug resistance and the need for new antibiotics.6

An additional respiratory pathogen, Moraxellacatarrhalis, frequently is resistant to b-lactams becauseof the high prevalence (> 80%) of b-lactamases and hasshown reduced susceptibility to macrolides and

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fluoroquinolones.8-13 Bordetella pertussis, H influenzae,and S pneumoniae are respiratory pathogens that havebeen targeted through vaccination. The incidence of Bpertussis has been reduced successfully; however, casesincreasingly are reported in certain countries, often withresistance toward macrolides, fluoroquinolones, andtetracyclines.14 In terms of H influenzae and Spneumoniae, vaccination has reduced certain serotypesalong with antibiotic resistance; however, otherserotypes with increasing resistance are now dominantin infections.15-17 In many countries, H influenzae ishighly resistant to ampicillin, with high levels ofb-lactamase production.10,12,13,18 S pneumoniaecontinues to show reduced susceptibility to b-lactamantibiotics globally, and treatment withfluoroquinolones and macrolides has resulted inincreased resistance to these agents.10,12,13,19 Finally, notonly is there an increase in rates of single agentresistance, multidrug-resistant (MDR) pathogens,including MDR S pneumoniae and MDR A baumannii,are becoming more prominent in the United States.20,21

Antibiotics: A Brief OverviewAntibiotics are compounds, either synthetically ornaturally produced, that kill bacteria (bactericidal) ordelay the growth of bacteria (bacteriostatic).22

Table 123,24 highlights important classes of antibioticdrugs and their mechanisms of action. Favoredmolecular targets of antibiotics include components ofessential cellular processes such as the synthesis andintegrity of the cell envelope, protein synthesis, DNAreplication, and RNA production.22 Most of the knownand currently used antibiotics are derived from naturalproducts synthesized by other microbes and wereidentified during the golden era of antibiotic discovery,roughly the 20 years after the development of penicillinin the early 1940s.25 Although there have been fewercompletely new antibiotic chemical scaffolds discoveredin the past 30 years, advances in medicinal chemistryand a greater understanding of antibiotic resistancemechanisms has facilitated the creation of moleculesbased on these golden era compounds that are highlyspecific, evade resistance mechanisms, and have reducedtoxicity.25,26 The following sections describe the majortargets of antibiotics, followed by how resistance to thesemolecules is manifested in pathogenic bacteria.

Antibiotics Targeting the Cell EnvelopeThe bacterial cell envelope consists of the cellmembrane; the peptidoglycan-containing cell wall; and,

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TABLE 1 ] Antibiotic Classes and Their Targets in the Bacterial Cell

Primary Target Antibiotic Class Example Chemical StructureMajor ResistanceMechanisms

Resistance Prevalent inRespiratory Pathogen

Cell wallbiosynthesis

b-Lactams (eg,penicillin G) NH

NO

HS

O

OOH

Modified penicillin-binding proteins(ftsI, mecA genes),efflux, b-lactamases

SA, KP, PA, AB,HI, MC

Glycopeptides (eg,vancomycin)

O

O

NHHN

Cl

NHO

ONH

O

HNNH

HOO H

N

O

O

O

HO OH

HO

Cl

OO NH2

OH

OH

OO

OHOH

OHO

OH NH2Modifiedpeptidoglycanprecursors (vangene family)

SA

Cell envelopeintegrity

Lipopetides (eg,colistin)

Increase in positivecharge on the cellsurface,(mprF, mcr-1)

SA, KP, MC

DNA replicationandtranscription

Quinolones (eg,ciprofloxacin)

HN

N N

O

FOH

O Mutations in targets:gyrAB,parEC; targetprotection (Qnr)

MT, SP, KP, HI,MC

Nucleotidesynthesis orfolatebiosynthesis

Sulfonamides (eg,sulfanilamide)

H2N S

O

O

NH

H

Targetoverexpression,mutation ormodified target: sulfamily

PA, KP, AB

Protein synthesis Aminoglycosides (eg,kanamycin A) H2N

OHOO

OHO

HO

OH

NH2

OOH

HO

H2NOH

NH2

Aminoglycoside-modifying enzymes;16S rRNAmethyltransferases

AB, PA, KP, SP

Oxazolidinones (eg,linezolid)

N

F

N

O

O

O

HN

O

23S rRNAmethyltransferase:Cfr; mutations inrRNA

SA

Tetracyclines (eg,tetracycline)

OHOH

NH2

O OOOH

HHOH N

Efflux, tet gene family,MexAB-OprM;ribosomalprotection proteins(TetM)

PA, SP

Macrolides (eg,erythromycin)

O

O

OHOH

HO

O O

O OCH3

HO

N

O

OCH3CH3OH

CH3

23S rRNAmethyltransferase:erm family; efflux(mef); 23S rRNAmutation

SA, SP, HI

Streptogramins (eg,pristinamycin IIa)

O

NH

O

O

O

N

OO

N

OH23S rRNAmethyltransferases;antibioticmodification

SA, SP

ChloramphenicolN+O

O-

NH

Cl

O

Cl OH

OH

23S rRNAmethyltransferase;efflux;acetyltransferase(cat)

SA, PA, KP

(Continued)

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TABLE 1 ] (Continued)

Primary Target Antibiotic Class Example Chemical StructureMajor ResistanceMechanisms

Resistance Prevalent inRespiratory Pathogen

RNA synthesis Ansamycins (eg,rifampin)

OH OH

NH

OH

O

O

O

OHOH

O

O

O

O

NN

N

Mutations in RNApolymerase (rpoBgene)

MT, SA

A representative structure of each class of antibiotic is shown. Major resistance mechanisms, including the respiratory pathogens in which resistance isprevalent, are highlighted.9,16,23,24,67,94,97,98 AB ¼ Acinetobacter baumannii; HI ¼ Haemophilus influenzae; KP ¼ Klebsiella pneumoniae; MC ¼ Moraxellacatarrhalis; MT ¼ Mycobacterium tuberculosis; PA ¼ Pseudomonas aeruginosa; rRNA ¼ ribosomal RNA; SA ¼ Staphylococcus aureus; SP ¼ Streptococcuspneumoniae.

in gram-negative bacteria, an outer membranecomposed of lipopolysaccharide (Fig 1). A major targetof antibiotics is the biosynthesis of the bacterial cell wall,which provides structural shape and protection fromosmotic forces in gram-positive and gram-negativebacteria. The common respiratory pathogens Kpneumoniae, H influenzae, M catarrhalis, and Paeruginosa are examples of gram-negative bacteriaprimarily characterized by an additional outermembrane embedded with porins. This feature providesa permeability barrier to components of the immunesystem and small molecules such as antibiotics.8,22,27

Multicomponent molecular machines known as “effluxpumps” that span both the inner cell membrane and theouter membrane facilitate efficient export of many

Figure 1 –Mechanisms of antibiotic resistance across gram-negative and grammembrane layer to their cell envelope that can be modified to provide furtherenvelope include active efflux, modified or reduced expression of porins, andpositive organisms (right) have a thick peptidoglycan layer that also can bechromosomally encoded proteins can modify or degrade antibiotics, includingb-lactamases. Other proteins or mutations can protect the target of a particulais implicated in macrolide and aminoglycoside resistance in which methylationoften is implicated in fluoroquinolone and rifampicin resistance.

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antibiotics.22 Gram-positive pathogens, such as Spneumoniae and S aureus, lack the outer membranestructure but have acquired extensive and diversemechanisms to avoid antibacterial effects.27 Finally,mycobacteria, including the major respiratory pathogenM tuberculosis, have a substantial cell envelopecomposed of mycolic acids, peptidoglycan, and apolysaccharide arabinogalactan that together greatlyreduce antibiotic entry into the cell.28

Antibiotics that target cell wall synthesis of bacteriainclude the b-lactams and the glycopeptides.22 The firstb-lactam to be developed into a drug was penicillin G,which subsequently was followed by many semisyntheticderivatives and three additional scaffolds: the

-positive bacteria. Gram-negative bacteria (left) have an additional outerbarriers to antibiotic entry. Mechanisms of resistance related to the cellchanges to the outer membrane by cell wall-modifying enzymes. Gram-modified to avoid certain antibiotics’ activities. Plasmid-encoded orthe acetylation of aminoglycosides and the widely disseminated family ofr antibiotic, thereby providing resistance. Target modification frequentlyprotects the ribosome target. Finally, mutational resistance in the target

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cephalosporins, the carbapenems, and themonobactams. These compounds covalently bind andinhibit the eponymous penicillin-binding proteins,which are transpeptidases that cross-link thepeptidoglycan component of the cell wall. b-Lactamactivity weakens the cell wall, ultimately leading to celllysis.29,30 Glycopeptide antibiotics, includingvancomycin and teicoplanin, are used for the treatmentof gram-positive bacterial infections and bind to theD-Alanyl-D-Alanine termini of membrane-linked cellwall precursors, blocking their addition to thepeptidoglycan chain and preventing cross-linking.31,32

Although these are examples of drugs that specificallytarget cell wall biosynthesis, additional importantantibiotics disrupt other elements of the cell envelope,including the integrity of the cell membrane throughelectrostatic interactions or pore formation.22

Antibiotics in this class include antimicrobial peptides,mammalian defensins, daptomycin, and polymyxins.The latter group includes colistin or polymyxin B, andthese typically are used as a last-resort treatment againstMDR gram-negative pathogens, including respiratorypathogens K pneumoniae, A baumannii, andP aeruginosa. These molecules are highly cationic andare attracted to the negative charges on thelipopolysaccharide moieties of the gram-negative outermembrane, ultimately disrupting the membrane.33

Antibiotics Targeting Protein SynthesisMany antibacterial drugs used in respiratory medicine,including the tetracyclines, macrolides, aminoglycosides,and oxazolidinones, block the synthesis of proteins atthe ribosomes.22 The bacterial ribosome is composed ofa large 50S subunit and a small 30S subunit, each withspecific ribosomal RNA (rRNA) sequences (23S rRNAand 16S rRNA, respectively) and protein substituents.34

Each subunit is targeted by a distinct group ofantibiotics. Linezolid is a member of the oxazolidinoneclass of antibiotics that block the peptidyl transferasecenter, the site of peptide bond formation.35 Thissynthetic molecule is active against gram-positivebacteria, including methicillin-resistant S aureus and Spneumoniae. Macrolides, such as erythromycin andazithromycin, also bind to the 50S subunit but block thepeptide exit tunnel where new proteins are extrudedfollowing peptide bond formation.34

Antibiotics that target the 30S subunit include theaminoglycosides gentamicin, tobramycin, andstreptomycin, the latter of which was used initially to treatM tuberculosis.36 These antibiotics function by binding to

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the 16S rRNA of the 30S subunit, preventing accuratecodon-anticodon matching, resulting in translationinfidelity with an ultimate bactericidal result.34

Tetracycline antibiotics target an adjacent region on the30S subunit and disrupt binding of aminoacyl transferRNAs, preventing translation.34 Finally, capreomycin, apeptide antibiotic that finds occasional use in thetreatment of tuberculosis, binds at the interface betweenthe 30S and 50S subunits of the ribosome, affectingtranslocation and peptide synthesis.37

Antibiotics Targeting DNA and RNADNA replication and transcription of DNA into RNA aretwo major processes targeted by many antibiotics. Thefluoroquinolones are a class of synthetic antibacterials thatinclude norfloxacin, ciprofloxacin, and levofloxacin.38

They target the type II topoisomerases DNA gyrase,encoded by gyrA and gyrB, and topoisomerase IV,composed of ParC and ParE.39 These enzymes modulatesupercoiling of DNA during replication, relieving tensionon the DNA by introducing and resealing double-strandedbreaks.39 Fluoroquinolones act by preventing the resealingof the cleaved DNA, resulting in cell death.38 Antibioticsalso can target enzymes involved in the folate biosynthesispathway, which is essential for the biosynthesis of theDNAbuilding block deoxythymidylate.22,40 Trimethoprim,almost universally used in combination with thesulfonamide drug sulfamethoxazole, targets dihydrofolatereductase, and the sulfonamides inhibit dihydropteroatesynthase.22,40 Finally, rifamycins, including rifampin, blockinitial stages of transcription by targeting RNA polymeraseand are a common first-line treatment for tuberculosis.22

How Antibiotic Resistance ArisesAntibiotic resistance can be acquired by horizontal genetransfer or arise in an organism through mutations topreexisting genes or regulatory sequences. Propagation ofbeneficial resistance mutations within a population isknown as “vertical gene transfer” and is the keymechanism of resistance development in Mtuberculosis.41 Acquisition of resistance throughhorizontal gene transfer occurs when genes are capturedor mobilized onto transferable elements, includingplasmids, which can be propagated easily among bacterialpopulations, even between species and genera, and thenmaintained through subsequent generations.42 Virusesknown as “bacteriophages” isolated from the sputum ofpatients with cystic fibrosis have been shown to harborantibiotic resistance genes and may be another majorroute of transfer of resistance among bacteria.43

Once a subset of a population of bacteria acquires a

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resistance-conferring mutation or gene, further antibiotictreatment will eliminate susceptible bacteria, leaving theresistant population to thrive and persist, often resultingin recurring infections. Certain mechanisms of resistanceare inducible in the presence of the correspondingantibiotic, including the expression of the b-lactamaseAmpC, tetracycline resistance through the expression oftet(M), and macrolide resistance through the Ermproteins.44,45 Although the excessive and inappropriateuse of antibiotics is a major factor in the rapiddevelopment and rise of antibiotic resistance, manyenvironmental and commensal organisms are innatelyresistant even in the absence of antibiotic use.46,47

Detecting Mechanisms of Antibiotic ResistanceMajor antibiotic resistance mechanisms that can beintrinsic, acquired through gene transfer, or aremutationally based are depicted in Figure 1. The set ofgenetic determinants within an individual bacterium orpopulation of bacteria that provides the potential forresistance is defined as the “resistome.”48 In a clinicalsetting, when a patient presents with a respiratoryinfection, it is imperative to understand not only thecausative agents but also the antibiotic susceptibilityprofile. The majority of respiratory tract infections arecaused by viruses; however, a large proportion of clinicor hospital visits for respiratory illnesses result inantibacterial prescription.49 Current standard diagnosticprocedures include attempting to culture the infectiveorganisms followed by strain identification andantibiotic susceptibility testing.50 More rapid approachesto diagnosing infections and resistance that do not relyon cultivation include polymerase chain reaction-basedassays, microarrays, and targeted sequencing efforts.50-53

Many of these tests still require further optimization andapproval before they can become reliable diagnosticassays at the point of care.

It has been well established that the antibiotic resistancegenes prevalent in clinical pathogens represent only a smallsubset of the overall resistome, with many new anduncharacterized resistance mechanisms continually beingdiscovered in environmental isolates, as well as commensalbacteria through techniques such as metagenomicsequencing and functional metagenomics.47,54-57 The hopeis that a greater understanding of all resistancemechanismsavailable to pathogens can aid in the treatment,surveillance, and prevention of the spread of antibioticresistance.46 Furthermore, rulingout viral infectionprior toantibiotic use can prevent unnecessary prescription andexposure to antibiotics.49,58

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Molecular Mechanisms of Resistance

Efflux Pumps and Porins

As introduced earlier, the complex cell envelopes ofboth gram-negative and gram-positive organisms can beembedded with membrane-spanning efflux pumps thatrecognize and export small molecules, whereas alteredexpression of porins reduces the permeability of the cellenvelope.59,60 Efflux pumps are prevalent in MDRorganisms and can confer resistance to b-lactams,macrolides, fluoroquinolones, tetracyclines, andaminoglycosides.59 P aeruginosa has an extensive set oftwo-component regulatory systems that can sensechanges in their environment, including the presence ofan antibiotic, and respond accordingly, often throughincreased expression of efflux pumps.61 Efflux systemsin P aeruginosa that have been implicated in antibioticresistance include MexAB-OprM, MexXY-OprM,MexCD-OprJ, and MexEF-OprN.59,62 Many of these areexpressed at basal levels until the presence of anantibiotic is sensed, thereby increasing geneexpression.59,63,64 Efflux systems are common in otherrespiratory pathogens, including A baumannii(AdeABC, AdeFGH, AdeIJK), the PatAB pump in Spneumoniae that confers fluoroquinolone resistance,and the AcrAB-OprM system in M catarrhalis thatconfers resistance to amoxicillin andclarithromycin.65-68

Whereas efflux systems result in the export of toxicmolecules, porins normally facilitate the entry ofantibiotics and other molecules into the cell.60 Ofparticular note is the OprD porin in P aeruginosa thathas been studied extensively for its role in resistancebecause of its reduction or elimination in expression,often because of mutations in regulatory elements.63,69

Carbapenem resistance in K pneumoniae and Abaumannii has been linked to loss of or changes inporins, including OmpK35/K36 and CarO,respectively.63 Other studies have implicated outermembrane porin M35 and porin-like protein CD in Mcatarrhalis, as well as porins in H influenzae withb-lactam resistance.8,9

Advances in the structural biology of efflux systemsprovide a greater understanding of the proteininteractions among efflux complexes and with theirtargeted antibiotics.63,70 Together with biochemicalcharacterization, this structural insight will help tofacilitate inhibitor design to prevent expulsion ofantibiotics before they can exert their effect on thebacterial cell.

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b-Lactamases

One of the most widespread, diverse, and well-studiedinactivation mechanisms is the hydrolysis of b-lactamantibiotics, including penicillins, cephalosporins, andcarbapenems.71 The b-lactamases constitute a family ofmore than 1,000 unique individuals, with most classesrelying on a serine residue to act as a nucleophile essentialfor the ring opening of the b-lactam scaffold through adeacylation reaction.22,71 b-Lactamases of the TEM, ROB,and VAT families are highly prevalent in H influenzaeisolates.7 In M catarrhalis, reports of > 90% of isolatescarrying chromosomally encoded b-lactamases(e.g., BRO) are frequent; thus, b-lactam treatment of thispathogen is limited.9,11,72 In P aeruginosa, AmpC is achromosomally encoded cephalosporinase that isinducible in the presence of b-lactams.73 Carbapenem-resistant Enterobacteriaceae are a major concern inrespiratory infections, with plasmid-borne KPC andOXAcarbapenemases resulting in broad-spectrumresistance.74-76 New variants are emerging continually,often with greater catalytic efficiency, broader substraterecognition, higher minimum inhibitory concentrations,and greater resistance to inhibitors.76

In 2009, a new mechanism of b-lactam resistance wasidentified in aKpneumoniae strain isolated fromaurinarytract infection inNewDelhi, India.77 NDM-1 is amemberof themetallo-b-lactamase class capable of hydrolyzing allb-lactam antibiotics except the monobactam aztreonam,which is of great concern because of its rapid spread acrossthe globe and among a variety of pathogens.78 At least 17variants of NDM-1 have been isolated and, althoughmany serine b-lactamase inhibitors have provenrestorative for b-lactams, inhibitors of metallo-b-lactamases have yet to be approved clinically.29,78,79

Aminoglycoside-Modifying Enzymes

The most prevalent mechanism of resistance toaminoglycosides is the modification of the antibiotic toreduce the affinity for or prevent interactions with the30S subunit of the ribosome.34 Additional mechanismsof aminoglycoside resistance include the increase inactive efflux and mutation or methylation of the 16SrRNA with which these antibiotics interact.34 Theseeffective bactericidal antibiotics are used frequently in Abaumannii infections in which the 6’-N-acetyltransferases and 3’-O-phosphotransferases are themost prevalent resistance enzymes.80 Amikacin often isused as a last effort to treat MDR- and b-lactam-resistant P aeruginosa strains. Unfortunately, modifyingenzymes able to inactivate amikacin have been identified

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in clinical isolates, demonstrating the breadth ofresistance available to pathogens and further threateningtreatment of MDR respiratory infections.81 Efforts todevelop antibiotics that are resistant to modifyingenzymes include the next-generation aminoglycosideplazomicin that avoids most aminoglycoside-modifyingenzymes.82 Although this aminoglycoside is not yetapproved for use in respiratory infections, it highlightsthat a better understanding of resistance can lead to therestoration of antibiotic activity.

Alteration of the Antibiotic Target

Alteration of an antibiotic target through modificationsthat produce a structure that is insensitive to or hasreduced affinity for a given antibiotic scaffold can occurthrough mutation or acquisition of a modifyingenzyme.22 Target modification ideally does not disrupt itscognate function but provides protection from antibioticaction. Methylation or mutations of 16S or 23S rRNAhave been implicated in resistance to many antibiotics,including aminoglycosides, macrolides, and linezolid.34

Common among A baumannii, K pneumoniae, and Paeruginosa are the 16S rRNA methyltransferases ArmAand RmtB-RmtH, which confer very high-levelaminoglycoside resistance.83 Methylation of specificresidues on the 16S rRNA disrupts the aminoglycosideaffinity for the ribosome, thus allowing translation toproceed without error.84 Methicillin-resistant S aureusand S pneumoniae respiratory infections often are treatedwith macrolides. Methylation of the 23S rRNA byerythromycin resistance methyltransferases (Erm)confers resistance to macrolide, lincosamide, andstreptogramin B antibiotics.22 These methyltransferasegenes are found onmobile genetic elements and are eitherconstitutively expressed or under macrolide-induciblecontrol.85 In the late 2000s, a new protein encoding achloramphenicol resistance mechanism was identified inan MDR S aureus isolate.86 Cfr is a 23S rRNAmethyltransferase that provides cross protection tolinezolid through a unique chemical mechanism, unlikeeither the 16S or 23S rRNA resistance methyltransferasesmentioned previously.87

Additional modifications to cellular targets that conferresistance to antibiotics include the covalent addition ofpositively charged groups onto anionic phospholipids ofcell membranes to reduce the affinity for or inducerepulsion of cationic antibiotics, including daptomycinand polymyxin.22 Colistin is often the last-resortantibiotic used to treat MDR infections, including Kpneumoniae and P aeruginosa. Until recently, resistance

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to polymyxins, in particular colistin, was limited tochromosomal mutations in two-component systems thatresult in upregulation of lipid A-modifying enzymes.88,89

In 2016, a new resistance gene, mcr-1, was discovered inEnterobacteriaceae.90 Mcr-1 is a phosphoethanolaminetransferase that modifies lipid A on the outer membraneof gram-negative organisms, preventing the action ofpolymyxins.91 Recently, the mechanisms of polymyxinresistance in M. catarrhalis were elucidated, and it isproposed that Moraxella species are a potential reservoirof mcr-like genes.92,93 Mcr-1 is found on plasmids andoften coexists with other resistance elements, includingextended-spectrum b-lactamases or carbapenemasessuch as NDM-1, thus compromising our last availableantibiotic therapies.91

Mutation of the Antibiotic Target

Fluoroquinolone resistance is mediated largely bymutations in either subunit of the targetedtopoisomerases. Mutations in gyrA are the mostcommon, with new mutations in the quinoloneresistance-determining region being identifiedfrequently, especially in M. tuberculosis and often in Mcatarrhalis and S pneumoniae.22,38,94,95 Less frequentlyencountered mechanisms of fluoroquinolone resistanceinclude the Qnr proteins, which bind to and protectGyrA, and a variant of aminoglycoside acetyltransferases(AAC(6’)-Ib-cr).96 Resistance to b-lactam antibioticscan arise through mutations in genes encodingpenicillin-binding proteins, including in S aureus(mecA), S pneumoniae (pbp), andH influenzae (ftsI).97,98

Macrolide resistance due to mutations in 23S rRNA andribosomal proteins is encountered frequently in Mcatarrhalis and S pneumoniae.53,99 Finally, protectionagainst aminoglycosides often results from 16S rRNAmutation; however, mutations in an elongation factorencoded by fusA1 involved in protein synthesis in Paeruginosa are also protective.100,101

Rifampin resistance due to mutations in the rpoB-encoded B subunit of RNA polymerase is very frequentand a major concern with S aureus and M tuberculosisbecause rifamycins commonly are used in first-linecombination therapies to treat tuberculosis.102 Mtuberculosis infections require lengthy combinationcourses of antibiotics to suppress resistance.22 Many ofthe antibiotics used to treat tuberculosis are prodrugsand require activation on entry into the cell. Mutationsin activating enzymes, as well as the targets of theantibiotic, can result in resistance.102 Large-scalegenomic studies have identified new mutations across

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many isolates that are associated with resistance;however, not all mutations have been characterized as totheir effect on the target of the antibiotic or the pathwayimplicated.102 Bedaquiline targets the production ofmajor cellular cofactor adenosine triphosphate and isone of few new antibiotics to be approved to treat Mtuberculosis.103 Resistance-conferring mutations in theadenosine triphosphate synthase gene, as well asmutations that upregulate efflux, have arisen in the shortperiod since bedaquiline was introduced.104 This is oftenthe case when a new antibiotic scaffold or next-generation antibiotics are introduced into clinicalpractice; resistance quickly and inevitably develops.

Implications of the Environmental andRespiratory ResistomesMany studies to date have probed the diversity andabundance of antibiotic resistance genes in a variety ofenvironments, including animals, the soil, water sources,and built environments such as hospitals.105 One majorconclusion of these surveys is that the genetic diversity ofresistance is much greater than previously predicted, and,perhaps unsurprisingly, these microorganisms havedeveloped many alternative routes to inactivate or avoidantibiotics. Many resistance mechanisms are proposed tobe homologs of resistance in antibiotic producers orenvironmental species that share a niche with antibioticproducers.46,48 The CTX-M b-lactamases are believed tohave transitioned into pathogenic bacteria from anenvironmental species of the genus Kluyvera.106

Pawlowski et al107 characterized new resistancemechanisms to many antibiotics in isolated cave bacteria.Although it is unlikely that the bacteria that employ theseresistance mechanisms will interact with pathogens orcause infection themselves, understanding the diversity ofresistance mechanisms can help to predict whichdeterminants are at risk of becoming mobilizable andlikely to appear in human pathogens and the clinic.108

In addition, the respiratory microbiome has beenimplicated in the development of host immunity and theoutcome of respiratory diseases, includingtuberculosis.109-111 In terms of antibiotic resistance,passive penicillin protection to susceptible respiratoryorganisms because of the production and encapsulationof b-lactamases in outer-membrane vesicles fromcoexisting bacteria has been detected.112,113 Finally, as inany given environment, commensal organisms in thelung may harbor antibiotic resistance elements that havethe potential to be transferred to opportunistic bacteriaor respiratory pathogens.114

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ConclusionsDiagnostic microbiology, surveillance, and genomicscontinually are providing an increased depth ofunderstanding of the resistance genes or mutations inrespiratory pathogens that are likely to contribute toreduced antibiotic susceptibility. As bacteria continueto adapt and acquire an increasingly abundant arsenalof resistance determinants, it becomes criticallyimportant to understand the molecular basis ofthese mechanisms. Furthermore, rapid diagnostictechniques to identify the causative agent of aninfection quickly, along with important resistantelements, are imperative to inform prescription ofantibiotics. Surveillance of environmental andcommensal organisms can provide information aboutthe most prevalent mobile antibiotic-resistant genesthat are at risk of becoming widespread in humanpathogens. Together these initiatives can promoteantibiotic stewardship, inform drug and inhibitordiscovery, and ultimately prevent the current trajectorytoward a postantibiotic era.

AcknowledgmentsFinancial/nonfinancial disclosures: None declared.

Role of sponsors: The sponsor had no role in the design of the study,the collection and analysis of the data, or the preparation of themanuscript.

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