Peterson 2018

REVIEW
published: 30 November 2018

doi: 10.3389/fmicb.2018.02928
Antibiotic Resistance Mechanisms in

Bacteria: Relationships Between
Resistance Determinants of
Antibiotic Producers, Environmental
Bacteria, and Clinical Pathogens
Elizabeth Peterson and Parjit Kaur*
Department of Biology, Georgia State University, Atlanta, GA, United States
Emergence of antibiotic resistant pathogenic bacteria poses a serious public health

challenge worldwide. However, antibiotic resistance genes are not confined to the clinic;
instead they are widely prevalent in different bacterial populations in the environment.
Therefore, to understand development of antibiotic resistance in pathogens, we need
Edited by:
to consider important reservoirs of resistance genes, which may include determinants
Jose L. Martinez, that confer self-resistance in antibiotic producing soil bacteria and genes encoding
Spanish National Research Council
intrinsic resistance mechanisms present in all or most non-producer environmental
(CSIC), Spain
bacteria. While the presence of resistance determinants in soil and environmental
Reviewed by:
Maite Muniesa, bacteria does not pose a threat to human health, their mobilization to new hosts and
University of Barcelona, Spain their expression under different contexts, for example their transfer to plasmids and
Francisco Dionisio,
Universidade de Lisboa, Portugal
integrons in pathogenic bacteria, can translate into a problem of huge proportions,
*Correspondence:
as discussed in this review. Selective pressure brought about by human activities
Parjit Kaur further results in enrichment of such determinants in bacterial populations. Thus, there
[email protected]
is an urgent need to understand distribution of resistance determinants in bacterial
Specialty section: populations, elucidate resistance mechanisms, and determine environmental factors
This article was submitted to that promote their dissemination. This comprehensive review describes the major known
Antimicrobials, Resistance
self-resistance mechanisms found in producer soil bacteria of the genus Streptomyces
and Chemotherapy,
a section of the journal and explores the relationships between resistance determinants found in producer soil
Frontiers in Microbiology bacteria, non-producer environmental bacteria, and clinical isolates. Specific examples
Received: 24 September 2018 highlighting potential pathways by which pathogenic clinical isolates might acquire
Accepted: 14 November 2018
Published: 30 November 2018 these resistance determinants from soil and environmental bacteria are also discussed.
Citation: Overall, this article provides a conceptual framework for understanding the complexity
Peterson E and Kaur P (2018) of the problem of emergence of antibiotic resistance in the clinic. Availability of such
Antibiotic Resistance Mechanisms
knowledge will allow researchers to build models for dissemination of resistance genes
in Bacteria: Relationships Between
Resistance Determinants of Antibiotic and for developing interventions to prevent recruitment of additional or novel genes into
Producers, Environmental Bacteria, pathogens.
and Clinical Pathogens.
Front. Microbiol. 9:2928. Keywords: self-resistance mechanisms, antibiotic resistance, Streptomyces, clinical pathogens, horizontal gene
doi: 10.3389/fmicb.2018.02928 transfer, resistance gene dissemination, environmental bacteria, producer bacteria
Frontiers in Microbiology | www.frontiersin.org 1 November 2018 | Volume 9 | Article 2928

Peterson and Kaur Antibiotic Resistance Mechanisms in Bacteria
INTRODUCTION: A BRIEF HISTORICAL Martinez, 2018). Indeed, with the global epidemic of antibiotic
PERSPECTIVE resistance unfolding before us, it is important to understand the
origin of these determinants in pathogens. This review article
Selman Waksman, a prominent researcher in the field of provides an up-to-date understanding of the antibiotic self-
actinomycetes in the early part of the twentieth century, resistance mechanisms found in producer soil bacteria of the
described the term antibiotic as a chemical compound generated genus Streptomyces and explores relationships between resistance
from microorganisms that inhibits or destroys other microbes determinants found in producer and non-producer soil and
(Hopwood, 2007; Davies and Davies, 2010). Most antibiotics in environmental bacteria and the clinical pathogenic bacteria.
use today originated from the phylum Actinobacteria with nearly The topic of self-resistance in producer bacteria has never
80% of actinobacterial-derived antibiotics produced by soil- before been reviewed in its entirety, while antibiotic resistance
dwelling bacteria of the genus Streptomyces (Barka et al., 2016). mechanisms in clinical isolates have been extensively described
Before the discovery of natural antibiotics, synthetic compounds, (Munita and Arias, 2016). Therefore, resistance mechanisms
including salvarsan, sulfa drugs and quinolones, were in use as of clinical isolates are not discussed in detail in this article.
chemotherapeutic agents (Aminov, 2010). Penicillin was the first Critical additional information about clinical isolates is, however,
natural antibiotic to be discovered accidentally by Alexander provided in a separate section following description of self-
Fleming in 1928 when the Penicillium fungus contaminated resistance in Streptomyces. These two sections were kept separate
a culture plate in his laboratory, however, penicillin was not in this review because resistance mechanisms of producers
developed for use until the late 1930s (Hopwood, 2007). and clinical isolates are currently at very different levels of
Penicillin inhibits cell wall synthesis and was found to be understanding. In the last sections of this review, origins
very effective against Gram-positive but not against Gram- of resistance determinants in clinical strains and potential
negative bacteria (due to the presence of the outer membrane) mechanisms for their mobilization are discussed. Although every
or the tubercle bacillus (because of the extra thick cell wall) attempt has been made to be inclusive of all available literature,
(Hopwood, 2007). Following the discovery of penicillin by the information on each topic addressed in this review is broad
Fleming, other scientists, including Rene Dubos and Selman and constantly growing, therefore any omission is unintentional.
Waksman, started a deliberate search for antibacterial agents Where possible, references to additional literature and review
among soil microorganisms, including bacteria and fungi. It was articles are provided for further reading.
soon realized that antibacterial activity was most often present
in actinomycete cultures and less often in other bacteria or
fungi. During this period, several antibiotics were discovered SELF-RESISTANCE MECHANISMS IN
in the screens designed by these scientists but many of these PRODUCER ORGANISMS
were of little use in the clinic due to their toxicity in animals.
The next biggest discovery came about in 1943, resulting in Antibiotic producing bacteria contain a variety of sophisticated
identification of streptomycin produced by Streptomyces griseus. mechanisms for self-defense against their own antibiotics
Streptomycin inhibits protein synthesis by binding to the 30S (Figure 1 and Table 1). Very often they contain multiple
subunit of the prokaryotic ribosome and was found to be mechanisms simultaneously to ensure complete protection from
effective not only against Gram-negative bacteria but also against the biologically active molecules produced by them. Interestingly,
the tubercle bacillus (Hopwood, 2007). With the discovery the genetic determinants for self-resistance are almost always
of streptomycin, the golden age of antibiotic discovery and clustered together with the antibiotic biosynthesis genes, and
development (1940–1990) ensued. This involved efforts of many their expression is co-regulated (Mak et al., 2014). The following
academic institutions and major pharmaceutical companies in section highlights major biochemical categories of self-defense
the United States and other countries. Currently, antibiotics mechanisms found in producer organisms with specific examples
affecting almost every process in the bacterial cell are known. provided for each category.
Based on their structure and mode of action, at least seven
major groups of antibiotics have been described. These include Antibiotic Modification or Degradation
β-lactams (inhibit cell wall synthesis), aminoglycosides (protein Antibiotic modification is a commonly used strategy for
synthesis), macrolides (protein synthesis), tetracyclines (protein rendering an antibiotic ineffective, especially in the case
synthesis), daptomycin (cell membrane function), platensimycin of aminoglycoside antibiotics (for example, kanamycin,
(fatty acid biosynthesis), and glycopeptides (cell wall synthesis). gentamycin, and streptomycin), chloramphenicol, and
It is only natural that organisms which produce antibiotics β-lactams. A large number of aminoglycoside modification
should also contain self-resistance mechanisms against their enzymes (AMEs), including N-acetyl transferases (AAC),
own antibiotics. In addition, co-existence of producer and non- O-phosphotransferases (APH), and O-adenyltransferases
producer bacteria is also believed to have resulted in co-evolution (ANT) that acetylate, phosphorylate, or adenylylate the
of resistance mechanisms in non-producing environmental aminoglycoside antibiotic, respectively, are known to exist in
bacteria. Resistance determinants found in these two groups producer bacteria. Although these enzymes were first identified
of bacteria have garnered significant attention in recent years in the producer Streptomyces species in the early 1970s, and
because of their possible link with the emergence of resistance they perform identical biochemical reactions to those seen in
in pathogenic clinical isolates (Surette and Wright, 2017; antibiotic resistant clinical strains (Walker and Walker, 1970;

FIGURE 1 | Schematic representation of different antibiotic resistance mechanisms in bacteria, shown with examples. (A) Antibiotic modification involves the
addition of acetyl, phosphate, or adenyl groups to aminoglycosides by N-acetyl transferases (AAC), O-phosphotransferases (APH), and O-adenyltransferases (ANT).
Other examples include chloramphenicol acetyl transferases (CAT) and bleomycin N-acetyltransferases (BlmB). (B) Antibiotic degradation is observed with
β-lactamases, which hydrolyze the antibiotic. (C) Antibiotic efflux pumps remove the antibiotic from the cell using energy from ATP hydrolysis in ABC pumps like
DrrAB, OtrC, TlrC, and MlbYZ, or proton gradients in MFS, MATE, SMR, and RND family pumps. (D) Target modification includes various target alterations, such as
23S rRNA or 16S rRNA methylation, alterations in the peptidoglycan precursors (for example, in the case of glycopeptides), or synthesis of alternate low-affinity
targets (PBPs) that reduce or completely block antibiotic (penicillins) from associating with the target. (E) Antibiotic sequestration involves proteins that can associate
with the antibiotic and block them from reaching their targets. (F) Target bypass involves generation of additional antibiotic targets or subunits that are not
susceptible to binding of the antibiotic. Meth, methylation.
Benveniste and Davies, 1973), a direct correlation between Other than the example of streptomycin, the biological
synthesis of aminoglycosides and the presence of modification function of AMEs in the producer organisms has been a
enzymes in producer Streptomyces is not always evident. For subject of unresolved debate for a long time. It has been
example, some species may not produce antibiotics but still speculated that these enzymes may not be directly involved
contain modification enzymes, and vice versa. One exception in resistance in producers, but instead may perform other
is streptomycin resistance, where a direct correlation between metabolic functions (Benveniste and Davies, 1973; Martinez,
antibiotic synthesis and the role of modification enzymes 2018). This claim is supported by comparative sequence analyses
in self-resistance has indeed been established. Streptomycin showing that the AMEs are quite diverse and are encoded by
resistance in the producer S. griseus involves the function of the a large group of unrelated genes, thus suggesting that they
modification enzyme streptomycin 6-phosphotransferase that might have originated by multiple convergent paths resulting
converts streptomycin to an inactive precursor streptomycin- in a similar function (Shaw et al., 1993). Other studies have
6-phosphate. Streptomycin 6-phosphotransferase is the last also pointed out potential structural and sequence similarities
enzyme in the biosynthetic pathway, and the expression of the between AMEs of producers and cellular metabolic enzymes,
gene encoding this enzyme is co-regulated with biosynthesis including similarity between APH and protein kinases and
genes (Shinkawa et al., 1985; Mak et al., 2014). between AAC and protein acylases (Heinzel et al., 1988;

TABLE 1 | Antibiotic self-resistance mechanisms in producer bacteria.
Mechanism of antibiotic Selected examples Gene location Reference

resistance
Antibiotic Aminoglycoside modifying Chromosome S. griseus (smk) Shinkawa et al., 1985; Mak
modification/degradation enzymes (AME):AAC; APH; et al., 2014
ANT Streptomycin-6-
phosphotransferase
β-lactamases Class A,B,C Chromosome Streptomyces Ogawara, 2016b
species
Antibiotic efflux ABC transporter DrrAB (Dox) Chromosome S. peucetius Yu et al., 2012; Li et al., 2014
OtrC (oxytetracycline) (drrAB) S. rimosus (otrC)
MFS transporter OtrB Chromosome S. rimosus (otrB) Ohnuki et al., 1985; Reynes
(oxytetracycline) Mfs1 S. chattanoogensis (mfs1) et al., 1988; Wang et al., 2017
(natamycin)
Antibiotic sequestration by Sequestration TlmA, BlmA, Chromosome S. hindustanus Gatignol et al., 1988; Sugiyama
special proteins ZbmA (bleomycin) (tlmA); S. verticillus (blmA); S. et al., 1994; Rudolf et al., 2015
flavoviridis (zbmA)
Antibiotic target modification Low affinity penicillin-binding Chromosome Streptomyces Ogawara, 2015, 2016a
proteins (PBP) Class A Class B species
Peptidoglycan remodeling Chromosome S. toyocaensis Marshall et al., 1998; Binda
(Glycopeptides) VanHst , DdlM, (vanHst, ddlM, vanXst ); et al., 2014
VanXst VanHaov , DdlN, VanXaov A. orientalis (vanHaov , ddlN,
vanXaov )
23S rRNA methylation (MLS) Chromosome S. caelestis (clr) Calcutt and Cundliffe, 1990;
Clr, PikR1, PikR2 S. venezuelae (pikR1, pikR2) Almutairi et al., 2015
16S rRNA methylation Chromosome S. pactum (pct) Ballesta and Cundliffe, 1991;
(Aminoglycosides) PCT, Sgm M. zionesis (sgm) Kojic et al., 1992
methylase
Antibiotic target bypass DNA gyrase subunit B Chromosome S. sphaeroides Schmutz et al., 2003
(novobiocin) (gyrBR )
Antibiotic target protection Antibiotic removal DrrC (Dox) Chromosome S. peucetius Doyle et al., 1991; Mak et al.,
OtrA (oxytetracycline) (drrC) S. rimosus (otrA) 2014; Prija and Prasad, 2017
AAC, N-acetyl transferases; APH, O-phosphotransferases; ANT, O-adenyltransferases; ABC, ATP-binding cassette superfamily; MFS, major facilitator superfamily; Dox,
doxorubicin; MLS, macrolides, lincosamides, and streptogramins; PCT, pactamycin methylase; Sgm, sisomicin-gentamicin resistance methylase.
Piepersberg et al., 1988; Davies and Wright, 1997), implying are also likely to be common in Streptomyces, only a few reports
that the modification enzymes might have been co-opted from of identification of CAT enzymes from Streptomyces species are
housekeeping metabolic enzymes for antibiotic resistance. Thus available (Murray et al., 1989).
many unanswered questions remain, which deserve a careful In contrast to the modification of antibiotics described above,
and systematic investigation. Future investigations should also resistance to β-lactam antibiotics is normally conferred by
determine if most aminoglycoside biosynthesis gene clusters antibiotic-hydrolyzing enzymes known as β-lactamases. These
found in producer Streptomyces contain genes for modification enzymes are widespread among Streptomyces, and, together
enzymes and whether these enzymes play a role in self-resistance. with similar enzymes found in pathogenic and non-pathogenic
Modification of the antibiotic as a mechanism for self- bacteria, they constitute the ‘β-lactamase superfamily’ of proteins
defense is also seen for other classes of antibiotics. For example, (Sattler et al., 2015; Ogawara, 2016b). β-lactamases are generally
the bleomycin (BLM) family members [bleomycin (BLM), grouped into four classes (A,B,C,D) based on their amino acid
tallysomycin (TLM), phleomycin (PLM) and zorbamycin (ZBM)] sequence and use of a catalytic serine or zinc ion (King et al.,
are subject to acetylation. BLMs and TLMs are produced 2016). In a recent phylogenetic screen conducted by Ogawara,
by Streptomyces verticillus and Streptoalloteichus hindustanus, it was found that diverse β-lactamases belonging to classes
respectively, and their biosynthesis gene clusters contain genes A, B, and C exist in many Streptomyces species. However, a
for N-acetyltransferases, BlmB and TlmB. These enzymes carry clear relationship between the level of β-lactamases and the
out acetylation of the metal-free forms of BLMs and TLMs, thus degree of resistance to β-lactam antibiotics in these species
preventing correct formation of the metal-binding domain of has not been established (Ogawara, 2016b). This is due to
these antibiotics (Coughlin et al., 2014). Finally, chloramphenicol the fact that most Streptomyces species produce β-lactamases
is another antibiotic that can be acetylated by a large and widely constitutively, and their production is not related to resistance
distributed group of enzymes known as chloramphenicol acetyl or synthesis of β-lactams. As discussed previously for AMEs,
transferases (CATs). Although these enzymes have been shown Streptomyces β-lactamases also exhibit diverse species-specific
to be very prevalent in clinical strains (Schwarz et al., 2004) and properties, again suggesting convergent evolution from different

proteins to perform the same function, i.e., hydrolysis of the efflux system that exhibits multidrug specificity. Self-resistance
β-lactam ring (Allen et al., 2009). The presence of β-lactamases in in S. rimosus is conferred by two efflux proteins: OtrB (previously
producers also presents an evolutionary conundrum – how can known as TetB) located in the biosynthesis cluster, and
β-lactams and β-lactamases co-exist simultaneously in producer OtrC located outside of the cluster (Mak et al., 2014). OtrB
cells? Perhaps these enzymes play alternative cellular functions belongs to the major facilitator superfamily (MFS) of transport
in Streptomyces, are expressed at low levels, or are expressed in proteins, but not much is known about its mechanism of
a growth phase different from biosynthesis? Overall, therefore, action or substrate specificity (Ohnuki et al., 1985; Reynes
it has been proposed that β-lactamases may not play an et al., 1988; Mak et al., 2014). OtrC protein is an ABC
important role in resistance in Streptomyces species, which may family protein, and like DrrAB, it also confers resistance to
instead involve the function of low-affinity penicillin binding multiple antibiotics and MDR substrates, including ampicillin,
proteins (PBPs) (Ogawara, 2015) discussed in Section “Target oxytetracycline, doxorubicin, ethidium bromide, ofloxacin and
Modification/Bypass/Protection Mechanisms” in this article. vancomycin (Yu et al., 2012; Mak et al., 2014). Interestingly,
the DrrAB and OtrC systems are quite homologous and
Antibiotic Efflux show high sequence conservation in the previously identified
Efflux of antibiotics is another commonly used mechanism for motifs, including the DEAD and the LDEVLF motifs of
self-resistance, although it usually occurs in conjunction with DrrA (Zhang et al., 2010, 2015) and the EAA-like motif in
other mechanisms, such as modification of the antibiotic or DrrB (Kaur et al., 2005; Yu et al., 2012), suggesting close
the target. The best studied example of antibiotic efflux among evolutionary links between efflux systems of different producer
producers is found in Streptomyces peucetius, which produces organisms.
two closely related anticancer antibiotics, daunorubicin (Dnr) It might be expected that efflux systems found in producer
and doxorubicin (Dox). These two antibiotics intercalate with organisms would be specific for the antibiotic that the system
DNA preventing further rounds of replication. Efflux of these is dedicated for. Surprisingly, however, the two examples
antibiotics in S. peucetius occurs by an ABC (ATP Binding (DrrAB and OtrC) discussed above suggest polyspecific drug
Cassette) family transporter DrrAB coded by the drrAB genes recognition in these systems. This raises interesting questions.
embedded within the gene cluster responsible for biosynthesis of Why is a multidrug transporter needed in a producer organism?
these antibiotics (Guilfoile and Hutchinson, 1991). The DrrAB What is the origin of DrrAB-like polyspecific antibiotic and
system has been studied in significant molecular and biochemical drug efflux systems? Are most efflux systems associated with
detail. The DrrAB pump is assembled from two subunits each biosynthetic gene clusters polyspecific? Did these systems evolve
of the ABC protein DrrA and the integral membrane protein from possibly even more ancient broad-spectrum efflux systems
DrrB. DrrA protein functions as the catalytic nucleotide binding that might have served as general defense mechanisms against
domain (NBD). DrrB protein functions as the carrier protein toxins in environmental bacteria? That transporters involved
and forms the transmembrane domain (TMD). In an in vitro in antibiotic resistance could have been repurposed from the
assay using inverted membrane vesicles, the DrrAB proteins general defense efflux systems has been suggested previously
were shown to carry out efflux of Dox in ATP or GTP- (Dantas and Sommer, 2012; Martinez, 2018). Such an origin
dependent manner (Li et al., 2014). Because of the location could explain why these systems are multi-specific, and how
of the drrAB genes in the Dox biosynthesis gene cluster, they could be easily adapted by different producer organisms to
this system is considered to be a dedicated transporter of transport individual antibiotics synthesized by them. Analysis of
Dnr and Dox in S. peucetius. Interestingly, however, recent many additional efflux systems found in biosynthesis clusters of
studies showed that DrrAB pump is a multidrug transporter producer organisms is needed to begin to formulate clear answers
with broad substrate specificity, and it can transport many to these questions.
previously known MDR (multidrug resistance) pump substrates Many other examples of ABC as well as MFS transporters used
such as ethidium bromide, Hoechst 33342, verapamil, and for conferring self-resistance in producer organisms to lantibiotic
vinblastine, among others (Li et al., 2014). In this regard, the NAI-107, polyene macrolide natamycin, tylosin, or actinorhodin
DrrAB system is similar to the mammalian ABC multidrug are known (Rosteck et al., 1991; Xu et al., 2012; Mak et al.,
transporter P-glycoprotein (Pgp), which is overexpressed in 2014; Pozzi et al., 2016; Wang et al., 2017). However, their
human cancer cells and is one of the major causes for molecular mechanisms and substrate specificities have not yet
failure of chemotherapy (Chufan et al., 2015). Recent studies been elucidated.
showed that critical aromatic residues, contributed by multiple
helices in DrrB, form part of a large (common) drug-binding Antibiotic Sequestration
pocket (Li et al., 2014; Brown et al., 2017). Mammalian Pgp Sequestration involves the function of drug-binding proteins,
also uses aromatic residues to provide flexibility in substrate which prevent the antibiotic from reaching its target. In
recognition, suggesting a common origin for these proteins and producers of the bleomycin family of antibiotics, the primary
an aromatic residue-based mechanism for polyspecificity that is mechanism of resistance involves sequestration of the metal-
conserved over large evolutionary distances (Chufan et al., 2015; bound or the metal-free antibiotic (Sugiyama and Kumagai,
Szewczyk et al., 2015). 2002) by binding proteins TlmA, BlmA, and ZbmA in
Interestingly, OtrC found in oxytetracycline producer S. hindustanus ATCC 31158 (Gatignol et al., 1988), S. verticillus
Streptomyces rimosus is another example of a self-resistance (Sugiyama et al., 1994, 1995), and Streptomyces flavoviridis,

respectively (Rudolf et al., 2015). Each bleomycin-family Marcone et al., 2014; Frasch et al., 2015). Other glycopeptide
producer member has one or more genes related to ABC producers, without an obvious vanHAX cluster, may have
transporters in their biosynthesis clusters (Du et al., 2000; currently unidentified or poorly understood van resistance genes,
Tao et al., 2007; Galm et al., 2009), which may be used to such as vanJ/staP (Hong et al., 2004; Novotna et al., 2012) and
remove the antibiotics bound to binding proteins. For additional vanK (Hong et al., 2005).
examples, see references (Sheldon et al., 1997, 1999; Pozzi et al., Target modification is also seen for MLS antibiotics, which
2016). bind to the 50S ribosomal subunit. This mechanism involves
methylation of 23S rRNA at residue A-2058 by 23S rRNA
methyltransferases (Douthwaite et al., 2004). Monomethylation
Target Modification/Bypass/Protection (MLS type I) typically provides moderate level of resistance,
Mechanisms while dimethylation (MLS type II) provides strong resistance
Target modification acts as a self-resistance mechanism (Fyfe et al., 2016). For further information on MLS resistance
against several classes of antibiotics, including β-lactams, mechanisms, see reviews (Matsuoka and Sasaki, 2004; Mast and
glycopeptides, macrolides, lincosamides, and streptogramins Wohlleben, 2014; Spizek and Rezanka, 2017). Finally, resistance
(MLS), and aminoglycosides. The β-lactam antibiotic has a against aminoglycosides by target modification uses 16S rRNA
similar structure to PBP substrates (peptidoglycan precursors), methyltransferases, which methylate at residue A1408 or G1405
thus allowing the antibiotic to associate and cause acylation (Shakil et al., 2008). This mechanism for self-resistance may work
of the active site serine resulting in its inhibition (Yeats in conjunction with the AMEs, which were described earlier.
et al., 2002). The producer Streptomyces species, despite Other resistance mechanisms bypass the original target by
being Gram-positive, are highly resistant to penicillins, producing additional low affinity targets. Examples include
which is due to either overproduction of PBPs or synthesis synthesis of additional B subunit of DNA gyrase for novobiocin
of low-affinity PBPs (Ogawara, 2015). Three classes of resistance, alternate resistant RNA polymerase for rifamycin
PBPs (A, B, and C) are found in bacteria (Ogawara, 2015). resistance, or an alternate fatty acid synthase for resistance to
Analysis of the biosynthesis clusters of β-lactam producing platensimycin (Blanco et al., 1984; Thiara and Cundliffe, 1988,
bacteria showed that they often contain genes for PBPs, 1989; Schmutz et al., 2003; Sanchez-Hidalgo et al., 2010; Peterson
suggesting their role in self-resistance (Liras and Martin, et al., 2014). Antibiotic removal from the target site provides
2006; Ogawara, 2015). Interestingly, Streptomyces species another protective resistance mechanism. In S. peucetius, DrrC
contain on average more than 10 PBPs, including both removes intercalated daunorubicin/doxorubicin from DNA
Classes A and B, a number much greater than found in resulting in normal transcription and replication (Prija and
other Actinobacteria. Some of these PBPs indeed have Prasad, 2017). In S. rimosus, the antibiotic oxytetracycline is
low affinity for β-lactams most likely due to the absence removed by OtrA from the ribosome (Doyle et al., 1991; Mak
of a serine/threonine protein kinase domain (STPK) et al., 2014).
(renamed PASTA) that binds β-lactams (Ogawara and
Horikawa, 1980; Nakazawa et al., 1981; Coque et al., 1993;
Paradkar et al., 1996; Yeats et al., 2002; Ishida et al., 2006; MULTIPLICITY OF RESISTANCE
Ogawara, 2016a). MECHANISMS IN PRODUCER
Glycopeptides, such as vancomycin and teicoplanin, inhibit ORGANISMS
cell wall transpeptidation and transglycosylation by associating
with peptidoglycan precursors (D-Ala-D-Ala) (Binda et al., Most producer organisms contain several mechanisms for self-
2014). Antibiotic resistance results from a change in the resistance. For example, S. peucetius relies on DrrAB to efflux
peptidoglycan precursor from D-Ala-D-Ala to D-Ala-D-Lac doxorubicin (Li et al., 2014; Brown et al., 2017), DrrC to remove
or D-Ala-D-Ser, which has a 1000- and 6-fold reduction the antibiotic from its target DNA (Prija and Prasad, 2017), and
in affinity for the glycopeptides, respectively (Bugg et al., DrrD is possibly used to modify the antibiotic to an inactive
1991; Billot-Klein et al., 1994). Genes conferring vancomycin form (Karuppasamy et al., 2015). In addition, there is also a
resistance were initially identified in clinical strains, with the serine protease capable of sequestering daunorubicin to prevent
vanA cluster (vanHAX) on the transposon Tn1546 being its re-entry into the cell following efflux (Dubey et al., 2014).
the most commonly seen. Some systems also use VanY, a Other examples of producers containing several mechanisms for
D , D -carboxypeptidase to produce tetrapeptides incapable of self-resistance include the following: Microbispora ATCC PTA-
glycopeptide binding (Binda et al., 2014). Related core vanHAX 5024 contains both an efflux pump (MlbJYZ) and a sequestration
clusters have been found in producer organisms, suggesting protein (MlbQ) to protect against NAI-107 (Pozzi et al., 2016);
an evolutionary relatedness of resistance within producers and S. rimosus has an ABC multi-drug efflux pump (OtrC) (Yu et al.,
pathogens (Marshall et al., 1997, 1998). The examples include 2012) and an MFS pump (OtrB) for efflux of oxytetracycline
similar vanH (Marshall et al., 1998), vanA (Marshall and (Mak et al., 2014) along with OtrA to protect the ribosome
Wright, 1997, 1998), and vanX (Lessard et al., 1998) sequences by antibiotic removal (Doyle et al., 1991); S. fradiae contains
in the glycopeptide producers Streptomyces toyocaensis NRRL several gene products (TlrA, TlrB, and TlrD) that modify
15009 and Amycolatopsis orientalis. Variants on the core cluster the ribosome to prevent tylosin binding and uses TlrC for
are also reported (Schaberle et al., 2011; Binda et al., 2012; efflux (Mak et al., 2014); and S. chattanoogensis L10 contains

several different efflux pumps for resistance against natamycin organism. A well-studied example of an intrinsic resistance
(Wang et al., 2017). system is the AcrAB/TolC efflux pump in Escherichia coli, which
has a very broad substrate specificity and can export different
classes of antibiotics, dyes, detergents, and disinfectants (Nikaido
DEVELOPMENT OF ANTIBIOTIC and Takatsuka, 2009). Vancomycin resistance in E. coli and other
RESISTANCE IN CLINICAL ISOLATES Gram-negative bacteria provides another example of intrinsic
resistance, which results from the permeability barrier imposed
Discovery of antibiotics and their development for treatment by the outer membrane (Arthur and Courvalin, 1993). Although
of infectious diseases is the biggest success story in the history intrinsic mechanisms confer low level antibiotic resistance in the
of chemotherapy. However, widespread and indiscriminate use original host, normal commensal flora or environmental bacteria
of antibiotics in the last 70 years has led to selection of containing intrinsic mechanisms can become opportunistic
resistant strains to every antibiotic that has been introduced pathogens in immunocompromised patients (Wright, 2007). The
so far. With the very first antimicrobial agents, such as acquired resistance mechanisms, on the other hand, are generally
sulfonamides, resistance was observed soon after in the late obtained by horizontal gene transfer (HGT, described later) and
1930s (Davies and Davies, 2010). Even before the widespread include plasmid-encoded specific efflux pumps (such as TetK and
use of penicillin in clinical practice, penicillinase was discovered TetL of S. aureus) and enzymes that can modify the antibiotic
in 1940 in Staphylococcus aureus and Streptococcus pneumoniae or the target of the antibiotic (Bismuth et al., 1990; van Hoek
providing evidence that the resistance mechanisms against et al., 2011). These mechanisms pose a more serious threat to
penicillin were already present in the natural environment human health because of a change in the context of the resistance
(Davies and Davies, 2010; Ogawara, 2016b). Similarly, after determinant from chromosomal to plasmid-mediated, resulting
the introduction of methicillin (a semi-synthetic penicillin) to in their enhanced expression and dissemination (Dantas and
treat penicillin-resistant S. aureus infections, resistance was once Sommer, 2012; Martinez, 2018). A well-documented example
again observed in strains now referred to as MRSA (Methicillin- of such a phenomenon is mobilization of the chromosomal
resistant Staphylococcus aureus) (Davies and Davies, 2010). β-lactamase gene ampC to a plasmid resulting in its worldwide
These observations suggest that the use of each and every dissemination (Dantas and Sommer, 2012).
antibiotic sooner or later results in appearance of resistant
strains. This is a testament to the extreme malleability and Distribution and Function of Resistance
plasticity of bacterial genomes and their vast potential for Determinants in Clinical Pathogens
adaptability. A high rate of spontaneous mutations and widely Interestingly, the biochemical mechanisms of resistance in
prevalent DNA exchange mechanisms in bacteria are critical clinical isolates are very similar to those found in producer
contributors to the emergence of this phenomenon. According organisms. Moreover, the resistance genes belong to the same
to the Centers for Disease Control and Prevention, antibiotic functional families as seen in the producers (Benveniste and
resistance leads to 23,000 deaths annually in the US alone. Davies, 1973; Marshall et al., 1998; Forsberg et al., 2012).
Recently, the development of MDR and XDR (extremely drug However, the distribution, expression, and genetic context of
resistant) strains of Mycobacterium tuberculosis, S. aureus, and resistance determinants in clinical strains are strikingly different.
Acinetobacter baumannii have become a cause for serious For example, resistance elements found in producer organisms
concern, leaving limited options for the treatment of infectious are embedded in the biosynthesis gene clusters, while in clinical
pathogens carrying these resistance mechanisms. These strains strains they are most often located on plasmids and transposons.
are commonly referred to as ‘superbugs,’ which can be normal For human health reasons, a lot more attention has been given to
human commensal flora that have acquired antibiotic resistance understanding the molecular and biochemical basis of antibiotic
and increased virulence, such as MRSA strains of S. aureus resistance in clinical isolates, and a large number of excellent
and vancomycin resistant enterococci (VRE), or intrinsically reviews have been written on this topic (Blair et al., 2015; Chang
resistant environmental bacteria that can become opportunistic et al., 2015; Munita and Arias, 2016). Therefore, the section
pathogens, such as Pseudomonas aeruginosa and A. baumannii below provides only relevant additional information about each
(Wright, 2007; Miller et al., 2014). resistance mechanism in clinical strains, allowing the reader to
compare and contrast our understanding of these determinants
in clinical strains vs. the producer organisms while providing a
MECHANISMS OF ANTIBIOTIC
more complete picture of the field of antibiotic resistance. Where
RESISTANCE IN CLINICAL ISOLATES available, examples of antibiotic resistance genes/mechanisms in
non-producing environmental bacteria are also provided, and
Intrinsic vs. Acquired Resistance their possible relationships with determinants in clinical strains
Intrinsic antibiotic mechanisms are normally chromosome- are discussed (Table 2).
encoded and include non-specific efflux pumps (which likely
evolved as a general response to environmental toxins), antibiotic Antibiotic Modification
inactivating enzymes, or mechanisms that serve as permeability As seen in producers, antibiotic modification is commonly used
barriers (Fajardo et al., 2008; Cox and Wright, 2013). These as a resistance mechanism for aminoglycosides in pathogenic
mechanisms are fixed in the core genetic make-up of an strains. Multiple types of AMEs (∼100), including a fusion

TABLE 2 | Antibiotic resistance mechanisms in clinical isolates.
Mechanism of Intrinsic Gene location Reference Acquired Gene location Reference

resistance resistance resistance
Antibiotic AME AAC(20 )-Ia Chromosome Macinga and AME AAC(60 )-Ib’ MGE P. aeruginosa Ramirez and
modification/ P. stuartii Rather, 1999 (aac(60 )-Ib0 Tolmasky, 2010
degradation (aac(20 )-Ia) integron)
β-lactamase AmpC Chromosome Jacoby, 2009 β-lactamase TEM-3 MGE Paterson and
E. coli (blaAmpC ) K. pneumoniae Bonomo, 2005
(blaTEM−3 plasmid)
Antibiotic efflux RND AcrAB/TolC Chromosome Thanassi et al., SMR QacC (MDR) MGE S. aureus Schindler and
(MDR) E. coli (acrAB/tolC) 1997 (qacC plasmid) Kaatz, 2016
MFS NorA (MDR) Chromosome Schindler and MFS TetK, TetL MGE S. aureus Bismuth et al.,
S. aureus (norA) Kaatz, 2016 (tetracycline) (tetK, tetL plasmid) 1990; van Hoek
et al., 2011
Antibiotic Sequestration Chromosome Sabnis et al., 2018 Sequestration MGE S. aureus (ble Sugiyama et al.,
sequestration with Lipocalin B.cenocepacia BLMS, BMLT on plasmid) E. coli 1995; Kumagai
special proteins (polymyxin B, (bcnA) (bleomycin) (ble on Tn5) et al., 1999
rifampicin,
norfloxacin,
ceftazidime)
Antibiotic target Low affinity PBP Chromosome M. Basu et al., 1996 Low affinity MGE S. aureus Fishovitz et al.,
modification PBP1 leprae (pon1) PBP PBP2a (mecA in SCCmec) 2014
Peptidoglycan Chromosome Binda et al., 2014; Peptidoglycan MGE E. faecalis Binda et al., 2014;
remodeling (GPAs) E. gallinarum (vanC Miller et al., 2014 remodeling (GPAs) (vanA cluster Miller et al., 2014
VanC, VanXYC , cluster) VanRS, vanHAXYZ Tn1546 on plasmid)
VanTC , VanRC,
VanSC
23S rRNA Chromosome M. Buriankova et al., 23S rRNA MGE S. aureus Roberts, 2008
Methylation (MLS) tuberculosis 2004 methylation (MLS) (ermC plasmids)
ErmMT (ermMT) ErmC
16S rRNA Chromosome Galimand et al., 16S rRNA MGE K. Doi et al., 2016
Methylation (AGs) E. faecium (efmM) 2011 methylation (AGs) pneumoniae (armA
EfmM ArmA on plasmid)
Antibiotic target Overproduction Chromosome Huovinen, 2001; Low affinity DHPS Chromosome Radstrom et al.,
bypass DHFR (TMP) E. coli (mutation in Munita and Arias, (sulfonamide) N. meningitidis 1992
promoter of dhfr) 2016 (dhps) by
transformation
Antibiotic target Antibiotic Chromosome E. Murina et al., 2018 Antibiotic removal MGE C. jejuni (tetO) Munita and Arias,
protection removal LsaA faecalis (lsa) TetO (tetracycline) plasmid, 2016
(lincosamide and transposon
streptogramin A)
AME, aminoglycoside modifying enzyme; AAC, N-acetyltransferase; MGE, mobile genetic element; RND, Resistance-Nodulation-Division; SMR, Small Multidrug
Resistance; MFS, Major Facilitator Superfamily; MDR, Multidrug resistance; PBP, penicillin-binding protein; GPAs, glycopeptide antibiotics; MLS, macrolides, lincosamides,
and streptogramins; AGs, aminoglycosides; DHFR, dihydrofolate reductases; DHPS, dihydropteroic acid synthase; TMP, trimethoprim.
enzyme containing both AAC and APH activities, have been conserved fold, these enzymes exhibit significant sequence,
identified in both Gram-positive and Gram-negative bacteria structural, and functional diversity, again implying convergent
(Schwarz et al., 2004; Ramirez and Tolmasky, 2010), and evolution of these enzymes from distinct housekeeping cellular
a detailed nomenclature has been developed (Ramirez and proteins (Stogios et al., 2017). Indeed, in the environmental
Tolmasky, 2010; Becker and Cooper, 2013). While these genes bacteria Providencia stuartii, physiological function of the
are commonly located on the mobile genetic elements (MGEs) chromosomally encoded AAC(20 )-Ia enzyme is thought to
in clinical bacteria, chromosomal determinants for AMEs have be acetylation and recycling of peptidoglycan although it can
also been found in a large number of environmental bacteria, also acetylate aminoglycosides (Macinga and Rather, 1999).
including Providencia and Acinetobacter species (Macinga Therefore, aminoglycosides may be ‘accidental’ substrates for
and Rather, 1999; Yoon et al., 2014), which are considered to these enzymes because of their similarity to cellular substrates
be the source of acquired determinants found on MGEs in containing amino sugars (Macinga and Rather, 1999). These
pathogenic strains. Of the known AMEs, AACs are the most studies further illustrate the plasticity of antibiotic modification
prevalent in clinical strains, and the AAC (60 ) enzymes, which enzymes (Fong et al., 2011; Stogios et al., 2017), as discussed
acetylate at the 60 position of the aminoglycoside scaffold, previously for the producers. In addition to AMEs, multiple
have been studied in detail. In spite of the presence of a CAT enzymes have been identified in both Gram-positive and

Gram-negative bacteria, which have been extensively reviewed in E. coli, norA in S. aureus, and lmrA in Lactococcus lactis.
(Schwarz et al., 2004). Of these, the best understood system is the tripartite RND
A third type of modification/degradation enzyme used by pump AcrAB/TolC. Although this system carries out efflux of
clinical bacterial strains is β-lactamase. While the role of a very broad spectrum of compounds, its biological function is
β-lactamases in producer bacteria is still debatable, they are believed to be export of bile salts in Enterobacteriaceae (Thanassi
known to play a critical role in β-lactam resistance in Gram- et al., 1997; Martinez, 2018). The RND pumps are unique in
negative clinical bacteria. Gram-positive bacteria instead prefer that they bridge the inner and outer membranes through a
PBP-based resistance mechanisms, likely due to differences in the fusion protein (AcrA in this case) and bring about export of
architecture of the cell wall/envelope between the two types of antibiotics from the inside to the outside in a single step. The
bacteria. More than 1000 β-lactamases have been identified from acquired antibiotic efflux determinants, often found on MGEs
clinical isolates, and this number continues to grow because of in clinical isolates, are exemplified by many different types of
the ever-new mutations in the active site allowing it to adapt to tet genes (at least 22 have been identified) located on plasmids
newer β-lactams. An example is the evolution of TEM-3, which in both Gram-negative and Gram-positive bacteria (Roberts,
can degrade 3rd generation cephalosporins, placing it into the 2005). Interestingly, RND pumps can act synergistically with
category of ESBLs (Extended Spectrum β-lactamases) (Paterson the simple Tet pump proteins (MFS family), resulting in a
and Bonomo, 2005), suggesting rapid evolution of β-lactamase significant increase in the minimum inhibitory concentration for
genes in clinical strains. Most β-lactamase genes are carried tetracycline (Lee et al., 2000). This likely occurs when tetracycline
on MGEs facilitating their rapid spread through populations; exported to the periplasm by a Tet protein can be captured by the
however, some β-lactamase genes are also found in chromosomes RND pump and exported to the outside (Nikaido and Takatsuka,
of members of the Enterobacteriaceae family where they are 2009), illustrating how acquired resistance mechanisms can be
poorly expressed and function as silent genes. Once again, it is augmented by the intrinsic mechanisms potentially resulting in
speculated that, as in the case of AMEs, β-lactamases may also major implications in the clinic.
perform dual functions, including housekeeping and antibiotic
resistance (Martinez, 2018). An interesting set of studies indeed Target Modification/Bypass/Protection
suggest that the biological function of β-lactamases may be A large number of target replacement and protection
peptidoglycan recycling (Wiedemann et al., 1998; Macinga and mechanisms are also found in clinical isolates. The classical
Rather, 1999), although their mobilization to a plasmid results in example of target modification is seen in MRSA strains where
high expression and high levels of antibiotic resistance (Jacoby, resistance to β-lactams is conferred by an exogenous PBP,
2009; Dantas and Sommer, 2012). known as PBP2a, whose transpeptidase domain is insensitive
to the action of several different β-lactams. Acquisition
Antibiotic Efflux of PBP2a facilitates bypass of the original sensitive target,
The second major mechanism of antibiotic resistance in clinical however, since it does not contain the transglycosylase activity
strains involves decreased permeability and/or efflux of the it functions together with the transglycosylase domain of
antibiotic. Decreased permeability is important for Gram- the native PBP2 to perform cross-linking reaction in the
negative bacteria because of the presence of the outer membrane, presence of β-lactams. PBP2a is coded by the mecA gene, which
which forms a permeability barrier and offers an intrinsic is located on a large MGE called SCCmec (Staphylococcal
mechanism for protection against hydrophilic antibiotics and chromosomal cassette) in S. aureus. Many different types of
other antimicrobial agents, such as vancomycin (Nikaido, 2003). SCCmec cassettes have been described, which contain varying
Mutations in the porin genes and/or changes in their expression numbers of accompanying resistance elements (Fishovitz
have been shown to further impact the susceptibility of Gram- et al., 2014; Liu et al., 2016). Another example of target
negative bacteria to hydrophilic antibiotics (Li et al., 2012). modification is vancomycin resistance, which results from
In addition, many types of active efflux pumps have been acquisition of the van gene cluster and is commonly a problem
described in Gram-positive and Gram-negative bacteria, which in enterococci (Miller et al., 2014). Of the many known
generally belong to one of the five families: ABC, MFS, RND types of van clusters, vanA and vanB, in particular, are a
(Resistance-Nodulation-Division), MATE (Multidrug and Toxin problem in clinical strains as they occur on MGEs. The
Extrusion), and SMR (Small Multidrug Resistance) (Sun et al., similarities in the sequence and arrangement of van genes in
2014; Schindler and Kaatz, 2016). Of these, only ABC proteins use producer and clinical strains suggest that they are evolutionarily
ATP as a source of energy, while the other four families couple linked.
transport of substrates to ion gradients. Normally transport Other target modification examples in clinical strains include
proteins carry out import or export of only one specific point mutations or enzymatic alteration of the target (Munita
substrate (for example, Tet proteins belonging to the MFS and Arias, 2016). For examples of point mutations in the target,
family). However, examples of multidrug/polyspecific exporters see (Hooper, 2002; Floss and Yu, 2005). Enzymatic alteration
have been found in each of these five families (Poole, 2005; of the target is best understood in the case of macrolide
Schindler and Kaatz, 2016), suggesting that polyspecificity is resistance conferred by a large group of erythromycin ribosomal
widely distributed and must be an ancient phenomenon. methylation (erm) genes. These enzymes methylate a specific
Genes encoding antibiotic efflux pumps can be either intrinsic adenine in the 23S rRNA (Weisblum, 1995). The erm genes
or acquired. Examples of intrinsic genes include acrAB/tolC in clinical strains are present on mobile genetic elements and

FIGURE 2 | Schematic showing reservoirs of antibiotic resistance genes found in nature and various pathways for their movement to the clinic. Transfer of resistance
genes to clinical isolates could occur by a variety of routes (shown by arrows), each using horizontal gene transfer mechanisms potentially involving plasmids,
integrons, or transposons. While direct transfer of resistance determinants from producers in the soil to clinical strains is possible (Route 1), a more likely route may
first involve movement from the producer soil bacteria to non-producer soil bacteria (for example Mycobacterium species) (Pang et al., 1994) (Route 2A), followed by
transfer to clinical pathogens through several carriers (Route 2B). Another, possibly more important route, could involve direct transfer from environmental bacteria
(found in bodies of water, aquaculture, livestock animals, wildlife, and plants) to clinical isolates (Route 3). Routes 2 and 3 are shown as thick red arrows, implying
greater probability of these pathways for dissemination of resistance genes to clinical strains.
are widespread among both Gram-positive and Gram-negative from antibiotic producer organisms by horizontal transfer was
bacteria (Roberts, 2008). Significant similarities between the originally proposed in the 1970s (Benveniste and Davies, 1973). It
methylation enzymes found in the clinical isolates and the was based on the observation that the aminoglycoside-modifying
producers have been observed, suggesting a common ancestral enzymes found in actinomycetes exhibit biochemical activities
origin (Uchiyama and Weisblum, 1985; Doi et al., 2016). Finally, similar to the enzymes found in pathogenic strains. Another
known examples of target protection in clinical strains include striking example of a strong connection between antibiotic
the Tet(M) and Tet(O) proteins commonly encoded by genes resistance genes in clinical isolates and those found in antibiotic
located on MGEs in S. aureus. Interestingly, these proteins are producing bacteria is provided by the vanHAX genes, which show
homologous to the elongation factors EF-G and EF-Tu, and their considerable protein sequence similarity as well as a conserved
binding to the ribosome facilitates removal of tetracycline in a arrangement and organization of genes within the cluster (Barna
GTP-ase activity-dependent manner (Burdett, 1996; Trieber et al., and Williams, 1984; Marshall et al., 1998).
1998). Despite strong indications that transfer from producer
Based on the discussion above, it is evident that our organisms to the pathogenic strains might occur (Figure 2,
understanding of the distribution and function of resistance Route 1) a direct link between producers and pathogens has,
determinants in clinical isolates is much more advanced as however, been hard to establish, and very rarely have the
compared to the producer organisms. It may also be concluded resistance genes of pathogens been tracked back to the producers.
that many (or most) of the antibiotic resistance mechanisms This is primarily due to the fact that resistance genes in
in producers, and possibly all organisms, appear to have been producers show high sequence divergence and a very different
repurposed from housekeeping/cellular functions or the intrinsic G+C content as compared to determinants in pathogens even
resistance mechanisms. Indeed, it is the incorporation of such when they use similar mechanisms (Forsman et al., 1990;
determinants into MGEs in pathogens that poses a serious threat Marshall et al., 1998). Altogether, these observations suggest
to human health. an evolutionary link between determinants of producers and
pathogens but not necessarily a direct recent gene transfer
from the producers (Forsman et al., 1990; Marshall et al.,
ORIGIN OF ANTIBIOTIC RESISTANCE IN 1998; Aminov and Mackie, 2007). Nevertheless, transfer from
CLINICAL ISOLATES producers could have occurred a long time ago through a
series of closely related carriers; for example, first transfer
Where do antibiotic resistance genes in the clinic come from? to closely related non-producing actinomycetes in the soil
This question continues to puzzle scientists and clinicians. (Figure 2, Route 2A) and then finally to proteobacteria and
The idea that resistance genes in pathogens may be acquired distant pathogenic strains (Marshall et al., 1998) (Figure 2,

Route 2B). The longer time horizon in this case could proposed in Figure 2 actually available’? Albeit limited in number,
explain a very different G+C content in the two groups of a few reports of direct genetic exchange from producer to
organisms. non-producer organisms and from environmental organisms to
An alternative school of thought and a growing body of recent clinical pathogens are indeed available. In one report, otrA and
literature, however, now seem to suggest that resistance genes otrB gene sequences, found in the oxytetracycline biosynthesis
found in non-producer environmental bacteria may have played cluster in Streptomyces, were identified in mycobacteria variants
a more important role in shaping the evolution of antibiotic (Pang et al., 1994). Mycobacterium is closely related to
resistance in pathogens (Figure 2, Route 3) (Aminov and Mackie, Streptomyces, and both are commonly found in the soil, therefore
2007). Indeed, resistance genes are much more widespread in the transfer of otrA and otrB to mycobacteria suggests their
environmental non-pathogenic microbial populations than was role as potential carrier organisms in the soil. Interestingly, the
originally believed (D’Costa et al., 2006; Nesme et al., 2014; same study also provided evidence for the presence of S. aureus
Surette and Wright, 2017). In an interesting study, which tested tetracycline resistance genes Tet(K) and Tet(L) in Streptomyces
500 Streptomyces strains enriched and isolated from soil against and mycobacteria variants. The sequences isolated from these
21 antibiotics (including natural, semisynthetic, synthetic as well variants were almost identical to the S. aureus genes and had
as recently introduced antibiotics), surprisingly all strains were a G+C content of only 35% as compared to the 70% G+C
multidrug resistant to 7 or 8 of the 21 tested antibiotics (D’Costa content normally seen in Streptomyces and mycobacteria, which
et al., 2006), suggesting widespread resistance mechanisms is a strong indication that these resistance elements originated
among modern organisms. The genome sequence analyses from low G+C Gram-positive bacteria (Pang et al., 1994). This
carried out in recent years have also shown that not only are the study therefore shows that resistance genes can move back and
intrinsic resistance mechanisms widely prevalent in all microbes forth between producer and non-producer organisms providing
(Fajardo et al., 2008; Cox and Wright, 2013), but that homologs support for Route 2A (Figure 2). In another study, bioinformatics
of the resistance determinants of clinical isolates are commonly analysis was used to obtain evidence for recent inter-phylum
present in non-pathogenic Gram-positive and Gram-negative transfer of chloramphenicol and lincomycin efflux genes cmx
bacteria (Seoane and Garcia Lobo, 2000; Mukhtar et al., 2001; and lmrA from Actinobacteria to Proteobacteria (Jiang et al.,
Sugantino and Roderick, 2002). Finally, there is also strong 2017), possibly also occurring through Route 2B, which may be
evidence showing that the antibiotic resistance gene sequences followed by transfer of these genes to clinical isolates (Figure 2).
are ancient and predate the use of antibiotics (D’Costa et al., 2011; The proposed mechanism for such inter-phylum exchange is
Bhullar et al., 2012; Warinner et al., 2014; Perron et al., 2015; discussed in (Jiang et al., 2017) and briefly described in Section
Kashuba et al., 2017). Analysis of microbial DNA isolated from “Role of HGT in Transfer of Antibiotic Resistance Genes” in this
the dental plaque of ancient human remains showed the existence article.
of gene sequences homologous to those conferring resistance The most compelling evidence of recent transfers from non-
to β-lactams, aminoglycosides, macrolides, tetracycline, and pathogenic environmental bacteria to clinical strains (Figure 2,
bacitracin in clinical strains (Warinner et al., 2014; Olaitan Route 3) comes from three independent reports (Dantas and
and Rolain, 2016). In another study, metagenomic analysis of Sommer, 2012; Forsberg et al., 2012). First report showed
ancient DNA derived from 30,000-year-old permafrost showed that the CTX-M ESBL gene found on plasmids in pathogenic
the presence of homologs of tetM, vanX, and bla genes bacteria worldwide is almost identical to CTX-M gene found
(D’Costa et al., 2011). Interestingly, the vanHAX cluster in in the genome of non-pathogenic environmental Kluyvera
permafrost DNA exhibited the same invariant organization species (Humeniuk et al., 2002; Canton and Coque, 2006),
as seen in modern vancomycin resistant isolates, confirming suggesting recent transfer of the gene to clinical strains. The
that these genes predate the use of antibiotics. Other similar second report shows that the quinolone resistance determinant
studies showing prevalence of resistance determinants in ancient qnr located on a conjugative plasmid in Klebsiella, originated
samples, or isolated caves, are also available (Bhullar et al., 2012; from the genome of non-pathogenic environmental Vibrio
Perron et al., 2015; Kashuba et al., 2017). Together these findings and Shewanella species (Poirel et al., 2005). And yet another
suggest that there is a continuum of resistance genes present in example provides evidence for transfer of the aph6 gene, which
the environmental, producer, and pathogenic organisms, leading codes for Aph (30 )-VI amikacin modification enzyme, from
to the concept of ‘resistome’ which is described as the collection of the chromosome of the environmental Acinetobacter guillouiae
antibiotic resistance genes found in all microorganisms (Wright, to a plasmid in A. baumannii and then to members of
2007). Therefore, it is proposed that to get a full understanding Enterobacteriaceae family and to Pseudomonas species (Yoon
of the origin of resistance, one must consider the pan-microbial et al., 2014). These examples provide definitive evidence
genome consisting of antibiotic producers, pathogens, cryptic of genetic transfer from environmental organisms and also
genes, and precursor genes (Wright, 2007; Nesme and Simonet, illuminate how an intrinsic resistance gene located in the
2015). genome of a non-pathogenic organism can result in a
Overall, it is safe to conclude that both producer and non- pandemic when mobilized to a conjugative plasmid or a
producing environmental organisms represent rich pools of phage and transferred to a clinically relevant strain. Overall,
resistance genes which could potentially be mobilized to the these examples suggest that both producer and non-producer
clinically relevant strains, leading to the question ‘is the evidence environmental bacteria play a role in dissemination of resistance
for transfer of resistance determinants using any of the routes genes although recent direct transfers to clinical strains seem

to have mainly occurred from non-producer environmental strains of S. aureus to methicillin-sensitive strains was shown to
bacteria. occur at low frequencies (10−9 to 10−10 ) (Scharn et al., 2013).
Another study, which used qPCR to quantify S. aureus genes
in viral particles, showed the presence of parts of the SCCmec
ROLE OF HGT IN TRANSFER OF element (specifically mecA and ccrA1) in phage particles at
ANTIBIOTIC RESISTANCE GENES relatively high frequency of about 10−4 (Maslanova et al., 2013).
Quantitative studies, however, do not take into consideration
Transfer of antibiotic resistance determinants between bacterial the transmission capability of the particles, therefore they likely
populations occurs by genetic exchange mechanisms involving reflect an overestimation of the transduction frequency (Torres-
transformation with free DNA, transduction by bacteriophages, Barcelo, 2018). Interestingly, other resistance and virulence genes
or conjugation involving plasmids (Wright, 2007; Hu et al., of S. aureus associated with special MGEs referred to as PICIs
2017), collectively referred to as the HGT mechanisms. (phage-induced chromosomal islands), which include SaPIs
All three HGT mechanisms are widely used in nature, (S. aureus pathogenicity islands), are known to be transduced by
although certain species of bacteria tend to employ one bacteriophages at remarkably high frequencies approaching 10−1
mechanism more heavily over the others (Barlow, 2009). For (Chen and Novick, 2009; Penadés and Christie, 2015). These
example, streptococci can become naturally competent and thus islands include many antibiotic resistance genes, suggesting
participate effectively in transformation, whereas enterobacteria that transduction may contribute significantly to variability
commonly use conjugative plasmids for exchange of genetic and evolution of resistance in S. aureus (Novick et al., 2010).
information. Transformation is best characterized in Gram- Interspecies and intergeneric transfer of SaPI elements has also
positive Streptococcus pneumoniae and Bacillus subtilis although been shown to occur between S. aureus, S. epidermidis, and
many Gram-negative bacteria also become competent (Johnston even Listeria monocytogenes, showing a broader host range of
et al., 2014). The factors that control competence generally staphylococcal phages (Maiques et al., 2007).
include the nutritional status of the bacterium (Claverys et al., In general, however, because of the difficulty in detecting
2006) and environmental stressors, such as antibiotics or recombination events outside of the laboratory, the contribution
DNA damaging agents (Prudhomme et al., 2006). Although of either transformation or transduction in transferring
the physiological role of transformation is still debated, its resistance genes in the clinic or the environment remains
main purpose is believed to be DNA repair or genetic unclear. Nevertheless, certain environments considered to be
diversification to enhance adaptability (Johnston et al., 2014). hot-spots for genetic exchange, such as sewage and wastewater
Indeed, transformation seems to have played an important role treatment plants, hospital effluents, aquaculture, agricultural and
in evolution of antibiotic resistance strains of Streptococcus and slaughterhouse waste, are prime locations for exchange events
Neisseria. For example, it is thought that the persistence of because of the high density of bacteria, phages, and plasmids in
penicillin resistance in S. pneumoniae may be related to the high these settings (Kenzaka et al., 2010; von Wintersdorff et al., 2016).
frequency of natural transformation in this organism (Hoffman- In one study, qPCR analysis showed that blaTEM , blaCTX−M , and
Roberts et al., 2005). Transformation of Neisseria gonorrhoeae mecA were indeed present in phage particles isolated from sewage
with DNA from resistant commensal Neisseria flavescens is samples (Colomer-Lluch et al., 2014). Other reports showing the
believed to have resulted in generation of a mosaic penA variant prevalence of phage carrying blaTEM and blaCTX−M genes in soil,
that confers resistance to β-lactams in clinical isolates (Spratt, water, and sewage are also available (Balcazar, 2014; Larranaga
1988; Spratt et al., 1992). Mosaic variants of antibiotic resistance et al., 2018; Mohan Raj et al., 2018). When combined with high
genes have also been reported in several Streptococcus species, selection pressure in these environments, resulting from the
implying the role of transformation in incorporating sections of presence of sub-inhibitory concentrations of antibiotics, metals,
foreign DNA (von Wintersdorff et al., 2016). and toxic materials, which can lead to induction of competence
Transduction is believed to play a major role in evolution (Prudhomme et al., 2006) as well as induction of prophages
of resistance in S. aureus, although it has been shown to (Motlagh et al., 2015), it further enhances the possibility of
occur in many bacteria at a low frequency ranging between HGT by these two mechanisms. Overall, these reports suggest
10−6 and 10−9 transductants/plaque-forming-unit (Ubukata that the original transfer of CTX-M from Kluyvera to the
et al., 1975; Mazaheri Nezhad Fard et al., 2011; Varga et al., clinic pathogens, referred to in Section “Origin of Antibiotic
2012). In S. aureus, which exhibits high strain variability and Resistance in Clinical Isolates,” might have been mediated by
carries a large accessory genome consisting of phages, plasmids, bacteriophages. Other settings suitable for genetic exchange
transposons, genomic islands, and SCCmec (most of which carry via transduction also include the colonized human or animal
resistance genes), it is generally accepted that HGT in general, host (McCarthy et al., 2014; Stanczak-Mrozek et al., 2015), gut
and transduction in particular, play a major role in antibiotic microbiome (Modi et al., 2013), and biofilms (Resch et al., 2005).
resistance gene transfer (Haaber et al., 2017). Indeed, moderate A recent report describing the phenomenon of auto-transduction
rates of transfer (about 10−5 or 10−6 ) of genes for penicillinase, in S. aureus provides further strong support for the important
metallo β-lactamase, and tetracycline resistance by transducing role of phages in delivering antibiotic resistance genes to the
phages have been reported in S. aureus (Varga et al., 2012; host bacteria (Haaber et al., 2016). Using in vitro and in vivo
Lee and Park, 2016; Varga et al., 2016). However, transduction virulence model, this study by Haaber et al. (2016) demonstrates
of even the small SCCmecs (20–25 kb in size) from MRSA how phages released from a subpopulation of lysogenic cells can

lyse other phage-sensitive cells in the same environment, recruit establish in a population also depends on whether it can replicate
beneficial genes from the killed competitors, and reintroduce autonomously and therefore get vertically transmitted. The most
these genes into the remaining lysogenic host cells, resulting in successful conjugative plasmids, such as the incompatibility
genetic diversity. group IncP, have a broad host range (Davies and Davies, 2010),
Plasmid-mediated conjugation as a gene transfer mechanism which facilitates their transfer to and maintenance in distantly
is, however, still considered to be far more prevalent related phyla (Klumper et al., 2015). The ability of MGEs or DNA
in disseminating resistance genes in nature than either to persist in the environment also determines success of HGT.
transformation or transduction. Plasmids are capable of For example, while cell-to-cell contact is essential for conjugation,
autonomous replication, and they carry genes for resistance it provides better protection to DNA. On the other hand, naked
against all major classes of antibiotics. In fact, plasmids can DNA is vulnerable to being degraded quickly, which reduces
carry a collection of resistance genes as part of transposons, the time period during which it remains intact to successfully
thus simultaneously conferring resistance to several classes encounter a competent cell. DNA packed in a phage particle is
of antibiotics and metal ions (Nikaido, 2009). Moreover, they more protected than naked DNA, although the narrow host range
can transfer genes over long genetic distances to different of a phage may determine if it will be in the gene pool long enough
species, genera, and even kingdoms depending on the host to infect a suitable host (von Wintersdorff et al., 2016).
range of the plasmid. Using mathematical modeling analysis, In spite of the limitations, bacterial genome sequencing
one study recently showed that conjugation may be 1000- efforts have made it abundantly clear that the HGT mechanisms
fold more common than transduction as a resistance gene have had a major impact on evolution of bacterial populations
transfer mechanism (Volkova et al., 2014). Since gene transfer (Nakamura et al., 2004; Andam et al., 2011; McDonald and
by conjugation can be easily tracked by DNA sequencing Currie, 2017). Our knowledge of the actual steps and carriers
and PCR-based approaches, there is sufficient evidence for involved in moving resistance genes from environmental and
its contribution to worldwide dissemination of antibiotic producer organisms to the clinic, or from the chromosome
resistance determinants both in community and hospital to the MGEs, is, however, still rather limited. In each of the
environments (Carattoli, 2013). Some of the most successful examples described in Section “Origin of Antibiotic Resistance
known plasmids are the ones that have resulted in the spread of in Clinical Isolates,” exchange was facilitated by conjugative
carbapenemase, blaCTX−M ESBL, and quinolone resistance genes plasmids (Humeniuk et al., 2002; Poirel et al., 2005; Yoon et al.,
among Gram-negative bacteria over very large geographical 2014) or by the presence of resistance genes on transposons
distances (Carattoli, 2013). In Gram-positive bacteria, other (Brisson-Noel et al., 1988). It is not clear, however, why and how
DNA elements, known as conjugative transposons or integrative resistance genes are captured or transferred from chromosome
conjugative elements (ICEs), can also mediate conjugation. These to the plasmids. In addition to the role of insertion sequences
elements integrate into the chromosome but contain the ability and transposons, mobilization of resistance genes may also be
to excise and transfer themselves by conjugation. ICEs often greatly aided by the presence of integrons. While they are not
carry resistance genes, for example Tn916 family members that self-mobile, they can be mobilized to plasmids or phages by
encode tetracycline resistance (Roberts and Mullany, 2011). The transposons, thus gaining the ability to move between cells by
known conditions for resistance gene transfer by conjugation HGT. Integrons typically contain three genetic elements, which
include high density settings, such as the human or animal gut, include a gene for site-specific recombination (IntI), a site-
biofilms, hospitals, and co-infection conditions (Weigel et al., specific recombination site (attI), and a promoter upstream of the
2003; Savage et al., 2013; Huddleston, 2014; Andersson and attI site used for expression of the recruited gene cassette (often
Hughes, 2017). Although some resistance determinants have containing resistance determinants) (Domingues et al., 2012).
been plasmid-associated for a long time (Barlow and Hall, 2002), Thus they are able to exchange and/or recruit gene cassettes by
others are mobilized to plasmids from chromosomes, and the site-specific recombination between the attC site on the cassette
rate at which these genes are being mobilized has increased since and the attI site on the integron, or they can excise gene cassettes
the widespread use of antibiotics about 70 years ago (Barlow by site-specific recombination, therefore conferring the ability
et al., 2008). Another worrisome emerging trend is the clustering on the host to rearrange resistance and virulence determinants
of antibiotic resistance genes on plasmids, perhaps as a response (Gillings, 2014). Class 1 integrons found on MGEs, in particular,
to selective pressures in the environment. A well-characterized are widely distributed in clinical settings and are often associated
mechanism of clustering is provided by the S. aureus conjugative with carrying and spreading antibiotic resistance genes (Naas
plasmid pSK41 that contains an insertion sequence IS257, which et al., 2001; Li et al., 2017). A rather large pool of circular gene
promotes capture of small resistance plasmids (Haaber et al., cassettes containing the attC site and the promoter-less resistance
2017). determinants for almost all classes of antibiotics used clinically
All three HGT mechanisms are subject to limitations imposed are also known to exist in bacteria (Partridge et al., 2009). These
by the host range of the incoming plasmid or the phage, the genes become functional after the cassettes are incorporated and
restriction modification systems of the host, ability to form expressed from the promoter sequence in the integron.
cell-to-cell contacts, fitness cost of acquiring a new gene, as Recently, a novel ‘carry-back’ mechanism for inter-phylum
well as the ability of the incoming DNA to recombine with exchange of genes was also proposed (Jiang et al., 2017). In
the host DNA (Thomas and Nielsen, 2005; Domingues et al., this mechanism, conjugation mediated by a broad-host range
2012). Further, the ability of a mobile genetic element to conjugative plasmid (Klumper et al., 2015) may transfer a carrier

sequence of DNA (a fragment from a widely spread class 1 persistence and possible reemergence of resistance genes in the
integron In4) from Proteobacteria to Actinobacteria, followed future (Ashbolt et al., 2013; Martínez et al., 2014; Bengtsson-
by recombination, resulting in actinobacterial DNA flanked by Palme et al., 2018). Recent studies have shown that antibiotic
proteobacterial DNA. Dead actinobacteria cells would release concentrations significantly below the minimum inhibitory
the actinobacterial DNA flanked by proteobacterial DNA into concentration for sensitive bacteria can be selective (Gullberg
the environment, and proteobacteria can take up this DNA et al., 2011, 2014). Moreover, other contaminants, such as heavy
by transformation and incorporate into their genome using metals, can also co-select for antibiotic resistance (Pal et al., 2015;
homologous recombination. Using such a mechanism, cmx and Andersson and Hughes, 2017).
lmrA genes are believed to have been recently transferred from There is indeed evidence that selective pressure caused by
Actinobacteria to Proteobacteria with the help of the broad-host human activities in the last 70 years has resulted in a significant
range conjugative plasmids and integrons (Jiang et al., 2017). enrichment of resistance genes in bacterial populations. One
Once these genes are transferred to proteobacteria, it is easy to study compared pre-antibiotic era microbes with modern
envision their transfer to pathogenic bacteria which also mostly environmental bacteria in archived soils collected from 1940
belong to the phylum Proteobacteria. Indeed the Proteobacterial to 2008 in the Netherlands and showed that genes conferring
Cmx protein identified in clinical isolates was found to be resistance to tetracycline, erythromycin, and β-lactams increased
52% identical to the self-resistance protein from producer in abundance over time (Knapp et al., 2010). Interestingly, an
S. venezuelae, and the cmx gene was found to be 99% identical increased rate of mobilization of β-lactamase genes from the
to genes from many non-Streptomyces actinobacteria, including chromosome to the plasmids was also reported (Barlow et al.,
Corynebacterium species, suggesting recent inter-phylum transfer 2008). A novel hypothesis advanced recently suggests that the
from Actinobacteria to Proteobacteria following Route 2B. use of antibiotics may provide a strong selection for ‘capture’ of
antibiotic resistance genes by mobile genetic elements (including
plasmids, transposons, and integrons) and acting as a strong force
ENRICHMENT OF ANTIBIOTIC in shaping evolution of microorganisms (Gillings, 2014; Surette
RESISTANCE GENES and Wright, 2017). Other reports also suggest that antibiotic
selection promotes competence in S. pneumoniae (Prudhomme
By now it is well-recognized that the environment itself plays et al., 2006), induction of prophages in S. aureus (Goerke
an important role in the acquisition of antibiotic resistance by et al., 2006), and enrichment of antibiotic resistance genes in
pathogenic organisms. This process is envisioned to go through phages present in the gut microbiome (Modi et al., 2013), all
four stages: emergence of novel resistance genes, mobilization, processes that could increase the rate of HGT. Interestingly, a
transfer to pathogens, and dissemination. While emergence and more recent study showed that the ratio of transducing particles
mobilization events likely occur all the time, environmental to virulent phages varies upon induction by sub-inhibitory
factors, such as selective pressure, fitness cost, and dispersal, concentrations of different antibiotics, suggesting that antibiotics
determine whether these events actually result in establishing affect packaging of genes into phage particles (Stanczak-Mrozek
novel genes in populations (Bengtsson-Palme et al., 2018). Of et al., 2017). Antibiotic exposure has also been shown to result
these, selection is perhaps the single most important factor which in increased rates of mutations and recombination as well as
plays a critical role in maintenance of resistance genes/MGEs an increase in integrase activity (Maiques et al., 2006; Lopez
at each stage of the acquisition process described above. What et al., 2007; Blazquez et al., 2012), thus compounding the
creates selective pressure strong enough to promote persistence multiple effects that excessive usage of antibiotics can have on
and longevity of resistance genes? Antibiotic producers present emergence and enrichment of antibiotic resistance in bacterial
one such scenario where resistance genes can be selected populations. In conclusion, mitigation strategies focused on
naturally in a competitive environment, thus preserving the pool limiting selective pressure, for example by reducing unnecessary
of resistance genes in that niche (Laskaris et al., 2010). The usage of antibiotics and avoiding settings which select for and
most important source of selective pressure, however, is the promote persistence, are needed to prevent further recruitment
widespread and indiscriminate usage of antibiotics by humans, of novel resistance genes into pathogens.
which results in dominance of resistant and multiply resistant
strains of bacteria not only among human pathogens but also
in environments where human activities (such as antibiotic CONCLUSION, RESEARCH GAPS, AND
manufacturing facilities) result in pollution with antibiotics FUTURE DIRECTIONS
(Larsson, 2014). Other settings, considered to be hot-spots
(described in section “Role of HGT in Transfer of Antibiotic Antibiotic producing bacteria of the genus Streptomyces as well as
Resistance Genes”), where human-associated and environmental non-pathogenic environmental bacteria are important reservoirs
bacteria co-exist, also provide significant opportunities for of antibiotic resistance determinants. These determinants may be
exchange of resistance genes as well as selection for resistance transferred to clinical strains by a variety of HGT mechanisms,
(Bengtsson-Palme et al., 2018). Such environments are ideal including transformation of naturally competent bacteria,
not only for transfer of resistance genes to pathogens, but phages, and the use of conjugative plasmids, transposons, and
they can also result in transfer of resistance from pathogens to integrons. Despite barriers to the exchange of genetic information
environmental bacteria or opportunistic pathogens, resulting in between different genera of bacteria, widespread transfer of

resistance genes from chromosomes of environmental and soil fill the gaps in our knowledge of intermediate stages and
bacteria to the mobilizable elements in clinical isolates seems to carriers for mobilization. Indeed two databases, the Antibiotic
have occurred. Indeed several examples of recent transfers from Resistance Database (ARDB) and the Comprehensive Antibiotic
environmental bacteria to the clinical strains are available (Route Resistance Database (CARD), assembled in the last decade
3, Figure 2); however, very limited evidence for recent direct (Liu and Pop, 2009; McArthur et al., 2013), are expected to
transfer from producers to clinical strains has been obtained provide computational tools for the rapid prediction of antibiotic
(Route 1, Figure 2). Nevertheless, transfer from producer bacteria resistance genes and their targets in newly sequenced genomes
to other actinomycetes in soil is possible (Route 2A), which could and establish phylogenetic relationships. This was demonstrated
provide a pathway for further transfer of these determinants to in a recent bioinformatics study using these databases (Jiang
proteobacterial clinical strains (Route 2B). Based on the available et al., 2017). It is expected that these bioinformatics tools will
evidence, we conclude that Routes 2 and 3 are much more unify information on resistance genes and their products found
prevalent in nature as compared to Route 1 for transfer of in thousands of bacterial species isolated from the clinic or the
resistance genes to pathogens. environment as well as their associated mobile genetic elements
To better understand factors that promote dissemination of and allow this information to be quickly mined by researchers in
resistance genes and to elucidate relationships between antibiotic this field.
resistance genes of producer, environmental, and pathogenic
bacteria, new and improved strategies for sampling and screening
of microbial populations and metagenomic libraries are needed. AUTHOR CONTRIBUTIONS
Moreover, better algorithms and the use of bioinformatics
approaches for determining relationships between resistance PK supervised the work, collected and reviewed literature, and
determinants of different environmental niches will be highly co-wrote the review article. EP collected and reviewed literature,
beneficial. Additional genome sequencing data will also help prepared the figures/tables, and co-wrote the review.
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6750-12-52
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Characterization of a novel domain ’GATE’ in the ABC protein distribution or reproduction is permitted which does not comply with these terms.

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published: 30 November 2018

Antibiotic Resistance Mechanisms in

Emergence of antibiotic resistant pathogenic bacteria poses a serious public health

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TABLE 1 | Antibiotic self-resistance mechanisms in producer bacteria.

Mechanism of antibiotic Selected examples Gene location Reference

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TABLE 2 | Antibiotic resistance mechanisms in clinical isolates.

Mechanism of Intrinsic Gene location Reference Acquired Gene location Reference

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