Antibiotic Resistance and Persistence-Implications For Human Health and Treatment Perspectives

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Review
Antibiotic resistance and persistence—Implications

for human health and treatment perspectives
Markus Huemer , Srikanth Mairpady Shambat , Silvio D Brugger & Annelies S Zinkernagel*
Abstract resulting in increased life expectancy in general (Adedeji, 2016).

Nevertheless, increasing numbers of bacteria are becoming resistant
Antimicrobial resistance (AMR) and persistence are associated with to multiple antibiotics currently in use resulting in multidrug-
an elevated risk of treatment failure and relapsing infections. They resistant (MDR) bacteria (Tanwar et al, 2014). Sir Alexander Flem-
are thus important drivers of increased morbidity and mortality ing, who was awarded the Nobel Prize in Medicine for discovering
rates resulting in growing healthcare costs. Antibiotic resistance is penicillin in 1945, warned about the potential risks of over- and
readily identifiable with standard microbiological assays, and the misuse of antibiotics resulting in resistance development (Fleming,
threat imposed by antibiotic resistance has been well recognized. 1929; Aminov, 2010). Since new antimicrobial drugs are scarce and
Measures aiming to reduce resistance development and spreading due to the increasing prevalence of MDR bacteria causing treatment
of resistant bacteria are being enforced. However, the phenomenon failures, antibiotic-resistant bacteria have become a major threat to
of bacteria surviving antibiotic exposure despite being fully suscep- modern health care (Spellberg et al, 2013).
tible, so-called antibiotic persistence, is still largely underestimated. Many governments together with the World Health Organization
In contrast to antibiotic resistance, antibiotic persistence is difficult (WHO) and the United Nations (UN) have joined efforts to reduce
to measure and therefore often missed, potentially leading to treat- and prevent resistance development, which is a fight against time.
ment failures. In this review, we focus on bacterial mechanisms The latest report from the World Economic Forum 2019 in Davos,
allowing evasion of antibiotic killing and discuss their implications Switzerland, highlights the necessity of taking actions to fight the
on human health. We describe the relationship between antibiotic “rapid and massive spread of infectious diseases”, one of the greatest
persistence and bacterial heterogeneity and discuss recent studies threats to human health (WorldEconomicForum, 2019). Infections
that link bacterial persistence and tolerance with the evolution of by antibiotic-resistant bacteria are responsible for around 700,000
antibiotic resistance. Finally, we review persister detection meth- deaths per year worldwide and estimated to be accountable for over
ods, novel strategies aiming at eradicating bacterial persisters and 10 million deaths per year in 2050 (O’Neill, 2014; Aslam et al, 2018).
the latest advances in the development of new antibiotics. Although AMR has been widely acknowledged as a pressing issue,
the incidence of infections and spread of MDR bacteria is still rising.
Keywords persistence; persistent infections; persisters; resistance; tolerance In addition, the increased use of implanted medical devices such as
Subject Categories Microbiology, Virology & Host Pathogen Interaction; joint prostheses, artificial heart valves, vascular endoprostheses and
Molecular Biology of Disease pacemakers leads to a higher incidence of biofilm-associated infec-
DOI 10.15252/embr.202051034 | Received 4 June 2020 | Revised 13 August tions, which in turn leads to another important phenomenon: antibi-
2020 | Accepted 2 November 2020 otic tolerance (Davidson et al, 2019; Gollan et al, 2019). Antibiotic
EMBO Reports (2020) 21: e51034 tolerance enables bacteria to survive antibiotic challenges even when
they are fully susceptible in standard microbiological assays
See the Glossary for abbreviations used in this article. (Brauner et al, 2016). By using in vitro experiments and mathemati-
cal modelling, Balaban and co-workers recently showed that toler-
The antibiotic resistance crisis ance can precede resistance and that tolerance mutations can lead to
a rapid evolution of antibiotic resistance (Levin-Reisman et al, 2017;
Antibiotics have paved the way for today’s modern medicine. The Windels et al, 2019). In order to combat AMR, it is important to
mid-20th century was even named the “antibiotic era”, and infec- understand both antibiotic tolerance and resistance.
tious diseases were believed to be eradicated by the end of the last
century. Similarly, antibiotics have been fundamental for successful
invasive and high-end surgeries including organ transplantation, Antibiotic resistance mechanisms
immunomodulatory treatments in rheumatology, oncology and
many other medical disciplines (Ventola, 2015). Availability of Antibiotic resistance frequently results in delayed adequate antibi-
antibiotic therapy has significantly reduced mortality in children otic treatment, increasing morbidity and mortality. AMR is the
Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland
*Corresponding author. Tel. +41 44 255 25 41; E-mail: [email protected]
ª 2020 The Authors EMBO reports 21: e51034 | 2020 1 of 24

14693178, 2020, 12, Downloaded from https://www.embopress.org/doi/10.15252/embr.202051034 by Algeria Hinari NPL, Wiley Online Library on [22/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
EMBO reports Markus Huemer et al
Glossary
AACs antibody–antibiotic conjugates MDK minimal duration of killing
ABDs antibacterial drones MDR multidrug resistant
AmpC b-lactamase MIC minimum inhibitory concentration
AMPs antimicrobial peptides MLST multilocus sequence type
AMR antimicrobial resistance MRSA methicillin-resistant S. aureus
ATP adenosine triphosphate NDM-1 New Delhi metallo-b-lactamase 1
blaZ b-lactamase nsSCs non-stable small colonies
C10 3-[4-(4-methoxyphenyl)piperazin-1-yl] piperidin-4-yl ParC DNA topoisomerase IV
biphenyl-4-carboxylate PBP2a penicillin binding protein 2a
CA community-acquired PJIs prosthetic joint infections
CCCP carbonyl cyanide m-chlorophenyl hydrazine pmf proton motive force
CDI C. difficile infections QRDR quinolone resistance determining region
CF cystic fibrosis REPTIS Replica Plating Tolerance Isolation System
CFUs colony-forming units RNA ribonucleic acid
CIP ciprofloxacin RND resistance nodulation division
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats ROS reactive oxygen species
DNA deoxyribonucleic acid RpoB RNA polymerase beta-subunit
dTMP deoxythymidine monophosphate SaPIs staphylococcal pathogenicity islands
dUMP deoxyuridine monophosphate SCCmec staphylococcal cassette chromosome mec
ECDC European Centre of Disease Prevention and Control SCVs small colony variants
EMBO European Molecular Biology Organization TA toxin/ antitoxin
ESBLs extended-spectrum b-lactamases TCA tricarboxylic acid
ESKAPE E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. TCS two-component systems
aeruginosa and Enterobacter species TD Tolerance Disk
EU European Union TMP-SMX trimethoprim-sulfamethoxazole
fabI/V enoyl-ACP reductase gene UN United Nations
GltX aminoacyl-tRNA synthetase UPEC uropathogenic E. coli
GyrA DNA gyrase USA United States of America
hip high persister UTIs urinary tract infections
HR heteroresistance UV ultraviolet
IMP imipenem VRE vancomycin-resistant Enterococci
KPC K. pneumoniae carbapenamases VRSA vancomycin-resistant S. aureus
MBC minimal bactericidal concentration WHO World Health Organization
mcr mobilized colistin resistance
inherited ability of microorganisms to grow at high antibiotic naturally susceptible to almost every antibiotic that has been devel-
concentrations (Brauner et al, 2016). It is usually quantified by oped, but it is well known for quickly acquiring antibiotic resistance
measuring the minimum inhibitory concentration (MIC) of a partic- by means of obtaining specific genetic modifications (mutations or
ular antibiotic wherein resistant bacteria are able to multiply and horizontal gene transfer), causing infections that come in epidemic
grow at concentrations of antibiotics, which are fatal to other strains waves (Chambers & Deleo, 2009).
of the same species. Antibiotic resistance comes in distinct flavours. Several mechanisms can lead to resistance and have been inves-
On the one hand, there is natural intrinsic resistance due to lack or tigated in detail. These molecular mechanisms are categorized in
presence of certain structures resulting in ineffectiveness of antibi- three main mechanistic groups of resistance: (i) reduction of intra-
otics. On the other hand, bacteria can acquire resistance via muta- cellular antibiotic concentrations, (ii) modification of the antibiotic
tions in chromosomal genes or via horizontal gene transfer of target and (iii) inactivation of the antibiotic (Blair et al, 2015).
chromosomes or plasmids that lead to antibiotic resistance. Intracellular concentrations of antibiotics can be kept low via
In Pseudomonas aeruginosa for example, antimicrobial decreased permeability of the bacterial cell membrane, e.g. by
substances such as triclosan, which inhibit fatty acid synthesis by reducing or mutating porins that would allow entry of antibiotics
targeting the enoyl-ACP reductase, are ineffective, as the bacteria into the cell. This has been well described for several Gram-negative
carry an insensitive homologue of fabI (enoyl-ACP reductase), fabV, pathogens such as carbapenem-resistant Enterobacter species,
that encodes an enzyme that is not inhibited by triclosan (Zhu et al, Escherichia coli and Klebsiella pneumoniae that display reduced
2010). In general, Gram-negative bacteria are less permeable than porin expression or mutated porins without expressing a carbapene-
Gram-positive and show an intrinsic resistance to many antibacte- mase (Wozniak et al, 2012; Baroud et al, 2013; Kong et al, 2018).
rial compounds such as the cell wall active glycopeptide vanco- Another way to decrease intracellular antibiotic concentrations is to
mycin (Zgurskaya et al, 2015). These large molecules are unable to increase the efflux of chemotherapeutics by using either substrate
cross the outer bacterial membrane of Gram-negative bacteria and specific or MDR efflux pumps. Although many bacteria carry multi-
thus cannot target the cell wall of Gram-negative bacteria. In ple genes that encode MDR efflux pumps on their chromosomes,
contrast, the Gram-positive pathobiont Staphylococcus aureus is some MDR efflux pump genes have been mobilized onto plasmids
2 of 24 EMBO reports 21: e51034 | 2020 ª 2020 The Authors

Markus Huemer et al EMBO reports
A E. coli B E. faecium C S. aureus

75,000 20.0
160,000 7 18
16,000
Vancomycin resistance [%]

Number of invasive isolates

Multi-drug resistance [%]
Methicillin resistance [%]

140,000 6 65,000 17.5
14,000
15
120,000
5 12,000
55,000 15.0
12
100,000 10,000
4 12.5
45,000
80,000 8,000 9
3
10.0
60,000 6,000 35,000
2012 2014 2016 2018 2012 2014 2016 2018 2012 2014 2016 2018
Year Year Year
D K. pneumoniae E Acinetobacter spp. F P. aeruginosa

21 6,500 30 20,000 16


35,000
18 6,000 17,500
27 14
30,000
5,500
15 15,000 12
24
25,000
5,000
12 12,500
20,000 21 10
4,500
9
15,000 4,000 10,000 8
2012 2014 2016 2018 2014 2016 2018 2012 2014 2016 2018
© EMBO
Year Year Year
Figure 1. Invasive bacterial isolates and resistance development over time in the European Union (EU) and European Economic Area (EEA).
(A) Total number of invasive E. coli isolates tested and percentage with combined resistance to fluoroquinolones, 3rd-generation cephalosporins and aminoglycosides,
including 95% confidence intervals (95% CI, shaded area). (B) Total number of invasive E. faecium isolates tested and percentage with resistance to vancomycin,
including 95% CI. (C) Total number of invasive S. aureus isolates tested and percentage with resistance to methicillin (MRSA), including 95% CI. (D) Total number of
invasive K. pneumoniae isolates tested and percentage with combined resistance to fluoroquinolones, 3rd-generation cephalosporins and aminoglycosides, including 95%
CI. (E) Total number of invasive Acinetobacter spp., including most of the disease-causing species (A. baumannii, A. pittii and A. nosocomialis) and the generally less
pathogenic A. non-baumannii group, isolates tested and percentage with combined resistance to fluoroquinolones, aminoglycosides and carbapenems, including 95% CI.
(F) Total number of invasive P. aeruginosa isolates tested and percentage with combined resistance (resistance to three or more antimicrobial groups among piperacillin
—tazobactam, ceftazidime, fluoroquinolones, aminoglycosides and carbapenems), including 95% CI. (A-F) Data are derived from European Centre for Disease Prevention
and Control’s yearly Surveillance of antimicrobial resistance in Europe (reports 2013-2018 were used). ECDC collects data from invasive isolated reported to the
European Antimicrobial Resistance Surveillance Network (EARS-Net) by 30 EU and EEA (Iceland and Norway) countries.
that can be transferred between bacteria (Lv et al, 2020). Genes gyrase GyrA or DNA topoisomerase IV ParC can cause ciprofloxacin
coding for a novel tripartite resistance nodulation division (RND) resistance in S. aureus (Sreedharan et al, 1990; Hooper & Jacoby,
pump were found to be carried on a plasmid together with genes 2015). Therefore, this domain has been named the quinolone resis-
encoding for the antibiotic-targeting enzyme New Delhi metallo-b- tance determining region (QRDR) (Yoshida et al, 1990). Although
lactamase 1 (NDM-1) (Dolejska et al, 2013; Lv et al, 2020). This is most of clinical methicillin-resistant S. aureus (MRSA) isolates are
especially worrying as it shows that different resistance mechanisms susceptible to rifampicin (Moran et al, 2006), resistance develop-
can be carried together on one plasmid and are thereby transmissi- ment occurs rapidly if rifampicin is used as monotherapy. Point
ble between bacteria, highlighting the importance of MDR efflux mutations in the gene encoding for the b-subunit of the DNA-depen-
pumps as a major resistance mechanism. Many clinical isolates that dent RNA polymerase (RpoB) cause decreased affinity for rifampicin
overexpress drug efflux pumps, including P. aeruginosa and and quickly confer resistance (Maranan et al, 1997).
S. aureus, have been isolated from patients with systemic infections Finally, resistance can be acquired by directly modifying antibi-
(Pumbwe & Piddock, 2000; Kosmidis et al, 2012). otics. Antibiotics can be inactivated by the transfer of chemical
Spontaneous mutations or post-translational modifications of an groups to vulnerable sites thus inhibiting target binding and func-
antibiotic target molecule can lead to conformational changes that tion or directly destroyed by hydrolysis. Inactivation of antibiotics
result in ineffective target binding and attenuation of antibiotic by addition of a chemical group, such as acyl, nucleotidyl,
activity. Single amino acid changes in close proximity to the active phosphate and ribitoyl groups, resulting in steric hindrance can
site tyrosine of the bacterial type II topoisomerase enzymes, DNA cause antibiotic resistance. Especially, the large molecules of

aminoglycosides with many exposed hydroxyl and amide groups 1940s, the prognosis of patients rapidly improved. Penicillin became
are susceptible to such a inactivation mechanism. A genomic island the first choice of treatment for S. aureus infections, but extensive
found in Campylobacter coli isolated from chicken in China harbours use quickly led to the evolution of b-lactamases (blaZ)-producing
genes for six aminoglycoside-modifying enzymes and confers resis- strains. Already in 1948, more than 80% of clinical isolates were
tance to aminoglycosides, including gentamicin (Qin et al, 2012). resistant to penicillin G (Appelbaum, 2007; Brown & Ngeno, 2007;
The development of new derivatives of already known antibiotic Wu et al, 2010). b-lactamase, a primarily extracellular enzyme,
classes led to the evolution of a diverse range of enzymes that can hydrolyses the b-lactam ring, rendering the b-lactam inactive
degrade different antibiotics within the same class. For example, the (Lacey, 1984). Already one year after the introduction of semi-
early b-lactamases were active against the 1st-generation b-lactams synthetic penicillins in 1961, MRSA strains were isolated and there
and were followed by extended-spectrum b-lactamases (ESBLs) that is even evidence that S. aureus developed methicillin resistance
are able to hydrolyse extended-spectrum oxyimino cephalosporins even before its clinical use (Harkins et al, 2017; Turner et al, 2019).
such as cefuroxime, cefotaxime, cefmenoxime and ceftriaxone The widespread use of first-generation b-lactams, such as penicillin,
(Johnson & Woodford, 2013). Together with the increasing numbers prior to the introduction of methicillin, selected for strains carrying
of bacteria carrying ESBL genes, the use of carbapenems the mecA determinant that confers resistance to methicillin (Harkins
has increased in the last two decades leading to the emergence of et al, 2017). Nowadays, MRSA prevalence ranges from < 1 to more
carbapenemase-producing strains (Queenan & Bush, 2007). than 40% in Europe, with very low levels of invasive MRSA isolates
Carbapenemases can degrade a variety of b-lactams, including in Scandinavian countries such as Iceland (0.0%) and Norway
extended-spectrum cephalosporins, and many carbapenemase-genes (0.9%), low levels, e.g. in the Netherlands (1.2%), in Switzerland
are now carried on plasmids and have been found in Enterobacteri- (4.4%), in Germany (7.6%) and in France (12.1%) and up to 34%
aceae, K. pneumoniae, P. aeruginosa and Acinetobacter baumannii in Italy, 38% in Portugal and 43% in Romania (Hassoun et al, 2017;
(Tzouvelekis et al, 2012) (Fig 1A). FOPH, 2018; ECDC, 2019) (Fig 1C). Methicillin resistance is medi-
ated by the gene mecA that is spread via horizontal gene transfer of
ESKAPE pathogens the mobile genetic element named staphylococcal cassette chromo-
The acronym ESKAPE describes nosocomial pathogenic bacteria some mec (SCCmec) (Katayama et al, 2000). The mecA gene
with growing levels of multidrug resistance and virulence. They encodes for a penicillin binding protein (PBP2a) that is responsible
place a significant burden on patient health, healthcare systems and for crosslinking peptidoglycans during cell wall synthesis and the
economies. ESKAPE stands for the Gram-positive bacterial species resistance arises through the low affinity of PBP2a to b-lactams
Enterococcus faecium and S. aureus and the Gram-negative bacteria (Hartman & Tomasz, 1984) rendering them ineffective. One
K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter prominent pandemic and hypervirulent clonal lineage of commu-
species. They all commonly cause hospital-acquired and potentially nity-acquired (CA)-MRSA is the multilocus sequence type (MLST) 8-
life-threatening infections in critically ill and immunocompromised MRSA-IV USA300 that started spreading in the USA 20 years ago
patients and are characterized by high levels of multidrug resistance and is a significant health threat and economic factor nowadays
(Rice, 2010). The WHO listed all ESKAPE pathogens among the (Tenover & Goering, 2009).
twelve bacterial species against which new chemotherapeutics are Colonization with MRSA increases the MRSA infection risk, as
desperately needed (Tacconelli et al, 2018). 50–80% of isolated invasive strains were found to originate from
colonizing strains as determined by molecular typing methods
E. faecium including whole genome sequencing (Benoit et al, 2018; Thomsen
The Gram-positive commensal E. faecium has a low virulence but is et al, 2019). It is a dynamic process, with strains persisting for
difficult to eradicate from the hospital environment. Therefore and long periods of time and others evolving or being replaced by
because of its potential to cause nosocomial infections and another strain within the same host (Azarian et al, 2016). In
outbreaks, it is strongly feared in the healthcare system (Elsner addition, transmission of S. aureus commonly occurs via hospital
et al, 2000). In the last decades, the incidence of drug-resistant ente- coats, mobile phones and tablets, regularly used in clinics (Frey
rococcal infections increased, especially for vancomycin-resistant et al, 2019; Turner et al, 2019). In clinics, glycopeptide antibiotics
Enterococci (VRE) reaching 14.9% in 2017 in the European Union such as vancomycin and teicoplanin, are used nowadays as first-
(EU) (ECDC, 2018b) (Fig 1B) and 30% in the United States of Amer- line treatment for MRSA infections. Shortly after the initiation of
ica (USA) (CDC, 2019). Treatment of these nosocomial VRE vancomycin therapy in the 1980s, MRSA strains with reduced
infections is very difficult and sometimes impossible due to the sensitivity to teicoplanin appeared in Europe (Kaatz et al, 1990).
resistance. In 2002, the first vancomycin-resistant S. aureus (VRSA) was
detected in the USA (Chang et al, 2003) and eleven years later in
S. aureus Europe (Melo-Cristino et al, 2013). Effective alternatives to vanco-
The Gram-positive bacterium S. aureus is part of the normal skin mycin include daptomycin, a lipopeptide bactericidal antibiotic,
microbiota, found especially in the nose. Carriage rates are high, linezolid, tigecycline, trimethoprim–sulfamethoxazole (TMP-SMX)
with around 50% of the population intermittently or permanently or a 5th-generation b-lactam (e.g. ceftaroline or ceftobiprole).
colonized (Lowy, 1998). Nevertheless, S. aureus is a leading cause Similarly, if a reduced daptomycin susceptibility is detected
of infective endocarditis, osteomyelitis, skin and soft tissue infec- together with a reduced vancomycin susceptibility, a combination
tions as well as bacteraemia. Before the antibiotic era, mortality or single use of the following is recommended; the streptogramin
rates for S. aureus bacteraemia were dramatically high (over 80%) antibiotics quinupristin–dalfopristin, TMP-SMX, linezolid or
(Skinner & Keefer, 1941). With the introduction of penicillin in the telavancin (Liu et al, 2011).

K. pneumoniae genes, like ESBLs and carbapenamases. Enterobacter species show

K. pneumoniae is a Gram-negative rod-shaped pathogen that is hyperproduction of AmpC b-lactamase that can hydrolyse broad-
often associated with nosocomial infections. Many K. pneumoniae spectrum penicillins and cephalosporins (Schwaber et al, 2003).
strains have acquired a variety of b-lactamases against penicillin, This overproduction is induced by b-lactams and carbapenems lead-
cephalosporins and carbapenems. Carbapenems are frequently used ing to the occurrence of resistance during the course of the treat-
to treat infections with Gram-negative bacteria, but the increase of ment and is associated with increased mortality rates (Schwaber
K. pneumoniae carbapenamases (KPC)-producing strains, carrying et al, 2003; Huh et al, 2014). Due to this ability to rapidly develop
blaKPC, renders such infections more and more difficult to treat resistances, many Enterobacter spp. strains are only susceptible to a
(Reyes et al, 2019) (Fig 1D). Additionally, some K.pneumoniae few last-resort antibiotics such as colistin and tigecycline (Pendleton
strains express the metallo-b-lactamase NDM-1, encoded by blaNDM- et al, 2013). Recently, even resistance to colistin, either due to a
1 (Yong et al, 2009). The presence of NDM-1 has increased the chromosomal mutation in genes of two-component systems (TCS)
prevalence of carbapenem-resistant K. pneumoniae strains, which such as pmrA/B and phoP/Q and their regulators mgrB and pmrD or
has made the use of other antibiotics such as aminoglycosides and via plasmid-mediated mobilized colistin resistance (mcr) genes, as
fluoroquinolones increasingly necessary. However, the more well as to tigecycline have been reported, highlighting the urgent
frequent use of these antibiotics may increase resistance against need for new antimicrobial drugs (Pournaras et al, 2016; Hong et al,
these drugs as well, making it even more difficult to treat these 2018; Berglund, 2019).
infections (Kumarasamy et al, 2010). Besides the ESKAPE pathogens, the Gram-positive and spore-
forming bacterium Clostridioides difficile (until 2016: Clostridium
A. baumannii difficile) is an emerging human pathogen with increasing resistance
The Gram-negative bacillus A. baumannii is an opportunistic towards metronidazole (Dingsdag & Hunter, 2018). The European
pathogen associated with nosocomial infections of primarily Centre of Disease Prevention and Control (ECDC) started in 2016 to
immunocompromised patients who have stayed in a hospital for monitor C. difficile infections (CDIs) and reported metronidazole
more than 90 days (Montefour et al, 2008). A. baumannii causes a resistance for 26/569 (4.6%) cases (ECDC, 2018a). The emergence
variety of infections including respiratory and urinary tract infec- of metronidazole resistance is of specific concern, since a new plas-
tions. Due to the high prevalence of antibiotic resistances, A. mid (pCD-METRO) conferring metronidazole resistance has been
baumannii poses a significant threat to patients especially in inten- found, occurring in both animal and human isolates of C. difficile
sive care units (Fig 1E). In addition, Carbapenem-resistant (Boekhoud et al, 2020). CDIs should be carefully monitored world-
A. baumannii strains carrying imipenem metallo-b-lactamases wide, and the blind use of broad-spectrum antibiotics has to be
(blaIMP) and oxacillinase serine b-lactamases (blaOXA) have been avoided, which should result in decreasing the incidence and spread
isolated. The combination of resistance genes enables them to evade of drug-resistant C. difficile.
the antimicrobial action of most conventional antibiotics (Vila et al, There are many excellent reviews already published focusing on
2007; Boucher et al, 2009). Because of the increasing incidence of antibiotic resistance (Turnidge & Christiansen, 2005; Spellberg et al,
multidrug-resistant A. baumannii strains globally, the WHO classi- 2013; Blair et al, 2015; Foster, 2017; Rather et al, 2017; Zaman et al,
fied A. baumannii as critical (in the priority group 1) for the devel- 2017; Aslam et al, 2018; Jorge et al, 2019; Lopez-Jacome et al,
opment of new antimicrobial agents (WHO, 2017). 2019). We would like to put our further focus on difficult-to-treat
infections, phenotypic heterogeneity, antibiotic tolerance and persis-
P. aeruginosa tence. We will discuss how they can cause treatment complications
The Gram-negative bacterium P. aeruginosa, also listed in the WHO and future strategies to fight recurrent bacterial infections.
priority group 1 of pathogens that urgently require new treatment
options, intrinsically shows low susceptibility to many antimicrobial
drugs and a high propensity to develop resistance. Reduced porin Antibiotic persistence—research then and today
permeability together with AmpC overproduction lead to increased
carbapenem resistance in P. aeruginosa (Fukuoka et al, 1993). In Already at the beginning of the antibiotic era in 1942, Hobby and
addition, P. aeruginosa can express KPCs, ESBLs and imipenem colleagues described a small subpopulation (< 1%) of S. aureus that
metallo-b-lactamases, resulting in high-level carbapenem resistance survived treatment with penicillin despite being susceptible to the
(Fig 1F). The increasing prevalence of multidrug-resistant isolates is drug (Hobby et al, 1942). J. Bigger confirmed this finding two years
challenging for successful treatments, and although the last-resort later and named this newly discovered phenotype “antibiotic persis-
antibiotic colistin is still effective in most cases, resistance was tence” (Bigger, 1944). J. Bigger did not only show that S. aureus
already reported due to previous widespread use of colistin in pig could survive penicillin treatment, but also that the surviving popu-
and poultry farming (Fukuoka et al, 1993; Pedersen et al, 2018; lation displayed the heterogeneous survival phenotype of a rapidly
Hameed et al, 2019). dying population and a surviving subpopulation. The fact that the
survivors did not grow under penicillin pressure clearly separated
the phenomenon of “antibiotic persistence” from “antibiotic resis-
Enterobacter species tance”. The rise of antibiotic resistance caught much more attention,
and the novel phenomenon of antibiotic persistence was almost
Enterobacter spp. are common Gram-negative pathogens usually forgotten for the next 39 years. After the discovery of the E. coli
causing infections in immunocompromised, hospitalized patients high-persistence mutant hipA7 in 1983 by Moyed and Bertrand
and are well known for harbouring many antibiotic resistance (Moyed & Bertrand, 1983), the scientific community refocused on

the phenomenon of susceptible bacteria surviving antibiotic chal- the production of the stringent response alarmone (p)ppGpp that
lenges. In the last 30 years, many mechanisms and characteristics inhibits DNA replication, and therefore, blockage of translation can
underlying bacterial persistence have been revealed. However, indirectly result in additional tolerance towards fluoroquinolones
research mainly focused on Gram-negative bacteria, e.g. E. coli, (Schreiber et al, 1995; Korch & Hill, 2006; Kaspy et al, 2013;
P. aeruginosa and S. enterica ser. Typhimurium, and little is known Wilmaerts et al, 2019). Next to hipA, hipB encodes an auto-
about the mechanisms and influence of bacterial persistence in repressor of hipBA transcription (Black et al, 1991; Black et al,
Gram-positive bacteria especially S. aureus and coagulase-negative 1994). HipB directly inhibits HipA, which suggested that hipBA
staphylococci (S. epidermidis), which are often associated with constitutes a toxin/ antitoxin (TA) locus (Korch et al, 2003). Today,
chronic and recurrent, biofilm-associated infections. The group of numerous publications support the model of TA loci cumulatively
Kim Lewis was the first to link bacterial persistence to biofilms in regulating antibiotic persistence in E. coli (Vazquez-Laslop et al,
P. aeruginosa (Brooun et al, 2000; Spoering & Lewis, 2001). Chronic 2006; Dorr et al, 2010; Page & Peti, 2016).
infections are associated with bacterial biofilms, and the newly
linked phenomenon of antibiotic persistence added a novel point of
view to recurring and difficult-to-treat infections and attracted the Toxin/ Antitoxin modules and persistence
interest of more and more scientists worldwide (Bjarnsholt, 2013;
Lebeaux et al, 2014). In 2018 at the European Molecular Biology TA modules are composed of a stable toxin (always a protein) that
Organization (EMBO) Workshop “Bacterial Persistence and Antimi- intoxicates the bacterial cell by inhibiting essential processes, e.g.
crobial Therapy”, 121 scientists studying antibiotic persistence and translation, transcription and DNA replication, and a labile antitoxin
tolerance came together in Ascona, Switzerland, and laid the foun- (either RNA or protein) that neutralizes the corresponding toxin
dations for a “Consensus Statement” on the definitions and guideli- (Harms et al, 2018). Up to now, there are six described classes of
nes for research on antibiotic tolerance (Balaban et al, 2019a, b). TA modules, defined by their neutralization mechanisms. Type I
Here, we follow the definitions published in the consensus state- antitoxins are antisense RNAs that inhibit the translation of the
ment (Balaban et al, 2019a). mRNA of their corresponding toxins, therefore inhibiting toxin
Antibiotic persistence or heterotolerance is defined as the ability production (Thisted et al, 1994). In the largest and best studied
of a bacterial subpopulation to survive high bactericidal drug group of TA modules, the type II TA systems, antitoxins are proteins
concentrations to which the bacteria are fully susceptible (no that directly bind their cognate toxins thereby neutralizing the toxin
change in MIC). One major characteristic of antibiotic persistence is (Pandey & Gerdes, 2005). Additionally, the antitoxin can be a toxin-
a biphasic killing, with a rapid killing of the susceptible population binding sRNA (type III) (Brantl & Jahn, 2015), which protects the
and a slower killing of the persister subpopulation. This phenom- toxin targets via binding to and directly inhibiting the toxin, instead
enon highlights the phenotypic heterogeneity in a bacterial culture of counteracting the toxins’ effect (type IV) (Masuda et al, 2012).
and that not all bacteria in a clonal population are killed at the same The antitoxin can also be an endoribonuclease that degrades the
rate. In contrast to antibiotic resistance and heteroresistance, where toxins’ mRNA (type V) (Wang et al, 2012) or a proteolytic adapter
a small subpopulation transiently displays a higher MIC, persistent of the toxin (type VI) (Aakre et al, 2013). Often, TA modules are
bacteria are not able to grow in the presence of antibiotics. Antibi- described as stress-responsive elements that are activated under
otic persistence and tolerance are often used interchangeably, and specific stress conditions (Ronneau & Helaine, 2019). As described
both describe an increased survival in the presence of an antibiotic before, the type II TA system toxin HipA7 can induce antibiotic
without a change in the MIC. However, persistence characterizes a persistence by phosphorylation of its target GltX in E. coli (Moyed &
bacterial subpopulation, whereas tolerance describes the ability of Bertrand, 1983). Therefore, TA modules were proposed to be perfect
an entire bacterial population to survive antibiotic exposure. Thus, candidates to trigger antibiotic persistence.
antibiotic persistence is a subcategory of tolerance, in which a small
subpopulation of persisters tolerates antibiotic exposure, reflected
by a biphasic killing curve. Environmental triggers of antibiotic persistence
Many environmental factors, such as nutrient limitation, antibiotic

Genetic basis of antibiotic persistence exposure, acidic pH, high cell numbers, heat shock and oxidative
stress, have been shown to increase persister levels in an isogenic
In 1983, Moyed and Bertrand isolated mutants of E. coli that were bacterial population (Fig. 2) (Dorr et al, 2010; Moker et al, 2010;
able to form high amounts of persister cells, and seven years later, Nguyen et al, 2011; Leung & Levesque, 2012; Vega et al, 2012;
mutagenesis experiments lead to the isolation of the high persister Amato et al, 2013; Bernier et al, 2013; Vulin et al, 2018; Rowe et al,
(hip) mutant (Moyed & Bertrand, 1983; Wolfson et al, 1990). The 2020). The stringent response reprograms bacteria from rapid
hipA7 allele enhanced antibiotic persistence in E. coli K-12 almost growth to metabolic homeostasis and is triggered under nutrient
1,000-fold and gave the first evidence that antibiotic persistence limitation. Mediated through the alarmone (p)ppGpp, found in
levels can be increased by an inheritable mutation. The hipA gene almost all bacterial species, the stringent response plays a crucial
encodes a eukaryotic-like Ser/Thr kinase that inhibits translation role in triggering antibiotic persistence. In E. coli, (p)ppGpp is
via phosphorylation of aminoacyl-tRNA synthetase GltX at its active produced either by the synthetase RelA that is activated by a lack of
site (Schumacher et al, 2009; Hansen et al, 2012; Germain et al, amino acids and heat shock or by the bifunctional synthetase/
2013). Inactivation of GltX leads to the formation of hungry codons, hydrolase SpoT during carbon, nitrogen, iron, phosphate and fatty
i.e. their cognate aminoacyl-tRNAs are in short supply. This induces acid starvation (Irving & Corrigan, 2018; Ronneau & Hallez, 2019).

Oxidative Antibiotic Nutrient

pH variation Heat shock
stress exposure limitation
SOS response Stringent response

SOS response
LexA genes • Limitation of
RelA amino acids
• Heat shock
RecA (p)ppGpp
Limitation of
• carbon,
SpoT • nitrogen,
LexA SOS response
• iron,
genes
• phosphate, and
• fatty acids.
PMF
Lex box containing Inhibition of
cellular functions T/A systems
ATP T/A systems
ATP tisAB/ istr1 • Transcription
symER • Translation
ATP hokE / sokE • DNA replication
yafN/ yafO •
… • Energy production
Drop in Accumulation of
ATP levels insoluble proteins
ATP T/A systems

© EMBO
High population Quorum sensing

density
Figure 2. Environmental triggers of antibiotic persistence and bacterial response mechanisms.

Environmental stressors such as oxidative stress and antibiotic exposure can trigger the bacterial SOS response via induction of RecA expression and the upregulation of
the Lex box containing TA systems leading to a drop in ATP levels and a downregulation of essential cellular functions, e.g. transcription, translation, DNA replication and
energy production. Other stressors such as pH changes, heat shock and starvation trigger the stringent response via production of (p)ppGpp, mainly by SpoT and RelA,
and activation of toxin/antitoxin systems leading to the inhibition of essential cellular functions. Low intracellular ATP levels can favour the accumulation of insoluble
proteins, a feature linked to increased antibiotic tolerance and dormancy. Additionally, high population densities can inhibit cellular functions via quorum sensing and T/
A systems. External triggers/signals are shown in red boxes. Abbreviations: T/A, antitoxin/toxin; PMF, proton motive force.
Accumulation of (p)ppGpp has been shown to upregulate or activate tisAB/istr1, symER, hokE/sokE and yafN/yafO (Pedersen & Gerdes,
TA systems during stringent response. For example, isoleucine star- 1999; Fernandez De Henestrosa et al, 2000; Vogel et al, 2004;
vation of E. coli increases (p)ppGpp levels and leads to an upregula- Kawano et al, 2007). The type I toxin TisB, a small 29 amino acid
tion of many type II TA modules, like MazEF, HicAB, RelBE and hydrophobic peptide, for example, binds to the bacterial cell
MqsRA as well as the type I toxin HokB (Fig. 2) (Traxler et al, 2008; membrane and disrupts the proton motive force (pmf), leading to a
Verstraeten et al, 2015; Shan et al, 2017; LeRoux et al, 2020). Muta- decrease in intracellular adenosine triphosphate (ATP) levels and a
tions that affect (p)ppGpp synthesis significantly reduce persister multidrug tolerant phenotype (Fig. 2) (Unoson & Wagner, 2008;
levels in both Gram-positive and Gram-negative bacteria, indicating Lewis, 2012). A drop in ATP levels has been associated with antibi-
the prominent role and connections of stringent response with otic tolerance in E. coli (Shan et al, 2017) and S. aureus (Conlon
bacterial stress response and antibiotic persistence (Amato et al, et al, 2016). In both studies, treatment of exponentially growing
2013; Amato et al, 2014). bacteria with arsenate, a drug known to inhibit ATP synthesis,
DNA damage is caused by many factors including oxidative resulted in elevated levels of antibiotic tolerance. Similarly, pretreat-
stress, UV light exposure and drugs and usually leads to the induc- ment with the metabolic poison carbonyl cyanide m-chlorophenyl
tion of the SOS response (Dorr et al, 2010; Baharoglu & Mazel, hydrazine (CCCP), an uncoupling agent that inhibits ATP synthesis,
2014). The SOS response not only activates DNA repair mechanism, resulted in increased persister levels (Kwan et al, 2013). Congruently
but also upregulates the Lex box containing TA systems such as with stringent response-mediated persistence, inhibition of

translation by pretreatment with the antibiotic tetracycline yielded 2013), recognition by immune cells (Leid et al, 2002; Jesaitis et al,
an increased fraction of persisters (Kwan et al, 2013). Recently, 2003; Vuong et al, 2004), killing by many antibiotics (Jolivet-
Conlon and co-workers showed that inactivation of the tricarboxylic Gougeon & Bonnaure-Mallet, 2014) and contains persister cells (Spo-
acid (TCA) cycle enzymes aconitase and succinate dehydrogenase ering & Lewis, 2001; Kamble Ekta, 2020). The increasing age of the
by reactive oxygen species (ROS) produced by human macrophages human population results in more tissue-related biofilm-associated
during infection, decreased ATP levels and increased persister levels infections such as osteomyelitis and endocarditis, but also in more
in S. aureus, further strengthening the link between reduced energy foreign body-associated biofilm infections due to the constantly
levels and antibiotic tolerance (Rowe et al, 2020). rising numbers of pacemaker and orthopaedic device implants.
Depletion or reduction of intracellular ATP levels could therefore Biofilm formation has been linked to persistent infections, especially
be a general mechanism of downregulating central bacterial to difficult-to-treat foreign body infections such as pacemaker-related
processes, such as transcription, translation and peptidoglycan infections and prosthetic joint infections (PJIs) (Jansen & Peters,
synthesis, thereby rendering antibiotics ineffective as their respec- 1993; Costerton et al, 1999; Dengler Haunreiter et al, 2019).
tive targets are not active (Fig. 2). PJIs are often caused by S. aureus or coagulase-negative staphy-
In addition, accumulation of insoluble endogenous proteins in lococci (e.g. S. epidermidis) and require prolonged antibiotic treat-
deep-stationary phase, starved E. coli, promoted by decreased levels ment and often surgical removal of the infected device (Tande &
of ATP, has been linked to dormancy depth and antibiotic tolerance Patel, 2014). The majority of PJIs occur in the first year after implan-
(Fig. 2) (Pu et al, 2019). Pu and co-workers showed that the tation and are typically caused by commensal microorganisms that
dormancy depth that correlates with antibiotic tolerance is linked to were inoculated during surgery (Del Pozo & Patel, 2009). Either
the formation of protein aggresomes. Resuscitation of these dormant physical contact or droplets lead to the contamination of the pros-
cells leads to degradation of protein aggregates and requires DnaK thesis surface and even very low inocula (< 102 CFUs for S. aureus)
and ClpB activity. The chaperone DnaK was previously identified to result in infection (Southwood et al, 1985). PJIs manifesting later
be involved in bacterial antibiotic persistence by analysing an E. coli than 2 years after surgery are typically caused by haematogenous
mutant library further strengthening the involvement of protein spread during bacteraemia. A study including 14,378 patients with a
aggregation in antibiotic persistence (Hansen et al, 2008). primary knee or hip replacement showed that 3.8% (542/14,378)
had at least one significant bacteremic episode and 8.3% (45/542)
developed a PJI during bacteraemia. The most common cause of PJI
Chronic and difficult-to-treat infections was S. aureus, with 21% PJIs resulting from bacteraemia (Honka-
nen et al, 2019). Many studies investigating PJI caused by S. aureus
There is a fundamental difference between the terms “persistent and other staphylococcal species found small colony variants
infection” and “antibiotic persistence”. Persistent infections are (SCVs) (Kahl et al, 2016). Proctor et al were the first to describe the
ongoing infections, which are not cleared by the host, whereas occurrence of SCVs in a patient suffering from MRSA bacteraemia
antibiotic persistence is used to describe a bacterial population that with a hip prosthesis infection (Proctor et al, 1995). However, SCVs
survives antibiotic exposure without being resistant (Balaban et al, are not only found in patients suffering from PJI, but there are many
2019a). Nevertheless, antibiotic persistence is thought to be studies reporting SCVs in other persistent biofilm-associated
involved in persistent infections, potentially impeding successful infections. Among these difficult-to-treat infections are pacemaker-
treatment (Fauvart et al, 2011). Persistent infections are also known related infections (Baddour et al, 1990; von Eiff et al, 1999),
as chronic infections and are often associated with antibiotic treat- endocarditis (Quie, 1969; Bhattacharyya et al, 2012), osteomyelitis
ment failure (Rhen et al, 2003; Fisher et al, 2017). These infections (Proctor et al, 1995), airway infections (Kahl et al, 1998; Besier
are not sufficiently cleared by the hosts’ immune system. Many dif- et al, 2007; Schneider et al, 2008) and skin and soft tissue infections
ferent factors, such as cell wall modification, capsule production, (Abele-Horn et al, 2000; Vulin et al, 2018).
antigenic mimicry and inhibition of effector proteins, contribute to
bacterial immune evasion (Hornef et al, 2002). Usually, the bacterial
cell wall is the first target of the hosts’ immune system and the first Colony size heterogeneity in persistent and difficult-
line of defence of the bacteria. For example, many Gram-negative to-treat infections
pathogens such as E. coli and S. enterica acetylate lipid A molecules
in their cell walls, which switches their negative surface charge to Even before the introduction of antibiotics and the first description
positive, repelling positively charged antimicrobial peptides (AMPs) of bacterial persistence, SCVs were reported for S. aureus and
from the host (Li et al, 2012). Others, like Borrelia spp. and Neisse- several other coagulase-negative staphylococci (Proctor et al, 2006).
ria gonorrhoea, vary their surface antigens during infection and thus Similar to bacterial persisters, SCVs have been associated with
evade the hosts’ humoral immune response (Nataro et al, 2000; recurrent and chronic infections (Proctor et al, 2006) and have been
Norris, 2006). Some pathogens such as Listeria monocytogenes and isolated from many different clinical specimens, e.g. abscesses and
Mycobacterium tuberculosis can trigger an anti-inflammatory soft tissue, bones and joints, the respiratory tract and
response by inducing the production of interleukin-10 that blood (Swingle, 1935; Goudie & Goudie, 1955; von Eiff et al, 1997a;
suppresses interferon-c production by macrophages, which in Kahl et al, 1998; Seifert et al, 1999; von Eiff et al, 1999; Rolauffs
general would inhibit bacterial growth (Redpath et al, 2001). et al, 2002).
Another way in which bacteria evade host recognition is by form- SCVs are characterized by their small colony size, reduced viru-
ing biofilms. Secreted polysaccharides and extracellular DNA form a lence and slow growth. Most studies were done on genetically deter-
protective matrix that blocks complement-killing (Domenech et al, mined stable SCVs that display a small colony size because of

genetic mutations (e.g. in hemB (von Eiff et al, 1997b), hemH et al, 2019a; Balaban et al, 2004; Van den Bergh et al, 2017). Many
(Schaaff et al, 2003), menD (Bates et al, 2003) or ctaA (Clements factors have been reported to trigger antibiotic persistence in bacte-
et al, 1999)), which either lead to an altered electron transport or ria, including nutrient limitation (Gutierrez et al, 2017), high cell
defect thymidine metabolism (Proctor et al, 2006; Proctor, 2019) density (Vega et al, 2012), oxidative stress (Wu et al, 2012; Rowe
(Fig. 3A). Defective hemin or menadione synthesis causes a reduced et al, 2020), an acidic environment (Lewis, 2007; Vulin et al, 2018),
electron transport and therefore decreased ATP levels. Low energy immune factors (Manina et al, 2015) and exposure to immune cells
levels and reduced membrane potential lead to a slower growth rate (Helaine et al, 2014).
of the bacteria and finally to smaller colonies. This defect can be Phenotypic heterogeneity is a complex quality that cannot be
overcome by supplementing with hemin or menadione leading to detected when analysing a single bacterial cell at a certain time
normal growing colonies. The second group of stable SCVs caused point. It needs the comparison to multiple other bacterial cells in a
by a thymidine biosynthesis defect has been isolated mainly from population or at least multiple observations of a single bacterium
cystic fibrosis (CF) patients that received a TMP-SMX treatment (Gil- over time (Ackermann, 2015). In case of bacterial growth analysis
ligan et al, 1987; Kahl et al, 1998). Kahl and co-workers showed that and determination of lag phase, Balaban and co-workers developed
a thymidine-auxotrophic clinical S. aureus could be complemented a method for E. coli to monitor the growth of many bacterial colo-
with thyA that encodes for a thymidylate synthase, enabling synthe- nies and lag times in parallel, the so-called ScanLag method (Levin-
sis of dTMP from dUMP (Chatterjee et al, 2008; Kriegeskorte et al, Reisman et al, 2010; Levin-Reisman et al, 2014). ScanLag generates
2014). a two-dimensional distribution of the lag time and of the growth
However, most small colonies recovered in vitro, directly from time of colonies on agar plates. We recently modified the ScanLag
patient material in clinical microbiology laboratories or from murine setup for the analysis of S. aureus lag time distributions after direct
infection models revert to a normal colony size upon subcultivation sampling from infection sites and in vitro stress exposure, named
(Tuchscherr et al, 2011; Vulin et al, 2018). Consequently, these ColTapp (Vulin et al, 2018; B€ ar et al, 2020). Automated agar plate
small colonies are named non-stable small colonies (nsSCs) imaging as well as single-cell microscopy revealed that the small
(Fig 3A). Usually, these small colonies occur at a very low colonies are the result of a prolonged bacterial lag phase and can be
frequency in a bacterial population, but increase after stress expo- linked to an increased antibiotic persistence in S. aureus (Fig 3B).
sure, e.g. acidic pH (Leimer et al, 2016; Vulin et al, 2018), exposure Therefore, these small colonies reflect a subpopulation of persis-
to an intracellular milieu in the host (Coffey & De Duve, 1968; Vesga ters and might serve as a read out for antibiotic persistence in clini-
et al, 1996; Tranchemontagne et al, 2016) or reactive oxygen species cal laboratories.
(Painter et al, 2015; Loss et al, 2019). NsSCs share many characteris-
tics with bacterial persisters: (i) they both occur at a low frequency
in a bacterial population; (ii) they display a revertible phenotype; Antimicrobial heteroresistance
and (iii) they show an increased tolerance towards antibiotics. A
clear and meaningful definition of these small colonies is still miss- Another bacterial bet hedging strategy to survive antibiotic treat-
ing. Usually, nsSCs are defined as colonies with an area 5 or 10 ment is heteroresistance (HR). In contrast to persistence, HR
times smaller than the area of the most common colony type (Proc- describes the ability of a subpopulation of susceptible bacteria to
tor et al, 2006). Instead of using a binary classification in “small” grow despite antibiotic pressure. The exact mechanisms underlying
and “normal” colonies, more and more focus is put on phenotypic HR are still unclear, but there is evidence that unstable amplification
heterogeneity and colony size distributions that give a detailed over- of antibiotic resistance genes is one way bacteria become heterore-
view on all recovered colony phenotypes. sistant (Hjort et al, 2016; Anderson et al, 2018; Nicoloff et al, 2019).
Phenotypic heterogeneity can be beneficial for the survival of a In order to survive antibiotic challenges and to grow even in pres-
bacterial population (Ackermann, 2015). Bacterial bet hedging via ence of antibiotics, the heteroresistant phenotype must be trans-
the formation of a subpopulation of persister cells in a clonal popu- ferred to daughter cells to ensure growth. Antibiotic resistance gene
lation of bacteria might be a great advantage in clinical infection amplification as the reason for HR can explain how this is achieved,
settings. While the actively growing part of a clonal bacterial popu- at least for genetic HR. It is still not clear though how phenotypic
lation is mostly responsible for host colonization and the successful HR that is not based on any genetic traits, is propagated in a popula-
establishment of an infection, the persister cell subpopulation tion under antibiotic treatment. One possible explanation for the
ensures the survival of the genotype under changing environments, maintenance of the heteroresistant phenotype by bacteria could be
e.g. under antibiotic therapy (Claudi et al, 2014). Nevertheless, epigenetic inheritance. There are several studies describing epige-
formation of these persisters is costly as they do not contribute to netic trait propagation (inheritance) of phenotypic variations over
the growth of the bacterial population but serve as a back-up plan generations (Veening et al, 2008; Ni et al, 2012; Ram & Goulian,
in case of emergencies. Persister cells form in two ways, either 2013). This heteroresistant phenotype is not maintained indefinitely
spontaneously or triggered by environmental factors (Balaban et al, but is lost as soon as the antibiotic pressure is removed, resulting in
2019a). The spontaneous formation of a small group of dormant a population consisting of antibiotic-susceptible bacteria and a small
bacterial cells in an overall growing population was previously fraction of heteroresistant bacteria creating the initial phenotypic
described as type II-persistence (Balaban et al, 2004). This sponta- heterogeneity.
neous persistence leads to a constant fraction of persisters in a The clinical relevance of heteroresistance and effect on treatment
population (usually between 0.001% and 1%), ensuring bacterial outcome is still under debate. Vancomycin HR in S. aureus has been
survival in the presence of antibiotics, but seems to be less common suggested to cause vancomycin treatment failure (Hiramatsu et al,
than triggered persistence (previously type I-persistence) (Balaban 1997; Moore et al, 2003), but there is also evidence that vancomycin

treatment is still effective for treating vancomycin heteroresistant vancomycin and may be another reason for vancomycin treatment
strains, depending on the antibiotic concentrations used (Khatib failure. Another study describes colistin HR in Enterobacter cloacae,
et al, 2011). Vancomycin heteroresistance might not be the only where a subpopulation (1–10% of the entire population) displayed
explanation for vancomycin treatment failure. In addition, intracel- 1,000-fold increased resistance to colistin (Napier et al, 2014). A
lularly residing S. aureus (Dziewanowska et al, 1999; Jevon et al, murine infection model demonstrated that colistin therapy failed to
1999; Fraunholz & Sinha, 2012; Lehar et al, 2015) are protected from successfully treat an infection with a heteroresistant strain of E.
A Genetically determined
Control Stress-exposed auxotrophic mutants
Exponential growth e.g. • pH • Abscess e.g. • ΔmenD
• ROS • Temperature • ΔhemB
• Intracellular
millieu
Larger colonies the of same size Non-stable small colonies (nsSCs) Small colony variants (SCVs)
100 100 100

of colonies
of colonies
of colonies
Proportion
Proportion
Proportion
50 50 50
500 1000 500 1000 500 1000

24h colony radius [µm] 24h colony radius [µm] 24h colony radius [µm]
100 100 100

of colonies
of colonies
of colonies
Proportion
Proportion
Proportion
75 75 75
50 50 50
25 25 25
Appearance time [h] Appearance time [h] Appearance time [h]
B
Single cell
Lag
level
Exponential
growth
Colony radius
Colony radius
Colony radius
Slow
Delayed growth
growth rate
© EMBO
Time [h] Time [h] Time [h]
Figure 3.

◀ Figure 3. Characterization of colony radius heterogeneity and single-cell growth dynamics of bacteria.
(A) Stress-exposed S. aureus and genetically determined auxotrophic mutants show small colony phenotypes. Top: The stress-exposed bacteria display colony radius
heterogeneity on agar plates by forming non-stable small colonies (nsSCs), whereas the auxotrophic mutants grow as genetically determined small colony variants
(SCVs). Middle: The colony radius histograms for stress-triggered bacteria show a broad distribution towards smaller colony sizes compared to exponentially growing
bacteria (most colonies have the same size) and SCVs (most colonies are small and the same size). Bottom: Translated into colony appearance times, the exponentially
growing bacteria almost all appear at the same time early after plating, whereas the stress-exposed bacteria show a broad lag time distribution with some colonies
appearing early and others later. In contrast, SCVs all appear later after a longer incubation time, but almost all at the same time. (B) The schemes show bacterial
growth dynamics on a single-cell level as well as different scenarios explaining the colony size at a certain time point. Short arrows indicate a fast division rate of wild
type bacteria (yellow), whereas longer arrows represent a slower growth rate for SCV forming mutants (green). Colonies can be small because of a late onset of growth
(lag time) or because of a slower growth rate.
cloacae, whereas infection with a fully susceptible strain could be lag and by slow growth (Ishida et al, 1982; Keren et al, 2004; Lewis,
treated with colistin and the mice survived, highlighting the impor- 2019).
tance of HR in infection settings and the adverse effect on treatment Balaban and co-workers used the standard disk diffusion assay
outcome (Band et al, 2016). as a basis to develop a semi-quantitative test for tolerance levels,
the so-called Tolerance Disk (TD) test (Gefen et al, 2017) (Fig 4B).
The TD test uses the diffusion and degradation of antibiotics in the
Detection of antibiotic tolerance and persistence in the agar after removal of the antibiotic-containing disc and enables
clinical setting the regrowth of tolerant and persistent bacteria that survived in the
inhibition zone after addition of fresh glucose. This test allowed
In the clinical microbiology laboratory, the options to test for antibi- detection of high levels of tolerance in clinical isolates of E. coli and,
otic persistence and tolerance are limited and typically focus on the most importantly, identified antibiotics that were able to kill these
detection of antibiotic resistance. Additionally, prevalence and pres- surviving bacteria. Therefore, the TD test might help tailoring the
ence of bacterial persisters and their clinical relevance in infection treatment regimens to treat persistent and recurrent infections.
per se are not well understood. One reason for this very limited test- However, this test cannot detect triggered antibiotic tolerance/per-
ing for tolerance in clinical microbiology laboratories is that there is sistence as this requires exposure to certain stressors like acidic pH
no quick and accurate testing protocol available so far. For resis- or nutrient limitation, as found in the host. Moreover, the standard
tance testing, the Kirby Bauer disc diffusion assay (Bauer et al, antibiotic discs used in clinical microbiology laboratories contain
1966) and the E-test (Joyce et al, 1992) are used to measure very high concentrations of antibiotics leading to high residual drug
increased MIC levels. These tests cannot be used for tolerance test- concentrations in the agar inhibiting regrowth of bacteria. Ideally,
ing, as antibiotic tolerance is defined as the ability of a bacterial discs should contain antibiotic concentrations high enough to create
population to survive antibiotic exposure without any changes in a large enough inhibition zone but low enough to fall below the
the MIC, meaning without resistance. MIC after 18 h. All these problematics make it difficult to standard-
Antibiotic tolerance can be determined by performing killing ize the TD test for clinical use.
assays in form of time–kill curves that are characterized by a Another way to test for antibiotic tolerance is to use the
bimodal killing in the case of antibiotic persistence, but this is “Replica Plating Tolerance Isolation System” (REPTIS) developed
very labour-intensive and shows high variability, which makes by Hiramatsu and co-workers to successfully identify and select
many repetitions necessary and undermines feasibility in routine ciprofloxacin (CIP)-tolerant mutants in S. aureus (Matsuo et al,
clinical microbiology laboratories (Fig 4A). In addition, the levels 2019) (Fig 4C). REPTIS does not require adjusting the antibiotic
of antibiotic persistence observed and evaluated in laboratories concentrations, overcoming this limitation of the TD test. Shortly,
under in vitro conditions might not correspond to the levels of instead of replacing the antibiotic disc by a glucose disc, this
persistence occurring in the patient from whom the strain was method uses a sterile silk cloth to transfer colony-forming units
isolated considering the complexities that contribute to this unsta- (CFUs) onto a fresh plate (replica plate) where surviving bacteria
ble phenomenon. Therefore, measurements of antibiotic persis- can regrow, and antibiotic tolerance is determined by the number
tence under laboratory conditions are only approximations of of growing bacteria in the former inhibition zone. Again, this test
bacterial phenotypes of infections in vivo. Yet, they provide impor- cannot detect triggered antibiotic persistence but has the potential
tant additional information that might help physicians in their to be adapted for an automated use in diagnostic microbiology
therapeutic decision-making. laboratories.
Many studies used the minimal bactericidal concentration (MBC) As mentioned above, the ScanLag method (Levin-Reisman
of an antibiotic necessary to kill 99.9% of the bacterial population et al, 2010) and ColTapp (B€ ar et al, 2020) can be used to detect
in 24 h to test for antibiotic tolerance (Handwerger & Tomasz, and measure colony growth heterogeneity and derive bacterial
1985). Although it was already known that tolerance is defined by a colonies’ lag times, which serve as a proxy for antibiotic toler-
decreased rate of killing, for clinical reasons a strain is considered ance (Fig 4D). To further validate and confirm the results from
tolerant, when the MBC is at least 32 times higher than the MIC. An the colony analysis at the single-cell level, single bacterial cell
MBC/MIC ratio of > 32 is defined as tolerance (Handwerger & microscopy can be used. However, these methods need specific
Tomasz, 1985; Jones, 2006; Gonzalez et al, 2013). However, the setups and equipment, laboratory space and are quite labour-
MBC/MIC ratio is not well suited to determine and measure antibi- intensive, which limits their usage in routine clinical microbiology
otic tolerance, and it especially fails to detect tolerance conferred by laboratories. For specific clinical cases of persistent and difficult-

A Time-kill curves (Bi-phasic killing) D Lag-phase determination
‘ScanLag’ ‘ColTapp’ Single-cell microscopy

1.00
0.01
Persistent
Susceptible
B Tolerance disk (TD) test Proportion of appearing colonies/

bacteria that initiated division
STEP 1 STEP 2
Antibiotic disk Glucose disk
Bacterial
infection
including
persisters
(blue)
Prolonged
lag
C Replica plating ‘REPTIS’

Time [h]
Master plate Replica plate
E Minimum Duration of Killing 99% – MDK 99

Duration [h]
MDK 99
© EMBO
Sterile velvet
on a block Concentration [mg/L]
Figure 4. Different detection methods of antibiotic tolerance and persister cells.

(A) The traditional method to test for antibiotic persistence is performing time–kill curves. Green line shows killing of a susceptible population and blue line displays the
biphasic kill curve of a mixed population consisting of susceptible rapidly killed bacteria and persisters killed at a slower rate. (B) Tolerance Disk (TD) test allows the
detection of bacteria that survive antibiotic exposure (persisters) by combining standard antibiotic disk assay (step 1) with regrowth after glucose addition (step 2). (C)
The replica plating tolerance isolation system (REPTIS) allows detection of bacterial survivors on antibiotic plates (master plates). Colony-forming units (CFUs) are
transferred via a sterile velvet from a master plate on a fresh replica plate that does not contain antibiotics and allows growth of bacteria. Regrowth of bacteria
indicates the presence of persister cells. (D) Detection of antibiotic persistence via lag phase determination. ScanLag, ColTapp and single-cell microscopy determine
colony and bacterial lag times that serve as a proxy for bacterial persistence (persistence by lag). (E) Determination of the minimum duration of killing 99% of a bacterial
population (MDK99) can be used to measure antibiotic persistence. High MDK99 values indicate high antibiotic persistence.
to-treat infections, where physicians suspect persister cell forma- MDK99 can be evaluated by a statistical analysis of measurements
tion, these methods could be used to improve and enable individ- performed manually or automated using a robotic system. This
ual patient-tailored treatments, but a broad standard testing might makes it interesting for standard diagnostic testing. Additionally,
not be suitable. the MDK99 evaluation has the inherent advantage of containing
Balaban and colleagues introduced a metric and an automated duration, an essential feature of persisters surviving stress over a
experimental framework for measuring antibiotic tolerance and period of time, is therefore superior to the MBC/MIC ratio evalua-
persistence, by determining the minimal duration of killing 99% tion, which is poorly linked to antibiotic tolerance (Keren et al,
of the population (MDK99) (Brauner et al, 2017) (Fig 4E). The 2004).

Future treatment regimens and novel antibiotics and exhibits bactericidal activity against a wide range
therapeutic approaches of pathogens including M. tuberculosis, carbapenem-resistant
Enterobacteriaceae, C. difficile and pan-resistant A. baumannii.
Despite the increase in antimicrobial resistance and the growing Interestingly, halicin disrupts the flow of protons across the bacte-
awareness of antibiotic persistence as a clinically relevant issue, the rial cell membrane (Fig 5A), a novel killing method compared to
development of new antimicrobial substances is declining. The conventional antibiotics. Additionally, this study identified eight
reasons for this are complex but are associated with the current other antibacterial substances that are structurally different from
focus of the pharmaceutical industry on medication for non- conventional antibiotics and highlights the opportunities and
infectious chronic diseases such as cancer, metabolic and cardiovas- impact machine learning can have on antibiotic discovery and
cular disorders and a lack of confidence in the profitability of new prediction of properties of potential drugs while decreasing the
antibiotics. However, there are novel strategies and substances in cost of screening efforts (Stokes et al, 2020). However, these
basic research and preclinical studies that might offer treatment results will have to be carefully evaluated and tested in animal
options in the near future (Fig 5). models including pharmacokinetic and toxicology studies before
moving to clinical trials.
Novel antimicrobials against a broad-spectrum of Based on the structure of a natural antibacterial peptide, Nicolas
multidrug-resistant bacteria and colleagues generated a new family of peptidomimetics, the
The recently discovered chimeric peptidomimetic antibiotics possess cyclic hepta-pseudopeptides that show antibacterial activity against
a broad-spectrum antimicrobial activity against Gram-negative Gram-positive and Gram-negative multiresistant pathogens while
bacteria, including all Gram-negative ESKAPE pathogens (Luther having limited potential to lead to resistance development (Nicolas
et al, 2019). Luther and colleagues showed that the bactericidal et al, 2019) (Fig 5A). They identified two peptide analogues that
activity of these chimeric antibiotics involves binding to both were active against MRSA and P. aeruginosa in severe sepsis and
lipopolysaccharide and the main component of the b-barrel folding skin infection murine models and exhibited low nephrotoxicity.
complex, BamA, that is required for the folding and insertion of b- Despite these promising results, their efficacy in deep-seated infec-
barrel proteins into the outer membrane of Gram-negative bacteria, tions is still to be shown. Both compounds are considered potential
explaining the specific targeting of Gram-negative bacteria while candidates for drug development; however, extensive pharmacoki-
leaving eukaryotic cell membranes intact (Fig 5A). Yet, the exact netic and toxicology studies will be necessary before starting phase I
mechanism driving the killing of Gram-negative bacteria remains an clinical trials (Nicolas et al, 2019).
open question. The leading candidate POL7306 underwent preclini- Another approach taken towards the effort of clearing antibiotic-
cal toxicology studies. Before moving into clinical trials, the focus is resistant Gram-positive and Gram-negative bacteria is the use of
currently on a new formulation of POL7306 as well as optimizing bacteriophages (Fig 5A), viruses that infect and lyse bacteria
peptide designs aiming to identify further candidates to broaden (Twort, 1915; Salmond & Fineran, 2015). Due to the success of
their therapeutic margins (Luther et al, 2019). antibiotics, bacteriophage therapy was abandoned in Western coun-
Generally, it is difficult to find new substances against Gram- tries, but is now being revived, because of increasing MDR bacteria
negative bacteria as they evolved an outer membrane that protects and studies show that bacteriophage therapy successfully treats
them from unwanted compounds (Li & Nikaido, 2004; Payne et al, urinary tract infections (UTIs) (Leitner et al, 2017) and burn wound
2007). Thus, Lewis and co-workers reasoned that compounds infections by P. aeruginosa (Jault et al, 2019). Nevertheless, there
against Gram-negative bacteria would be present in microorganisms are several potential disadvantages of phage therapy: (i) not all
that need to compete with Gram-negative bacteria. They screened phages are suitable for use as therapeutics. Temperate or toxin-
isolates of Photorhabdus, symbionts of entomopathogenic nematode carrying phages and those with poor killing potential against target
microbiomes, for antimicrobial substances against Gram-negative bacteria should be avoided. (ii) The narrow host spectrum, wherein
bacteria and isolated a new compound named darobactin (Imai different species or even different strains from the same species,
et al, 2019). In general, antimicrobial drugs target bacterial might need specific phages for eliciting efficient killing. Although
enzymes, but darobactin has been shown to stabilize the closed broadly acting phage cocktails are normally more selective in their
conformation of the chaperone BamA, inhibiting insertion of its spectrum of activity than conventional narrow-spectrum antibiotics,
substrates into the membrane (Imai et al, 2019) (Fig 5A). This it might be challenging to produce or at least provide all the needed
newly discovered antimicrobial drug might provide novel treatment phage cocktails. It might be even difficult to find the exact phage
options for infections with multidrug-resistant Gram-negative patho- needed for therapy. (iii) Phages are protein-based, biological agents
gens and shows that there are diverse molecules against pathogens that can actively replicate, evolve and interact with the hosts’
already available in nature. immune system. Especially, Western countries are not familiar with
Using a machine learning approach, Collins and co-workers phages and there is still hesitancy to use such biological entities in
discovered completely new kinds of antibiotics, without using any humans (Loc-Carrillo & Abedon, 2011).
previous human assumption (Stokes et al, 2020). Screening a
pool of more than 100 million molecules led to the discovery of Novel antimicrobials against a narrow-spectrum of multidrug-
c-Jun N-terminal kinase inhibitor SU3327 (De et al, 2009), a resistant bacteria
preclinical nitrothiazole under investigation as a treatment for Because of the discussed disadvantages of phage therapy, research
diabetes, exhibiting a strong antimicrobial activity. Halicin, has focused more specifically on phage-encoded peptidoglycan
renamed after HAL, the intelligent computer in the film “A Space hydrolases, the endolysins, which lyse bacterial cells (Haddad
Odyssey” from 2001, is structurally divergent from conventional Kashani et al, 2018) (Fig 5A). One of the biggest advantages of

A Targeting multi-drug resistant bacteria
Darobactin Chimeric Antibacterial drones Bacteriophages Endolysins

peptidomimetic (ABD)
antibiotics
BamA
Lysis
CRISPR-Cas9
Bactericidal cargo or
Virulence-blocking cargo
Membrane permeability
H+ Leakage H+
H+ Cyclic H+
Halicin ΔpH hepta-pseudopeptides ΔpH Lugdunin + LL-37 or + DCD-1(L)
B Persister targeting approaches
Cell-wall targeting Simple sugars Bacterial opsonisation

substances • Mannitol • C10
• Endolysins • Glucose Antimicrobial drug • CDA
• NT-61 • Fructose (Cis-2-decenoic acid)
• XF-73 Linker
• NCK10 Aminoglycosides
PMF Antibody
H+
Growth stimulation
Stringent Translation
response Antimicrobial-susceptible
Transcription state
Translation blockade
Mistranslation
Non-specific and lethal
(p)ppGpp protease activity
ClpP
DNA crosslinking Membrane

RelA permeabilization
ATP
ATP
(p)ppGpp analogue Mitomycin C ADEP4 Retinoid derivatives
e.g. relacin • CD437
© EMBO
• CD1530
•…
Figure 5.

◀ Figure 5. New approaches to target multidrug-resistant bacteria and bacterial persister cells.
(A) Scheme showing novel strategies to target multidrug-resistant Gram-negative and Gram-positive bacteria. New approaches to target multiresistant bacteria include
darobactin, chimeric peptidomimetic antibiotics, antibacterial drones (ABD), bacteriophages, endolysins, halicin and cyclic hepta-pseudopeptides and lugdunin. (B)
Scheme of persister-targeting approaches. Different strategies have been suggested to specifically target bacterial persister cells, e.g. addition of cell wall targeting
substances like endolysins, NT-61, XF-73 or NCK10, simple sugars, bacterial opsonization by antibody–antibiotic conjugates (AACs), 3-[4-(4-methoxyphenyl)piperazin-1-
yl] piperidin-4-yl biphenyl-4-carboxylate (C10), cis-2-decenoic acid, (p)ppGpp analogues like relacin, mitomycin C, acyldeptipeptide (ADEP4) or retinoid acid derivatives
(e.g. CD437 and CD1530).
endolysins is their species specificity, avoiding selective pressure on Rather than directly targeting the pathogen, an alternative
commensal bacteria (Schmelcher et al, 2012). Another advantage is approach is to attenuate their pathogenicity by targeting specific
the low probability of bacteria evolving resistances against endoly- virulence factors involved in the infection process. The blocking of
sins, as phages and host bacteria coevolved and endolysins target virulence factors also helps avoiding resistance development by the
highly conserved structures in the bacterial cell wall (Borysowski bacteria, as less antibiotics would have to be used (Keyser et al,
et al, 2006). However, to overcome certain limitations, such as 2008). An important strategy is to hinder bacterial dissemination
immunogenicity, short serum half-lives, low penetration into and tissue invasion by blocking bacterial attachment to host cells
eukaryotic cells to kill intracellular bacteria and toxicity, many and translocation into host tissue. Mannosides have been shown to
research groups focus on engineering improved variants that might inhibit the FimH component of type I fimbriae of uropathogenic
offer novel treatment options in the future (Schmelcher et al, 2012; E. coli (UPEC) thereby significantly decreasing colonization of UPEC
Haddad Kashani et al, 2018; Ro €hrig et al, 2020). in a murine infection model (Klein et al, 2010).
Independent of discovering new antibiotic substances and phages, The synthetic antibody ScFv-Fc KP3 blocks the type 3 fimbrial
a new method using CRISPR-Cas9, was recently developed to kill or subunit in K. pneumonia and has been shown to confer protection
block multidrug-resistant S. aureus (Ram et al, 2018). In this study, in murine pneumonia models (Wang et al, 2016). By neutralizing
they replaced the staphylococcal pathogenicity islands’ (SaPIs) toxin toxin B from C. difficile, the monoclonal antibody bezlotoxumab
genes with antibacterial cargos to generate antibacterial drones prevents recurrent C. difficile infections in combination with antibi-
(ABDs) that target S. aureus in the host (Fig 5A). By constructing otics and has already been approved for the use in humans
ABDs with either a CRISPR–Cas9 bactericidal or a CRISPR–dCas9 (Mullard, 2016). Similarly, the monoclonal anti-a-toxin antibody,
virulence-blocking module, bacteria are either killed or disarmed, MEDI4893, has been shown to decrease the S. aureus burden and
leading to the abrogation of the infection. This was also shown in a increase wound healing in diabetic foot ulcers and phase II clinical
murine abscess model, where mice were rescued by treatment with studies have just been concluded (ClinicalTrials.gov, 2019). Despite
these ABDs (Ram et al, 2018). However, the ABD approach comes promising novel strategies and molecules targeting pathogens and
with some potential obstacles, e.g. simultaneous packaging of host their virulence factors, further research and alternative approaches
genes, induction of or recombination with resident SaPIs or recombi- are needed to ensure effective treatment regimens against multi-
nation with plasmids carrying virulence or resistance genes. Addi- drug-resistant bacteria in the future.
tionally, resistance to the ABDs can occur (Ram et al, 2018).
Taking a different approach, Krismer, Peschel and co-workers Antipersister strategies
identified the potent antimicrobial compound lugdunin from The recognition of the phenomenon of antibiotic persistence and its
S. lugdunensis (Zipperer et al, 2016). Rather than treating an already clinical importance in chronic and relapsing infections including its
existing infection, they suggest using lugdunin or lugdunin-produc- connection to resistance development has led to both basic and
ing commensal staphylococci to prevent S. aureus colonization and application-oriented research. Today, there are three main strategies
invasive infections in high-risk patients such as immunocompro- for treatment and prevention of infections associated with bacterial
mised or before elective surgeries. The macrocyclic thiazolidine persisters: (i) directly targeting persister cells; (ii) inhibiting forma-
peptide antibiotic lugdunin is active against a wide range of Gram- tion of bacterial persisters; and (iii) resuscitating persisters and
positive pathogens including MRSA and VRE with a high barrier to sensitizing them to conventional antibiotic therapy.
resistance by mutation (Zipperer et al, 2016) (Fig 5A). It exhibits
immunomodulatory activity by inducing the expression of the host Directly targeting persister cells
AMP LL-37 and the pro-inflammatory chemokines CXCL8 and MIP-2 As mentioned above, persisters are often characterized by slow
in human keratinocytes, which leads to the recruitment of mono- growth or dormancy rendering antibiotic targets inactive. One of the
cytes and neutrophils potentially contributing to effective bacterial obvious targets for directly attacking persisters without the require-
eradication (Bitschar et al, 2019). Moreover, lugdunin acts synergis- ment of active targets is the bacterial membrane and cell wall.
tically with the human AMPs LL-37 and DCD-1(L) in killing Phage-derived endolysins and other synthetic lysins degrade the
S. aureus. The antimicrobial activity against S. aureus is strongly peptidoglycans in the cell wall independent of the presence of
associated with the disruption of the membrane potential without metabolically active and growing bacteria, targeting both growing
lysing the bacterial cells, demonstrating that the mode of action is and non-growing populations (Gutierrez et al, 2014; Rodrıguez-
translocation of protons (Schilling et al, 2019). Lugdunin could be €hrig et al, 2020) (Fig 5B).
Rubio et al, 2016; Ro
used either directly as a topically applied drug to inhibit growth of Synthetic retinoid acid derivatives, specifically CD437 and
S. aureus or indirectly applied with a safe probiotic strain such as an CD1530, have been shown to penetrate and disrupt the lipid bilayer
avirulent S. lugdunensis or an exclusively commensal species of the cell membrane of MRSA thereby killing growing and
containing the lug genes (Zipperer et al, 2016). non-growing bacteria (Fig 5B). Additionally, CD437 alone or in

combination with gentamicin exhibited high efficacy in a murine

In need of answers
thigh infection model highlighting the potential of retinoid deriva-
tives as a new class of antibiotics and antipersister drugs (Kim et al, i How can the identification of resistance in diagnostics be acceler-
2018; Kim et al, 2019). Various other membrane-active small mole- ated to ensure a targeted therapy from the beginning? Moreover,
can novel narrow-spectrum antibiotics prevent microbiota distur-
cules like NT-61, a fluoroquinolone (Hu et al, 2010), XF-73 (Ooi
bance and selection for resistance?
et al, 2010) or NCK10 (Ghosh et al, 2015) have shown activity
ii What is the persister status in an infected patient?
against S. aureus persisters (Fig 5B). Another approach to eradicate
iii How homogeneous is a persister population during infection? Do
persisters directly is to use compounds that interact with inactive
we see differences in their transcriptome/ proteome on a single
targets in the bacterial cell. The acyldeptipeptide ADEP4, for exam- bacterial cell level?
ple, activates the protease ClpP in S. aureus, leading to non-specific
iv How well do in vitro findings correlate with the in vivo situation?
and lethal protease activity (Conlon et al, 2013) (Fig 5B). In combi-
v How does the hosts’ immune system and milieu influence persister
nation with conventional antibiotics, ADEP4 was able to clear a cell formation and interact with persisters?
deep-seated murine S. aureus biofilm infection (Conlon et al, 2013).
vi Can a bacterial population including persisters be targeted and
Recently, repurposing of anticancer drugs leads to the discovery of eradicated by combination treatments? Will a subpopulation of
their antipersister activity. Mitomycin C, for example, causes DNA extremely dormant persisters survive?
crosslinking independently of bacterial growth and exhibits bacteri- vii How can we better implement testing for antibiotic persistence in
cidal activity against E. coli, P. aeruginosa and S. aureus growing clinical microbiology laboratories?
and non-growing cells (Kwan et al, 2015) (Fig 5B). As of their viii Will a better characterization of persisters in diagnostics allow
nature as anticancer drugs, these agents are quite cytotoxic for the predicting the clinical course, e.g. difficult-to-treat/ chronic or
host and certainly not specific to pathogens, making their use as relapsing infections?
antipersister compounds questionable.
Inhibition of persister formation

Ideally, we would be able to inhibit persister formation directly from addition, cis-2-decenoic acid was shown to induce biofilm dispersion
the beginning and thereby limit the problem of difficult-to-treat and and might provide a new potential adjuvant drug to treat biofilm-
chronic infections. As described above, different cellular pathways, associated infections (Davies & Marques, 2009) (Fig 5B). However,
e.g. stringent response and SOS response, can lead to persister no follow-up studies for this molecule showing in vivo efficiency in
formation. Synthetic ppGpp analogues such as relacin, have been reducing bacterial persisters were published and toxicity studies are
shown to efficiently inhibit Rel proteins, thereby decreasing long- missing. A different approach that targets bacteria irrespectively of
term survival potential of S. aureus (Wexselblatt et al, 2012) their growth state and metabolic activity is to use antibody–antibiotic
(Fig 5B). However, persister cell formation might happen already at conjugates (AACs) (Fig 5B) (Lehar et al, 2015). Such constructs were
the very beginning of an infection, when the bacteria encounter dif- designed to target and opsonize extracellular S. aureus directly
ferent stressors in the host, meaning that it requires an intervention promoting phagocytosis. The conjugated antimicrobial drug is only
at the same time as the infection happens or as a prophylactic treat- active and kills bacteria inside phagolysosome preventing the transfer
ment. For these reasons, the most promising approach, using the of bacteria between host cells (macrophages) during infection. Addi-
hosts’ first line of defence, might be vaccination. tionally, the drug killed persister cells and targeted non-dividing
MRSA sequestered inside murine macrophages (Lehar et al, 2015). In
Resuscitate and sensitize persisters summary, novel approaches and strategies will help boosting our
Awakening and stimulating the regrowth of persisters can render capacity to treat microbial infections, especially therapy of persistent
them again sensitive to conventional antibiotics that usually target and chronic infections.
actively growing bacteria. Some compounds that are able to wake up
persister cells and resensitize them to conventional antibiotics have
already been identified. Simple sugars like mannitol, fructose and Concluding remarks
glucose combined with gentamicin can decrease persister levels more
than 1,000-fold in E. coli and S. aureus not via activating bacterial The rise of antibiotic resistance, as well as the increase of difficult-to-
growth, but by increasing the pmf that in turn increases the uptake of treat and biofilm-associated infections caused by antibiotic-suscepti-
aminoglycosides (Allison et al, 2011) (Fig 5B). This effect was amino- ble pathogens, led to intensive research on new antimicrobial
glycoside-specific, and this approach failed to reduce persister levels strategies including persister-targeting approaches. Recent develop-
in combination with other classes of antibiotics like b-lactams and ments in antibiotic discovery give hope for new treatment options of
fluoroquinolones. Kim and colleagues identified a compound, 3-[4-(4- infections caused by multiresistant bacteria. However, there is still a
methoxyphenyl)piperazin-1-yl] piperidin-4-yl biphenyl-4-carboxylate desperate need for further antibiotic research and discovery to over-
(C10), that shortens the lag time of E. coli that survived in vitro ampi- come the imminent post-antibiotic era. In addition, several mecha-
cillin or ciprofloxacin exposure and facilitated their killing, suggesting nisms involved in persister formation in different bacterial species
that this compound is able to stimulate bacterial regrowth thus accel- have been revealed. Global cellular dormancy and target inactiva-
erating the reversion of persisters to sensitive cells (Kim et al, 2011) tion, including reduced ATP levels, blocked translation, protein
(Fig 5B). Similarly, the fatty acid signalling molecule cis-2-decenoic aggregation and arrested DNA replication, have been linked to bacte-
acid is able to activate E. coli and P. aeruginosa persisters growth and rial survival despite antibiotic treatment. Although the field of antibi-
metabolism resensitizing them to antibiotics (Marques et al, 2014). In otic persistence research is growing and great advances have been

made, we still do not fully understand how bacteria form persisters. Azarian T, Daum RS, Petty LA, Steinbeck JL, Yin Z, Nolan D, Boyle-Vavra S,
Clearly, the interplay of multiple mechanisms and the resulting Hanage WP, Salemi M, David MZ (2016) Intrahost evolution of methicillin-
phenotypic heterogeneity is beneficial for the survival of a bacterial resistant Staphylococcus aureus USA300 among individuals with
population during infection. Recent advances show novel therapeu- reoccurring skin and soft-tissue infections. J Infect Dis 214: 895 – 905
tic strategies aiming to eradicate such persisters, desperately needed Baddour LM, Barker LP, Christensen GD, Parisi JT, Simpson WA (1990)
to shorten treatment duration and thus reducing morbidity remark- Phenotypic variation of Staphylococcus epidermidis in infection of
ably. However, further research and focus on modulating the host transvenous endocardial pacemaker electrodes. J Clin Microbiol 28:
environment is needed to successfully tackle antibiotic resistance 676 – 679
and persistence. Novel technologies including the development of Baharoglu Z, Mazel D (2014) SOS, the formidable strategy of bacteria against
cutting-edge techniques such as single-cell analysis, microfluidics, aggressions. FEMS Microbiol Rev 38: 1126 – 1145
dual-RNAseq and bacterial sorting as well as bacterial epigenetics Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI,
are being used and will be used more and more to track, enrich and Brynildsen MP, Bumann D, Camilli A, Collins JJ et al (2019a) Definitions
analyse rare, non-growing bacteria that are able to survive antibiotic and guidelines for research on antibiotic persistence. Nat Rev Microbiol 17:
therapy without being resistant. 441 – 448
Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B, Andersson DI,
Acknowledgements Brynildsen MP, Bumann D, Camilli A, Collins JJ et al (2019b) Publisher
We thank the members of the Zinkernagel laboratory for feedbacks and proof- correction: definitions and guidelines for research on antibiotic
reading of the manuscript, especially Judith Bergada Pijuan for her help with persistence. Nat Rev Microbiol 17: 460
the presentation of the resistance data. The authors apologise to colleagues in Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S (2004) Bacterial
the field for being unable to cite all relevant studies and thank the reviewers persistence as a phenotypic switch. Science 305: 1622 – 1625
for their helpful comments. This work was funded by the SNSF project grant Band VI, Crispell EK, Napier BA, Herrera CM, Tharp GK, Vavikolanu K, Pohl J,
31003A_176252 (to A.S.Z); by the Swedish Society for Medical Research (SSMF) Read TD, Bosinger SE, Trent MS et al (2016) Antibiotic failure mediated by
foundation grant P17-0179 (to S.M.S) and the Promedica Foundation 1449/M a resistant subpopulation in Enterobacter cloacae. Nat Microbiol 1: 16053
(to S.D.B). B€
ar J, Boumasmoud M, Kouyos RD, Zinkernagel AS, Vulin C (2020) Efficient
microbial colony growth dynamics quantification with ColTapp, an
Conflict of interest automated image analysis application. Sci Rep 10: 16084
The authors declare that they have no conflict of interest. Baroud M, Dandache I, Araj GF, Wakim R, Kanj S, Kanafani Z, Khairallah M,
Sabra A, Shehab M, Dbaibo G et al (2013) Underlying mechanisms of
carbapenem resistance in extended-spectrum beta-lactamase-producing
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14693178, 2020, 12, Downloaded from https://www.embopress.org/doi/10.15252/embr.202051034 by Algeria Hinari NPL, Wiley Online Library on [22/03/2023].

Antibiotic resistance and persistence—Implications

Abstract resulting in increased life expectancy in general (Adedeji, 2016).

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A E. coli B E. faecium C S. aureus

Vancomycin resistance [%]

Number of invasive isolates

Number of invasive isolates

Methicillin resistance [%]

D K. pneumoniae E Acinetobacter spp. F P. aeruginosa

Number of invasive isolates

Number of invasive isolates

Multi-drug resistance [%]

Year Year Year

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K. pneumoniae genes, like ESBLs and carbapenamases. Enterobacter species show

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Many environmental factors, such as nutrient limitation, antibiotic

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Oxidative Antibiotic Nutrient

SOS response Stringent response

ATP T/A systems

High population Quorum sensing

Figure 2. Environmental triggers of antibiotic persistence and bacterial response mechanisms.

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100 100 100

500 1000 500 1000 500 1000

100 100 100

Appearance time [h] Appearance time [h] Appearance time [h]

Time [h] Time [h] Time [h]

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A Time-kill curves (Bi-phasic killing) D Lag-phase determination

‘ScanLag’ ‘ColTapp’ Single-cell microscopy

B Tolerance disk (TD) test Proportion of appearing colonies/

C Replica plating ‘REPTIS’

E Minimum Duration of Killing 99% – MDK 99

Figure 4. Different detection methods of antibiotic tolerance and persister cells.

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A Targeting multi-drug resistant bacteria

Darobactin Chimeric Antibacterial drones Bacteriophages Endolysins

B Persister targeting approaches

Cell-wall targeting Simple sugars Bacterial opsonisation

DNA crosslinking Membrane

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combination with gentamicin exhibited high efficacy in a murine

Inhibition of persister formation

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