The Indian Journal of Pediatrics

https://doi.org/10.1007/s12098-019-03113-0

REVIEW ARTICLE

Antibiotics: From the Beginning to the Future: Part 2

Nancy Khardori 1 & Cecilia Stevaux 1 & Kathryn Ripley 1

Received: 30 October 2019 / Accepted: 31 October 2019

# Dr. K C Chaudhuri Foundation 2019

Abstract
We read, write, and discuss the option of adding new agents to the armamentarium of antibiotic therapy very frequently.
However, the past and present has taught us that resistance is likely to develop to any and all kinds of antibiotics. Here we start
with an overview of potential future antibiotics from novel sources and targets that may circumvent most known resistance
mechanisms. The other future options for antibiotic discovery include antibiotic hybridization, harvesting, and modifying natural
antimicrobial peptides from eukaryote and prokaryote organisms. Non bacteriostatic and bactericidal agents that have the
potential of becoming therapeutic agents include bacterial attachment inhibitors, bacteriophages, and live microbial vectors. In
this review, we have incorporated all the possible avenues that might be useful in the future. However, none is more important
than relearning the judicious use of antibiotics based on microbiology, pharmacology, and genetics.

Keywords Antibiotics . Resistance . Antibiotic stewardship . Bacteriophages . Antimicrobial peptides

Novel Targets and Sources of Antibiotics
Once a threshold of high microbial cell density or “quorum” is
reached, bacteria sense one another’s presence, resulting in the
formation of a protected biofilm or an orchestrated production
of virulence factors. The promising role of anti-quorum sensing
agents as adjuncts to standard antimicrobial therapy has be-
come an area of exploration. A group of enzymes with ability
to duplicate damaged DNA and create new mutations has been
reported [1]; the spread of antibiotic-resistant bacteria can po-
tentially be slowed by agents that cause suppression of these
enzymes. The pathogenic strains of E. faecalis that are
vancomycin-resistant or multi-drug resistant are known to con-
tain a 150-kb long “pathogenicity island” [2]. None of the
genes in this island are known to code for antibiotic resistance.
The virulence factors they encode include the enterococcal tox-
in cytolysin (a surface protein that aids colonization) and fac-
tors that enhance bacterial clumping, host tissue adherence, and
resistance to phagocytosis. Agents that target this island can be
designed to prevent colonization by the virulent strains.
* Nancy Khardori
nkhardori@gmail.com
1
Infectious Diseases, Solid Organ Transplant Program at Sentara
Norfolk General Hospital, Norfolk, Virginia, USA
A small molecule, virstatin, inhibits the activation of chol-
era toxin genes and abolishes both the production of cholera
toxin and toxin-regulated pili [3]. This molecule was shown to
be a potent inhibitor of intestinal colonization by Vibrio
cholerae in an infant mouse model without interfering with
the growth of organisms in laboratory media. It also reduced
the burden of Vibrio cholerae in already-colonized mice.
Combining antivirulence factor drugs as described above with
conventional antibiotics may prove to be a safe as well as
efficacious approach, with the potential of extending the use-
ful life of both types of agents [4].
The first new antibiotic discovered in 30 y has been hailed as a
paradigm shift in the approach to management of antibiotic re-
sistance. The concept is based on the fact that most antibiotics
that have come into clinical use since the 1940s were discovered
by screening cultivable soil microorganisms. We saw an end to
the initial era of antibiotic discovery by 1960s due to over mining
of this limited resource [5]. Synthetic approaches to antibiotic
development have fallen short of expectations [6, 7]. But a prom-
ising source of new antibiotics is the 99% of all species in exter-
nal environments that do not grow under laboratory conditions
[8]. Ling et al. [5] developed a number of methods to grow
unculturable organisms by cultivation in situ or by using specific
growth factors. They reported a new antibiotic, Teixobactin, dis-
covered during a screen of unculturable bacteria. It binds to a
highly conserved motif of lipid II (precursor of peptidoglycan)
and lipid III (precursor of cell wall teichoic acid), and inhibits cell

wall synthesis. The researchers did not obtain any mutants of
Staphylococcus aureus or Mycobacterium tuberculosis resistant
to Teixobactin. The characteristics of this compound suggest a
path towards avoiding development of resistance in microbes.
Another investigational compound named The Sheffield-RAL
Compound has several modes of action making it more difficult
for microbes to become resistant to its activity.
It is well understood that Staphylococcus aureus is able to
invade and survive inside mammalian cells including the
phagocytic cells that are expected to kill the bacteria. The
phagocytic cells containing uncleared S. aureus then act as
Trojan horses for dissemination outside the initial site of in-
fection. There is evidence that disseminated disease is more
likely to occur in patients with normal neutrophil counts com-
pared to those with reduced counts. Lehar et al. [9] have re-
ported the concept “homed to the hideout” using an antibiotic
antibody-conjugate (AAC). They studied a construct in which
an antibiotic (a rifalogue) is linked to an antibody that binds to
the surface of Staphylococcus aureus. By itself, this prodrug is
inactive. When the bacteria coated with the prodrug (AAC)
enter host cells, the active antibiotic is released by enzymatic
activity. This strategy was shown to be much more potent than
standard antibiotic treatment in a mouse model [9].
Antibiotic Hybrids
It is possible to combine two antibiotics in a single molecule
[10]. This could overcome problems that arise with the use of
combinations as two separate drugs, which may not allow
simultaneous effective concentrations at the target site. Beta
lactam antibiotics (cephalosporin and carbapenems) and
fluoroquinolones have been studied in hybrid combinations,
and some of the hybrids retain antibacterial effects of both
types. Bacterial enzymes like beta-lactamases may be used
to produce two separate antibiotics from a prodrug.
Although none of the hybrid antibiotics have become avail-
able for clinical use, some have been administered safely to
humans in clinical trials.
A very novel approach to hybridization of antibiotics is the
use of gold nanoparticle-targeted laser therapy for enhance-
ment of antibiotic efficacy against MRSA in biofilms [11]. It
is well understood that bacterial persistence on wounds, tis-
sues, and devices is significantly enhanced by formation of
biofilms. Bacteria within the biofilm (sessile mode) exhibit a
500 to 5000 fold increase in antibiotic tolerance compared
with free floating (planktonic) bacteria. The tolerance in the
biofilm-associated bacteria is due to their encasement by a
dense extracellular matrix (glycocalyx) that functions as an
impenetrable barrier against antimicrobials. The collateral
damage caused by biofilms is the establishment of a nidus that
becomes source for recurrent infections and dissemination of
the infectious process.
Indian J Pediatr
Antimicrobial Peptides and Bacteriocins
The ubiquitous naturally-occurring peptides such as beta-
defensins and cathelicidins are expressed on epithelial sur-
faces and in neutrophils of mammals [12, 13]. They demon-
strate a broad spectrum of activity against bacteria, fungi, vi-
ruses, and cancer cells. Insects have existed and thrived for
millions of years by relying on the transient synthesis of potent
antimicrobial peptides to survive microbial infections; they do
not possess lymphocytes, immunoglobulins, or any other
components of an acquired immune system. Typically, natural
antimicrobial peptides are found at sites that have direct con-
tact with the environment, such as the eyes, ears, oral cavity,
respiratory tract, and gastrointestinal tract. The lipid in the
bacterial membrane interacts with the peptide, followed by
binding and ingestion of the peptide into the cytoplasmic
membrane. The disruption of the cytoplasmic membrane leads
to lysis and the resultant bactericidal effect. Induction of single
strand breaks in the bacterial DNA has been shown to be
caused by defensins.
Peptide antibiotics with high molecular weight are pro-
duced by bacteria, fungi, and Streptomyces. From this class,
Bacitracin, Gramicidin S, Polymixin B, and Polymixin E
(Colistin) have been in clinical use. Their non-selective activ-
ity causes toxic side effects, requiring drastic modification of
the drugs. Gramicidin from Bacillus brevis was used commer-
cially to treat bacterial infections more than a decade before
the introduction of penicillin. Until recently, these agents were
declared to be too unsafe for systemic use, and served only as
topical antibacterials. Colistin is now being used to treat in-
fections caused by multidrug resistant Gram-negative bacteria
that have lost susceptibility to the safer classes. Currently, a
number of other polymixins and modifications of the current
polymixins are being altered to improve the toxicity profile.
Designed antimicrobial peptides are synthesized based on
modifications of existing natural peptides or by using novel
designs. Among the agents studied are D2A21 and D4E1, but
none are available for use at this time.
Bacteriocins are potent protein toxins known to be pro-
duced by every bacterial and archeal species examined.
These bactericidal peptides play an important role in the nat-
ural microbial systems by regulating competitive interactions.
They have a narrow target range, high activity, stability, and
low toxicity which make them viable alternatives and/or com-
pliments to existing small molecule antibiotics. This is the
cutting edge of a new approach to antibiotic discovery and
design in the twenty-first century since we have lost a lot of
agents discovered in the twentieth century.
The search for more active, stable, and nontoxic variants of
naturally occurring peptides has led to the synthesis of a
macedocin from Streptococcus macedonicus, originally iso-
lated from Greek cheese. This compound is a food-grade bac-
teriocin that could prevent food spoilage and the growth of
Indian J Pediatr
pathogenic microbes [14]. Nisin is currently the only bacteri-
ocin approved as a food additive.
Zipperer et al. [15] reported that nasal Staphylococcus
lugdunesis strains produce a novel thiazolidine-containing cyclic
peptide antibiotic that prevents colonization by S. aureus.
Lugdunin is a rare example of a non-ribosomally synthesized
compound from human associated bacteria that is active against
pathogenic bacteria. It is bactericidal against major pathogens,
effective in animal models, and less likely to become ineffective
through development of resistance. Human nasal colonization by
S. lugdunesis has been shown to reduce carriage rate by
S. aureus. Therefore, Lugdunin producing human commensal
bacteria or synthesized Lugdunin may be valuable in preventing
colonization and subsequent infection by Staphylococci.
Antibiotic biosynthesis has been identified in human
metagenomes leading to the discovery of Lactocillin.
Lactocillin is a ribosomally synthesized thiopeptide antibiotic
produced by human vaginal commensal flora.
The Post-Antibiotic Approach
Bacterial Attachment Inhibitors
The process of initiating an invasive infection begins with
recognition of and attachment to epithelial/mucosal cells, spe-
cifically to oligosaccharide complexes. These oligosaccha-
rides can be competitively replaced by noncellular oligosac-
charides to saturate the bacterial receptors leading to preven-
tion of attachment and therefore, infection [16]. The free oli-
gosaccharides produced by mucosal surfaces may be insuffi-
cient in quantity to alter the level of attachment and microbial
invasion. The large amounts of free oligosaccharides present
in breast milk may be responsible for protection against neo-
natal infection. The release of bound bacteria from target cells
by large amounts of specific oligosaccharides may dissociate
them from the target and prevent further invasion. This ap-
proach would likely not lead to development of resistance due
to the process being non-lethal and “non-interfering.” E. coli
infections have been prevented in newborn calves and mon-
keys with some success using this concept.
The significant role of bacterial attachment inhibitors is
based on the sequence of events that take place during an
infectious process i.e., contamination followed by coloniza-
tion followed by invasion leading to local/focal infection with
or without dissemination to other areas. The interruption of
this process i.e., biofilm formation would obviously prevent
the development of infection. The biofilm also provokes a
severe and prolonged inflammatory response with production
of proteolytic enzymes that degrade newly formed human
extracellular matrix and delay the healing process.
Polyhexamethylene biguanide (PHMB) is a positively
charged polymer that interacts with negatively charged
phospholipids in the microbial membranes punching holes.
This results in bacterial death because of the loss of cellular
integrity while the toxicity to surrounding human cells is low.
A novel technology now available combines the broad spec-
trum and non-cytotoxic PHMB with a purified Type 1 native
collagen matrix. This combination is able to manage
bioburden as well as support healing of complex wounds.
PHMB has been combined with Benzalkonium chloride in a
gel formulation. Benzalkonium chloride is a biocide belong-
ing to the quaternary ammonium group. In addition to being
an effective surfactant, Benzalkonium chloride is bactericidal
due to disruption of cellular membrane lipid biolayers. Dialkyl
carbomol chloride (DACC) is a highly hydrophobic agent as
are the outer membranes and cell walls of pathogenic bacteria.
DACC technology uses the physical property of hydrophobic
interaction to promote bacterial binding just like oil droplets
attached to each other in water. Bacteria are kept from
attaching to the wounds and then removed with the dressing
change with no chemical or metabolic interaction to which
pathogens could develop resistance.
Bacteriophages
Viruses with bacteria as their hosts are among the most com-
mon organisms on planet Earth. Killing caused by
bacteriophage-induced lysis is highly efficient and specific.
The discoverers of bacteriophages during World War I
(1917) believed that nature had provided us with a living
weapon against bacteria [17], but the manufacture of thera-
peutic phages for the United States market in the 1930s was
quickly replaced by penicillin. The simple molecular structure
of bacteriophages allowed them to become an ideal tool for
DNA research and advancement of molecular biology. The
Eliava Institute in Tbilisi, Georgia, became the world’s leading
center for therapeutic phage research. It supplied a powerful
dysentery phage to the Red Army and subsequently provided
it to targeted Soviet bloc hospitals. The most significant con-
tribution of Eliava Institute has been the creation of an intra-
venous phage preparation against Staphylococcus aureus.
With the growing need for alternatives to conventional an-
tibacterial agents, bacteriophage therapy is looking promising
again. In 2015, a major scientific meeting was hosted by the
U.S. National Institute of Allergy and Infectious Diseases
(NIAID) on phage-based therapeutics. The participants in-
cluded phage biologists, public and governmental research
organizations, practicing clinicians, pharmaceuticals, and the
federal regulatory agencies. There was a consensus about the
potential of phage therapy and funding of appropriate investi-
gation. The lytic phages injected into a bacteria undergo a
replicative cycle resulting in bacterial cell lysis and liberation
of hundreds of virions ready to infect more bacterial cells. This
form of treatment modality is therefore self-replicating. The
other phage related potential therapeutic option is the use of

muralytic enzymes such as lysins and tailocins. Lysins are
used by phages to enable the DNA to penetrate the bacterial
cell and in the phage-induced lysis process. Tailcins or phage
tail-like bacteriocins or headless phages cause lethal damage
to the bacterial cell envelope in a single hit.
Wild phages are expelled by the body’s filtering system
quickly. However, engineered mutant phages have been
shown to remain viable for much longer in experimental ani-
mals. Their replicative potential is high; 400,000 daughter
cells can be created from a single phage in an hour. If the
potential of bacteriophage therapy is realized, they will be
the only drugs to make more of themselves within the infected
hosts. The potential for development of resistance to specific
bacteriophages is projected to be low to non-existent.
Living Therapeutics
Honey, a natural product, has been used for human infections
for millennia without sufficient scientific evidence. Olofsson
et al. [18] discovered a unique lactic acid bacterial (LAB)
microbiota which is in symbiosis with honeybees and present
in large amounts in fresh honey. The LAB was tested against a
number of wound pathogens including MRSA, Vancomycin-
resistant enterococcus (VRE), and Pseudomonas aeruginosa.
A strong activity and synergistic effect was demonstrated
against all the tested pathogens. The mechanisms of action
seems to be dependent on the production of a number of active
compounds such as proteins, fatty acids, anesthetics, organic
acids, volatiles, and hydrogen peroxide.
The field of living therapeutics is the most recent and fasci-
nating approach to the present crisis of antibiotic resistance. The
concept is based on the current day interest in the microbiome as
well as growing study on bacteria as probiotics. Unlike the un-
regulated use of probiotics such as in yogurt, kimchee, and other
foods, genetically modified bacteria will be studied and regulated
as drugs [19]. The U.S. Food and Drug Administration (FDA) is
reviewing the use of such products calling them “live microbial
vectors” to reflect drug delivery or live biotherapeutic products.
Informal names of designer probiotics and probiotics 2.0 have
also been given to these agents.
In the Meantime…
While awaiting potential breakthroughs like the ones de-
scribed above, we must still find ways to improve the man-
agement of infectious diseases with currently available modal-
ities. This includes:
1. The use of pharmacokinetic and pharmacodynamic prin-
ciples of antimicrobial therapy in addition to in vitro an-
timicrobial sensitivity testing. In this regard, beta lactam
agents like cefepime and piperacillin-tazobactam are
Indian J Pediatr
being used currently as extended infusions to optimize
the killing kinetics for the bacteria.
2. Combining antistaphylocccal beta lactams with vancomy-
cin has been shown to shorten the duration of treatment
for MRSA bacteremia. Combination therapy for carba-
penem resistant gram negative bacterial infections is also
being recommended. The most frequently used agents in
combination are carbapenems, tigecycline, fosfomycin,
aminoglycosides, and rifampicin.
3. Antibiotic cycling has been studied in the past as part of
antibiotic stewardship to maintain efficacy of agents left
out of use for defined periods of time. Kim et al. [20] used
whole genome sequencing and experimental evolution of
antibiotic resistance to present evidence that alternating
drugs slows the rate of resistance in bacteria compared
to same drug treatment. The authors speculate that alter-
nating therapy may impede horizontal acquisition of anti-
biotic resistance in addition to the major effect on
constraining de novo mutations.
4. Use of immunopropyhlaxis (active/vaccines and passive/
polyclonal and monoclonal antibodies) is a vast and di-
verse area. Used for prevention and treatment,
immunoprophylaxis has been undoubtedly more useful
for containment of human infectious diseases than the
antibiotics. Detailed discussion of these agents is beyond
the scope of this review.
5. Optimal and appropriate use of microbiological testing and
its interpretation is certainly an area that can be improved
upon. Its significance in the management of infectious dis-
ease is undisputable. The recent inclusion of faster method-
ology has certainly been helpful; however, we are lagging
behind in “point of care testing” that would give confidence
to the prescribers in not using antibacterials for viral infec-
tions and fevers of unknown origin which is a major area of
antibiotic overuse and/or misuse [21].
6. The role of regulatory agencies in restricting the use of
antibiotics as growth promotors for animals has already
been proven useful in European countries especially
Denmark [22]. As of today, the FDA is depending on
voluntary guidelines in the form of veterinary feed direc-
tive rather than laws.
Back to the Basics
Our experience, over-expectation and disappointment with
antibiotics clearly sends us toward the path of starting all over
again; i.e., to focus on understanding pathophysiology and
mechanisms of disease rather than memorizing a few antibi-
otic names. The most important aspect of this scientific ap-
proach is the understanding of steps involved and their inter-
ruption during host-pathogen interaction. Most infections
Indian J Pediatr
involve the evolution of a sessile biofilm where bacteria exist
in a viable but non-culturable state. It requires actual removal
or spontaneous dispersal to make them planktonic and
culturable. When we do have the luxury of a specimen grow-
ing bacteria, it is often lost to inattention, distrust, and lack of
understanding by the prescribers who often are the ones to
send the specimen to the laboratory. Instead they play
Russian roulette in order to choose an antibiotic and depend
on their preferences. This non-serious and random approach to
the management of infectious disease is the reason behind the
loss of the miracle of antibiotics. In the long term, the curricula
for medical and related professional education need to be
reassessed with renewed attention to pathophysiology, applied
elements of microbiology content, and pharmacology of anti-
microbial agents [23]. This is definitely easier said than done
especially in an era where going to classes is a “choice”.
Even if we stay optimistic for change and do find the best
antibiotics from all the approaches described above, this quote
will still be true. “Infectious Diseases is one of the great tragedies
of living things–the struggle for existence between two different
forms of life...Incessantly the pitiless war goes on, without quar-
ter or armistice–A nationalism of species against species.”
-Hans Zinsser–Rats, Lice, and History, 1935.
Compliance with Ethical Standards
Conflict of Interest None
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