An Overview of Multi-Antibiotic Resistance in Pathogenic Bacteria - From Selected Genetic and Evolutionary Aspects - A Review

TL;DR: This review is aimed at informing newcomers, coming from the field of biology and genetics, about problems related to rapidly emerging, new multi-drug resistant, pathogenic, bacteria.

Abstract: The challenge posed by multi-drug resistance (MDR) of pathogenic organisms, spectacularly manifested in the 6 “ESKAPE” bacterium (two Gram-positive, four Gram-negative) species, should invoke new comprehensive strategies, and needs cooperation of scientists with medical, veterinary and natural science background. This review is aimed at informing newcomers, coming from the field of biology and genetics, about problems related to rapidly emerging, new multi-drug resistant, pathogenic, bacteria. Unlike persistence, the antibiotic resistance is inherited. A functioning “resistance gene” makes a susceptible organism resistant to a given antibiotic, encoding for polypeptides capable of acting either as decomposing enzymes, or acting as trans-membrane pumps, or membrane structure components capable of modifying the permeability implementing a «by pass» mechanism enabling the antibiotic molecule to reach its cellular target(s). A functioning “sensitivity gene” encode for a polypeptide, capable (directly or indirectly) of transferring toxic molecules into target cells, or of metabolizing non-transferable to transferable, or non-toxic molecules to toxic derivatives. A gene of a normal function could act as a “sensitivity” gene in the presence of antibiotics of chemical structures similar to the natural substrate of the gene product, (enzyme or binding/ trans-membrane protein). The Agrocin 84 story is a good example. Multi-drug resistance is a phenotypic consequence of the sequential accumulation of mutations, and/or up-take of plasmids or genomic islands carrying resistance genes from the environment via horizontal gene transfer, mediated by conjugative plasmid or bacteriophage carrying mobile genetic elements. Both multi-drug resistance and collateral sensitivity are evolutionary products. Some revealed evolutionary process and their Lamarckian and Darwinian interpretations are discussed. Toolkits of comparative full-genome sequencing, genomics, experimental evolution and population genetics may provide perspectives for overcoming the invincibility of multi-drug panresistance. The status of some recently emerging pathogenic bacterium species with zoonic features and of veterinary background is also discussed.

Summary

1. Introduction

• (2) Some genetic and evolutionary mechanisms, leading to increase or decrease in the frequencies of multi-drug.
• This subsection is restricted only to the species "officially" registered in the ESKAPE "club" and tries to draw the attention of the respected Reader to some new candidates.

2.2.1. Methicillin resistant Staphylococcus aureus, MRSA

• From this important discovery, one concludes pessimistically that genomic sources of intrinsic antibiotics resistances in genus Staphylococcus are non-exhaustible.
• The reviewers hope that both the options provided by the possibly very high number of structure/activity combinations, as well as by the natural sources of antimicrobial peptides effective in MRSA, are also non-exhaustible.

2.2.4. Acinetobacter baumannii, the "queen" of multi-drug resistant bacteria

• The other Gram-negative opportunistic human pathogen bacterium species creating an extraordinarily large scale of MDR phenotypes is A. baumannii.
• This species is probably even a worse pathogen than P. aeruginosa, with an extremely large pangenome and heterogeneous accessory genome.
• It has a continuously enlarging set of virulence and resistance armament, as first reviewed by Bonomo and Szabó in the literature [89] .
• Since then A. baumannii has been considered as a threatening source of resistance coding genes transmittable via horizontal gene transfer.
• The WHO declared that A. baumannii is one of the most serious ESKAPE organisms that effectively escape the effects of antibacterial drugs [116] .

2.2.4.5.3. Efflux pumps

• The EmrAB-TolC efflux pump is also present in A. baumannii and responsible for resistance to netilmicin, tobramycin and imipenem [201] .
• A1S-1535 confers resistance to gentamicin, kanamycin, chloroxylenol, oxytetracycline, 1, 10-phenanthroline, and chloramphenicol [202] .
• A1S-2795 is responsible for resistance to the sulphonamide sulfathiazole, and ABAYE-0913 is associated with resistance to chloramphenicol and fusidic acid [202] .

2.2.4.5.5. Biofilm formation

• Biofilm formation plays an important role not only in the immune evasion by A. baumannii [220] , but persistence as well.
• Imipenem treatment of the imipenem-resistant A. baumannii isolate induces expression of important genes responsible for synthesis of type IV pili [221] , the existence of which is needed for biofilm formation.

2.2.5.2.4. Resistance mechanisms to antibiotics that interfere with protein synthesis

• Cross-resistance with all macrolides is a result of the modification of the 23S rRNA target.
• Resistance to tetracyclines and glycylcyclinesis is mediated by multiple genes, but follows two general strategies: efflux of the antibiotics and ribosomal protection.
• Mutations in the genes, gyrA and parC, (present in E. faecium and E. faecalis, but absent from E. gallinarum and E. casseliflavus) affect the quinolone resistance-determining regions, which presumably alter the binding affinity of the antibiotic quinolones, the target enzymes (DNA gyrase and topoisomerase IV) that are responsible for DNA supercoil relaxation [53, 228, 261] .
• Rifampicin resistance arises from a variety of mutations in the rpoB gene that encodes for the β-subunit of the RNA polymerase [228] .
• Trimethoprim and sulfamethoxazole (inhibitors of bacterial enzymes involved in the folate synthesis pathway) are ineffective in vivo to enterococci, because they are those extreme rare bacteria which can utilize exogenous sources of folate [228, 53] .

2.3.1. Mycoplasma bovis

• But, there were 3 of the 35 Hungarian field strains for which the fluoroquinolone MICs were high [272] .
• Fluoroquinolone-resistant mutants were also selected in vitro for danofloxacin, enrofloxacin and marbofloxacin and each showed complete cross-resistance with the others.
• The respective mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were mapped into genes encoding 23S rRNA [273] .

2.3.2. Bacillus anthracis

• Correlations were not observed between the isolation date, location, host species, genotype, and antibiotic susceptibility profile of the strain.
• A similar study in Cameroon showed that Bovine B. anthracis isolates from there showed a strong homogeneity, and they belong, together with strains from Chad, to a cluster Aβ, which appears to be predominant in western Africa [282] .
• One strain that belongs to a newly defined clade (D) and cluster (D1) was penicillin resistant.

2.3.3. Francisella tularensis

• Each isolate belonged to F. tularensis subsp.
• Each strain was susceptible to those antibiotics which have commonly been used in therapy, such as aminoglycosides, gentamicin, streptomycin, tetracycline, doxycycline, quinolones, ciprofloxacin, levofloxacin, and chloramphenicol, and in addition tigecycline and rifampicin.
• Naturally, they were resistant to erythromycin and linezolid.

2.3.4. Escherichia coli

• The latest British simulation experiments have supported this prognosis [288] .
• The authors developed an in vitro chemostat system to approximate the chicken caecal microbiota, simulated colonization by an MDR Salmonella pathogen and examined the dynamics of transfer of its MDR plasmid, harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1.
• They also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity.
• Bacterial community profiles, obtained by culture-independent methods, showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered.
• MDR plasmid transfer from Salmonella to commensal E. coli was demonstrated by the polymerase chain reaction (PCR) technique, and whole-genome sequencing of isolates purified from agar plates containing cefotaxime.

3.3.1. Morbidostat and experimental evolution of intrinsic antibiotic multiresistance

• As the authors saw, in the "war" against antibiotic resistance, they have just been losing battle after battle.
• It has had moderate success, usually in combination with a carbapenem.
• Unfortunately, colistin resistance has been evolving at the clinical level [310] .
• As a wonderful example of justifying the validity of the classical Mendelian genetics, strong and unambiguous intergenic epistasis seems to limit the number of possible evolutionary pathways for antibacterial peptide resistance.
• These dominant suppressor mutations serve then as kind of "nodes potentiating further steps in the evolutionary process leading to higher resistance" by increasing/channelizing the effects of other mutations, see https://www.ncbi.nlm.nih.gov/ pubmed/27694971, (available publicly) [310] .

3.3.2. Experimental evolution of intrinsic antibiotic resistance and collateral sensitivity

• They sequenced the whole-genome of each of their laboratory-evolved aminoglycoside-resistant strains, and demonstrated multiple mechanisms underlying aminoglycoside resistance, including a reduced proton-motive force (PMF) through the inner membrane.
• They suggested that, as a pleiotropic consequence, these mutations diminished the activity of PMF-dependent antibiotics efflux pumps (such as the AcrAB transporter), resulting in hypersensitivity toward other antibiotics.
• The authors believe that this Hungarian discovery is of science historic value, allowing the fight against resistance development to new antibiotics, to be compensated by "reactivating" some previously used ones.
• The authors consider that the discovery of collateral sensitivity has an extremely great theoretical and practical impact, but probably relevant only for mutation-based, "intrinsic" resistances [311] .

3.4. MDR Revolution in genus Enterococcus

• While the pathogenesis of ES remains poorly understood, recent evidence suggests that the increased incidence of enterococcal-associated disease in poultry may be due to horizontal spread of dominant clones of E. cecorum that exhibit increased pathogenicity [327, 328] .
• A recent study was directed at investigating the genetic relatedness and antimicrobial resistance of isolates recovered from spondylitis lesions and caeca of affected/ unaffected birds from geographically and temporally distinct outbreaks of ES in the southeastern United States [327] .
• From these 22 epidemiologically distinct outbreaks, 326 isolates of E. cecorum were recovered.
• Phenotyping used GenIII MicroPlate™ (Biolog; Hayward, CA, USA), microbial identification plates, and antimicrobial sensitivity testing.
• Isolates from spinal lesions were incapable of mannitol metabolism, and the majority of these isolates were genetically clonal.

Full text

Fodor A. et al (2018) Multidrug resistance in bacteria…a review (Preprint)
1
An overview of multi-antibiotic resistance in pathogenic bacteria - from selected genetic 1
and evolutionary aspects - A review 2
András Fodor
1,
*
,$
, Birhan Addisie Abate
2
, Péter Deák
1
, László Fodor
3
, Michael G. Klein
4,&
László Makrai
3
, 3
Josephat Muvevi
5
, and Dávid Vozik
6,#
4
5
1
Department of Genetics, University of Szeged, H-6726 Szeged, Középfasor 52; (
$
Present mail address: 6
5651 Fredericksburg Road, Wooster, OH 44691, USA); E-mail (András Fodor): csingicsangi@gmsil.com; 7
fodorandras@yahoo.com; E-mail (Péter Deák): deakp@bio.u-szeged.hu 8
9
2
Ethiopian Biotechnology Institute, Agricultural Biotechnology Directorate; E-mail: Birhan Addisie 10
birhanaddisie@gmail.com 11
12
3
Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, 1581 Budapest, P.O. Box 13
22, Hungary; E.mail (László Fodor): Fodor.Laszlo@univet.hu; E.mail (László Makrai): Makrai.Laszlo@univet.hu 14
15
4
Department of Entomology, The Ohio State University, Madison Avenue, Wooster, OH-44691, USA; (
&
Present 16
mail address: O. Box 1104, Heber, AZ 85928, USA). E.mail: klein10@osu.edu 17
5
Plant Biotechnology Research Division, Addis Ababa, Ethiopia;
4
National Cereals and Produce Board, P.O. Box 18
84696-80100, Mombasa, Kenya. E.mail: jmuvevi@gmail.com 19
20
6
Research Institute on Bioengineering, Membrane Technology and Energetics, Faculty of Engineering, University of 21
Pannonia, Egyetem utca 10, 8200 Veszprém, H-8200, Hungary; (
#
Present mail address: Transdanubian Regional 22
Waterworks Co., Department of Technology, Siófok, Hungary); E.mail: vozikd@gmail.com 23
24
25
*Corresponding author: csingicsangi@gmail.com; fodorandras@yahoo.com, ORCID 0000- 0003-3495-00154 26
. 27
28
ABSTRACT 29
The challenge posed by multi-drug resistance (MDR) of pathogenic organisms, spectacularly manifested in the 6 30
“ESKAPE” bacterium (two Gram-positive, four Gram-negative) species, should invoke new comprehensive strategies, 31
and needs cooperation of scientists with medical, veterinary and natural science background. This review is aimed at 32
informing newcomers, coming from the field of biology and genetics, about problems related to rapidly emerging, new 33
multi-drug resistant, pathogenic, bacteria. Unlike persistence, the antibiotic resistance is inherited. A functioning 34
“resistance gene” makes a susceptible organism resistant to a given antibiotic, encoding for polypeptides capable of 35
acting either as decomposing enzymes, or acting as trans-membrane pumps, or membrane structure components 36
capable of modifying the permeability implementing a «by pass» mechanism enabling the antibiotic molecule to reach 37
its cellular target(s). A functioning “sensitivity gene” encode for a polypeptide, capable (directly or indirectly) of 38
transferring toxic molecules into target cells, or of metabolizing non-transferable to transferable, or non-toxic 39
molecules to toxic derivatives. A gene of a normal function could act as a “sensitivity” gene in the presence of 40
antibiotics of chemical structures similar to the natural substrate of the gene product, (enzyme or binding/ 41
trans-membrane protein). The Agrocin 84 story is a good example. Multi-drug resistance is a phenotypic consequence of 42
the sequential accumulation of mutations, and/or up-take of plasmids or genomic islands carrying resistance genes 43
from the environment via horizontal gene transfer, mediated by conjugative plasmid or bacteriophage carrying 44
mobile genetic elements. Both multi-drug resistance and collateral sensitivity are evolutionary products. Some revealed 45
evolutionary process and their Lamarckian and Darwinian interpretations are discussed. Toolkits of comparative 46
full-genome sequencing, genomics, experimental evolution and population genetics may provide perspectives for 47
overcoming the invincibility of multi-drug panresistance. The status of some recently emerging pathogenic bacterium 48
species with zoonic features and of veterinary background is also discussed. 49
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 August 2018 doi:10.20944/preprints201808.0036.v1
© 2018 by the author(s). Distributed under a Creative Commons CC BY license.

Fodor A. et al (2018) Multidrug resistance in bacteria…a review (Preprint)
2
KEYWORDS: ESKAPE-bacteria; Persistence; Resistance; Intrinsic/Acquired/ Multidrug (MDR) and Pan – 50
Resistance; Genetic background; Experimental Evolution; Collateral sensitivity; Agrocin. 51
52
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Fodor A. et al (2018) Multidrug resistance in bacteria…a review (Preprint)
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TABLE OF CONTENTS 53
1. Introduction 54
2. The “Card Game” of antibiotics research scientists and antibiotic resistance 55
2.1. Multi-drug resistance: Definitions and 56
nomenclature 57
2.2. The “ESKAPE Club” of omnipotent 58
multi-drug resistant bacterium species 59
2.2.1. Methicillin resistant Staphylococcus 60
aureus, MRSA 61
2.2.2. Extended spectrum β-lactamase 62
(ESBL) producing Enterobacteriaceae 63
and Klebsiella pneumoniae 64
2.2.3. Pseudomonas aeruginosa, a pan- 65
genomic hot bed of multi-drug 66
resistance 67
2.2.4. Acinetobacter baumannii, the queen 68
of multi-drug resistant bacteria 69
2.2.5. Enterococci: The Gram-positive 70
“Vanguards” of the “MDR 71
movement” 72
2.3. Zoonic and veterinary pathogen candidates 73
for the “ESCAPE Club” 74
2.3.1. Mycoplasma bovis 75
2.3.2. Bacillus anthracis 76
2.3.3. Francisella tularensis 77
2.3.4. Escherichia coli 78
3. Evolutionary aspects 79
3.1. Prelude: Lamarck and Darwin 80
3.2. Tolerance, persistence, & resistance 81
3.3. Evolution of antibiotic multi-resistance 82
and collateral sensitivity 83
3.3.1. Morbidostat and experimental 84
evolution of intrinsic antibiotic 85
resistance 86
3.3.2. Experimental evolution of intrinsic 87
antibiotic resistance and collateral 88
sensitivity 89
3.4. MDR Revolution in genus Enterococcus 90
4. “Dialectics” of resistance and sensitivity: the Agrocin story in genus Agrobacterium 91
5. Closing remarks 92
ACKNOWLEDGEMENT 93
CONFICT OF INTEREST STATEMENT 94
REFERENCES 95
96
97
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Fodor A. et al (2018) Multidrug resistance in bacteria…a review (Preprint)
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1. Introduction 98
Multi-drug resistance (MDR) of pathogenic bacteria is an extremely complex field of life sciences that needs the 99
expertise of physicians, (docs, vets), microbiologist, biochemists, theoretical and preparative organic chemists, 100
bioinformatics, geneticists, and evolutionary biologists. This review is prepared first of all for geneticists and biologists 101
who are newcomers to life science background without clinical or veterinary experience. It is well known today, that 102
although the antibiotics are extremely important therapeutic tools in human, veterinary and even plant medicine, 103
their use has gradually become limited because of resistance problems. The phenomenon of antibiotic-resistance was 104
first discovered as early as 1940 [1]. Whenever pathogenic microorganisms are exposed to the selective pressure of 105
antimicrobials, either in the laboratory, medicine, or agriculture, it is favorable for the development, survival and 106
spread of resistant clones [2]. Resistance means non-susceptibility to given antibiotics. When an isolate of a given 107
pathogenic bacterium is resistant to more than one antibiotic, the options for antibiotic therapy of the disease caused by 108
this pathogen is decreased. The emergence of antibiotic multi-resistance in pathogenic bacteria has become alarming in 109
the recent decades. As an example, 1481 patients died in Hungarian hospitals in 2016. In 174 of those deaths, 110
infection was the cause of death, or was involved in it, according to the (Hungarian) National Epidemiological 111
Center. Last year, 4830 MDR infections were reported, compared with 4187 in 2015 and 3998 in 2014. Mostly urinary 112
tract infections occurred, followed by infected wounds, blood vessel infections, and hospital-related pneumonia. A 113
majority of the patients were above 60 [3]. 114
Infections caused by multi-resistant bacteria have dramatically increased not only in Hungary, but all over the 115
world, invoking an enormous public concern. There are not human clinical, [4-7] but zoonic [8] and veterinary [9-116
15], as well as plant health aspects [16-18] come forward alarmingly. 117
A spectacular plant example is the increasing number of streptomycin-resistant Erwinia amylovora isolates, (the 118
pathogen of the “fire blight” of Rosacea, including apple trees) causing serious difficulties in the treatment of severe 119
plant infections both in the USA [19] and in Europe [20]. Although application technology has been improving 120
revolutionarily [21], the trend is that the application of antibiotics for clinical use as plant medicines has been 121
increasingly more restricted [22]. 122
All this has been motivating research to introduce not only new antibiotics, but environmentally friendly plant 123
medicines as well, with novel modes of action. A rational approach for elaborating effective therapies has been 124
based on the better understanding of the different bacterial mechanisms of drug resistance, especially for Gram-125
negative pathogens, [4, 5, 7]. 126
When reporting a radical and continuous decrease in the number of new antibiotics in the market, Canadian authors 127
[23] asked in 2005: 128
- “Where are all the new antibiotics?” 129
Eleven-years later, a late answer appeared in ‘Nature’: 130
- “Antibiotics” (are) “right under our nose”! [24]. 131
It is good news and may be true. But unfortunately they still have not been in the market; at least not in the required 132
numbers. Their number seems to be much less than needed for really effective control of multi-resistant pathogens 133
[4]. In the period 2003-2007 only 5 new antibiotics appeared. In 2009 there were 16 new molecules listed as being in 134
clinical trials phase, with only 2 in the pipeline; including 3 glycopeptides, 4 quinolones, 2 oxazolidinones, 2β-135
lactams, 1β-lactamase inhibitor, 1 trimethoprim, 1 macro-ketolide, 1 streptogramin, and 1 glycyl-cycline [25]. 136
It is encouraging that well-qualified top scientists have been working on better understanding of the process, solving 137
the newly appearing and spreading resistance problems, and working on new approaches all over the world [26]. The 138
scientific approach based on a better understanding of the different bacterial, genetic and evolutionarily mechanisms 139
of drug resistance [5], aimed at “reducing the bottle neck in the discovery of new antibiotics” [27]. This new approach is 140
based on transcriptome analysis, and exploiting the options provided by using RNA sequencing (RNASeq) to 141
identify promising novel antimicrobial compounds from microbial extracts. 142
The analysis of the reasons of the moderate interest of the pharmaceutical industry toward new antibiotics is not in the 143
scope of this article. 144
This review intends to focus on a few selected aspects, such as: 145
(1) A short overview of the list of the most significant bacterial pathogens which cause the most striking examples of 146
MDR outbreaks; 147
(2) Some genetic and evolutionary mechanisms, leading to increase or decrease in the frequencies of multi-drug 148
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Fodor A. et al (2018) Multidrug resistance in bacteria…a review (Preprint)
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resistant pathogen bacteria around us; 149
(3) Some evolutionary and coevolutionary mechanisms (co-existence, horizontal gene transfer) channelizing these 150
two-way movements, weighted by the genetic load of newly acquired antibiotic resistance. 151
2. The “Card Game” of antibiotics research scientists and antibiotic resistance. New antibiotic drugs 152
invoke new resistances; it is just question of time. 153
2.1. Multi-drug resistance: Definitions and nomenclature (Based on phenotype and origin of multi-drug 154
resistance) 155
The resistance to an antimicrobial compound means non-susceptibility to a given antibiotic molecule. From practical 156
aspects, one can distinguish between multi-resistant pathogens based on qualitative and quantitative profiles. A recent 157
classification defines (i) multi-drug resistant (MDR) strains and isolates, which are not susceptible to (at least) one 158
representative of each of three categories of antimicrobial compound families; (ii) extreme drug resistant, (XDR), 159
which are not susceptible to (at least) one representative of all but very few categories of antimicrobial compound 160
families; and (iii) pan-drug resistant (PDR) ones, which are not susceptible to any of the tested representatives of all 161
known antimicrobial compound families [28]. 162
The resistance to an antimicrobial compound is an inherited character (phenotype) determined by the presence and 163
expression of a respective “resistance gene”. This gene can be localized in the bacterial chromosome, or in an 164
extrachromosomal element, which is most frequently a plasmid, and in a rarer, but worse case, an episome, capable 165
of being inserted into the chromosome permanently. The origin of the resistance could be a mutation, changing the gene 166
which had originally been present, resulting in structural and functional changes of the original gene product. When 167
this is the case, the literature calls it “intrinsic” resistance. If the resistance to an antimicrobial compound is a 168
phenotypic consequence of the activity of a resistance gene that has been harbored by a plasmid taken-up from the 169
environment, the literature calls it “acquired” resistance [29]. An antibiotic resistance gene is most frequently a coding 170
gene, (an open reading frame) located, organized and regulated in a so-called antibiotics resistance cassette, which is 171
most frequently harbored and transferred by some mobile genetic element. Considering that mobile genetic elements 172
are capable of separating from, and integrating into, any available bacterial DNA (chromosome, plasmid or even 173
phage), and vice-versa, that event, called horizontal gene transfer (HGT), is possible in more than one step between 174
bacteria, (including pathogens of rather different taxa), on condition that the plasmid is compatible with the new 175
“host”. The gastro-intestinal track of humans (and of course that of the animals), has a densely populated mixed 176
microbial community (“microbiota”), and therefore it is an optimal “market place” for such exchanges during horizontal 177
gene transfer In addition, hospitals are ideal meeting place for pathogens harboring different resistance genes [30]. 178
2.2. The “ESKAPE Club” of omnipotent multi-drug resistant bacterium species 179
In 2006 The Antimicrobial Availability Task Force (AATF) of Infectious Diseases Society of America (IDSA) 180
prepared a review that highlighted frequently resistant pathogens to licensed antimicrobials, and for which only a few, 181
if any, potentially effective drugs are shown in late-stage drug development [31]. This “Six Bad Bugs” originally 182
comprised a notorious group of 5 pathogen bacterium species, and Aspergillus, characterized by an enormously high 183
rate of antibiotic resistance, and extremely versatile MDR phenotypes, that are responsible for the majority of 184
nosocomial infections [31]. 185
The present list (without the fungus Aspergillus), includes 6 bacterium species called the ESKAPE Pathogen 186
Bacterium Species list. The name of the six letters involve the initials of genus names of these bacteria: Enterococcus 187
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and 188
Enterobacter [32]. The explanation: these groups of bacteria may produce omniresistant (panresistant) pathogen 189
strains, against which there is “NO DRUG” (no protecting antibiotics), and therefore there is “NO ESKAPE” [32]. 190
The “club” of the worst 6 “bad bugs” includes 4 extended spectrum β-lactamase (ESBL)-producing Gram-negatives - 191
A. baumannii, P. aeruginosa; Enterobacteriaceae species (such as E. coli), Klebsiella pneumoniae – and 2 Gram 192
positive - methicillin resistant S. aureus (MRSA) and vancomycin-resistant gastrointestinal Enterococci, (E. 193
faecalis, E. faecium) – “club-members”. 194
This internationally accepted list which has been refreshed yearly, and should be considered still authentic, but 195
probably will be expanding soon, and may not be considered as complete. This authentic list has been renewing 196
from time-to-time [4], and has been expected to be expanded. The Clostridium genus for instance, which provides 197
examples of MDR pathogens, (in C. difficile: see [33, 34]; in C. perfringens: see [35]), is not included, but is a potential 198
candidate. Similarly, the Salmonella genus has not been included in the ESKAPE list, despite alarming publications 199
related with signs of MDR pathogen evolution in this taxonomic group [36-40]. 200
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Frequently asked questions

Q1. What are the contributions in this paper?

This review is aimed at 32 informing newcomers, coming from the field of biology and genetics, about problems related to rapidly emerging, new 33 multi-drug resistant, pathogenic, bacteria. Some revealed 45 evolutionary process and their Lamarckian and Darwinian interpretations are discussed. 20944/preprints201808. 0036. v1 © 2018 by the author ( s ).