Phosphonopeptides revisited, in an era of increasing antimicrobial resistance

Article abstract of DOI:10.3390/molecules25061445

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess

Full text of DOI:10.3390/molecules25061445

molecules
Article
Phosphonopeptides Revisited, in an Era of Increasing
Antimicrobial Resistance
Emma C.L. Marrs 1,2, Linda Varadi 3 , Alexandre F. Bedernjak 3, Kathryn M. Day 1, Mark Gray 3,
Amanda L. Jones 2, Stephen P. Cummings 2, Rosaleen J. Anderson 3,† and John D. Perry 1,
*
1
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK;
e.marrs@nhs.net (E.C.L.M.); notthisearth@hotmail.com (K.M.D.)
Department of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK;
2
amanda.l.jones@northumbria.ac.uk (A.L.J.); S.Cummings@tees.ac.uk (S.P.C.)
Sunderland Pharmacy School, University of Sunderland, Sunderland SR1 3SD, UK;
3
linda.varadi@rmit.edu.au (L.V.); abedernjak@reviral.co.uk (A.F.B.); mark.gray@sunderland.ac.uk (M.G.);
roz.anderson@sunderland.ac.uk (R.J.A.)
Correspondence: john.perry5@nhs.net
Deceased.
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 February 2020; Accepted: 20 March 2020; Published: 23 March 2020
Abstract: Given the increase in resistance to antibacterial agents, there is an urgent need for
the development of new agents with novel modes of action. As an interim solution, it is also
prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in
the context of widespread resistance to conventional agents. In the 1970s, much work was
performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin.
We investigated the activity of alafosfalin, di-alanyl fosfalin and β-chloro-L-alanyl-β-chloro-L-alanine
against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128),
methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci
(GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates
of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and
alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE.
Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis
(MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active
against MRSA (MIC90; 8 mg/L), whereas β-chloro-L-alanyl-β-chloro-L-alanine was slightly more
active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have
a therapeutic role to play in an era of increasing antibacterial resistance.
Keywords:
phosphonopeptides; alafosfalin; carbapenemase; antimicrobial resistance;
glycopeptide-resistant enterococci; MRSA
1. Introduction
The increasing resistance of pathogenic bacteria to antimicrobial agents is a substantial challenge to
microbiologists and the medical community in general. There is a worrying lack of new antimicrobials
in the pharmaceutical ‘pipeline’ and the lack of options for treatment of multi-resistant Gram-negative
bacteria, such as those producing carbapenemases, has been recognised for some years as a particular
concern [1–3]. a number of proposals have been made to address this issue, including the introduction
of new incentives for the pharmaceutical industry to return their attention to antibacterial drug
development. As an interim solution, it may be expedient to revisit old or abandoned antibacterial
compounds to re-evaluate their efficacy against infections for which there are few remaining treatment
Molecules 2020, 25, 1445; doi:10.3390/molecules25061445
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options [4]. The increasing role of agents such as colistin and fosfomycin as treatment options for
carbapenemase-producing Enterobacterales lends credence to this approach [5,6].
In the late 1970s, there was much interest in the use of peptide ‘mimetics’ as antibacterial agents.
These typically consisted of an antibacterial compound covalently linked to one or more amino acids
to facilitate uptake via the microbial peptide transport system. Intracellular hydrolytic cleavage
(via aminopeptidase activity) would then release the antibacterial ‘warhead’ to interact with its
target. Probably the best known agents of this type were synthesised by Roche and exemplified by
alafosfalin (L-alanyl-L-1-aminoethylphosphonic acid). Bacterial uptake of alafosfalin is accomplished
via LL-dipeptide permeases and subsequent hydrolysis yields fosfalin (L-1-aminoethylphosphonic
acid) which binds with alanine racemase; thus preventing synthesis of D-alanine, an essential ingredient
for peptidoglycan biosynthesis [
7]. Alafosfalin was shown to have a broad spectrum of antibacterial
activity [ ] against certain Gram-positive aerobes (Staphylococcus aureus, Enterococcus faecalis, but not
8
Group a streptococci and Streptococcus pneumoniae), anaerobic bacteria (Bacteroides spp. and Clostridium
perfringens, but not Clostridium difficile) and many species of Enterobacterales (but not Pseudomonas
or Acinetobacter). Against Gram-negative bacteria, alafosfalin is bactericidal, causing rapid lysis of
susceptible strains [9].
Other synthetic antibacterials developed around the same time exploited a similar route of
delivery and mode of action. For example, Cheung et al. explored the antibacterial activity of a range
of halogenated dipeptides exemplified by L-β-chloroalanyl-L-β-chloroalanine. The β-chloroalanine
released by hydrolysis interacts with a number of enzyme systems including alanine racemase and
also shows broad spectrum antibacterial activity [10].
In this study, we report the evaluation of three peptide mimetics: alafosfalin, di-alanyl
fosfalin and
β-chloro-L-alanyl-β-chloro-L-alanine (compounds A–C; see Figure 1 for structures,
full chemical names and abbreviations). These compounds were evaluated for antibacterial activity
with a collection of 297 bacteria that included a predominance of multi-drug resistant strains,
including carbapenemase-producing Enterobacterales (n = 128), methicillin-resistant Staphylococcus
aureus (n = 37) and glycopeptide-resistant enterococci (n = 43). Fosfomycin, a naturally occurring
antibiotic also containing a phosphonic acid group (D), was included for comparison.
Figure 1.
abbreviations used in this study) are as follows:
acid (alafosfalin); L-alanyl-L-alanyl-L-1-aminoethylphosphonic
fosfalin); -chloro- L-alanyl- -chloro-L-alanine -Cl-Ala- -Cl-Ala);
[(2R,3S)-3-methyloxiran-2-yl]phosphonate (fosfomycin).
Structures of four compounds used in this study.
Systematic names (and
(A
) L-alanyl-L-1-aminoethylphosphonic
(
B
)
acid
(di-alanyl
Disodium
(
C
)
β
β
(
β
β
(D)
2. Results
Tables 1 and 2 shows the minimum inhibitory concentrations (MICs) for the four antimicrobials
against the major groups of bacteria tested. Alafosfalin showed good activity against most isolates of
Enterobacterales, although different species showed different degrees of susceptibility. Strong activity
was shown against 53 isolates of Escherichia coli, of which 35 isolates (66%) were carbapenemase
producers. The MIC90 for E. coli was 0.25 mg/L and all isolates were inhibited by 2 mg/L. The activity

Molecules 2020, 25, 1445
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of alafosfalin was around fourfold higher than that of fosfomycin. Klebsiella pneumoniae was less
susceptible to all of the test compounds when compared with E. coli, although many isolates showed
relatively low MICs, e.g., 87% of K. pneumoniae isolates were inhibited by 8 mg/L alafosfalin. All isolates
of Enterobacter cloacae (n = 27) were inhibited by 4 mg/L alafosfalin, which was typically 16-fold more
active than fosfomycin against this species. Other species of Enterobacterales are not summarized in
Table 1 as less than 10 isolates were tested. For Citrobacter species (n = 9), Klebsiella aerogenes (n = 1),
Kluyvera sp. (n = 1), and Serratia marcescens (n = 1) all isolates were susceptible to 4 mg/L alafosfalin.
≤
One of five isolates of Klebsiella oxytoca required a MIC of >8 mg/L alafosfalin. Eight isolates of
Proteus mirabilis and two isolates of Providencia rettgeri required MICs of ≥8 mg/L for all agents tested
(including fosfomycin). Three isolates of Salmonella species required MICs of 8 mg/L for alafosfalin,
≥
whereas fosfomycin was more active against Salmonella (MICs: 0.125–0.25 mg/L).
Table 1. Minimum inhibitory concentrations of various antimicrobial agents against groups of
Gram-negative bacteria including isolates with defined resistance mechanisms.
Organism (No. Tested) and
Antimicrobial Agent
Concentration (mg/L)
Mode
MIC50
MIC90
Range
Enterobacterales (n = 197)
Alafosfalin
2
2
8
> 8
4
> 8
> 8
> 8
≤0.031–>8
≤ 0.031–> 8
2–> 8
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
> 8
> 8
4
> 32
0.125–> 32
E. coli (n = 53)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
0.063
0.25
8
0.125
0.5
8
0.25
2
> 8
1
≤ 0.031–2
≤ 0.031–> 8
2–> 8
0.5
0.5
0.125–8
K. pneumoniae (n = 87)
Alafosfalin
2
2
> 8
> 8
> 8
0.25–> 8
0.5–> 8
8–> 8
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
> 8
> 8
4
> 8
> 8
8
> 32
2–> 32
E. cloacae (n = 27)
Alafosfalin
1
4
> 8
16
1
1
0.125–4
0.25–> 8
8–> 8
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
> 8
> 8
16
> 8
> 8
32
4–> 32
CPE (n = 128)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
2
1
8
> 8
4
4
≤ 0.031–> 8
0.063–> 8
2–> 8
> 8
> 8
16
> 8
> 8
32
0.125–> 32
ESBL (n = 47)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
2
2
8
> 8
8
> 8
> 8
> 8
≤ 0.031–> 8
≤ 0.031–> 8
2–> 8
> 8
> 8
> 32
> 32
0.125–> 32
AmpC (n = 22)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
> 8
> 8
> 8
32
4
> 8
> 8
> 8
0.063–> 8
0.063–> 8
8–> 8
> 8
> 8
8
> 32
0.125–> 32
Abbreviations: MIC50: concentration of antimicrobial required to inhibit 50% of isolates. MIC90: concentration
of antimicrobial required to inhibit 90% of isolates. CPE: carbapenemase-producing Enterobacterales; ESBL:
Enterobacterales with extended spectrum β-lactamase; AmpC: Enterobacterales with acquired AmpC β-lactamase.

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Table 2. Minimum inhibitory concentrations of various antimicrobial agents against groups of
Gram-positive bacteria including isolates with defined resistance mechanisms.
Organism (No. Tested) and
Antimicrobial Agent
Concentration (mg/L)
Mode
MIC50
MIC90
Range
All S. aureus (n = 50)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
4
4
2
8
4
8
2
4
8
16
4
0.125–16
0.5–32
0.125–16
0.5–> 32
16
MRSA (n = 37)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
4
4
2
8
4
8
2
4
8
16
4
0.125–16
0.5–32
0.125–16
0.5–> 32
16
MSSA (n = 13)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
4
16
1
4
8
1
4
8
16
2
0.25–16
0.5–32
0.5–2
4
16
2–16
All Enterococci (n = 50)
Alafosfalin
8
0.5
16
16
0.5
16
> 32
2
32
4–> 32
≤ 0.016–> 32
2–16
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
> 32
> 32
> 32
16–> 32
E. faecalis (n = 11)
Alafosfalin
8
0.031
8
8
0.063
8
32
0.5
16
4–> 32
≤ 0.016–> 32
4–16
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
32
32
> 32
32–> 32
E. faecium (n = 34)
Alafosfalin
16
0.5
16
16
0.5
16
16
2
32
4–32
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
≤ 0.016–4
2–> 32
> 32
> 32
> 32
16–> 32
GRE (n = 43)
Alafosfalin
Di-alanyl fosfalin
β-Cl-Ala-β-Cl-Ala
Fosfomycin
16
0.5
16
16
0.5
16
> 32
> 32
> 32
> 32
4–> 32
≤ 0.016–> 32
4–> 32
> 32
> 32
32–> 32
Abbreviations: MIC50: concentration of antimicrobial required to inhibit 50% of isolates. MIC90: concentration of
antimicrobial required to inhibit 90% of isolates. MRSA: methicillin-resistant S. aureus; MSSA: methicillin-susceptible
S. aureus; GRE: glycopeptide-resistant enterococci.
The results of chequerboard synergy testing are shown in Table 3. For 16 of 20 Enterobacterales
producing the five most common carbapenemases, synergy was observed between alafosfalin and
meropenem as determined by a FICI ≤ 0.5.

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Table 3. Interaction between alafosfalin and meropenem against carbapenemase-producing
Enterobacterales as determined using a chequerboard technique.
Meropenem MIC
Species
Carbapenemase Alafosfalin MIC (mg/L)
FICI
Interpretation
(mg/L)
K. oxytoca
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
E. coli
E. coli
E. cloacae
K. pneumoniae
E. coli
E. cloacae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. oxytoca
K. pneumoniae
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
KPC-2
KPC-3
KPC-3
KPC-4
KPC
NDM-1
NDM-1
NDM-1
NDM-1
NDM-1
VIM-1
VIM-1
IMP
IMP
IMP
OXA-48
OXA-48
OXA-48
OXA-48
OXA-48
4
1
1
0.5
0.25
1
16
4
0.25
8
0.125
4
2
16
4
16
1
1
0.5
8
0.25
1
4
2
0.16
0.25
0.19
0.38
0.38
0.75
0.76
1
0.27
0.25
0.19
0.28
0.37
0.50
0.13
0.38
0.37
0.5
Synergy
Synergy
Synergy
Synergy
Synergy
No interaction
No interaction
No interaction
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
Synergy
1
0.125
0.031
1
2
0.125
2
2
0.25
0.25
1
1
0.125
2
2
0.26
0.63
1
No interaction
Abbreviation: FICI; Fractional inhibitory concentration index.
Compared to alafosfalin, β-Cl-Ala-β-Cl-Ala showed relatively poor activity against Gram-negative
bacteria but it showed the highest activity against Staphylococcus aureus; however, there was
little difference overall between the three peptide-based antimicrobials. Ninety percent of
methicillin-resistant S. aureus (MRSA) isolates were inhibited by 8 mg/L alafosfalin. Against enterococci,
the most notable observation was the high activity of di-alanyl fosfalin, for which the MICs were
(on average) 16-fold lower than those of alafosfalin and in some cases 256-fold lower (Table 2).
Of 34 isolates of Enterococcus faecium (including 31 glycopeptide-resistant isolates), all were inhibited
by 32 mg/L alafosfalin or 4 mg/L Di-alanyl fosfalin.
3. Discussion
The re-evaluation of previously abandoned antibacterial agents is a credible interim solution to the
problem of dwindling treatment options created by increasing bacterial resistance. To our knowledge,
alafosfalin was never licensed for clinical use and consequently there are no approved breakpoints
to define susceptibility, but there are a number of published reports on the experimental use on
alafosfalin in animals and volunteers [
animal models, such as the mouse septicaemia model employed by Allen et al. that showed successful
treatment of E. coli, K. pneumoniae and E. faecalis using alafosfalin [ ]. In a study of human volunteers,
9,11–14]. Treatment of infections has been demonstrated in
9
Allen and Lees reported that oral doses ranging from 50–2500 mg of alafosfalin were well absorbed
and well tolerated [12]. However, after oral administration of the drug, some hydrolysis prior to
absorption resulted in a bioavailability of approximately 50%. In a further study with volunteers,
Welling et al. confirmed that alafosfalin is rapidly absorbed with peak serum concentrations of 4–9 mg/L
appearing within 1 h of a 500 mg oral dose [14]. It is also rapidly eliminated by the kidneys with
a serum half-life of 1 h or less. They also reported that bioavailability following oral dosing could
be significantly improved if the dose was given with milk rather than water. More recent work has
shown that alafosfalin is actively transported across the intestinal epithelium due to its high affinity for
H+ / peptide symporters, PEPT1 and PEPT2 [15]. In animal models, Allen et al. have reported that
alafosfalin shows no significant binding to serum proteins and is distributed in most tissues except
for liver and brain. In human volunteers, the same group reported that administration of 200 mg
(intramuscular) or 500 mg (oral) doses consistently resulted in peak plasma levels of 5–10 mg/L with
mean urine concentrations in excess of 50 mg/L for the first 6 h after dosing [11]. These concentrations
were maintained on chronic administration but urine levels were adversely affected in patients with

Molecules 2020, 25, 1445
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impaired renal function. The rate of absorption and elimination of alafosfalin was found to be similar
for published data on -lactam antibiotics [11] and synergy with -lactams such as cephalexin and
β
β
mecillinam, as well as other cell-wall inhibitors (e.g., D-cycloserine), has been well documented due to
a ‘multi-blockade’ of cell wall biosynthesis [9,13,16,17].
Alafosfalin has a number of advantageous properties as an antimicrobial including a broad
spectrum of activity, low toxicity, good absorption, low protein binding, a bactericidal mode of action,
and a target site that is distinct from that of other licensed antimicrobials. It also has a number of
limitations including its tendency to be metabolized (thereby reducing bioavailability), a reduced
activity at alkaline pH, a relatively high ‘inoculum-effect’, and, according to some reports, the relative
ease with which bacteria can acquire resistance [8,18]. Despite this latter claim, in a comprehensive
review Ringrose concluded that spontaneous mutants with resistance to alafosfalin arise at a relatively
low frequency of 10−8 to 10−9 due to the need for mutations in multiple permeases [19]. Given the
limitations, it is understandable that alafosfalin was not actively pursued in an era when a wide range
of alternative antimicrobial agents were available for most infections.
However, some of these limitations are shared by other antimicrobials that are relied upon for
routine treatment. The ease with which bacteria can develop resistance to alafosfalin can be reduced
by combining it with other established antimicrobials as demonstrated previously [13]. This study has
shown that alafosfalin in combination with meropenem has a synergistic interaction against many
highly resistant isolates of carbapenemase-producing Enterobacterales. We have shown that alafosfalin
(and other peptide-based antimicrobials) retains strong activity against some bacterial isolates that
are now difficult to treat using conventional antimicrobials. Given the large amount of data already
available on the pharmacokinetics and safety of alafosfalin, it may be worth revisiting this agent as
a potential treatment for infections caused by isolates that are otherwise difficult to treat.
Phosphonopeptides such as alafosfalin have a unique target site that is not shared by any other
antimicrobial agent in current use, and further research is warranted to ensure that this does not
remain unexploited. As we have shown in this study, other peptide mimetics, such as di-alanyl
fosfalin, may show substantial antimicrobial activity against E. coli and particularly enterococci.
However, at present there is insufficient information on the pharmacokinetics and toxicity of other
compounds to speculate further on their therapeutic potential. Atherton et al. have recommended
other phosphonopeptides with improved potency and broader antibacterial spectrum [20] and
these are also worthy of further investigation in the era of widespread antimicrobial resistance to
conventional antimicrobials.
4. Materials and Methods
4.1. Antibacterial Agents and Media
Fosfomycin, alafosfalin, glucose-6-phosphate and all ingredients of the antagonist-free agar were
purchased from the Sigma Chemical Company, Poole, UK. IsoSensitest agar was purchased from
Oxoid, Basingstoke, UK. The synthesis and characterisation of the two other antimicrobial agents is
described in Supplementary section S1.
4.2. Bacterial Isolates
Enterobacterales (n = 197) were obtained from diverse international sources and all possessed
β-lactamases that had been defined at a molecular level by reference laboratories and/or recognized
experts in the field. These included Citrobacter freundii (n = 5), other Citrobacter species (n = 4),
Klebsiella aerogenes (n = 1), Enterobacter cloacae (n = 27), Escherichia coli (n = 53), Klebsiella oxytoca (n = 5),
Klebsiella pneumoniae (n = 87), Kluyvera species (n = 1), Proteus mirabilis (n = 8), Providencia rettgeri
(n = 2), Salmonella species (n = 3), and Serratia marcescens (n = 1). Of these 197, there were 128
(65%) carbapenemase producers including isolates with NDM-1 (New Delhi Metallo
β-lactamase;
n = 87), IMP (imipenemase; n = 9), KPC (Klebsiella pneumoniae carbapenemase; n = 11), OXA-48

Molecules 2020, 25, 1445
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(oxacillinase-48; n = 14) and VIM (Verona integron-encoded metallo-β-lactamase; n = 7). Most of the
carbapenemase-producers co-produced ESBL or AmpC
here for the sake of clarity. Of the remaining isolates of Enterobacterales, 47 produced extended
spectrum -lactamases including isolates with CTX-M (n = 20), SHV-type (n = 19), TEM-type (n = 8),
and 22 had acquired AmpC -lactamase including isolates with ACC-1 (n = 3), CMY-type (n = 6),
DHA-1 (n = 6), FOX-type (n = 3) and LAT-type (n = 4). Isolates with acquired AmpC -lactamase
β-lactamases, but these are not documented
β
β
β
included K. pneumoniae (n = 11), P. mirabilis (n = 5), E. coli (n = 3), Salmonella species (n = 2) and K. oxytoca
(n = 1).
A collection of 50 isolates of Staphylococcus aureus included 36 strains of MRSA frequently
encountered in Europe, including strains isolated in Belgium, Finland, France, Germany, and the
United Kingdom. Another MRSA strain, NCTC 11939, was included as a control, as well as
a methicillin-susceptible control (NCTC 6571). a further twelve isolates of methicillin susceptible
S. aureus (MSSA) recently recovered from blood cultures were included. Finally, 50 isolates of enterococci
included two control strains (Enterococcus faecalis NCTC 775 and Enterococcus faecium NCTC 7171) and
48 isolates from clinical samples obtained from at least three different hospitals. The clinical isolates
included E. faecalis (n = 10), E. faecium (n = 33), Enterococcus casseliflavus (n = 3), and Enterococcus
gallinarum (n = 2). Of the 50 isolates, 43 were resistant to vancomycin as demonstrated by MIC testing
and confirmation of resistance genes by PCR.
4.3. Determination of MICs
All MICs were determined using an agar dilution method [21]. This necessitated the use of a defined
antagonist-free medium (peptone-free), prepared as previously described [7] with the inclusion of 2%
saponin-lysed horse blood, 25 mg/L NAD and 25 mg/L haemin. Test compounds were dissolved in
sterile deionised water and incorporated into agar at a concentration range of 0.031–8 mg/L (0.016–32
mg/L for Gram-positive bacteria). All isolates were prepared at a density equivalent to 0.5 McFarland
units in sterile deionised water using a densitometer (bioM
é
rieux, Basingstoke, UK) to produce an
inoculum of approximately 1.5
×
108 CFU/mL, and then diluted 1 in 15. a 1
µL aliquot of each diluted
suspension was then delivered onto plates with a multipoint inoculator (Denley Instruments Limited,
Cambridge, UK) to give a final inoculum of 10,000 CFU/spot as recommended [21]. Fosfomycin MICs
were performed using the same method except that IsoSensitest agar (Oxoid, Basingstoke, UK) was
used supplemented with 25 mg/L glucose-6-phosphate (Sigma, Poole, UK) and an extended range of
fosfomycin concentrations. All plates, including the antimicrobial-free control, were incubated for 22 h
at 37 ◦C.
4.4. Study of the Interaction between Alafosfalin and Meropenem Against Carbapenemase-Producing Enterobacterales
Twenty isolates were selected that showed varying susceptibility to meropenem (susceptible,
intermediate and resistant) as defined by CLSI guidelines [22]. These included isolates with KPC
(n = 5), NDM-1 (n = 5), OXA-48 (n = 5), IMP (n = 3) and VIM (n = 2) carbapenemases. Interaction was
examined using a checkerboard technique with agar plates incorporating various concentrations of
meropenem (0, 0.031–64 mg/L) and alafosfalin (0, 0.016–8 mg/L). The medium used was antagonist-free
agar (as described above) supplemented with 70 mg/L zinc sulphate to ensure optimal activity
of metallo β-lactamases [23]. Plates were inoculated and incubated as described for MIC testing.
Fractional inhibitory concentration indices (FICI) were calculated as follows: [(MIC of meropenem in
combination with alafosfalin)/(MIC of meropenem alone)] + [(MIC of alafosfalin in combination with
meropenem)/(MIC of alafosfalin alone)], and a FICI of 0.5 was used to define synergy. All experiments
≤
(including MIC testing) were performed on at least two separate occasions to ensure that only
reproducible results were reported.

Molecules 2020, 25, 1445
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5. Concluding Remarks
In light of the emerging threat posed by antimicrobial resistance it is imperative that we find
not only new treatments for resistant bacterial infections, but also new diagnostic tools so that
the physician can rapidly identify a suitable course of treatment for the presenting patient [24,25].
This paper focuses upon the first of these requirements, and serves as a reminder that there are
many useful compounds for treatment of bacterial infections that are already known, but have lay
dormant in some cases for decades because at the time of their initial discovery there were many
other options available. However, the situation has changed markedly in recent years. We show
here that the phosphonopeptides are potentially useful weapons for treating drug resistant infections,
including those posed by some of the pathogens that clinicians find to be most challenging at the
present time.
As mentioned above it is also important for the clinician to be armed with suitable tools to help
distinguish key pathogens so as to be able to determine a suitable course of treatment in a minimal
timeframe. One other use of antimicrobial agents is deployment within selective culture media,
wherein selectivity in antimicrobial activity can be exploited to suppress the growth of commensal
bacteria present within a patient derived sample, thus allowing a non-inhibited pathogen to grow
freely which in turn allows for clear and prompt diagnosis [26]. In the period subsequent to the work
disclosed within this paper we performed systematic modifications to the structures of the inhibitors
with this other goal in mind. The results of these subsequent investigations form the basis of our
second and third papers which may be found within this Special Issue of molecules dedicated to
microbial detection and identification [27,28].
Supplementary
L-alanyl-L-alanyl-L-1-aminoethylphosphonic acid (di-alanyl fosfalin) and
(β-Cl-Ala-β-Cl-Ala) is available online at http://www.mdpi.com/1420-3049/25/6/1445/s1.
Materials:
Supplementary
section
S1
describing
the
synthesis
of
β
-chloro- L-alanyl-
β-chloro-L-alanine
Author Contributions: Conceptualization, J.D.P., E.C.L.M. and R.J.A.; methodology, J.D.P., E.C.L.M. and R.J.A;
formal analysis, L.V., A.F.B., M.G.; investigation, E.C.L.M., K.M.D.; resources, J.D.P., R.J.A., A.L.J. and S.P.C.; data
curation, E.C.L.M. and J.D.P.; writing J.D.P., E.C.L.M., R.J.A. and M.G.; writing—review and editing, J.D.P. and
M.G.; supervision, J.D.P., R.J.A., A.L.J., M.G. and S.P.C.; project administration, J.D.P., R.J.A., A.L.J. and S.P.C.;
funding acquisition, J.D.P., R.J.A., A.L.J. and S.P.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded, in part, by bioMérieux, La-Balme-les-Grottes, France.
Acknowledgments: The authors are most grateful to the following collaborators who generously shared isolates of
Enterobacterales with characterized resistance mechanisms: Patrice Nordmann, Hôpital de Bicêtre, France; Gilles
Zambardi, bioM
érieux, La Balme les Grottes, France; Enno Stürenburg, Universitätsklinikum Hamburg-Eppendorf,
Germany; and Jaroslav Hrab
á
k, Plzen Charles University, Prague. We are also grateful to Public Health England,
Colindale, UK for providing a collection of diverse MRSA strains.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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