1. A composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa comprising at least one antibiotic against Pseudomonas aeruginosa, and at least one antibody or antibody fragment thereof against Pseudomonas aeruginosa wherein said antibody or antibody fragment is of Seq. D 1 targeting C4 dicarboxylate transporter of Pseudomonas aeruginosa; said ß lactam antibiotic is selected from the group consisting of carbapenems, amoxicillin, and pipericillin; said antibody or antibody fragment of Seq. D 1 shows MIC-50 at a concentration of 6.25 µg/mL, MIC-90 at a concentration of 12.5 µg/mL, and MIC-99 at a concentration of 25 µg/mL against P. aeruginosa model strain ATCC 27853 and ß-lactam antibiotic-resistant strain MCC 50428.

2. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises a carbapenem selected from the group consisting of meropenem, and imipenem.

3. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 16 µg/ml concentration of meropenem, and 6.25 – 50 µg/ml concentration of Seq. ID 1.

4. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 8 µg/ml concentration of meropenem, and 6.25 – 50 µg/ml concentration of Seq. ID 1.

5. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 4 µg/ml concentration of meropenem, and 50 µg/ml concentration of Seq. ID 1.

6. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 32 µg/ml concentration of meropenem, and 1.58 – 50 µg/ml concentration of Seq. ID 1.

7. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 8 µg/ml concentration of imipenem, and 12.5 µg/ml concentration of Seq. ID 1.

8. The composition effective against antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 64 µg/ml amoxicillin and 12.5 µg/ml concentration of Seq. ID 1.

9. The composition effective against antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 64 µg/ml pipericillin and 12.5 µg/ml concentration of Seq. ID 1

Description:FIELD OF THE INVENTION
The present disclosure relates to a composition effective against multi-antibiotic-resistant Pseudomonas aeruginosa.
BACKGROUND OF THE INVENTION
Worldwide, infection due to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. (ESKAPE) group that are multidrug-resistant (MDR) pathogens in critical care settings have emerged as a new health care crisis. In 2013, the Centres for Disease Control and Prevention and World Health Organization (2017) listed these pathogens as a serious threat. Among them, the carbapenem resistant Enterobacteriaceae, Pseudomonas, and Acinetobacter were placed in the highest threat priority category. Some of the P. aeruginosa strains are resistant to nearly all antibiotics, including carbapenems and are common in hospitalized patients especially the immuno-compromised with a lung and urinary tract infections, skin infections in burn patients, diabetic ulcer and cystic fibrosis that cause increased morbidity and mortality due to suboptimal efficacy and cross-resistance to multiple drugs.
Carbapenems (meropenem and imipenem) and ß Lactams are frontline drugs that have been widely used against many drug-resistant bacterial infections. Although carbapenems are widely used for treating P. aeruginosa, the prevalence and surge of resistant strains in recent years have made them less effective which necessitates the need to develop newer therapeutic options.
One of the ways of overcoming this problem is to develop newer antibiotics to which the existing bacteria are not resistant to. Newer small molecule antibiotics are constantly being developed but these suffer from the same fatal flaw, mutation-based resistance in the bacteria. Antibiotics often attach to a small target inside a bacterial cell, and bind to it and inactivates it. A simple point mutation in the target protein often is sufficient to inactivate this mechanism as a result of which the antibiotic becomes ineffective. This happens rapidly after the antibiotic is deployed with the first cases of resistance appearing within a few months.
Alternative strategies are also being developed to control drug resistant Pseudomonas. These are biologicals that are antibodies, peptides, phages or vaccines. As they have a much more complex mechanism of action as compared to the small molecules, these molecules are resilient to forces of evolution that cause resistance in the first place and have an extended product life cycle.
Camelids (camels, llamas, and alpacas) are known to produce unconventional single heavy chain antibodies that are devoid of light chain known as VH from which the antigen binding domain VHH can be isolated for various applications. Despite their small size (one-tenth) than conventional antibodies, they have a high affinity for antigens due to their unique structural arrangement. They have additional advantages over conventional antibodies such as ability to bind to cryptic domains on the antigen, higher thermal and chemical stability and ease of production by simple microbial systems that has enabled their use for therapeutic and diagnostic applications. VHH fragments have been exploited as drug candidates against many pathogens and parasites, such as Listeria monocytogenes, Leishmania infantum and Coronaviruses as well.
Combinatorial molecules have been developed to increase the efficacy and druggable properties of antibodies which include drug-antibody conjugates, antibiotic-antibody conjugates or pegylated antibodies etc. These are mostly covalently linked conjugates either at the nucleic acid level to directly produce fusion proteins or are linked in vitro later. These conjugates comprise one molecule each of drug or antibiotic, and the antibody. Such conjugates have been effective against resistant pathogens. However, the major limitation is that it is 1:1 molecule ratio as well as the complex chemistry involved to generate them which may not always be useful in terms of production costs.
Therefore, the need of the hour is to develop antibody drug conjugates that are simple in structure and can be produced and purified by a single step chromatography procedure in adequate quantities which not only limit the cost of production, but are more effective due to novel targets they attach to and enable to tackle the pathogen resistance problem.
The present invention takes into consideration the drawbacks of the prior art and provides a synergistic composition effective against multidrug resistant Pseudomonas aeruginosa.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a novel composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa.
Another object of the invention is provide a synergistic composition comprising 1) antibody or antibody fragment or a variant thereof targeting Pseudomonas aeruginosa of Seq. ID 1, and 2) ß-lactam antibiotic against Pseudomonas aeruginosa to reverse antibiotic -resistance of Pseudomonas aeruginosa.
Yet another object of the invention is to provide a solution to increasing antibiotic drug resistance seen in Pseudomonas aeruginosa by providing a composition capable to reverse antibiotic -resistance of Pseudomonas aeruginosa.
BRIEF SUMMARY OF THE INVENTION
The problem underlying the present invention is solved by the subject matter of the attached independent claims. Preferred embodiments may be taken from the attached dependent claims.
The present invention relates to a composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa. The composition comprises 1) antibody or antibody fragment or a variant thereof targeting Pseudomonas aeruginosa, and 2) ß-lactam antibiotic against Pseudomonas aeruginosa.
In one embodiment, the invention provides a composition effective against multi antibiotic-resistant Pseudomonas aeruginosa comprising neutralising antibody fragment targeting C4 dicarboxylate transporter of Pseudomonas aeruginosa to block the dicarboxylate transporter. This transporter is usually present on the cell membrane of P. aeruginosa and other bacteria and facilitates the uptake of C4 carbon sources that is fed into the glyoxylate shunt that is operative under conditions of oxidative stress in the host. Under oxygen limiting conditions when the TCA cycle is not fully operative glyoxylate shunt is used for energy generation and anabolic activities and has a critical survival value in pathogenic bacteria. It is present in bacteria but is absent in the eukaryotic host, making it a suitable target for drug development. C4 dicarboxylate transporter is involved in efflux of antibiotics thus rendering the pathogen resistant to multiple drugs. The binding of the antibody fragment to its target in Pseudomonas aeruginosa results in blocking of efflux mediated resistance. This makes the composition of the antibody fragment against C4 dicarboxylate transporter and the antibiotic a synergistic combination which mitigates the problem of antibiotic resistance to the two classes of antibiotics.
In one embodiment, the invention provides a composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa comprising antibody fragment of Seq. ID 1 targeting a component of C4 dicarboxylate transporter of Pseudomonas aeruginosa.

In another embodiment, the invention provides a composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa comprising antibiotic selected from the group consisting of carbapenems and other ß lactams.
In another embodiment, the composition comprises ß lactam antibiotic such as amoxicillin, and piperacillin.
In yet another embodiment, the composition comprises carbapenem antibiotic selected from the group consisting of meropenem, and imipenem.
A pharmaceutical composition for any of the uses described herein may be formulated for administration to a patient who show antibiotic-resistance towards ß lactams and carbapenems in Pseudomonas aeruginosa. Further, the pharmaceutical composition may comprise an anti-C4 dicarboxylate transporter of Pseudomonas aeruginosa, more specifically antibody fragment of Seq. ID 1 that is formulated for administration orally, intravenously, intramuscularly, subcutaneously or by insufflation. Further, in any of the uses described herein where a pharmaceutical composition is administered to a patient, the antibiotic may be formulated for topical application, intravenous, intramuscular, intradermal subcutaneous or intratracheal insufflation.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts a graphical representation of neutralization experiment of P. aeruginosa with the supernatant of induced E. coli secreting anti-Pseudomonas VHH fragments from pADL vector as quantitated by measurement of OD625 after 16 hours;
Fig. 2a depicts graphical representation of quantitation of binding specificity and cross-reactivity of purified PsC23 VHH by whole-cell ELISA to both strains of P. aeruginosa model strain ATCC 27853 and the antibiotic-resistant strain MCC 50428, and other pathogens from the ESKAPE group namely- S. aureus, K. pneumoniae, A. baumanni, and E. faecalis;
Fig. 2b depicts graphical representation of specificity of the neutralization action of PsC23 VHH as quantitated by their effect on the growth of P. aeruginosa model strain ATCC 27853 and the antibiotic-resistant strain MCC 50428, and different human gut bacteria namely- E. coli, L. casei, L. plantarum, L. fermentum, L. acidophilus, and B. subtilis;
Fig. 2c depicts graphical representation of specificity of the neutralization action of PsC23 VHH as quantitated by their effect on the growth of P. aeruginosa model strain ATCC 27853 and the antibiotic-resistant strain MCC 50428 and different human pathogens namely- S. aureus, K. pneumoniae, A. baumanni, E. faecalis, S. paratyphae, and S. haemolyticus;
Fig. 3a depicts graphical representation of growth kinetics of P. aeruginosa ATCC 27853 strain after treatment with 25 µg/mL, 50 µg/mL and 100 µg/mL PsC23 VHH or Seq. ID 1 over a period of 24 hrs;
Fig. 3b depicts graphical representation of growth kinetics of antibiotic-resistant P. aeruginosa MCC 50428 strain after treatment with 25 µg/mL, 50 µg/mL and 100 µg/mL PsC23 VHH or Seq. ID 1 over a period of 24 hrs;
Fig. 4a depicts graphical representation of growth kinetics of P. aeruginosa strain ATCC 27853 in buffer (negative control), P. aeruginosa strain ATCC 27853 in the presence of meropenem (8 µg/mL) (M + P. aeruginosa 27853), antibiotic-resistant MCC 50428 strain in the presence of meropenem (8 µg/mL) (M + P. aeruginosa 50428), antibiotic-resistant MCC 50428 strain in the presence of PsC23 or Seq. ID 1 (12.5 µg/mL) and meropenem (8 µg/mL) (PsC23 + M + P. aeruginosa 50428);
Fig. 4b depicts graphical representation of growth kinetics of P. aeruginosa strain ATCC 27853 in buffer (negative control), P. aeruginosa strain ATCC 27853 in the presence of imipenem (8 µg/mL) (I + P. aeruginosa 27853), antibiotic-resistant MCC 50428 strain in the presence of imipenem (8 µg/mL) (I + P. aeruginosa 50428), antibiotic-resistant MCC 50428 strain in the presence of PsC23 or Seq. ID 1 (12.5 µg/mL) and imipenem (8 µg/mL) (PsC23 + I + P. aeruginosa 50428);
Fig. 4c depicts checker board analysis to evaluate the combination of meropenem and PsC23 or Seq. ID 1 antibody effective in inhibition of P. aeruginosa antibiotic-resistant MCC 50428;
Fig. 5 is graphical representative of total ion current image of the mass spectrometry-based analysis of Seq. ID 1 antibody target;
Fig. 6a depicts graphical representation of growth kinetics of P. aeruginosa MCC 50428 strain under anaerobic conditions in minimal medium supplemented with 40mM of C4 carbon source- fumarate alone, or fumarate in combination with PsC23 or Seq. ID 1;
Fig. 6b depicts the effect of PsC23 or Seq. ID 1 on the growth of P. aeruginosa MCC 50428 strain under anaerobic conditions in minimal medium supplemented with 40mM of C4 carbon source- malate alone, or malate in combination with PsC23 or Seq. ID 1;
Fig. 7 is a pictorial depiction of the hypothetical role of C4 dicarboxylate transporter and glyoxylate shunt in efflux mediated drug resistance,
Fig. 8a depicts effect of meropenem (8 µg/mL), PAßN (20 µg/mL), and PsC23 or Seq. ID 1 (25 µg/mL) individually, and in combinations (meropenem with PsC23, and meropenem with PAßN) on the growth of meropenem resistant P. aeruginosa MCC 50428;
Fig. 8b depicts effect of imipenem (8 µg/mL), PAßN (20 µg/mL), and PsC23 or Seq. ID 1 (25 µg/mL) individually, and in combination (imipenem with PsC23, and imipenem with PAßN) on the growth of carbapenem-resistant P. aeruginosa MCC 50428;
Fig. 9 depicts a graphical representation of effect of beta-lactam antibiotics on growth of antibiotic-resistant P. aeruginosa MCC 50428 over 8 hours - in absence of any antibiotics, in presence of amoxicillin (64 µg/mL) alone, or in combination with PsC23 or Seq. ID 1 (12.5 µg/mL) and amoxicillin (64 µg/mL), pipericillin (64µg/mL) alone, or in combination with PsC23 or Seq. ID 1 (12.5 µg/mL) and pipericillin (64 µg/mL);
Fig. 10a depicts bacterial load estimation in blood of BALB/c mice (Log10 CFU/mL) after 24 hrs of infection with antibiotic-resistant P. aeruginosa MCC 50428 without treatment (buffer control), with meropenem (5mg/kg) alone, PsC23 or Seq. ID 1 (5mg/kg) alone, and combination of PsC23+ meropenem (5 mg/kg each); and
Fig. 10b depicts percentage survival of uninfected BALB/c mice, or antibiotic-resistant P. aeruginosa MCC 50428 infected neutropenic BALB/c mice when treated with meropenem (5 mg/kg) alone, PsC23 or Seq. ID 1 (5 mg/kg) alone, combination of PsC23 with meropenem (5mg/kg each), or no treatment (buffer treated) monitored over a period of 72 hrs.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
In the description that follows, a number of terms are used and the following definitions are provided to facilitate understanding of the present invention.

As used herein, “antibody fragment” refers to polypeptides or proteins that bind to specific antigens. It also means immunoglobulins, not limited to polyclonal, monoclonal, chimeric, humanized antibodies, Fab fragments, F(ab’)2 fragments and likewise.
A “minimum inhibition concentration', or “MIC' refers to the lowest dose of a drug or antibiotic that will inhibit the visible growth of a microorganism after overnight incubation.
The term “MIC-99” as used herein refers to minimal inhibitory concentration for killing 99% microorganisms.
The term “MIC-90” as used herein refers to minimal inhibitory concentration for killing 90% microorganisms.
The term “MIC-70” as used herein refers to minimal inhibitory concentration for killing 70% microorganisms.
The term “MIC-50” as used herein refers to minimal inhibitory concentration for killing 50% microorganisms.
As used herein, “synergy” refers to an effect in combination where the end result is greater than the effect obtained with the sum of each of the parts of the combination taken separately.
The term “VHH” as used herein refers to an antigen binding fragment of antibody derived from camels which is composed only of fragments of heavy chains and does not comprise any light chains; it is also called as nanobody. Typically, an IgG antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region (encoded by VHH, D and J elements) and a constant region.
The term “PsC23” as used herein refers to polypeptide of Seq. ID 1 which is an antibody fragment (VHH) targeting C4 dicarboxylate transporter of P. aeruginosa.

Hereinafter, preferred modes for carrying out the present invention are described. The embodiments described below are given merely for illustrating one example of a typical embodiment of the present invention and are not intended to limit the scope of the present invention.
The main object of the present invention is to provide a composition that is effective against antibiotic-resistant Pseudomonas aeruginosa, which is non-toxic to host or other beneficial gut residing host bacteria.

Accordingly, in the main embodiment, the present invention provides a composition effective against antibiotic-resistant Pseudomonas aeruginosa comprising:
1) antibody or antibody fragment or a variant thereof targeting Pseudomonas aeruginosa, and
2) antibiotic against Pseudomonas aeruginosa,
wherein,
the antibody or antibody fragment or a variant thereof targets C4 dicarboxylate transporter of Pseudomonas aeruginosa, and
the antibiotic is selected from the group consisting of ß lactams, and or carbapenems, which are commonly used antibiotics to treat P aeruginosa infections.

In one embodiment, the invention provides a composition effective against antibiotic-resistant Pseudomonas aeruginosa comprising antibody fragment of Seq. ID 1 targeting C4 dicarboxylate transporter of Pseudomonas aeruginosa with high specificity.

Seq. ID 1 alone shows growth inhibition towards the model strain P. aeruginosa ATCC 27853 and the antibiotic-resistant strain MCC 50428. The MIC-50, 90 and 99 that correspond to growth inhibition by 50%, 90% and 99% are 6.25, 12.5, and 25 µg/mL respectively for both the model strain ATCC 27853 and the antibiotic-resistant strain MCC 50428. Treatment of P. aeruginosa ATCC 27853 and the antibiotic-resistant strain MCC 50428 with Seq. ID 1 alone at MIC 99 concentration 25 µg/mL and above decreased and restricted the bacterial growth up to 2 hours. After 2 hours both the strains recovered from the growth inhibition and showed logarithmic growth. This indicated that the antibody fragment alone is not sufficient for arresting bacterial growth for long.
Further, antibiotic-resistant Pseudomonas aeruginosa, are known to resistant to most of the available antibiotics.

Therefore, in another embodiment, the present invention provides a novel composition comprising:
1) Seq. ID 1 antibody fragment or a variant thereof targeting C4 dicarboxylate transporter of Pseudomonas aeruginosa, and
2) ß lactam antibiotic selected from the group consisting of carbapenems and other ß lactams.
ß lactams, and carbapenems (a sub-group of ß lactams) are generally effective against Pseudomonas aeruginosa but are ineffective against the multi drug resistant P. aeruginosa MCC 50428
In yet another embodiment, the invention provides a composition comprising 8-16 µg/ml concentration of meropenem, and 6.25 – 50 µg/ml concentration of Seq. ID 1 for 100% bactericidal effect against ß lactam antibiotic-resistant Pseudomonas aeruginosa.
In yet another embodiment, the invention provides a composition comprising 4 µg/ml concentration of meropenem, and 50 µg/ml concentration of Seq. ID 1 for 100% bactericidal effect against ß lactam antibiotic-resistant Pseudomonas aeruginosa.
In yet another embodiment, the invention provides a composition comprising 8 µg/ml concentration of imipenem, and 12.5 µg/ml concentration of Seq. ID 1 for 100% bactericidal effect against ß lactam antibiotic-resistant Pseudomonas aeruginosa.
In yet another embodiment, the invention provides a composition comprising 64 µg/ml amoxicillin and 12.5 µg/ml concentration of Seq. ID 1 for 100% bactericidal effect against ß lactam antibiotic-resistant Pseudomonas aeruginosa.
In yet another embodiment, the invention provides a composition comprising 64 µg/ml piperacillin and 12.5 µg/ml concentration of Seq. ID 1 for 100% bactericidal effect against ß lactam antibiotic-resistant Pseudomonas aeruginosa.
In yet another embodiment, the invention provides a composition comprising Seq. ID 1 which shows high binding affinity towards ATCC 27853 model strain and MCC 50428 ß lactam antibiotic-resistant strain of P. aeruginosa, but minimal affinity towards other ESKAPE pathogens such as K. pneumoniae, S. aureus. E. faecalis, and A. baumannii, E. coli and Salmonella sp.
In yet another embodiment, the invention provides a composition comprising Seq. ID 1 which shows specificity towards P. aeruginosa including ATCC 27853 model strain and MCC 50428 ß lactam antibiotic-resistant strain and minimal specificity towards beneficial probiotic gut bacteria thereby, providing a composition which has minimum effect when consumed orally.
EXAMPLE 1
Generation of Seq. ID 1
a) Construction of P. aeruginosa VHH library and isolation of the neutralizing hits
A 7–10-year-old Indian dromedary camel (Camelus dromedarius) was infected with a meropenem-resistant P. aeruginosa (strain MCC 50428). Briefly, the overnight grown culture of meropenem-resistant P. aeruginosa (strain MCC 50428) was kept for inactivation in PBS and a mixture (1:1 v/v) of inactivated P. aeruginosa (~1 x 109 CFU) and alum mineral oil adjuvant was injected subcutaneously on 1st, 30th and 45th day. Heparinized blood (60 mL) was collected from the jugular vein on 60th day. Peripheral blood mononuclear cells (PBMCs) were isolated and a buffy coat was collected. Total RNA was extracted from PBMCs and used for cDNA generation. Using the camelid antibody-specific primers VHH fragments were amplified. The amplified VHH fragments were then digested and cloned in phagemid and transformed in E. coli ER2738. The library size was determined by plating various dilutions on 2X YT agar containing?carbenicillin (100 µg/mL) and 2% glucose. 10 randomly selected colonies were subjected to colony PCR to check the presence of the VHH insert. Freshly ligated VHH-vector constructs were transformed into E. coli ER 2738 by electroporation, a library (CIL-Ps) of 2×109 clones were obtained. Colony-PCR screening revealed that over 90% of these colonies contained VHH gene constructs.
a) Enrichment and selection of P. aeruginosa binding VHH hits by phage display
Enrichment of phages expressing hits was carried in immunotubes pre-coated with whole-cell P. aeruginosa. Three consecutive rounds of bio-panning enriched the population of Pseudomonas-specific binding phages. These phages were allowed to infect E. coli ER 2738 and screened for antigen binding hits by phage-ELISA using anti-M13-HRP conjugated antibodies.
Colonies that gave 3-5 folds more reading than negative control were identified as optimal binding hits. 3-8 such hits were isolated from each plate and categorized into strong, moderate and non-binders. The strongest binders were tested for their neutralizing activity by leaky expression method developed in house. Out of the 32 binders, five clones namely, PsC11, PsC23, PsC30, PsC56 and PsC79 were found to have a neutralizing activity.
As depicted in Fig. 1, PsC11, PsC23, PsC30, and PsC79 inhibited the growth of P. aeruginosa, whereas PsC56 was less effective comparatively. The uniqueness of the neutralizing hits was determined by RFLP. Three of the neutralizing hits PsC11, PsC23 and PsC56 showed a unique restriction digestion pattern, while two clones namely PsC30 and PsC79 were found to be identical. One of the clones PsC23 that distinctly inhibited both strains of P. aeruginosa was identified as a hit for further characterization.
b) Identification of P. aeruginosa neutralizing VHH hits by leaky expression analysis
Hits having a high reading in phage-ELISA compared to negative control were further evaluated for their neutralization ability against P. aeruginosa. One of the clones (PsC23) which showed consistent neutralization and strong binding in ELISA was sub-cloned into E. coli expression vector pET-28c (+).
PsC23 antibody is of Seq. ID 1 of the present invention.
Table 1 Sequence information
Seq. ID No. Name Amino acid Sequence
1 PsC23 HMDVQLQESGGASVQPGGSLLISCEASGLASYSNYCIM
WFRQPPGKEREGVAGINLRSGITYYAEAVRPRFTISADS
VDGRFAISQDNARNTVYLQMNSLKPEDTAIYYCAAGNL
CGGSWSGYRYWGQGTQVTVSSLE

c) Cloning, expression and purification of Seq. ID 1
Seq. ID 1 was cloned in pET-28c expression vector and expressed in E. coli C41(D3). Seq. ID 1 was purified from culture and the yield was 3-4 mg/L.
The purified Seq. ID 1 protein was stored in 50 mM phosphate-citrate buffer at -20°C for further use.
d) Antigen binding and cross-reactivity of Seq. ID 1
The antigen-binding activities were measured by modified whole-bacterial cell ELISA. The binding affinity of Seq. ID 1 to ESKAPE pathogens (P. aeruginosa, K. pneumoniae, S. aureus. E. faecalis, and A. baumannii, with the exclusion of Enterobacter spp) was evaluated. For this purpose, the model strain ATCC 27853 and the antibiotic-resistant strain MCC 50428 of P. aeruginosa were used.
As depicted in Fig. 2a, the intensity of binding in terms of OD450 was higher for both strains, ATCC 27853 and MCC 50428 strains of P. aeruginosa as compared to other bacteria. This indicated that Seq. ID 1 has a high binding specificity to P. aeruginosa and has a nominal cross-reactivity with other bacterial pathogens from the ESKAPE group.
Since Seq. ID 1 is an antibody fragment, it should have specificity towards a particular pathogen. To check the neutralizing specificity of Seq. ID 1 towards other bacteria associated with human health, a purified Seq. ID 1 antibody (25 µg/mL) was incubated with Pseudomonas aeruginosa as well as different bacteria (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, and Staphylococcus haemolyticus) at 1 x 106 CFU/mL in MH broth at 37°C and growth (OD 625 nm) was measured after 8 h.
As depicted in Fig. 2c, purified Seq. ID 1 inhibited Pseudomonas sp. within 8 hours but did not significantly affect the growth of other tested pathogens (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella enterica, Staphylococcus haemolyticus). Further, as depicted in Fig. 2b Seq. ID 1 did not inhibit the growth of normal human gut bacteria also. This indicated that the Seq. ID 1 was very specific and did not have much off target activity and also did not affect gut bacteria which are beneficial to human health. Thus, it could be used for development of P. aeruginosa specific targeted therapeutics and diagnostics and it would be gut friendly when administered internally, unlike almost all antibiotics which disturb the gut bacteria extensively.
e) Minimal Inhibitory Concentration (MIC) and pathogen-specificity and time kill kinetics of PsC23
Minimal Inhibitory Concentration (MIC) can be defined as the lowest concentration of antimicrobial agent required for the inhibition of the bacterial growth. The MIC-50, 90 and 99 of Seq. ID 1 for both P. aeruginosa strains (ATCC 27853 and MCC 50428) were determined. Various concentrations 1X (25 µg/mL), 2X (50 µg/mL), 4X (100 µg/mL) times MIC-99 of Seq. ID 1 were inoculated with 1 x 106 CFU/mL of both P. aeruginosa strains and evaluated for the inhibitory activity.
The MIC of Seq. ID 1 for two strains of P. aeruginosa, ATCC 27853 and antibiotic-resistant strain MCC 50428 was evaluated in vitro after 4 hrs of treatment. MIC-50, 90 and 99 that correspond to growth inhibition by 50%, 90% and 99% were found to be 6.25, 12.5, and 25 µg/mL respectively for both these test strains. This suggests that the purified antibody fragment (Seq. ID 1) is efficacious irrespective of the resistance profile and might be effective against other antibiotic-resistant P. aeruginosa strains as well.
f) Carbapenem and effect of the efflux channel blocker on antibiotic-resistant P. aeruginosa MCC 50428
A time-kill assay of Seq. ID 1 against both strains (ATCC27853, MCC50428) of P. aeruginosa at 1X (25 µg/mL), 2X (50 µg/mL) and 4X (100 µg/mL) of the effective dose was performed and monitored the number of viable cells over the period 2, 4, 8, and 24 hrs. Fig. 3a depicts the graphical representation of the time kill assay of ATCC 27853 strain, and Fig. 3b represents MCC 50428 ß lactam antibiotic resistant strain. The difference in the growth profiles was not significant at these concentrations indicating that a threshold might have been obtained at 25 µg/mL, the most pronounced effect was seen within 2 hrs after which the cells recovered and a gently upward sloping curve was observed. Moreover, the effect of the antibody seems to be maximum around 2 hrs, after which the population seems to recover slightly.
However, as depicted in Fig. 4a and 4b, when the ß lactam antibiotic-resistant P. aeruginosa MCC 50428 strain was treated with combination of Seq. ID 1 (12.5 µg/mL) and carbapenems (8 µg/mL meropenem and imipenem), a bactericidal effect was observed to be more pronounced and no further growth was seen after 4 hrs.
This clearly showed that the combination of Seq. ID 1 (12.5 µg/mL) and carbapenems had a synergistic effect over Seq. ID 1 alone, and Seq. ID 1 alone as shown in Fig. 3a and Fig. 3b could not induce severe bactericidal effect.
To evaluate the effective combination of meropenem and Seq. ID 1 antibody in reversing resistance in ß lactam antibiotic resistant P. aeruginosa MCC 50428, 106 cells were grown under various combinations of meropenem (32µg/ml - 0.5µg/ml) and Seq. ID 1 antibody (50µg/ml - 0.79µg/ml) at 37°C for 24 hrs and optical density was recorded periodically. A checkerboard was made to indicate the probable combinations of these two drugs that were most effective in inhibiting the growth.
As depicted in Fig. 4c, ß lactam resistance was completely reversed in the following combinations:
1. 16 µg/ml of meropenem, and 6.25 – 50 µg/ml of Seq. ID 1, where 6.25 µg/ml of Seq. ID 1 was sufficient to reverse meropenem resistance in P. areuginosa MCC 50428;
2. 8 µg/ml concentration of meropenem, and 12.5 – 50 µg/ml concentration of Seq. ID 1, where 12.5 µg/ml of Seq. ID 1 was sufficient to reverse meropenem resistance in P. aeruginosa MCC 50428;
3. 4 µg/ml concentration of meropenem, and 50 µg/ml concentration of Seq. ID 1;
4. 32 µg/ml concentration of meropenem, and 1.58 – 50 µg/ml concentration of Seq. ID 1, where 1.58 µg/ml of Seq. ID 1 was sufficient to reverse meropenem resistance in P. aeruginosa MCC 50428.
This showed that only 4 µg/ml of meropenem and 50 µg/ml of the Seq. ID 1 is sufficient to reverse meropenem resistance in P. aeruginosa MCC 50428 in vitro. When the amount of meropenem was increased to 8, 16 and 32 µg/ml, the Seq. ID 1 dose went down to 12.5, 6.25 and 1.58 µg/ml. These data give an idea of the range of concentrations Seq. ID 1 can be used to reverse meropenem resistance, the optimal ratio of which can be fixed depending on the druggable properties of the combination and economy factor.
g) The target of Seq. ID 1 is a component of the C4 dicarboxylate transporter as identified by mass spectrometry.
Mass spectrometry analysis identified a component of C4 dicarboxylate transporter as a binding target of Seq. ID 1 VHH with high confidence. Fig. 5 depicts the Representative total ion current image of the mass spectrometry-based analysis of antibody target. This transporter is usually present on the cell membrane of P. aeruginosa and other bacteria and facilitate the uptake of C4 carbon sources that is fed into the glyoxylate shunt that is operative under conditions of oxidative stress in the host. Under oxygen limiting conditions when the TCA cycle is not fully operative glyoxylate shunt is used for energy generation and anabolic activities and has a critical survival value in pathogenic bacteria. It is present in bacteria but is absent in the eukaryotic host, making it a suitable target for drug development.
h) Seq. ID 1 completely inhibits growth of P. aeruginosa in anaerobic conditions with C4 as sole carbon source
It was further reasoned that blockage of C4 dicarboxylate transporter by Seq. ID 1 should be optimally assessed if P. aeruginosa is grown in minimal media supplemented with C4 carbon source. As depicted in Fig. 6a and 6b, when P. aeruginosa was grown in a minimal medium supplemented with a combination of C4 carbon source fumarate alone, or malate alone a pronounced growth was seen, and when Seq. ID 1 (25µg/ml) was added the growth was found to be inhibited in both conditions, which could be probably due to blockage of the import of C4 carbon substrates by Seq. ID 1.
i) Seq. ID 1 reverses efflux mediated resistance in a ß lactam resistant P. aeruginosa
The hypothesis behind the role of C4 dicarboxylate transporter and glyoxylate shunt in efflux mediated drug resistance is as follows: the transporter is responsible for the uptake of C4 succinate, fumarate and malate that are fed into the shunt, the energy generated is used to drive ATP or PMF pump mediated carbapenem and ß lactam efflux, and PsC23 or Seq. ID1 binds to one of the components of the transporter blocking the efflux, thus reversing the resistance.
As one of the mechanisms of acquired resistance to multiple drugs is through active efflux of drugs as depicted in Fig. 7, the effect of Seq. ID 1 on drug efflux in P. aeruginosa MCC 50428 ß lactam antibiotic-resistant strain was studied by comparing the change in resistance profile of carbapenems with efflux blockers Phenylalanine-Arginine-ß-naphthylamide (PAßN). P. aeruginosa was incubated with either
a. carbapenems- meropenem or imipenem (8 µg/mL),
b. carbapenems (8 µg/mL) and Seq. ID 1 (12.5 µg/mL),
c. carbapenems (8 µg/mL), and PAßN (20 µg/mL), and
d. PAßN (20 µg/mL)
and growth was monitored for 24 hrs.
As depicted in Fig. 8a and 8b, the carbapenems had no effect on the resistant strain as seen in earlier experiments whereas the combination of PAßN and meropenem or imipenem reversed this effect within 2 hrs which was almost similar to the inhibition pattern of growth of resistant strain in the presence of combination of meropenem or imipenem and Seq. ID 1 (also depicted in Fig.4a, and Fig. 4b). It indicates a possible efflux-mediated resistance to carbapenem that is reversed by PAßN or Seq. ID 1. There is a similarity in the mode of action of PAßN and Seq. ID 1, which is the blocking of drug efflux that prevented carbapenem efflux from P. aeruginosa. The other possibility may be that antibody fragment targets an energy transduction mechanism that energizes an efflux pump by a yet uncharacterized mechanism, otherwise Seq. ID 1 would not have affected the growth of resistant P. aeruginosa when applied with carbapenems.

j) Seq. ID 1 reverses ß-lactam antibiotic resistance of P.aeruginosa MCC 50428
Further, Seq. ID 1 was tested for reversal of ß-lactams in P. aeruginosa MCC 50428 in vitro. As depicted in Fig. 9, treatment of P. aeruginosa MCC 50428 with 64 µg/ml amoxicillin or piperacillin had no effect on the growth, whereas, combination of 64 µg/ml amoxicillin or piperacillin with 12.5 µg/ml of Seq. ID 1 significantly reduced the growth of P. aeruginosa (almost 100% reduction) within 2 hours and no significant growth was seen upto 8 hours.

k) In vivo efficacy of Seq. ID 1 and meropenem against P. aeruginosa MCC 50428
Pathogen free 6-8 weeks old BALB/c mice (female) were housed in polypropylene cages and maintained under controlled conditions (23°C –25°C, 50–55% humidity and 12 hrs dark and light cycles). They were fed with chow diet and water ad libitum. Before infection, neutropenia was induced in these mice by injecting two intra-peritoneal doses of cyclophosphamide at day - 4 (150 mg/kg) and day -1 (100 mg/kg). On the day of infection, an overnight grown culture of meropenem resistant P. aeruginosa MCC 50428, 0.5 McFarland standard suspension (equivalent to a density of 108 CFU/mL) was prepared in a sterile lactate ringer’s solution. Infection was induced by injecting 50 µL bacterial suspension into tail vein of mice using 27-guage-needle.
A total of five groups (n=4) of mice were used. One hour post infection mice received various treatments for 72 hrs as described in Table 1 below.
Table 2
Group Treatment Dose (mg/kg)
1 Infection Control PBS, pH 7.2
2 Infection + Meropenem 5 mg/kg
3 Infection + Seq. ID 1 5 mg/kg,
4 Infection + Seq. ID 1 + Meropenem 5 mg/kg each
5 No infection + Seq. ID 1 5 mg/kg

The survival of the control and treated animals was monitored. Heparinized blood samples were collected aseptically after 24 hrs, centrifuged at 2,000 x g for 5 min and a suitable dilution was plated on MH agar, and incubated at 370C, 24 hrs for determination of the bacterial load (CFU/mL). The 5th group of mice was administered purified Seq. ID 1 (equivalent to 5 mg/kg of body weight) without inducing any infection and examined for the preliminary signs of toxicity.
As depicted in Fig. 8a, a significant increase in bacterial counts (approx. 12-logs) in the blood of control mice (buffer treated) was noted, suggesting a rampant systemic infection. Similarly high bacterial counts were seen in mice administered with 5 mg/kg body weight dose of meropenem. Both these groups of mice appeared sick and morbid, their movements were sluggish. Treatment of the infected mice with Seq. ID 1 alone reduced the colony counts by 8-logs, and treatment with combination of Seq. ID 1 with meropenem reduced the colony counts by 10-logs compared to control mice. The lower bacterial counts in the later is due to synergistic effect of combination of meropenem and Seq. ID 1.
Percentage survival of meropenem-resistant P. aeruginosa MCC 50428 infected neutropenic BALB/c mice when treated with meropenem (5 mg/kg), Seq. ID 1 (5 mg/kg), combination of Seq. ID 1 with meropenem (5mg/kg each), or no treatment (buffer treated) was monitored over a period of 72 hrs. As depicted in Fig. 8b, the untreated mice showed 100% mortality within 72 hrs due to a high bacterial load in blood. Further, meropenem treatment also had very little effect on survival of the infected mice due to antibiotic resistance achieved by the bacteria. Whereas, Seq. ID 1, and Seq. ID 1 and meropenem treated mice showed no mortality till 72 hrs. The 5th group of mice that were administered with Seq. ID 1 equivalent to 5 mg/kg body weight without infection also survived for 72 hrs indicating non-toxic property of the Seq. ID 1. Seq. ID 1 is a new molecule with a unique target and in combination with meropenem is useful for controlling carbapenem resistance in P. aeruginosa if the two are co-administered.
, Claims:Claims
We claim:
1. A composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa comprising at least one antibiotic against Pseudomonas aeruginosa, and at least one antibody or antibody fragment thereof against Pseudomonas aeruginosa
wherein
said antibody or antibody fragment is of Seq. D 1 targeting C4 dicarboxylate transporter of Pseudomonas aeruginosa;
said ß lactam antibiotic is selected from the group consisting of carbapenems, amoxicillin, and pipericillin;
said antibody or antibody fragment of Seq. D 1 shows MIC-50 at a concentration of 6.25 µg/mL, MIC-90 at a concentration of 12.5 µg/mL, and MIC-99 at a concentration of 25 µg/mL against P. aeruginosa model strain ATCC 27853 and ß-lactam antibiotic-resistant strain MCC 50428.

2. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises a carbapenem selected from the group consisting of meropenem, and imipenem.
3. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 16 µg/ml concentration of meropenem, and 6.25 – 50 µg/ml concentration of Seq. ID 1.
4. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 8 µg/ml concentration of meropenem, and 6.25 – 50 µg/ml concentration of Seq. ID 1.
5. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 4 µg/ml concentration of meropenem, and 50 µg/ml concentration of Seq. ID 1.
6. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 32 µg/ml concentration of meropenem, and 1.58 – 50 µg/ml concentration of Seq. ID 1.
7. The composition effective against ß-lactam antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 8 µg/ml concentration of imipenem, and 12.5 µg/ml concentration of Seq. ID 1.
8. The composition effective against antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 64 µg/ml amoxicillin and 12.5 µg/ml concentration of Seq. ID 1.
9. The composition effective against antibiotic-resistant Pseudomonas aeruginosa as claimed in claim 1, wherein, the composition comprises 64 µg/ml pipericillin and 12.5 µg/ml concentration of Seq. ID 1