Immunological active proteins I-spga. The specific reaction against Colistin resistance Klebsiella Pneumoniae strains.

Ionel-Victor Pătraşcu, Liliana Viasu, Maria-Anca Petriu, Mircea Penescu, Carmen Balotescu-Chifiriuc, Maria Serdaru, Rareş-Ilieş Preutu, Sabina Petrică. Immunological active proteins I-spga. The specific reaction against Colistin resistance Klebsiella Pneumoniae strains. One Health International Journal, 3(2),120-126,2017. One Health New Medical Concept Association, Romania

  1. Imunomedica-P, Bucharest, Romania
  2. Carol Davila Nephrology Hospital, Bucharest, Romania
  3. Faculty of Biology, University of Bucharest, Bucharest, Romania

Abstract

Colistin resistance of bacteria called superbug, triggered a warning about the risks of everyone on infection with E.coli or Klebsiella pneumoniae containing genes that make them resistant to all antibiotics including Colistin. Immunologically active proteins (PIA) made with the immunogen PC2, the group name Imunoinstant proved that can inhibit the growth of antibiotic-resistant bacteria, including E.coli, K.pneumoniae, Staph aureus MRSA. The new generation of PIA made by I-SPGA immunogen have been shown to act specifically on E.coli resistant to all current antibiotics used in hospitals in Romania, the K. pneumoniae resistance to Colistin, and on Candida spp., antibiotic resistance. PIA first generation cured urinary infection and no relapses have occurred in 17 of 19 women treated orally with IMUNOINSTANT. Preliminary results allow us to report the possibility of using passive immunity to produce the active immunity and effective use of these mechanisms in the prevention and the treatment of the antibiotic resistance.

Key words: pasive immunity, active immunity, antibiotic resistance, superbug, Colistin Klebsiella pneumoniae resitance strains, immunological active proteins

Superbug E.coli, resistant to last-resort antibiotic, show up in China 2015. The drug resistance gene known as mcr-1 – which has the capacity to move from one bacterium to another – was found in about 1% of E.coli bacteria and 1% of a bacteria known as Klebsiella pneumoniae, that can cause pneumonia, bloodstream infections, and wound infections. The mcr-1 gene confers transferable colistin resistance. Mcr-1-positive Enterobacteriaceae (MCRPE) have attracted substantial medical attention, clinical associations of mcr-1-positive Escherichia coli (MCRPEC) infection, and risk factors for MCRPEC carriage. Of 17,498 isolates associated with infection,

mcr-1 was detected in 76 (~1%) of 5,332 E.coli isolates, 13 (<1%) of 348 Klebsiella pneumoniae, one (<1%) of 890 Enterobacter cloacae, and one (1%) of 162 Enterobacter aerogenes. But with resistance to better drugs on the rise, Colistin has taken on increasing importance in medicine [1].

Acquired resistance due to chromosomal mutation and selection is termed vertical evolution, since the advantage will be conferred to a bacterial line. Bacteria also develop resistance through the acquisition of new genetic material from other resistant organisms through horizontal transfer, and may occur between strains of the same species or between different bacterial species or genera sharing a same ecological niche. Mechanisms of genetic exchange include conjugation, transduction, and transformation. For each of these processes, transposons facilitate the transfer and incorporation of the new resistance genes into the genome of the bacterial host or into plasmids [2].

Superbugs are present in Europe. E.coli resistant to the Colistin was find out in the pig meet [3] and Klebsiella pneumoniae strain Colistin resistance were isolated form 18 patients in Romania, and there are existing in the bacterial collection of the Bucharest Biology Faculty and Immunomedica-P collection.

Colistin resistance has been seen before, but not in this form. What makes this situation unique – and unsettling – is that the gene that makes the bacteria Colistin-resistant is contained in a plasmid, a mobile piece of DNA. Plasmids can easily move from one bacterium to another, both within a family of bacteria and to other families, as well. That means E.coli carrying mcr-1 can share it with other E.coli, as well as pass it to bacteria like Klebsiella pneumoniae [5].

A report on antimicrobial resistance in bacteria by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) said some 25,000 people die from such superbugs in the European Union every year. “Antimicrobial resistance is an alarming threat putting human and animal health in danger,” said Vytenis Andriukaitis, the EU’s health and food safety commissioner. “We have put substantial efforts to stop its rise, but this is not enough. We must be quicker, stronger and act on several fronts.” [6].

Bacteria found in humans, animals and food continue to show resistance to widely used antimicrobials, says the latest report on anti-microbial resistance (AMR) in bacteria by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC). The findings underline that AMR poses a serious threat to public and animal health. Infections caused by bacteria that are resistant to antimicrobials lead to about 25,000 deaths in the EU every year [7].

MATERIALS AND METHODS Microorganisms

In this study strains isolated from patients form Romanian hospitals, were used for immunization of Rhode Island red chickens. The isolated strains were: seven strains of E.coli, seven strains of Klebsiella pneumoniae resitance of colistin, 7 strains of Proteus mirabilis (PR 31 – Proteus mirabilis, PR 33 – Proteus mirabilis, PR 34 – Proteus vulgaris, PR 35 – Proteus mirabilis, PR 36 – Proteus vulgaris, – PR 37 – Proteus mirabilis, PR 38 – Proteus mirabilis), 7 strains of Candida (C1 – Candida albicans, C2 – Candida tropicalis; C3 – Candida glabrata; C4 – Candida albicans; C5 – Candida albicans; C6 – Candida krusei – ATCC 14243; C7 – Candida parapsilosis – ATCC 22019). Stock cultures were kept at -80°C in BHI (Brain heart infusion – Oxoid) and glycerol. Prior to each experiment, the strains were streaked on BHI agar (Oxoid) plates with incubation at 37°C for 24 h.

The bacterial strains isolates form Romanian patients a resistant to following antibiotics: Cefepime, Cefixim, Trimetroprim/Sulfamethaxazol, Fosonicin, Gentamicin. Imipenem, Cefuroxim, Meropenem, Ciprofloxacina, Amoxicilina, Clavulianic acid, Ceftibuten. The Klebsiella pneumoniae strains are Colistin resistance, and were isolated from Romanian patients.

 

Immunogen I-spga

In the presented method, the strains from E.coli, Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis and Candida are grown under appropriate conditions in a liquid culture medium to a predetermined density, followed by harvesting and suspension of the bacterial culture in saline, where upon formalin is added to the suspension under slight agitation to a final concentration of 0.2 M formaldehyde, followed by incubation under continuous agitation at 37°C, for approximately 2 hours, followed by incubation at 4°C for 24-48 hours, resulting in a formalin-killed E.coli strain having substantially preserved antigenic properties. The inactivated strains were was three times with distillated water, and mixed with SPGA as immuno-modulator, 10 mg/ml for each bacterial strain and mixed with QS 21 as adjuvant.

 

Chicken immunization

20 weeks of age Rhode Island red chickens form the Mihailesti Poultry Farm, Romania were immunizated with 2 ml of Rhode Island red hens, at 20 weeks of age, were immunized by three intramuscular injections with I-spga (200 mg/dose/animal). After the first injection, the second one was administrated after 30 days and the third intramuscularly administration was done after 14 days after the second one.

 

IgY and white egg proteins preparation

IgY and white egg proteins were purified from 6 eggs laid prior to immunization and from 12 eggs after 8-10 days after the first immunization. The IgY and white egg proteins as holo-ovotransferrin, ovomuicin, ovoalbumin and lysozyme were prepared by the method described previously [9-11].

 

RESULTS AND DISCUSSIONS

Serological assay

Rapid seragglutination assay (SAR)

The SAR was performed using “in house” reagents. The antigens were the formalin inac-tivated bacterial suspension at the 10 optical density. The IgY and immunological active proteins were used as undiluted or double fold dilution in PBS. It was added 30μL of antigen at 30μL of IgY or PIA samples and homogenized for 10 seconds to form a circle about two inches in diameter, after which it was kept at rest for two minutes. At the end of reaction time, the test was considered positive if it had the presence of lumps and the absence of it, the test was considered negative (Table 1, Table 2).

 

Agglutination assay

Each bacterial suspension was adjusted with PPBS to an optical density at 525 nm of 0.3 with a Coleman Junior spectrophotometer. This suspension was used both in the rapid or slow Agglutination assay. The agglutination procedure used was basically that developed by Schaefer

[12], with some key modifications. To the culture tubes (12 mm) with 0.5-ml portions of the working dilution of IgY-poly I-spga or PIA I-spga were added, followed by 0.5-ml portions of the bacterial suspension. Control tubes containing 0.5 ml of PPBS and 0.5 ml of bacterial suspension were also included. The tubes were shaken and placed in an incubator at 37″C, and after 3 and 24 h they were removed to analyze the extent of agglutination (Table 3).

Agglutination was graded from “no aggluti-nation” to “complete agglutination” on a scale of 0 to 4+. In most instances type-specific agglutination occurred within 3h; however, sometimes the reaction took 24h to develop.

 

Proteins identification

The IgY-poly I-spga, active immunological proteins-poly I-spga (ovotransferrin-apo ovo-transferrin-holo, ovomucin, ovoalbumin and lysozyme extracted from hyper immune eggs I-spga) were indentified by ELISA assay. IgY-poly I-spgawas identificated by Chicken IgY ELISA Kit (ab189577) ABCAM, ovotransferrin by Chicken Ovotransferrin ELISA Kit (ab157694) ABCAM, ovomucine by Chicken MUC6 (Mucin 6) ELISA kit Elabscience E-EL-Ch0799, ovoalbumine by Ovoalbumina [OVA slG1) ELISA kit Blue Genue, lysosime by Lysosime (LZM) Chicken (Gallus) SEB193Ga ELISA kit Cloud-Clone Corp.

 

ELISA quantitative assay for IgY-poly I-spga

The samples of IgY-poly I-spga extracted from hyper immune eggs 8-10 days after first chicken immuization with the imunogen I-spga were tested by ELISA under the intruction of use of Chicken IgY ELISA Kit (ab189577) ABCAM.

The IgY-poly has the positive reaction at the concentration of 20-40 ng of IgY specific per 0.1 ml of IgY-poly for the antigen of K. pneumoniae, Candida albicans, E.coli and 0.80 ng/0.1 ml of IgY-poly for Proteus mirabilis.

 

ELISA quantitative assay for IgY-poly

Samples of IgY-poly I- spga extracted form hyper immune eggs 8-10 days after the first chicken immunization with immunogenic I-spga, were tested by ELISA usind the Chicken IgY ELISA Kit (ab189577) ABCAM. The IgY-poly has 320 up to 400 mg of IgY per egg extracted by the new tehnique (Patrascu unpulished data).

 

Table 1

Rapid seragglutination assay for the IgY-poly I-spga and immunological active proteins (PIA) with bacterial antigens

Bacterial line IgY-polyI-spgaa IgY IgYI-PC2c)
Quantitative Positive tested Positive tested
E.coli +++ 7/7 1/1
K.pneumoniae ++++ 7/7 1/1
Proteus mirabilis +++ 7/7 ND
Candida albicans ++++ 2/2 1/1
Candida tropicalis ++++ 1/1 ND
Candida glabrada ++++ 1/1 ND
Candida krusei ATCC 14243 ++++ 1/1 ND
Candida parapsilosis ATCC 22019 ++++ 1/1 ND

 

  1. IgY-poly I-spga extracted from eggs of hyper immunized hens, 8 days after the first injection of the immunogenic I-spga chicken contain antibiotic resistance E.coli, Proteus mirabilis, 7 strains of Candida and 7 strains of Colistin resistant K.pneumoniae
  2. IgY extracted from the same eggs before the first injection of immunogenic I-spga c. IgY-poly IMUNOINSTANT immunogenic I-PC2 as reference sample [10] ND – not determined

 

Table 2

Rapid seragglutination assay for the proteins immunological active (PIA) with bacterial antigens

OTf-polyI-spgaa IAP-poly I-
Bacterial line OTfb) spgac)
Positive tested Positive tested
E.coli 7/7 1/1
K.pneumoniae 7/7 1/1
Proteus mirabilis 7/7 1/1
Candida albicans 2/2 1/1
Candida tropicalis 1/1 1/1
Candida glabrada 1/1 1/1
Candida krusei ATCC 14243 1/1 1/1
Candida parapsilosis ATCC 22019 1/1 1/1

Ovotransferrin holo poly I-spga extracted from white eggs of hyper immunized hens, 8 days after the first injection of the immunogenic I-spga chicken contain antibiotic resistance E.coli, Proteus mirabilis, 7 strains of Candida and 7 strains of Colistin resistant

K.pneumoniae

  1. OTf extracted from the same eggs before the first injection of immunogenic I-spga
  2. IAP -poly I-spga whole sample with immunological active ovotranseferrin, ovomucin, ovoalbumin and lysozyme

Grow inhibition assay for Klebsiella pneumoniae

In vitro grow inhibiton assay for Klebsiella pneumoniae strain 55,717 ESBC by IgY-poly imunogenic I-PC2. The test was done at the Victor Babes Hospital, Bucharest [6,9,10,13-15].

The new generation of immunologically active proteins (IAP) prepared on red Rhode Island chickens inoculated with a new I-spga immunogenic containing G-type immuno-modulators, are considered the 2nd generation IAP and in preclinical evaluations proved to act specifically, directly on antibiotic-resistant bacteria including Colistin-resistant Klebsiella pneumoniae strains.

The second generation IAP (I-spga IAP), which exhibit for identification two biologic markers, G and A, were prepared using as antigens the bacterial strains resistant to all antibiotics currently used in Romanian hospitals, isolated from patients during 2016-2017, from the Carol Davila Nephrology Hospital collection,

Bucharest, Romania and the Faculty of Biology Bucharest collections. The I-SPGA immunogenic contained also a mixture of seven Colistin-resistant Klebsiella pneumoniae strains from the collection of the Faculty of Biology, Bucharest, Romania. Grow inhibition assay of Klebsiella pneumoniae strain 55717 antibiotic resitance (fig.1). Experiment was done at the Hospital Vuctor Babes Laboratory (8,13).

Immunoglobulin (Ig) samples extracted from the yolk (Y) and immunologic active proteins (IAP I-spga) produced from the eggs whites obtained from hens hyperimmunized with I-SPGAimmunogen prepared from a collection of bacteria (K.pneumoniae, E.coli,

Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei – ATCC 14243, Candida parapsilosis

22019 – ATCC) resistant to all antibiotics, isolated from patients with urinary infections, reacted positively in rapid and slow agglutination tests with antibiotic-resistant pathogens corresponding to the bacteria used for immunization.

For confirmation of the specificity of the agglutination tests, the poly-IgY samples and I-spga IAP samples, containing immunologically active ovotransferrin, immunologically active ovoalbumin, immunologically active ovomucine, and immunologically active lysozyme, were tested by ELISA technique using the ABCAM kits. The poly-IgY I-spga samples were found to contain 250-500 mg of specific poly-IgY I- spga anti-K.pneumoniae and 250 mg specific IgY anti-pollution C.albicans.

 

Table 3

Seroaglutination assay for IgY I-spgaa with bacterial antingens

Bacterial line Positive reaction
Eserichia coli
1 : 320
Klebsiella pneumoniae
1 : 320
Proteus mirabilis 1 : 40
Candida albicans 1 : 160

 

  • IgY poly I-spga extracted from white eggs of hyper immunized hens, 8 days after the first injection of the immunogenic I-spga chicken contain antibiotic resistance coli, Proteus mirabilis, 7 strains of Candida and 7 strains of Colistin resistant K.pneumoniae

 

Immunologically active proteins (I-spga IAP) and poly-IgYI-spga were retested using the exhaustion test by direct contact with the bacteria present in 18h old cultures. To this purpose, antibiotic-resistant E. coli, Colistin-resistant K. pneumoniae, antibiotic-resistant P.aeruginosa and antibiotic-resistant Candida albinans. C.glabrata, C.tropicalis, C.krusei were tested.

IgY I-spga and IAP I-spga were put in contact with the pathogens directly in the culture medium, incubated at 37 °C for 60 minutes, and afterward centrifuged for 15 minutes at 3000 rpm. The supernatant was tested with ABCAM ELISA kits and compared to samples that were not in contact with the pathogenic bacteria. In the presence of specific pathogenic germs, the content in I-spga IAP and that poly-IgY I-spga decreased by two logarithms as compared to the controls.

Second generation poly-IgY, at a concentration of 2,5 mg /ml of specific poly-IgY, specifically inhibited the growth of antibiotic-resistant

E.coli, K.pneumoniae, P.aueruginosa, Acineto-bacterbaumani, Salmonella enteritidis and Salmonella typhimurium [8,9].

Recent studies at Carol Davila Nephrology Hospital showed that the treatment with I-spga IAP and poly-IgY from the I-PC2 IMUNOINSTANT immunogenic administered orally, by gargling and afterwards swallowing, at an amount of 200 ml / 80 ml of sterile suspension in PBS, has cured the urinary infections due to antibiotic-resistant E.coli in women. 17 patients healed without recurrences. Two female patients showed recurrent infections, one of which was re-infected with a new strain of a susceptible to antibiotics E. coli (Patrascu et al. unpublished data).

Preliminary studies concerning the direct action of I-spga IAP and poly-IgY I-spga demonstrate their ability to react specifically with pathogens resistant to antibiotics, including Colistin-resistant K.pneumoniae strains.

These results compel us to expand the researches concerning the in vitro and in vivo direct action of IAP and IgY I-spga on one hand, and on the other hand, the information must be taken over and checked by other specialized institutions for acknowledgment. Confirmation of these results will enable the urgent work out of special programs to prevent and treat infections with specific pathogenic germs in humans, including the prevention and treatment of urinary infections with E.coli and K.pneumoniae superbugs.

 

CONCLUSIONS

 

  1. Klebsiella pneumoniae Colistin resistant strains isolated from Romanian patients react serologicaly by seroaglutination and by indirect ELISA test with the chicken immunolgobulins (IgY) and with immunologically active proteins I-spga form white egg of hyper immune eggs of the chickens immunized by I-spga immuno-genic.

 

  1. Immunized chickens with I-spga immuno-genic with formalin inactivated Klebsiella pneumoniae Colistin resistant strains produce a specific immunological response. The IgY-poly and immunologically active proteins react in vitro with the pathogenic Klebsiella pneumoniae

 

Colistin resistant strains, by rapid agglutination, slow agglutination and by competitive ELISA assay.

 

  1. The oral passive immunological treatment with IgY-poly and immunological active proteins of patients with urinary tract infections by antibiotic resistant bacteria is a new opportunity to treat the antibiotic resistance.

 

AKNOWLEDGEMENTS

The authors acknowledge the Laboratory of immunology of IMUNOMEDICA-P and Laboratory of Carol Davila Nephrology

Hospital, Bucharest, Romania, for the help done to perform the presented researches investigations.

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