Biological resistance to beta-lactam antibiotics, methods of registration
Semenov V. M., Zhiltsov I. V., Dmitrachenko T. I., Zenkova S. K., Skvortsova V. V., Veremey I. S.
Nowadays antibiotic resistance of bacteria is one of the most important and actual problem of the infectiology. Almost all bacteria known to science which are the causative agents of infectious diseases (with rare exception) possess more or less significant resistance to antibacterial preparations.
Since the epochal opening of penicillin in 1928 antibiotics had great influence on the quality of human life. The possibility to treat and cure lethal bacterial infections changed the medicine forever. It brought a pain relief and cardinally alleviated suffering and reduced the mortality. Penicillin and its numerous derivations dominated among other antibiotics, showing an unprecedented success in treatment of bacterial infections all over the world.
Semenov Valeriy Mikhaylovich is aDoctor of Medicine, professor, dean of medical faculty in Vitebsk State Medical University, Chairman of Scientific Society of Infectious Diseases Specialists in Republic of Belarus, a member of International Alliance for the Prudent Use of Antibiotics (APUA), Chairman of byelorussian department APUA, a member of Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (IACMAC), Chairman of Byelorussian department of International society of chemotherapeutists (ISC), a member of ISC and European society of Clinical Microbiology and Infectious Diseases (ESCMID), vice-chairman of International Euro-Asian Society for Infectious Diseases.
Zhiltsov Ivan Viktorovich is acandidate of Medicine, associate professor of department of infectious diseases of Vitebsk State Medical University, a member of Scientific Society of Infectious Diseases Specialists in Republic of Belarus, a member of International Alliance for the Prudent Use of Antibiotics (APUA), secretary of Byelorussian department APUA, a member of Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (IACMAC), secretary of Byelorussian department of International society of chemotherapeutists (ISC), a member of ISC and European society of Clinical Microbiology and Infectious Diseases (ESCMID), a member of International Euro-Asian Society for Infectious Diseases.
Dmitrachenko Tatyana Ivanovna is a Doctor of Medicine, professor, a head of department of infectious diseases of Vitebsk State Medical University, a member of Scientific Society of Infectious Diseases Specialists in Republic of Belarus, a member of International Alliance for the Prudent Use of Antibiotics (APUA), a member of Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (IACMAC), a member of ISC and European society of Clinical Microbiology and Infectious Diseases (ESCMID), a member of International Euro-Asian Society for Infectious Diseases.
Zenkova Svetlana Konstantinovna is a candidate of Medicine, an assistant of department of infectious diseases of Vitebsk State Medical University, a member of Scientific Society of Infectious Diseases Specialists in Republic of Belarus, a member of International Alliance for the Prudent Use of Antibiotics (APUA), a member of Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (IACMAC), a member of ISC and European society of Clinical Microbiology and Infectious Diseases (ESCMID), a member of International Euro-Asian Society for Infectious Diseases.
Skvortsova Viktoriya Valeryevna is a candidate of Medicine, an assistant of department of infectious diseases of Vitebsk State Medical University, a member of Scientific Society of Infectious Diseases Specialists in Republic of Belarus, a member of International Alliance for the Prudent Use of Antibiotics (APUA), a member of Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (IACMAC), a member of ISC and European society of Clinical Microbiology and Infectious Diseases (ESCMID), a member of International Euro-Asian Society for Infectious Diseases.
Veremey Igor Svyatoslavovich is a leading laboratory assistant of department of infectious diseases of Vitebsk State Medical University.
Taking benzylpenicillin as a base, pharmaceutists developed a number of its derivations with the extended spectrum of activity, making a class of beta-lactam antibiotics. Beta-lactams are the set of antibiotics, which have more than 6 structural varieties, and every variety includes 2-azetidinone ring. They showed very high activity against a wide spectrum of bacterial pathogens, herewith having low (if not zero) toxicity for mammals` cells. It is considered that antibiotics of beta-lactam group are the most successful antibacterial preparation from the beginning of an era of antibiotics. Nevertheless for the past 60 years the frequency and level of bacterial resistance to beta-lactams have being increased up until now, when many people consider that soon beta-lactams will become unfit for fighting with heavy bacterial infections. Resistance of bacteria to beta-lactam antibiotics and inhibitors of beta-lactamases is continuously increasing problem, which leads to decrease of clinical efficiency of medicines forming a pillar stone of an arsenal of antibiotics.
Antibiotic resistance to any antibacterial preparation including beta-lactams can be associated with four main mechanisms: 1) prevention from drug interaction with its target (as a rule because of a change of structure of penicillin-binding proteins due to mutation in appropriate genes), 2) an emission of antibiotic from cell, 3) immediate destruction or modification of antibiotic ( i.e. its fermentative degeneration), and 4) deterioration of permeability of outer membrane for antibiotics, which is provoked by a change of structure of membranous lipopolysaccharides and proteins (porines). Hydrolysis of antibiotics has the greatest significance for a clinic, especially in point of beta-lactams. At the same time the method of antibiotic modification is distinguished by the greatest variety of variants and includes acetylation, phosphorylation, glycosylation, ribosylation, fusion with thiols and/or nucleotide. A unique property of enzymes, being capable of physically modifying antibiotics – their ability to actively reduce a concentration of appropriate preparations in local surrounding. A fermentative degeneration of beta-lactams in consequence of beta-lactamases production by the causative agent of different diseases is very important but not the only known mechanism of bacterial resistance to these antibacterial preparations.
1. Beta-lactamases production is a principal mechanism of bacterial resistance to beta-lactam antibiotics.
Natural or acquired ability of producing beta-lactamases - enzymes being capable of hydrolyzing an endocyclic peptide bond in beta-lactam antibiotics, - is a principal mechanism of permanently increasing resistance of bacteria to this class of antibacterial preparations. 4 main classes of beta-lactamases are known by now – А, В, С and D. The enzymes belonging to classes А, С and D contain a lateral radical of serine, which is a donor of electrons in the mechanism of catalysis, while the enzymes of class B need Zn2+ for realization of their biological function. In the number of famous beta-lactamases are SHV and TEM of class A and their derivatives –extended-spectrum beta-lactamase (ESBL) and also inhibitor resistant enzymes, so-called neither TEM, nor SHV and carbapenemases. Class B includes metallo-beta-lactamases, class C includes chromosomal beta-lactamases, and ESBL, carbapenemases and oxacillinases of OXA-type belong to class D. There are known 255 types of beta-lactamases and new modifications permanently appear. New beta-lactamases appear in response to a wide clinical use of new classes of beta-lactam antibiotics. It produces the evolutional pressure which is needed for the process of selection.
The ability to produce the different types of beta-lactamases in various concentrations was detected in a great number of bacteria, both in gram(+) and (especially) gram(-). Almost all known bacteria can synthesize these enzymes. Microorganisms can have natural ability to produce beta-lactamases because of having the appropriate genes in their chromosome, or they can gain the possibility after a successful transduction of DNA from other microorganism (usually in composition with the interweaving R-plasmids). Probably the genes of antibiotic resistance existed in a limited quantity even before the era of antibiotics began, but wide antibiotic introduction to a clinical practice led to their selection and appearance in great number of new stable strains. The level of beta-lactamases production can be stable, non inductible (statical production of enzymes) or it can intensify because of influence of some beta-lactam antibiotics (inducible production of enzymes).
Allowedly that the formation of new beta-lactamases with modified structure and mechanism of effect is the most widespread mechanism of microorganisms` adaptation to introduction of new beta-lactam preparations in a clinical practice (i.e. actually bacterial response to the evolutional pressure, originating from the use of inappropriate therapy schemes), especially among gram(-) bacteria. Traditional explanation of this phenomenon are the accidental mutations and horizontal transfer of genes of resistance with their following amplification. A typical example of point mutations leading to expansion of substrate spectrum of beta-lactamases are the beta-lactamases TEM-1, widely known and spreading everywhere, and they are transmitted by interweaving plasmids of gram(-) bacteria. Traditional plasmid beta-lactamases were primarily found out only among members of the Enterobacteriaceae family, but by the present time they spread among other genuses and species including P. aeruginosa, H. influenzae и N. gonorrhoeae. For the first time in enterobacteriaceae (especially in Klebsiella spp. и E. coli) plasmid ESBL were detected. They could hydrolyse natural and semisynthetic penicillins and also cefalosporines of 2nd and 3rd generations. Natural variants of TEM-1 with the higher ability to hydrolyse broad-spectrum beta-lactams as ceftazidime and/or resistant to beta-lactamases inhibitors appear in response to a wide use of corresponding antibiotics. A typical mutation appearing in such settings is a substitute of single amino acid M182T which is found only in combinations with other point mutations, which probably can restore biological functionality broken by other mutations, and specificity of this enzyme. A similar phenomenon is retraced by the example of SHV enzyme typical for the family of Enterobacteriaceae which nowadays includes the largest quantity of beta-lactamases known. The first described beta-lactamase SHV had narrow spectrum of activity, but accumulation of point mutations led to appearance of new forms of mentioned enzymes with a modified active center – so-called SHV-1. Representatives of SHV-1 group have extended spectrum of activity including cefalosporines of third generation or they are resistant to beta-lactamases inhibitors. There are more than 150 variants of ESBL described. Carbapenems (imipenem / cilastatin at the first place) are still the preparations of choice fit enough for treatment of diseases caused by ESBL-producing strains.
From a variety of well-known beta-lactamases the greatest clinical importance is possessed by the enzymes of classes A and C. A wide use of carbapenems led to the growth of significance of metallo-beta-lactamases of class B, which can hydrolyze carbapenems. In general Zn2+-dependent beta-lactamases destroy a majority of well-known beta- lactam antibiotics. In class A metal-independent carbapenemase had been recently discovered. It has wide spectrum of possible substrates and also can destroy carbapenems. The existence of a huge variety of antibiotic-modifying enzymes explains why there are so few effective inhibitors for these enzymes as yet.
As a worldwide tendency for the last decades we can note a permanent growth in the level of acquired bacterial resistance (predominantly among Enterobacteriaceae family) to the stable broad-spectrum beta-lactams (as cephalosporins of 3-4 generations, monobactams and carbapenems) nowadays having a great clinical use. Herewith different plasmids, gene cassettes or integrons, coding new beta-lactamases, are detected permanently. Genetic processes responsible for the evolution of resistance can be divided in 2 types: A) the evolution of «old» beta-lactamases by means of point mutations in appropriate genes, and B) appearance of new resistance genes, quite often taken from nonpathogenic bacteria of the environment. Besides mutant variants of classic penicillinases TEM and SHV, the resistance to cephalosporins of the third generation is mediated by: 1) some variations of ESBL of class A, mainly of CTX-M type (for example, CTX-M-40), and also more rare forms as BES, GES, PLA, PER, VEB, and 2) interweaving cephalosporinases of class C (AmpC), usually encoded by chromosome genes, but nevertheless they can move between the strains of Enterobacter spp., Citrobacter freundii, Hafnia alvei, Morganella morganii, Aeromonas caviae, Proteus vulgaris, Pseudomonas spp., Providencia spp., Serratia spp., and also among nonfermentative bacteria including Acinetobacter spp. However the genes responsible for beta-lactamases AmpC production can be localize not just in chromosome but also in plasmids. These genes encode beta-lactamases of the following types: ACC-1, ACT-1, CFE-1, CMY group, DHA-1, FOX group, MIR-1, and MOX-1. AmpC cephalosporinases have broader spectrum of substrate specificity than ESBL which includes cephalosporins of the third generation, cephamycins and inhibitors of beta-lactamases of A class. The production of these enzymes can be induced by the influence of certain beta-lactam antibiotics (for example, ceftazidime or cefoxitin). Accidental mutations can lead to hyperproduction of chromosomal cefalosporines, which makes the strains of Enterobacteriaceae resistant to the majority of widely used beta-lactam antibiotics. The alternative preparations for treatment of appropriate infections are cephalosporins of the 4th generation and carbapenems, but the combination of the highest level of ESBL productions and nonfermentative mechanisms of resistance leads to the resistance to these reserve preparations also.
It is generally considered that resistance to carbapenems is limited by P. aeruginosa, but then this phenomenon was expanded to Acinetobacter baumannii and, finally, to other Enterobacteriaceae spp. The resistance of such a type was specified by the production of new enzymes (of А, В and D classes), namely IMP, VIM SME, GIM, OXA, KPC. An outstanding example of evolution in the direction of steady increase of antibiotic resistance are the representatives of Salmonella genus. E. coli and K. pneumonia can also produce plasmid ESBL belonging to the class A. Frequently ESBL-producing strains of the bacteria mentioned above are detected during infective episodes caused by these bacteria, especially in an intensive care units what leads to the growth of the cost of treatment and duration of the hospital stay. Herewith reservoirs of ESBL-producing strains can be not just the patients from semiclosed collectives but also the ambulatory patients with the chronic illnesses.
The resistance of pathogenic staphylococci to beta-lactam antibiotics is mediated not just by penicillin binding proteins (PBP) 2а with lower affinity to penicillins but also by beta-lactamase production, intrinsically plasmid (detected not less than in half of the examined MRSA strains). Herewith this mechanism specifies a formation of temperate level of Staphylococcus resistance to beta-lactams. Moreover some of the strains of Staphylococcus can produce ESBLs.
At the same time there are many exceptions from the general rule as few pathogenic microorganisms which are absolutely sensitive to beta-lactam antibiotics by now because of inherent inability to produce beta-lactamases (and an absence of other known nonfermentative mechanisms of resistance). For example Streptococcus agalactiae still keeps sensitivity to beta-lactams; it causes infections among pregnant women and newborn children.
Unsuccessful efforts to develop the inhibitor of beta-lactamases have been undertaken since the end of the 40th. In the early 50th it was found out that some semisynthetic penicillins can inhibit beta-lactamases, but there wasn`t found any clinical application to this phenomenon. In 1967 a search program of natural inhibitors of beta-lactamases producing microorganisms had been started. That investigation led to discovery of olivanic acids and clavulanic acid. Clavulanic acid combined with amoxicillin and later with ticarcillin became a part of clinical practice in 1981. Later other inhibitors of beta-lactamases were found: sulbactam and tazobactam. It is still unclear do they have any advantages over the clavulanic acid or not. Only above-mentioned combination are available for the clinical use by now. Clavulanic acid represents natural clavam, and sulbactam and tazobactam – a sulphone of penicillanic acid. These three substances structurally are similar to beta-lactam antibiotics, it is a basis of their functioning as suicide inhibitors of class A beta-lactamases. The principle of action of described combinations is the so-called “mechanism-based” (or “suicide”) inhibition, i.e. irreversible binding to key Ser70 residue in an active center of the enzyme which structure becomes irreversibly changed preventing the enzyme of doing it’s biological function. Key amino acids to which the inhibitors bind are the Met 69, Ser 70, Arg 244, Arg 275 and Asn 276. The blocking of Ser70 is a key moment of the inhibition. Point replacements of the mentioned amino acids lead to deterioration of effectiveness of inhibition. Inhibitor-resistant beta-lactamases differ from initial TEM-1 и TEM-2 of substitutes by the one, two or three amino acids of different localizations.
Above-mentioned inhibitors significantly improve the effectiveness of beta-lactams combined with them in treatment of serious infections which caused by Enterobacteriaceae spp. and penicillin-resistant staphylococci. Combinations of beta-lactams with inhibitors of beta-lactamases are widely used in clinical practice. Although there are many good variants of these combinations, the majority of suicide inhibitors effectively suppresses beta-lactamase activity of class A enzymes but are useless for classes В, С and D, respectively.
Nowadays there are 4 beta-lactam combinations with the inhibitors of beta-lactamases which are widely used in clinical practice: ampicillin- sulbactam, amoxicillin- clavulanate, ticarcillin- clavulanate and piperacillin- tazobactam. Ticarcillin- clavulanate and piperacillin- tazobactam have the moast broad-spectrum activity including Pseudomonas aeruginosa. Many factors can influence the activity of such combinations including inhibitor pharmacokinetics, a type and a quantity of beta-lactamases producing by specific microorganism, and also the possibility of inhibitor to induce an expression of chromosomal cephalosporinases. Although ticarcillin- clavulanate and piperacillin- tazobactam have equal spectrum of activity, they have some differences. The most essential are improved activity of piperacillin against Enterobacteriaceae and P. aeruginosa and also heightened activity of piperacillin- tazobactam against gram(-) causative agents producing beta-lactamases of class PSE or other plasmid beta-lactamases. Moreover pharmacokinetics of tazobactam is better than clavulanic acid. In treatment of Pseudomonas infections clavulanate has the ability to induce an expression of chromosomal cephalosporinases which destroy ticarcillin while tazobactam haven`t this ability. Besides ticarcillin- clavulanate has significant activity against many bacteria including streptococci, Staphylococcus aureus, Bacteroides fragilis and numerous Enterobacteriaceae. Amoxicillin- clavulanate and ampicillin- sulbactam demonstrate clinically significant activity against streptococci (including enterococci), S. aureus, B. fragilis and some Enterobacteriaceae. Ticarcillin- clavulanate is prescribed for a treatment of severe infectious diseases including septicemia. Amoxicillin- clavulanate is useful in the treatment of bacterial infections of upper respiratory tract, urinary system, skin and soft tissues. Ampicillin- sulbactam can be used in treatment of abdominal and gynecological infections, urinary tract diseases, skin and soft tissues infections. A selective pressure produced by the redundant use of antibiotic combinations with the inhibitors of beta-lactamases precipitated the development of resistance to these combinations at the expense of formation of new mutant extended spectrum beta-lactamases, which are resistant to combined preparations. Recent investigations of the structure of class A beta-lactamases (namely TEM and SHV) widened their spectrum: so-called ESBLs and carbapenemases (Sme, NMC-A, IMI-1) were discovered and it led scientists to the understanding of progress of resistance to inhibitors of beta-lactamases. Moreover the extension of chromosomal enzymes of class C (CMY, MIR), a growing clinical role of enzymes of class В (IMP, VIM), the appearance of inhibitor- resistant broad- spectrum beta-lactamases of class D (OXA) and at the same time the ability of single causative agent to produce the beta-lactamases of different classes induced the investigations directed to create universal inhibitors. As a result some new broad-spectrum inhibitors were created as cephem-sulfones and oxapenems (incidentally, cephem-sulfones probably can inhibit metallo-beta-lactamases of class B), and also boronates and phosphonates, which are the principally new classes of inhibitors based on the common structural and functional characteristics of serine beta-lactamases.
2. Macroorganisms` factors lowering the effectiveness of etiotropic therapy by beta-lactam antibiotics.
Up to date antibiotic resistance of pathogenic bacteria has been considered only as adaptive reaction of microorganisms. Herewith scientists and clinicians traditionally don`t appreciate that human organism, for its part, also is not indifferent to introduction of antibiotics. For the macroorganism antibiotics are foreign substances which must be released from within by the different mechanisms.
Thus, there is well-known system of oxidative degradation of foreign compounds including beta-lactam antibiotics under the influence of cytochrome systems Р450 (mainly 1A2, 2C9, 2C19, 2D6 and 3A4), principally located in a liver; nevertheless this destructive way isn`t essential for beta-lactams. There are other degradation ways of antibiotics in an organism. In particular it is showed that laked blood can destroy 3-acetoxymethyl-cephalosporins (as cefalotin and cefotaxime) by deacetylation of 3-acetoxymethyl group. This observation shows that beta-lactam antibiotics can be destroyed by some components of a whole blood, plasma or blood serum. Besides it has been known for a long time that imipenem is destroyed by renal dehydropeptidases and decay products are neurotoxic; that's why commercial preparation imipenem includes the inhibitor of renal dehydropeptidases cilastatin. It was also demonstrated that analogs of carbapenems (namely 2-metilpenem-3-rhodizonic acid or BCL-98) are hydrolyzed by albumin of human blood and this activity is mediated by albumin sites containing lysine amino acids and L-tryptophan.
A phenomenon of beta-lactamase activity of human blood has been known for a long time. Thus, in 1972 the group of scientists of Glaxo Research Ltd company explored the characteristics of their recently synthesized chromogenic cephalosporin nitrocefin, and this group described a significant destruction of beta-lactam bond of this antibiotic under the influence of human blood serum and what this activity was mainly mediated by albumin fraction.
In 1994 a scientific group headed by B. Nerli described a phenomenon of intensive destruction of nitrocefin under the influence of human serum albumin (HSA). But clinical significance of the phenomenon wasn`t analyzed that time and finally the phenomenon of unusually high beta-lactamase activity of human blood was left unnoticed by scientific community. Moreover an attempt to localize an active center of albumin molecule failed: the spatial structure including Lys 199, Tyr 411, Trp 214, Lys 195, Lys 225, Lys 240 and His 146 in fact couldn`t be an active center because the listed amino acids actually were in different sites of reconstructed albumin molecule which were far away from each other.
In the next few years a profound research of this phenomenon was undertaken. It was persuasively proved that beta-lactamase activity is an intrinsic property of human blood. Besides HSA the majority of other protein blood fractions possesses beta-lactamase activity making about 9,6% from the total serum activity. E.g., polyclonal IgG (so-called “abzymes”) also have some beta-lactamase activity.
We have determined a number of characteristics of serum beta-lactamase activity, as optimal рН (9,0) (pic.1), the correlation of reaction rate with the surrounding temperature (increases exponentially with an increase of the temperature to 39-40°С) (pic.2) and ionic strength of solution (decreases with the growth of ionic strength above the level typical for blood plasma) (pic.3). Kinetics of cephalosporin destruction catalyzed by HSA was also investigated (first-order reaction with Кm=0,115). The level of beta-lactamase activity of HSA critically depends on integrity of its tertiary structure but doesn`t depend on the presence of cofactors. We’ve proved the presence of active center in an albumin molecule, and this active site is responsible for binding of beta-lactam antibiotics and its decay. Its tertiary structure and amino-acid content are modeled, and catalysis mechanism is reconstructed (it is not fully similar to bacterial beta-lactamases of class A). This site with a high level of probability includes amino-acid residues GLN 196, ALA 291, ARG 257, LYS 195, HIS 288, SER 192, GLU 153, GLU 292, TYR 150, GLU 188 and ARG 160 (pic. 4). Besides, the composition of activity center probably can include also HIS 242, PHE 157, ASP 451, LYS 199, CYS 245 and TYR 148. All above-listed amino acids are located close to each other in HSA molecule at a specific formation called «hydrophobic pocket». A median of a quantity of amino-acid residues directly interacting with one antibiotic molecule is 15 (95% CI: 14…16). Molecules of different beta-lactam antibiotics can interact with above-mentioned active site with the different ways including binding to the following aminoacids: ASP 108, HIS 146, PRO 147, PHE 149, SER 193, CYS 200, TRP 214, ARG 218, ARG 222, TYR 452 and other. Sulbactam and tazobactam are bind to the albumin molecule in other site composed by the amino-acid residues PRO 384, LEU 387, ILE 388, ASN 391, GLY 434, ALA 449 and ARG 485. In contrast to sulbactam and tazobactam clavulanic acid binds to the HSA molecule in the same site with beta-lactam antibiotics. This phenomenon allows to explain half effectiveness of inhibition of beta-lactamase activity of HSA by tazobactam if compared to clavulanic acid of the same concentration.
A comparison of amino-acid sequences of HSA and various beta-lactamases demonstrated that they have common sites namely short sequence KQRLK (lysine – glutamine – arginine – leucine – lysine); in the primary albumin structure these amino acids have positions from 195 to 199. In penicillinase blaZ molecule similar amino acids are located in positions from 140 to 144. Above-listed results of molecular simulation indicate that at least three aminoacids from this sequence (LYS 195, GLN 196 and LYS 199) take part in formation of active site of HSA molecule which is responsible for binding of beta-lactam antibiotics. These amino acids are probably relevant to catalysis process because of the KQRLK sequence is conservative and constantly detected in structure of different beta-lactamases.
We’ve demonstrated the fact of destruction of beta-lactam antibiotics (imipenem, benzylpenicillin, aztreonam, cephalexin and others) under the influence of HSA by means of HPLC.
We’ve also described the phenomenon of formation of polyclonal immunoglobulins G having beta-lactamase activity (so-called “abzymes” or “catalytic antibodies”) in human organism. Leading scientists in many countries convincingly demonstrated that antibodies can participate in ligand-receptor interaction of any type, i.e. actually they can act as any molecular transmitter of biological regulatory signal, and also as enzymes. According to modern conceptions catalytic antibodies («abzymes») develop in an organism in natural settings as one of parts of immunological net of Jerne (so-called second-order antibodies). A scheme of their formation looks like this: an antigen getting in an organism leads to formation of first-order antibodies i.e. antibodies which can specifically bind the corresponding antigen. As a rule these antibodies belong to different pools and have different antigenic specificity and avidity. For the rise of humoral immune response a great number of various antibodies is synthesized which are commonly can cover («opsonize») and neutralize embedded antigen and separately react with different antigenic determinants of mentioned antigen. Generally it leads to formation of antibodies to virtually all antigenic determinants of a certain antigen. In case of antigen is an enzyme among other antibodies directed against its antigenic determinants there will inevitably appear some antibodies to the active site of the enzyme. Up to the Jerne`s theory of immunological net the second-order antibodies are generated to binding sites of the first-order antibodies. Thus, binding site of the second-order antibodies is more or less similar to initial antigen. Jerne considered that existence of second-order antibodies is necessary for the prolonged maintenance of immune response. From the point of view of admirers of the theory of immunological net the second-order antibodies are «the effective models of the true antigens» for the system of immunological memory. If antigenic determinant for development of the first-order antibodies was an active site of an enzyme a binding site of the second-order antibody would represent a structural analog of an active site of initial enzyme. It can be expected that the second-order antibody will be capable of showing an appropriate enzymatic activity. This activity will be as more expressed as more accurate structural analog of the active site of initial enzyme will be represented by the binding site of the second-order antibody.
At present almost the all known abzymes were got artificially (in vitro or in vivo). Only in the last decade a few and uncoordinated publications appeared describing the detection of catalytic antibodies in vivo in patients with different pathology (predominantly autoimmune diseases or illnesses with a significant autoimmune component in their pathogenesis as systemic lupus erythematosus, rheumatic arthritis, autoimmune thyroiditis, bronchial asthma). DNA-hydrolyzing antibodies were found out in blood of sick persons in a number of infectious diseases. So in spite of small number of publications rather weighty proofs were got: 1) in human organism in several pathological conditions we can detect abzymes with different specificity of activity, and 2) these abzymes can play an appreciable role in pathogenesis of corresponding diseases.
It’s experimentally proved that in infectious diseases caused by penicillinase- producing causative agents (for example, pneumonia caused by P. aeruginosa) the antibodies develop against bacterial penicillinase of corresponding type. They have also confirmed the possibility of formation of abzymes having penicillinase activity in vivo by immunization of laboratory mice with penicillinase.
Taking into account the above-mentioned points the new approaches appear which can explain some mysterious phenomena observed in the clinic. Thus, in some infectious diseases the doctors observe a clinical (i.e. in vivo) ineffectiveness of different beta-lactam antibiotics which in vitro effectively put down vital activity of causative agents of appropriate diseases including autostrains. This phenomenon can be explained by peculiarities of localization of causative agents in human organism and also by pharmacokinetics of a certain antibacterial preparations; other explanation assumes the presence of significant titer of catalytic antibodies in patient`s blood which can effectively hydrolyze corresponding antibiotics. Moreover, prolonged organism exposure to bacterial beta-lactamases which can occur in some severe and progressive infections (especially complicated by tissue destruction) can lead to increase of «biological» antibiotic resistance because it results in arising of high titers of abzymes with beta-lactamase activity and accordingly to destruction of additional quantities of antibiotics been introduced into the organism.
3. Detection of “biological” resistance to beta-lactam antibiotics.
In order to detect and measure the beta-lactamase resistance to antibiotics one can use test-system «BioLactam» intended for detection and quantitative assessment of beta-lactamase activity in biological substrates which can produce transparent extractions, manely blood serum, sputum, cerebrospinal fluid (liquor), oral fluid (saliva), urine and bacterial suspension.
The basis of the functioning of the test system «BioLactam» is the spectrophotometric technique based on the change of color of synthetic antibiotic of cephalosporin line after its beta-lactamase bond cleavage. Thus there is a bathochromic shift in chromophoric molecule system, and the color of reaction mixture changes from yellow to red-orange. Maximum of light absorption of the reaction product changes from 390 Nm to 486 Nm and it makes possible a spectrophotometric detection. Beta-lactamase activity is evaluated in % of decay of standard quantity of cephalosporin added to the tested specimen.
For automation of test results a special program «Intercom» is developed (actual version is 1.5). This program can operate a flatbed reader connected with personal computer with the operating system Windows 98, 2000, ХР, Vista or 7. This program automatically measures optical density in cells of immunological plate and produces output results as a table, and also estimates the level of beta-lactamase activity in every sample (if that samples were placed in immunological plate in accordance with attached scheme) and emphasizes all activity values exceeding the threshold level specified by researcher. This program can keep a record: immediately before the measurements one can indicate the numbers of processed samples according to their real position in immunological plate and also include researcher`s surname, name and patronymic name and the name of organization; current date and time are added automatically. All the results of measurements and computations can be saved in a file with *.csv format which is compatible with all versions of MS Excel. The program is provided with a standard scheme of specimens placement and concise instruction manual.
3.1. Detection and quantitative evaluation of beta-lactamase activity of bacterial suspension with the use of «BioLactam» test-system.
The initial material for making of suspensions is pure bacterial cultures growing on the corresponding culture medium (Endo or Russell medium was commonly used for culturing of Enterobacteriaceae spp., and egg yolk agar – for staphylococci) in tubes. Then 2-3 ml of sterile physiologic solution of sodium chloride was put into the tubes with bacterial cultures. After this the tubes were energetically shaken till the fluid above agar becomes opalescent. Then this fluid was taken from the tube and (if needed) diluted with sterile physiologic sodium chloride solution to 0,5 McFarland turbidity units for the standardization. Sensitivity of the analysis obviously improves if bacterial suspension undergoes freezing – thawing cycle before the analysis.
If the level of beta-lactamase activity of bacterial suspension is equal or exceeds 8,7% one can conclude that the corresponding causative agent produces clinically significant quantity of beta-lactamase what results in significant lowering of effectiveness of beta-lactam antibiotics (except cephalosporins of 3-4th generation, carbapenems, monobactams and inhibitor-protected beta-lactams). If compared to the disc- diffusion method as a reference the sensitivity of the above technique is 76,4% (95% CI: 64,9…85,6) and specificity is 82,8% (95% CI: 70,6…91,4), р<0,0001.
If beta-lactamase activity of bacterial suspension revealed by the experiment is equal or above 13,4% we can conclude the corresponding microorganism is also resistant to inhibitor-protected beta-lactams. If compared to disc- diffusion method as a reference the sensitivity of above technique is 79,2% (95% CI: 65,0…89,5) and specificity is 79,3% (95% CI: 68,9…87,4), р<0,0001.
Test-system «BioLactam» allows to get an important and highly reliable information about the level of resistance of agents of infectious diseases to beta-lactam antibiotics what is necessary for prescription of rational antibacterial therapy.
Test-system «BioLactam» doesn`t compete with disc- diffusion method but complements it allowing to get important information about the nature of antibiotic resistance of microorganisms- causative agents of infectious diseases. One of advantages of test-system «BioLactam» is short time needed for the analysis (0,5-1 hour) in contrast with disc- diffusion method (24 hours or more). It is also characterized by the high reproducibility of results what is a weak point of disc- diffusion method.
The test-system is optimal for testing of resistance to beta-lactam antibiotics of Gram(-) bacteria (i.e., different strains of intestinal bacilli as Pseudomonas app., Salmonella spp., Shigella spp., Citrobacter spp., Proteus spp., Klebsiella spp., Haemophilus spp. and others) as their resistance to beta-lactam antibiotics is mainly mediated by production of different beta-lactamases.
3.2. Detection and quantitative evaluation of beta-lactamase activity in sputum with the use of «BioLactam» test-system.
Specimens of sputum for testing should be got by the fiber-optic bronchoscopy to avoid contamination with the microflora of oral cavity which mainly can produce beta-lactamases. The sputum obtained by the fiber-optic bronchoscopy may be stored at ?20°С before the procedure.
Before the investigation the tubes with the specimens of sputum 1) must be kept in ultrasonic bath for 30 minutes at 37°С to disintegrate dense clots of sputum and pus and to release its contents into the solution; 2) are intensively shaken on vortex for 5 minutes for the full mixing of contents, and 3) are centrifuged at 12.000 rpm for 5 minutes. After centrifugation a transparent fluid above the sediment is taken for the analysis, and viscous part of sputum (sediment) is disposed. If final volume of transparent fluid is too small one can put 0,5 ml of sterile physiologic solution of sodium chloride into the tube and after this the sample processing should be repeated from the point 2.
According to the results of our research beta-lactamase activity of sputum is caused by beta-lactamases produced by bacteria- causative agents of diseases of respiratory tract, and the level of the activity is proportional to intensity of beta-lactamases production. A high level of beta-lactamase activity of sputum of patients with bacterial infections of respiratory tracts results in lower effectiveness of empiric antibacterial therapy including beta-lactam preparations of the first line. It leads to the common need to prescribe beta-lactam preparations which are stable against the majority of beta-lactamases (e.g., carbapenems, 3-4 generation cephalosporins, inhibitor-protected preparations) or the non-beta-lactam antibiotics.
It was proved the level of beta-lactamase activity of sputum above 20% increases the risk of failure of starting empiric therapy (including beta-lactam preparations of the first line) in 2,0-3,1 times if compared to the patients having low level of beta-lactamase activity of sputum (here we used for comparison a lot of criteria of effectiveness assessment of antibacterial therapy). As we were able to obtain the sputum cultures of pathogenic microorganisms in only 37% of cases the advantage of test-system «BioLactam» over the classic bacteriological methods is a possibility to detect beta-lactamase activity in biological fluids without the mandatory stage of isolation of pure culture of causative agents what reduces the time needed for the analysis and significantly increases the probability of obtaining of valid results of testing eligible for further analysis.
If the level of sputum beta-lactamase activity is higher than 20% we recommend to prescribe inhibitor-protected beta-lactams, 4th generation cephalosporins, carbapenems or (if needed) non-beta-lactam preparations from other pharmacological groups with the similar spectrum of antimicrobial activity to the patients with bacterial pathology of respiratory tract.
3.3. Detection and quantitative evaluation of beta-lactamase activity in cerebrospinal fluid with the use of «BioLactam» test-system.
Samples of cerebrospinal fluid (CSF) are taken by the diagnostic lumbar puncture. Obtained samples of cerebrospinal fluid can be stored at ?20°С and should be thawed once before testing. If obvious turbidity of CSF samples is present one should use the centrifugation at 3000 rpm for 10 minutes to obtain the transparent supernatant.
High (above 40%) beta-lactamase activity of cerebrospinal fluid increases 1,9-3,2 fold the probability of failure of starting empiric antibacterial therapy of purulent meningitis what generally results in change of treatment scheme by adding of reserve antibiotics as new beta-lactam preparations (carbapenems, 4th generation cephalosporins) or second-line antibiotics of other pharmacological groups (glycopeptides, oxazolidinones, rifampicin, chloramphenicol and others). Moreover, there is a tendency to lowering of cell count in CSF of patients under the starting antibacterial therapy who has the low level of liquor beta-lactamase activity in contrast to the people with high CSF beta-lactamase activity level who commonly presents with high cell count (if that initial therapy scheme includes first line beta-lactam preparations).
High beta-lactamase activity of liquor corresponds to growing of S. аureus, Haemophilus influenzae or Enterobacteriaceae spp. which are capable of producing beta-lactamases; these causative agents cause about 21,2% of all notified cases of purulent meningitis. Bacterial agents responsible for CNS lesions are rarely isolated from cerebrospinal fluid so the significant advantage of «BioLactam» test-system over the classic methods of bacteriological analysis is that this test-system can quantitatively evaluate the beta-lactamase activity of CSF without the mandatory step of obtaining of pure culture of bacterial pathogen what strongly decreases the time needed for the analysis and significantly improves the probability of obtaining of valid testing results suitable for further analysis.
If the level of beta-lactamase activity of CNS is above 40% it is recommended to prescribe antibiotics stable to the impact of beta-lactamases and easily passing through the blood-brain barrier (as carbapenems except imipenem, fluoroquinolones, aminoglycosides, vancomycin, linezolid, etc.) to the patients having bacterial CNS pathology. Unfortunately, inhibitor-protected beta-lactams hardly penetrate the blood-brain barrier so their use in bacterial meningitis/ meningocephalitis is limited.
3.4. Detection and quantitative evaluation of beta-lactamase activity in blood serum with the use of «BioLactam» test-system.
Blood serum of examined patients was obtained from the whole blood by the centrifugation at 3000 rpm for 15 minutes after formation of fibrin clot. The samples were kept at the fridge at +4°С for 4-6 hours until fibrin clot develops. This blood serum can be stored under ?20°С and should be thawed once before testing.
Beta -lactamase activity of blood serum in most cases is caused by human serum albumin (HSA). Besides HSA the majority of blood proteins possess beta-lactamase activity which level is about 9,6% of the whole serum one. Polyclonal IgG also have beta-lactamase activity, their contribution in common serum level is 10 to 15%. In severe infectious diseases the role of above mentioned proteins is rebalanced: the contribution of HSA decreases but this one of polyclonal IgG grows what may be explained by the modification of molecular 3D-conformation of HSA because of acidosis (decline in pH) and (possibly) because of change of redox-potential of blood.
Serum beta-lactamase activity depends on рН, temperature (significantly increases at the temperature about 39-40°С) and ionic strength of solution (drops with the increase of ionic strength above the level typical for human plasma).
As a rule, high level (above 68,2%) of blood serum beta-lactamase activity in patients having severe bacterial diseases (as pneumonia, meningitis or meningoencephalitis, etc.) is strongly associated with significant duration of antibacterial therapy, frequent changes of antibacterial therapy schemes including common prescription of reserve antibiotics of all groups. For such patients the substitution of first line beta-lactam preparations to reserve antibacterials (e.g., inhibitor-protected beta-lactams, 4th generation cephalosporins, carbapenems, glycopeptides, oxazolidinones, respiratory fluoroquinolones, etc.) results in shortening of hospitalization period by an average of 4,6 days (p=0,003).
«BioLactam» test-system is useful for qualitative and quantitative analysis of beta-lactamase activity of biological fluids, in particular blood serum, sputum and cerebrospinal fluid and also bacterial suspensions. The level of beta-lactamase activity of sputum and cerebrospinal fluid above 40% and blood serum above 68,2% demands cancellation of treatment with first line beta-lactam preparations and prescription of reserve antibiotics instead (especially inhibitor-protected beta-lactams, carbapenems or preparations of other pharmacological groups with the similar spectrum of antibacterial activity).
Analysis of bacterial suspension with the use of «BioLactam» test-system enables us to reliably detect and confirm:
- The fact of production of clinically significant amounts of beta-lactamases (and, correspondingly, the resistance to penicillins and cephalosporins of 1st and 2nd generations) if the level of beta-lactamase activity of bacterial suspension is equal or above 8,7% (sensitivity of this test is 76,4%, specificity – 82,8%);
- The fact of resistance to inhibitor-protected beta-lactams if the level of beta-lactamase activity of bacterial suspension is equal or above 26,5% (sensitivity of this test is 79,2%, specificity – 79,3%);
- The fact of resistance to 3rd generation cephalosporins if the level of beta-lactamase activity of bacterial suspension is equal or above 81,2% (sensitivity of this test is 82,4%, specificity – 94,7%).