BioLactam test system was approved at the state level

Ministry of Health of the Republic of Belarus recommends public health organizations to consider the necessity to determine beta-lactamase activity level.

Ministry of Health of the Republic of Belarus points out the following: in the context of Belarusian Innovative Week held in November 15-18, 2011, in Minsk the project of developing BioLactam test system took the first place in the nomination «The Best Innovative Project». BioLactam test system was developed by educational institution «Vitebsk State Medical University of Order of Friendship of Peoples». The test system is intended for identification and quantitative estimation of ?-lactamase activity in biological substratums. This procedure is very important for deciding if it is appropriate to use ?-lactam antibiotics in a patient’s treatment.

Test system was registered by Ministry of Health of the Republic of Belarus (registration number Мн-7.115570-1011). Presently, there is an industrial domestically produced turnout (TY BY 391353648.001-2011).

BioLactam test system is based on the methodology of color changing of synthetic cephalosporin antibiotic in the decay of its ?-lactam compounds. Herewith, bathochromic change in chromophoric  molecule system occurs, and the color of reaction mixture is changing from yellow to red-orange color. The maximum of reaction product absorption is changing from 390 nanometers to 486 nanometers, and it makes possible a spectrophotometric detection. ?-lactamase activity is estimated in % of decay of standard cephalosporin quantity, inserted in a sample.

The optical density measuring is carried out using ELISA (enzyme-linked immunosorbent assay).

Nowadays, the most frequently used remedies in infection of different localization treatment in hospitals of the Republic of Belarus are ?-lactam antibacterial agents (aminopenicillin, cephalosporins, carbapenems, monobactams).

If there is no affect while using ?-lactam antibiotics in patient treatment, we can speak about two main causes:

  1. There is a biological resistance connected with ?-lactam antibiotics destruction in blood serum and other biological fluids (pleural fluid, peritoneal fluid, etc.) under ?-lactamase activity impact, due to albuminous and globulin fractions.
  2. A microbiological resistance appears due to antibiotics destruction in biological fluids and connects with an active increase and reproduction of  microorganisms producing ?-lactamase, destroying ?-lactam ring which is the basis of ?-lactam antibiotics structure (cerebrospinal fluid, phlegm, urine, saliva, pleural fluid, peritoneal fluid, daily plating culture of infected material).

The capability for producing ?-lactamase in different concentrations is revealed in a great number of bacteria as gram-positive, as gram-negative. Almost all bacteria are capable to synthesize these enzymes. There are two ways for microorganisms to produce ?-lactamase: to have this capability naturally and produce ?-lactamase due to appropriate genes presented in their chromosome, or they can gain this ability after successful DNA transduction from other microorganism (usually composed of interweaved R-plasmid). At the moment there are 4 main classes of ?-lactamase: A, B, C, D. Producing of ?-lactamase means enzymes capable to hydrolyse an endocyclic peptide bond in ?-lactam antibiotics. Natural or acquired capability to produce ?-lactamase is the basic mechanism of constantly increasing resistance of bacteria to this or that class of antibacterial agents. This leads to clinical inefficiency of using antibiotics and reasonable complications, and also to disease prognosis worsening, term treatment prolongation and patient’s hospitalization. Allowedly, the forming of new ?-lactamase with changed structure and mechanism of action is the most widespread mechanism of microorganisms adaptation to introduction of new ?-lactam agents into clinical practice. Thus, it could be considered as the bacteria answer to an evolutional pressure created due to the use of corresponding treatment regimen (especially, among gram-negative bacteria).

1. The detection and quantitative estimation of ?-lactamase activity of bacterial emulsion using «BioLactam» test-system.

The initial material to make suspensions is pure bacterial cultures growing on the nutrient medium surface (Endo or Russell medium is commonly used for culturing of Enterobacteriaceae spp., and yolk salt agar is used for staphylococci) in tubes with «slant agar». Then, 2-3 ml of sterile physiologic solution of sodium chloride is put into the tube with nutrient medium. After this, the tube is energetically shaken till the fluid above agar becomes opalescent. Then, this fluid is taken from the tube and (if it’s needed) is diluted with a sterile physiologic sodium chloride solution up to 0,5 McFarland turbidity units for the standardization. The analysis sensitivity is raised, if the bacterial emulsion has been frozen and defrosted once before the analysis.

If the ?-lactamase activity level of bacterial suspension is equal or exceeds 8,7%, the corresponding causative agent produces clinically significant quantity of ?-lactamase. As the result, we can observe a great self-tapering action of beta-lactam antibiotics (except cephalosporins of third and fourth generation, carbapenems, monobactams and inhibitor-protected agents). If the ?-lactamase activity level of bacterial emulsion is equal or exceeds 13,4%, the corresponding causative agent is also persistent to inhibitor-protected ?-lactams. «BioLactam» test system allows to get highly reliable data about the resistance of agent of infection to ?-lactam antibiotics. And this is very necessary condition for providing a rational antibacterial treatment.

«BioLactam» test system doesn't compete with a disk diffusion method but amplifies it. Thus, it allows to get an important information about the nature of  microorganisms antibiotic resistance. One of the main advantages of «BioLactam» test system is the shorter time needed for the analysis (30 min – 1 hour) in comparison with the disk diffusion method (24 hours). And other advantage is high results reproducibility, when there is a traditionally weak spot of disk diffusion method.

The test system is appropriate for determining the resistance of bacteria (different strains of intestinal bacilli as Pseudomonas app., Salmonella spp., Shigella spp., Citrobacter spp., Proteus spp., Klebsiella spp., Haemophilus spp. and others) to ?-lactam antibiotics because their resistance to ?-lactam antibiotics is mainly mediated by different ?-lactamase production.

2. The detection and quantitative estimation of ?-lactamase activity in phlegm using «BioLactam» test-system.

Before the analysis, the tubes with phlegm samples are being kept in a sonicator bath for 30 minutes at 37°С in order to disintegrate dense clots of phlegm and pus and the following transformation of their contents into a solution. Then, the tubes are being intensively shaken on vortex for 5 minutes till the full mixing of all contents. Then, they are being centrifuged at 12 000 rpm for 5 minutes. After the centrifugation, a generated pellet is used for the analysis and the viscous part of phlegm (sediment) is annulled.

The phlegm ?-lactamase activity is caused by ?-lactamase bacteria production which are infestants of bronchopulmonary diseases. And the activity level is proportional to ?-lactamase production intensity.  A high level of ?-lactamase phlegm activity of patients with bacterial infection of respiratory tract necessitates to prescribe reserve antibiotics including ?-lactam agents stable for the majority of ?-lactamase (carbapenems, fourth generation cephalosporins, inhibitor protected agents) and also reserve antibiotics which aren’t related to ?-lactam group. If the level of phlegm beta-lactamase activity is higher than 20%, the risk of wrong starting empiric therapy using first-line ?-lactam agents is increasing in 2,0 – 3,1 times in comparison with patients having a low level of phlegm ?-lactam activity. At this, another advantage of «BioLactam» test system as compared to classical  bacteriological methods is that the test system allows to detect ?-lactamase activity of biological fluids omitting the phase of causative agent pure culture. And due to this the time costs are reduced, therefore, the expenses are also reduced but the probability to get the applicable result for the analysis from the first time increases.

If the level of phlegm beta-lactamase activity is higher than 20%, it is recommended to prescribe inhibitor-protected beta-lactams, 4th generation cephalosporins, carbapenems or (if needed) antimicrobials of other pharmacological groups with similar antimicrobial activity spectrum for patients with bacterial respiratory tract pathology.

3. The detection and quantitative estimation of ?-lactamase activity in cerebrospinal fluid using «BioLactam» test system.

Cerebrospinal fluid (CSF) samples are collected using a lumbar puncture.

The collected CSF samples can be kept at  ?20°С  on condition of a one-time defrost just before the investigation. If there is a clearly defined turbidity in CSF samples, it is necessary to separate a pellet by centrifugation at 3000 rpm during 10 minutes before the analysis.

Quite high (more than 40%) ?-lactamase CSF activity corresponds to (in 1,9 – 3,2 times) a low probability of a correct starting empiric antibacterial therapy in purulent meningitis treatment. It means that it is necessary to change the primary therapeutic regimen for reserve antibiotics including ?-lactam agents (carbapenems, 4th generation cephalosporins) and second-line antibiotics of other pharmacological groups (glycopeptides, oxazolidinones, rifampicine, chloromycetin, etc.).

A high ?-lactamase activity in CSF is closely connected with S. aureus reproduction in it as well as bleeders and gram-negative bacteria capable to produce ?-lactamase. The mentioned causative agents cause 21,2% of all registered purulent meningitis. Often, causative agents of pyoinflammatory processes in central nervous system (CNS) aren’t evolved from CSF. And it emphasizes one of the most important «BioLactam» test system advantage over classic method of bacteriological analysis. The test system allows to quantify ?-lactamase activity in biological fluids without pure culture isolation of pathogen, thereby the time needed for the analysis is reduced and obtained results informativity increases.

If the level of beta-lactamase CNS activity is above 40%, it is recommended to prescribe antibiotics resistant to ?-lactamase taking into account their capability to pass through the brain-blood barrier (carbapenems excepting imipenem, flourquinolones, aminoglycosides, vancomycin, linezolid, etc.). Inhibitor protected  ?-lactams partially infiltrate through the brain-blood barrier  whereupon their use in bacterial meningitis / meningoencephalites treatment is limited.

4. The detection and quantitative estimation of ?-lactamase activity in blood serum, pleural and peritoneal fluids using «BioLactam» test system.

 The examined patient’s blood serum is insulated by centrifugation at 3000 rpm during 15 minutes of freshly obtained whole blood kept in a cooling chamber at  +4°С during 4-6 hours until a fibrin clot is formed.

The obtained blood serum can be kept in a cooling chamber at  -20°С in condition of a one-time defrost just before the investigation. Blood serum, pleural and peritoneal fluids ?-lactamase activity usually is conditioned by an albuminous fraction. At a heavy disease course we can observe the changing of factors roles, mediated serum beta-lactamase activity: a relative human serum albumin (HSA) contribution decreases and  polyclonal one increases. This phenomenon is explained by HSA molecule conformation changing due to some pH decrease or blood redox-potential changing. Serum beta-lactamase activity depends on pH, temperature (at the temperature more than 39-40°С it increases) and on solution ionic strength  (it decreases at ionic strength above the normal level increase). As a rule, a group of people with high (68,2%) beta-lactamase blood serum or pleural and peritoneal fluids activity, with a heavy infectious, bacterial disease course, with long duration of antibacterial therapy, with frequently antibiotics change and reserve antibiotics prescription is detected. The replacement of first-line ?-lactam agents for reserve antibiotics (inhibitor protected ?-lactams, 4th generation cephalosporins, carbapenems, glycopeptides, oxazolidinones, respiratory fluorquinolones) for such patients leads to hospitalization time reduction on average for 4-6 days.

The «BioLactam» test system use doesn't require a bacteriological laboratory in health organization. Detecting beta-lactamase activity using «BioLactam» test system means high response (70%), specificity (90%) and reproducibility. It’s process is simple, it doesn’t require any specific equipment and specially trained staff.

The detection of biological and microbiological resistance to ?-lactam antibiotics at early stages of a disease allows to correct  the treatment timely and to lower frequency of  an unfounded beta-lactam antibiotics prescription by 20-30%.  It leads to hospitalization time reduction and significant cost cutting.

The Ministry of Health of the Republic of Belarus recommends public health organizations to consider the necessity to determine beta-lactamase activity level before prescribing antibacterial therapy.