APPROPRIATE TEMPERATURE VALUE AT WHICH THE MICROORGANISM IS

INHIBITED

Subject of the Invention

The present invention is related to a method for antimicrobial hyperthermia detecting minimum temperature value that inhibits the pathogenic bacteria that infects the patient.

Prior Art

Hyperthermia (thermotherapy) is the process in the clinical medicine in which the tissues and the organs of the body is heated a few degrees over the normal body temperature. Hyperthermia treatment is applied specifically to cancer patients. For example, it has been observed that some cancer types regress upon certain body tissues and organs are heated up to 38 to 50°C via various methods. These methods focusing on certain organs can be applied by ultrasound waves, microwave focusing, heating with electric energy, heating with radiant heater or heating by wrapping with local heating bags. Besides cancer, scientific researches have been conducted in relation to treatment of some systemic infection diseases stemming from microbial, local apses and tissue, organ infections with hyperthermia .

In medicine, in the discipline of infectious diseases, the bacteria type that infects the patient is isolated via culturing before the antibiotic treatment is began for the patient and this bacteria type subjected to antibiogram. With antibiogram, it is ensured that the medicine group to which the bacteria is sensitive is administered in the dose the bacteria is sensitive to. However, a method for detecting the appropriate temperature for the hyperthermia treatment via analysing the structural thermal resistance/sensitivity of the microorganism that infects the patient or the subject does not exist in the prior art. In this case, the treatment begins without analysing the potential benefit for the patient or the subject will obtain from the hyperthermia treatment. Therefore, the patient or the subject is exposed to heat that exceeds the required level for the pathogenic bacteria to be inhibited, resulting in the potential observation of a heat damage in the patient or subject.

A non-patent document named "The effect of thermal dose on hyperthermia-mediated inhibition of DNA repair through homologous recombination" (Van den Tempel, Nathalie, et al . "The Effect of Thermal Dose on Hyperthermia-Mediated Inhibition of DNA Repair through Homologous Recombination." Oncotarget, 15 May 2017, pp . 44593-44604.) in relation to the current state of art discusses thermic dosage's effect on the hyperthermia-mediated inhibition of the DNA repair via homolog recombination. In the said document, it has been stated that hyperthermia has a number of biological effects that sensitize tumours to radiotherapy in the range between the temperatures of 40-44°C. One of these effects is heat-induced degradation of BRCA2 and in turn causes reduced RAD51 focus formation, which results in an attenuation of DNA repair through homolog recombination. In the treatment temperatures over 41°C, a decrease in cell survival, an increase in sensitization towards irradiation, a decrease of BRCA2 protein levels and altered RAD51 focus formation are observed. It has been found that when the temperature exceeded 43°C, hyperthermia alone killed more cells directly and the processes other than homologous recombination were affected by the heat. This study demonstrates that the optimal inhibition of HR is achieved by subjecting cells to hyperthermia at 41-43°C for 30 to 60 minutes. Said document analyses the experimental effect of hyperthermia in cancerous diseases, in particular, the effect of hyperthermia to BRCA2 protein. Therefore, it does not provide configuration for the sensitivity analysis of the pathogenic bacteria before the hyperthermia in infectious, microbial diseases.

As another example for the current state of art, non-patent document named Influence of Temperature on Escherichia Coli Growth in Different Culture Media (Noor, Rashed, et al . "Influence of Temperature on Escherichia Coli Growth in Different Culture Media." Journal of Pure and Applied Microbiology, 7(2), June 2013, pp . 899-904.) can be given. In said document, the effect of temperature on Escherichia Coli growth in different culture environments is studied. According to the said document, cellular response against environmental stresses is one of the most highly conserved regulatory features among all organisms. Cells being subjected to heat shock causes accumulation of partially or totally denatured proteins that inhibit the normal cellular function. Said study has been conducted to analyse the growth and physiology of Escherichia coli at different temperatures in laboratory conditions. Patching and spot tests were carried out. The effect of different temperature values (25°C, 30°C, 37°C and

45°C) in the laboratory conditions on the growth of E. coli medium in different media varying in nutrients was studied. The results indicated that the growth of E. coli is inhibited in 45°C and its normal phenotypic colonial characteristics are disabled. Said study indicates that the growth of E. coli laboratory strain decreases by temperature increase. In the research discussed in said document growth analysis is being performed on some thermal levels in an incubator and probability of E. coli strain in high temperature for solely physiological purposes is analysed. In addition, growth is being analysed in only one temperature level that can be deemed to be in hyperthermia temperature. However, a configuration to detect that minimum inhibiting temperature for hyperthermia treatment was not provided. To resolve the disadvantages in the art discussed above, developing a method that detects the minimum inhibiting temperature value of the said bacteria by analysing the thermal resistance/sensitivity level of the bacteria isolated from the subject or patient for hyperthermia treatment is intended .

Detailed Description of the Invention

The invention relates to a method for detecting the minimum inhibiting temperature value of the pathogenic bacteria, in particular for the hyperthermia treatment.

One object of the invention is to detect the minimum temperature level that can be treated with hyperthermia for the bacteria that spread over in the infected patient/subject, before the hyperthermia treatment. Thus in the hyperthermia, thermal tissue damage caused by applying extra heat on patient/subject is prevented.

The invention is based on determining pathogenic bacteria isolate being thermal resistant or thermal sensitive by detecting whether there is growth in the solid or liquid medium of the said pathogenic bacteria isolate that is placed in the serial incubators (1) in constant temperature, the thermal values being at increasing level in relation to one another and that infects the patient/subject, and in the case it is thermal sensitive, detecting the minimum inhibiting temperature level.

The invention is, in the most general terms, a method to detect the temperature level at which the hyperthermia treatment will be applied and comprises the step of preparing of the microbiological solid or liquid culture mediums (known to be produced by the related bacteria type) inoculated with a colony taken from a pathogenic bacteria isolate, placing each prepared medium in serial incubators (1) set to constant temperature values with an increasing level, incubating the mediums for a certain period of time, removing the mediums from the incubators (1) at the end of the said period, detecting whether or not growth (proliferation) occurs in the mediums and detecting the minimum temperature level in which the growth does not occur by defining it as the minimum inhibiting temperature level for the said bacteria.

One embodiment of the invention is called as thermobiogram method .

In one embodiment of the invention, the incubator (1) is a microbiological heat oven.

In one embodiment of the invention, the serial incubator (1) consists of at least 10 incubators (1) .

In one embodiment of the invention, medium plates and medium tubes (2) are inoculated with the same bacteria isolate as much as the number of incubators (1) .

In one embodiment of the invention, mediums are incubated at the same time.

In one embodiment of the invention, incubation period is 24 hours. Said period can vary depending on the bacteria type. For example, it may be 24 hours for aerobe-facultative anaerobes, at least 48 hours for fastidious/reluctantly growing bacteria and anaerobes or at least 1 month for tuberculosis bacilli.

In one embodiment of the invention, whether or not growth occurs in the mediums is determined by bare eye.

In one embodiment of the invention, inoculation is made to unique solid or liquid medium of the bacteria at a certain population based on the type characteristics of the bacteria whose thermal sensitivity level is analysed and that isolated from the patient or the environment. In one embodiment of the invention, not only bacteria isolated from humans, but also bacteria isolated from animal/plant pathogens and from environment and food samples are analysed. For example, performing thermobiograms of the bacteria that are animal/plant pathogens, it will be ensured that the hyperthermia treatment is also effectively performed on these animates as well.

In one embodiment of the invention, the thermal sensitivity of the pathogenic bacteria is determined in temperature degrees (degree centigrade=degree Celsius (°C)). Later, the detected value is defined as the minimum inhibiting temperature (MIT) value .

In one embodiment of the invention, if the bacteria cannot be inhibited by the heat, said bacteria is classified as thermo- resistant. Therefore, it can be determined that the patient infected by the said bacteria will not benefit from hyperthermia treatment.

One embodiment of the invention is applied to the bacteria isolated from the infected patient simultaneously with the post-culture antibiogram. Said embodiment is applied preferably before the hyperthermia treatment.

One embodiment of the invention comprises the steps of inoculation and suspension preparation from one single bacterium isolate colony to liquid medium, incubation for a certain period and determining the minimum temperature level inhibiting the bacteria growth at the end of the said period. Said period is preferably 24 hours. Said temperature level is minimum inhibitor temperature (MIT) level.

An assembly pertinent to one embodiment of the invention is shown in Figure 1. Said assembly involves 7 serial incubators (1) and the temperature levels of these are set to 38°C, 39°C, 41°C, 42°C, 43°C, 44°C, 45°C, respectively. Each of the liquid medium tubes (2) prepared from a single bacterium isolate is placed in one of the serial incubators (1) . At the end of 24 hours of incubation, it is seen the bacteria growth is inhibited in the incubators (1) at the temperature levels of 44°C and 45°C. Therefore, minimum inhibiting temperature (MIT) level is determined as 44°C.

In one embodiment of the invention, serial incubators (1) involve at least 10 incubators (1) and are used simultaneously, inoculation is made to unique solid or liquid medium of the bacteria at a certain population based on the type characteristics of the bacteria isolated from the patient or the environment and thermal sensitivity level of which is being analysed. Medium plates/medium tubes (2) inoculated from the same bacteria isolate at a number equal to the number of incubators (1) are simultaneously incubated in the at least 10 incubators (1) the temperature level of which are gradually rising. Said temperature range is between 37°C-46°C. For example, incubator (1) temperatures for the serial incubators (1) are 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, respectively. Growth times in their unique mediums are taken into consideration. At the end of incubation period, medium tubes (2) /medium plates are taken off of the incubator (1) and non-growth minimum temperature level is determined by bare eye. For example, if there has not been any growth at 42°C and over, minimum inhibiting temperature (MIT) is detected and recorded as 42°C for the said bacteria isolate. As another example, if inhibition is achieved at a low dose like 39°C, applying a high temperature like 45°C to the patient/subj ect and causing thermal damage in the patient/subj ect is prevented. As another example, if inhibition cannot be achieved in none of the temperature levels (in none of the ten incubators (1)), as hyperthermia over 46°C may be hazardous, the bacteria can be reported as hyperthermia-resistant or thermo-resistant. By this way, treating resistant bacteria type with hyperthermia unnecessarily is prevented. One embodiment of the invention is developed to plan the treatment of infectious diseases with hyperthermia and first of all thermo-sensitive pathogenic bacteria types are selected and these pathogenic bacteria are treated with heat. It is projected that thermo-resistant ones are not going to benefit from hyperthermia. As bacteria thermal sensitivity is measured in centigrade with the method of the invention, the patient is administered with only therapeutic dose of heat. Also, only the patients infected with pathogens with potential to answer the hyperthermia treatment will be treated and therefore loss of time and high costs will be prevented.

The results of the method (thermobiogram method) applied with the invention to different bacteria types can be recorded to data tables statistically and then can be used as a new parameter in defining the bacteria types.

By the developed method, in the infectious diseases and clinic hyperthermia discipline, the thermal sensitivity of the pathogenic bacteria can be detected before and after the hyperthermia treatment in centigrade degrees. The patient is treated with hyperthermia application over the detected degree so to ensure the maximum benefit.

With the method subject to the invention, thermo-resistant and thermo-sensitive pathogenic bacteria isolate differentiation can be achieved. Later, this provides an insight on whether the patient will benefit from hyperthermia treatment.

The invention may also be applied only by science-people who do not perform routine patient treatment but instead do only in vivo hyperthermia experiments and researches for scientific purposes. By selecting the bacteria types likely to answer to hyperthermia treatment with the method subject to the invention, studies can be conducted on these bacteria groups, saving time and reducing the costs. In microbial diseases (e.g., dermal abscesses, bone infections) while the pathogenic bacteria are isolated in culture by the expert and antibiogram is performed for medicine treatment, the method subject to invention (thermobiogram method) can be applied simultaneously. By this way, the most effective and the least damaging heat dose to be applied to the patient is detected. In addition, thermo- resistant pathogenic bacteria are detected, and patients infected with said bacteria is prevented from being administered unnecessary hyperthermia treatment.

Brief Description of Drawings

Figure 1: An assembly that has the operation method subject to the invention.

Definition of Reference Number in the Drawings 1. Incubator

2. Medium tube