The potential of therapeutic hyperthermia to eradicate Staphylococcus aureus bacteria; an in vitro study.

Staphylococcus aureus is one of the most common infectious agents, causing morbidity and mortality worldwide. Most pathogenic bacteria are classified in the group of mesophilic bacteria and the optimal growth temperature of these bacteria changes between 33 and 41 °C. Increased temperature can inhibit bacterial growth and mobility, which in turn, can trigger autolysis and cause cell wall damage. Hyperthermia treatment is defined as a heat-mediated treatment method applied using temperatures higher than body temperature. Nowadays, this treatment method is used especially in the treatment of tumours. Hyperthermia treatment is divided into two groups: mild hyperthermia and ablative or high-temperature hyperthermia. Mild hyperthermia is a therapeutic technique in which tumour tissue is heated above body temperature to produce a physiological or biological effect but is often not aimed at directly causing significant cell death. The goal of this method is to achieve temperatures of 40-45 °C in human tissues for up to 2 h. Hyperthermia can be used in the treatment of infections caused by such bacterial pathogens. In addition, using hyperthermia in combination with antimicrobial drugs may result in synergistic effects and reduce resistance issues. In our study, we used two different temperature levels (37 °C and 45 °C). We assessed growth inhibition, some virulence factors, alteration colony morphologies, and antimicrobial susceptibility for several antibiotics with three methods (Kirby-Bauer, E-test and broth microdilution) under hyperthermia. In the study, we observed that hyperthermia affected the urease enzyme, antibiotic sensitivity levels showed synergy with hyperthermia, and changes occurred in colony diameters and affected bacterial growth. We hypothesise that hyperthermia might be a new therapeutic option for infectious diseases as a sole agent or in combination with different antimicrobials.

[Analysis of Mycobacterium tuberculosis Complex Strains Isolated from the Samples of Patients Living in Northern Syria]. / Suriye'nin Kuzeyinde Yasayan Hastalarin Örneklerinden Üretilen Mycobacterium tuberculosis Kompleks Izolatlarinin Analizi.

Tuberculosis causes serious mortality and morbidity worldwide each year. A lot of effort and money is spent for the diagnosis and treatment of tuberculosis all over the world. The importance that countries give to health policies and public health is inversely proportional to the incidence of tuberculosis and multidrug resistant tuberculosis. The aim of our study was to evaluate the resistance profiles of Mycobacterium tuberculosis complex strains which were isolated from sputum samples, collected by World Health Organisation from patients living in the northern region of Syria, where health services were disrupted due to the civil war. According to the protocol signed between the World Health Organization and our hospital; sputum samples taken from tuberculosis patients living in Afrin, Azez and Idlib regions or suspected of being resistant to anti-tuberculosis drugs were studied in our hospital. The cultivation process was performed in our laboratory using Löwenstein Jensen media and MGIT-960 system. The susceptibility tests for primary anti-tuberculosis drugs were performed using MGIT-960 system for M.tuberculosis complex isolates. The isolates identified as MDR/RD-TB (multi-drug-resistant-rifampicin-resistant tuberculosis) were sent to National Tuberculosis Reference Laboratory of Public Health Institution of Türkiye for susceptibility testing to first and second line drugs. Mutation and wild-type determination were studied by "Line Probe Assay (LPA)" method to investigate the susceptibility of the isolates to isoniazid, rifampicin, fluoroquinolone and aminoglycoside/cyclic peptide. The results obtained from the patients were collected and evaluated retrospectively from the records. Growth was observed in 18 samples out of 171 sputum samples from 67 patients; 13 isolates were detected as MDR-TB while one isolate was detected as mono RR-TB. The rate of mono RR-TB was 1.5% and the rate of MDR-TB was 19.4%. MUT3 causing rifampicin resistance was detected in 17.9% of the patients, katG/MUT1 causing isoniazid resistance in 17.9% and WT loss causing aminoglycoside/cyclic peptide resistance were detected in 19.4% of the patients. Neither fluoroquinolone resistance nor a mutation leading to fluoroquinolone resistance was detected in the study. When the sputum samples taken from the patients living in Northern Syria were examined, the frequency of MDR-TB was found to be quite high. MDR-TB, which is an important public health problem, was found at high rates due to the internal turmoil in the region and poor accessibility to health services. Since the gene mutations causing drug resistance with the LPA method differ with the conducted studies, it is important to evaluate the dominant gene mutations for determining the TB treatment strategies in the region.

In vitro activity of hyperthermia on swarming motility and antimicrobial susceptibility profiles of Proteus mirabilis isolates.

AIM: Swarming motility is a virulence factor for Proteus mirabilis and is a coordinated multicellular movement of bacteria. In this study, we investigated the inhibitory effect of hyperthermia on bacterial swarming motility and antimicrobial resistance. METHODS: Thirty-one P. mirabilis isolates were included in the study. Seven inoculated agar plates were incubated inside incubators with increasing temperature levels: at 36 °C (control) and 40-45 °C. On the next day, inhibition of swarming was evaluated and minimum paralyzing temperature (MPT) values were determined. An antimicrobial susceptibility test (antibiogram) is performed by exposing bacteria to increasing concentrations of antibiotics, in vitro. Thus, we used the Kirby-Bauer disk diffusion test as a screening method to analyze the antibiogram profiles of the isolates at 36 °C and 42 °C. Finally, a time-kill assay was performed to analyze the killing effect of hyperthermia (42 °C) on planktonic bacteria, in combination with the antibiotic meropenem at the first and third hours. A Wilcoxon signed-rank test was used to compare the killing effects of meropenem, hyperthermia and their combinations. RESULTS: The median MPT value was determined as 44 °C. In the disk diffusion assay, susceptibility development was observed in 94% of isolates for at least one antibiotic. In the time-kill assay, we observed a significant killing effect of hyperthermia in combination with meropenem. Under the microscope, we observed the formation of spherical cells by the effect of heat. CONCLUSION: We conclude that these findings might be useful when employing the hyperthermia method to treat infectious diseases caused by P. mirabilis in the future.

Inhibition of swarming motility using in vitro hyperthermia.

Hyperthermia is a therapeutic technique in which body tissue is exposed to temperatures in the region of 40-45 °C to induce a physiological or biological effect. Swarming motility is an important virulence factor for Proteus mirabilis and Pseudomonas aeruginosa and swarming phenomenon is a coordinated multicellular movement of differentiated bacterial population over semi-solid surfaces. In this study, we aimed to investigate the inhibitory effect of hyperthermia on bacterial swarming motility using a modified thermobiogram method and show the potential of this thermal method to treat bacterial infections. Ten P. mirabilis and 10 P. aeruginosa clinical isolates were included in the study. Sheep blood agar (SBA) plates were prepared and inoculated with bacterial suspensions of clinical isolates. Inoculated SBA plates were incubated inside 2 different incubators; at 37 °C and 45 °C for 20 h. The diameter of bacterial growing zones (swarming diameters) were measured every 2 h and noted. Finally, Gram stains of the isolates were prepared for microscopic examination. Wilcoxon signed-rank test was used to compare the swarming inhibition rates of the isolates incubated at 37 °C and 45 °C. Regarding P. mirabilis species, a significant difference was found between two different temperatures (P = 0.0078). So, a temperature at the level of hyperthermia significantly inhibited the swarming motility of P. mirabilis isolates. In addition, transformation to coccus form was observed at 45 °C. We speculate that these findings might be useful for employing thermal therapies including hyperthermia method to treat infectious diseases caused by swarming bacterial pathogens in the future.

In vitro activity of methylene blue on Mycobacterium tuberculosis complex isolates.

Methylene blue is used for bacterial staining in microbiology and as an antidote drug in medicine. In this study, we aimed to investigate the antimicrobial effects of methylene blue against Mycobacterium tuberculosis complex clinical isolates. Seventeen stored M. tuberculosis complex clinical isolates were included in the study. The isolates were inoculated into Mycobacteria Growth Indicator Tubes and incubated in Automated Mycobacterial Detection System. Mycobacteria Growth Indicator tubes containing methylene blue at critical concentrations of 0.2, 2, 20, 1000 µg ml-1 and control tube were prepared. Antimicrobial susceptibility testing was performed using Automated Mycobacterial Detection System which is gold standard for second line anti-tuberculosis drug testing. At the end of the study, six clinical isolates were susceptible to methylene blue at all critical concentrations. Five isolates were susceptible to only 1000 µg ml-1 methylene blue. Three isolates were susceptible to 1000 and 20 µg ml-1 methylene blue. Susceptibility rate was found as 94% when the critical proportion was accepted 400 GU (1/100 of control). Significant relationship was observed between the administered methylene blue concentrations and bacterial survival rate in statistical analysis. We conclude that methylene blue may become a potential anti-tuberculosis agent due to its well-known side effects and dosing regimens.

Are infectious diseases and microbiology new fields for thermal therapy research?

Antimicrobial chemotherapy and surgery are classical methods for treating infectious diseases. However, there is a need for alternative methods to cure infections caused by antibiotic-resistant pathogens, recurrent or chronic infections, and unreachable local infections in which the use of drugs or surgery is anatomically and physically restricted. Several micro-organisms are known to be sensitive to mild hyperthermia, and this sensitivity is one of the potential benefits proposed for the host during an episode of fever. Additionally, some immunological or biophysical changes occur during hyperthermia. These changes may be useful for eliminating thermo-susceptible microbial pathogens using local heat therapy. There are several experimental studies proposing the use of hyperthermia to treat local infections. The infected organs or tissues may be heated up to a temperature that can inhibit invading microorganisms. Here, it is hypothesised that local heat therapy may become an alternative or adjuvant method for curing local infections. Here, we highlight the potential for local hyperthermia in the treatment of bacterial infections caused by thermo-susceptible pathogens in a systematic plan. If the proposed thermal-microbiology concepts and local thermal therapies can be adapted to clinical microbiology and infectiology, new medical fields, such as thermo-microbiology and thermo-infectiology, may be created in the future.

In Vitro and Ex Vivo Investigation of the Antibacterial Effects of Methylene Blue against Methicillin-Resistant Staphylococcus aureus.

Methylene blue (MB) is a water-soluble dye that has a number of medical applications. Methicillin-resistant Staphylococcus aureus (MRSA) was selected as a subject for research due to the numerous serious clinical diseases it might cause and because there is a significant global resistance challenge. Our main goal was to determine and analyze the antibacterial effects of MB against S. aureus both in vitro and ex vivo to enhance treatment options. A total of 104 MRSA isolates recovered from various clinical specimens were included in this study. Minimum inhibitory concentration (MIC) values of MB against MRSA isolates were determined by the agar dilution method. One randomly selected MRSA isolate and a methicillin-susceptible S. aureus strain (S. aureus ATCC 25923) were employed for further evaluation of the antibacterial effects of MB in in vitro and ex vivo time-kill assays. A disc diffusion method-based MB + antibiotic synergy assay was performed to analyze the subinhibitory effects of MB on ten isolates. MICs of MB against 104 MRSA isolates, detected by the agar dilution method, ranged between 16 and 64 µg/mL. MB concentrations of 4 and 16 µg/mL showed a bactericidal effect at 24 h in the ex vivo time-kill assays and in vitro time-kill assays, respectively. We observed a significant synergy between cefoxitin and methylene blue at a concentration of 1-2 µg/mL in two (20%) test isolates. Employing MB, which has well-defined pharmacokinetics, bioavailability, and safety profiles, for the treatment of MRSA infections and nasal decolonization could be a good strategy.

[Epidemiological evaluation of a rapidly-prevented tularemia outbreak in Canakkale province, Turkey]. / Çanakkale'de Hizla Önlenen Bir Tularemi Salgininin Epidemiyolojik Olarak Degerlendirilmesi.

Tularemia is a disease caused by Francisella tularensis and widely seen at northern hemisphere of the world. In Turkey, oropharyngeal infections caused by a less virulent serotype F.tularensis subsp. Holarctica are more prevalent. The aim of this study was to present the results of an epidemiological research performed after the detection of tularemia cases from Biga county of Canakkale province, Turkey, in December 2009. Following the report of two tularemia suspected cases from two villages (Baliklicesme and Sinekci) of Biga, an epidemiological investigation was undertaken to inspect the situation in this area. Water samples, clinical samples as throat swabs, wound swabs and serum samples were collected. Samples were cultured on heart agar supplemented with sheep blood, cysteine and antibiotics. Cultures were incubated at 37°C in 5% CO(2) and followed for 10 days. Suspected colonies were identified by slide agglutination test using F.tularensis antisera. F.tularensis antibodies were investigated by standard tube agglutination method. Positive results obtained with agglutination test were also checked for a probable crossreaction with Brucella antibodies by Rose-Bengal test. Water and wound samples were investigated using real-time polymerase chain reaction (RT Taqman PCR; Quantica, Techne Inc, UK) with probe and primers specific for ISFtu2 gene. All of the cultures yielded negative results, however eight of 16 water samples, one lymph node aspirate and one throat sample were found positive in F.tularensis TaqMan RT-PCR test. In tube agglutination test positive antibody titers between 1:20-1:1280 were detected in 36 of 115 serum samples. Two cases with antibody titers of 1:1280 and accompanying acute clinical findings, were diagnosed as tularemia and treated accordingly. Lymphatic drainage fluid samples obtained from one of these patients yielded positive result in PCR, however clinical sample could not be obtained from the other patient. The only epidemiological linkage between these acute cases (n= 2) and the other seropositive subjects (n= 34) was the use of local water supply system. It was learned that water obtained through reverse osmosis system had been used as drinking water at Baliklicesme village. Pre- and post-reverse osmosis system water samples from Baliklicesme village and samples from water supply of Sinekci village revealed positive results for F.tularensis by PCR. Since the only epidemiological relation between these two villages was using local water supply, tularemia cases encountered in this area were attributed to a waterborne epidemic and an automatic chlorination system was set up at each water reservoir in these villages. The establishment of these preventive measures curbed the growth of the epidemic. The cases presenting with throat sore, fever, lymphadenopathy (more than 2 cm), non-responsive to beta-lactam antibiotics, should be further investigated for tularemia. This work emphasizes that systematic setup and control of water disinfection systems are crucial to prevent tularemia outbreaks. Community and related authorities should be educated about the importance of water sanitation and chlorination.

Investigation of heteroresistant vancomycin intermediate Staphylococcus aureus among MRSA isolates.

INTRODUCTION: Heteroresistant vancomycin intermediate Staphylococcus aureus (hVISA) testing is recommended when therapeutic failure is suspected in the clinics. In our research, we aimed to investigate the prevalence of hVISA among methicilline-resistant S. aureus (MRSA) isolates in our university hospital and compared three methods for detection of hVISA. METHODOLOGY: One hundred MRSA clinical isolates were collected in our medical microbiology laboratory between 01.04.2018 and 01.10.2019. For screening of hVISA, we used two screening agar plates and used one commercial medium; brain heart infusion agar (BHI) plates containing 4 µg/mL vancomycin and 16 g/Lt casein (BHIA-VC; Satola's test), BHI agar plates containing 4 µg/mLvancomycin (BHIAV), and commercially obtained vancomycin resistant Enterococci (VRE) agar for detetection of hVISA. Colonies which could grow on plates were counted manually at 24th and 48th hours. RESULTS: Among 100 MRSA isolates, 43 (43%) were found as hVISA using Satola's test. BHIAV and VRE agar screening test results were found 70% and 4%, respectively. Finally, at the step, MIC values of 20 (47%) hVISA isolates reduced to 2 µg/mL after sub culturing for the gradient test. CONCLUSIONS: We found higher rates of hVISA comparing other studies in Turkey. Both VRE agar and BHIAV screening test failed to detect hVISA properly. Meropenem in combination with vancomycin inhibited the growth of 90% hVISA isolates in our study.

Identification of Recent Tuberculosis Exposure Using QuantiFERON-TB Gold Plus, a Multicenter Study.

We investigated whether the difference of antigen tube 2 (TB2) minus antigen tube 1 (TB1) (TB2-TB1) of the QuantiFERON-TB gold plus test, which has been postulated as a surrogate for the CD8+ T-cell response, could be useful in identifying recent tuberculosis (TB) exposure. We looked at the interferon gamma (IFN-γ) responses and differences in TB2 and TB1 tubes for 686 adults with QFT-plus positive test results. These results were compared among groups with high (368 TB contacts), low (229 patients with immune-mediated inflammatory diseases [IMID]), and indeterminate (89 asylum seekers or people from abroad [ASPFA]) risks of recent TB exposure. A TB2-TB1 value >0.6 IU·ml-1 was deemed to indicate a true difference between tubes. In the whole cohort, 13.6%, 10.9%, and 11.2% of cases had a TB2>TB1 result in the contact, IMID, and ASPFA groups, respectively (P = 0.591). The adjusted odds ratios (aORs) for an association between a TB2-TB1 result of >0.6 IU·ml-1 and risk of recent exposure versus contacts were 0.71 (95% confidence interval [CI], 0.31 to 1.61) for the IMID group and 0.86 (95% CI, 0.49 to 1.52) for the ASPFA group. In TB contact subgroups, 11.4%, 15.4%, and 17.7% with close, frequent, and sporadic contact had a TB2>TB1 result (P = 0.362). The aORs versus the close subgroup were 1.29 (95% CI, 0.63 to 2.62) for the frequent subgroup and 1.55 (95% CI, 0.67 to 3.60) for the sporadic subgroup. A TB2-TB1 difference of >0.6 IU·ml-1 was not associated with increased risk of recent TB exposure, which puts into question the clinical potential as a proxy marker for recently acquired TB infection. IMPORTANCE Contact tuberculosis tracing is essential to identify recently infected people, who therefore merit preventive treatment. However, there are no diagnostic tests that can determine whether the infection is a result of a recent exposure or not. It has been suggested that by using the QuantiFERON-TB gold plus, an interferon gamma (IFN-γ) release assay, a difference in IFN-γ production between the two antigen tubes (TB2 minus TB1) of >0.6 IU·ml-1 could serve as a proxy marker for recent infection. In this large multinational study, infected individuals could not be classified according to the risk of recent exposure based on differences in IFN-γ in TB1 and TB2 tubes that were higher than 0.6 IU·ml-1. QuantiFERON-TB gold plus is not able to distinguish between recent and remotely acquired tuberculosis infection, and it should not be used for that purpose in contact tuberculosis tracing.

In vitro activity of methylene blue and eosin methylene blue agar on colistin-resistant A. baumannii: an experimental study.

Introduction. Colistin is a last-resort antibiotic used against carbapenem-resistant Acinetobacter baumannii (AB); however, colistin resistance has been reported recently. Methylene blue (MB) is used in microbiology for staining, and in medicine as an antidote drug.Aim. We aimed to investigate the antimicrobial effects of MB and eosin methylene blue (EMB) agar against colistin-resistant AB strains.Methodology. Firstly, a standard strain and AB clinical isolate were included in the study. After determining MICs, two strains were transformed into colistin-resistant forms, using Li's method. At each step, new MICs were determined and subcultures were inoculated onto EMB and sheep blood agar (SBA). Colistin MICs of the subcultures were also determined using Mueller-Hinton agar (MHA) containing MB. Secondly, colistin-resistant clones from 31 multidrug-resistant AB clinical isolates were screened to investigate their susceptibilities to EMB agar.Results. In the first round, MICs of both strains had risen to 64 µg ml-1. Subpopulations with high colistin resistance were inhibited by MB and EMB agar, but could grow well on SBA. In MHA plates containing MB, the MICs decreased to 0.5 µg ml-1 for colistin-susceptible or moderately resistant clones. Additionally, clones with high colistin resistance showed atypical colony morphology on SBA. In the second round, 35 % of the clinical isolates, which had gained resistance to colistin, were inhibited by EMB agar.Conclusion. MB may have inhibitory effects against colistin-resistant AB. Secondly, using only EMB agar for subculturing may cause missing of colistin-resistant strains.