Doubling Dilutions Research Articles

In vitro evaluation of Morinda lucida root extracts against multi-drug resistant bacterial pathogens isolated from diabetic foot ulcers

Background: The continuous rise in microbial resistance to orthodox antimicrobial drugs has led to the search for alternative sources with proven efficacy to solve the challenges of antimicrobial resistance (AMR). The preferred alternatives are plant sources, and this has led to the evaluation of constituents and potency of medicinal plants to provide scientific justification for their use. Methodology: The root of Morinda lucida plant was dug up from the ground, washed clean, and cut into smaller pieces and dried. The root was then ground into fine powder and extracted with water (aqueous), methanol, ethyl acetate and n-hexane. Multi-drug resistant (MDR) Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa were isolated from wound swab samples collected from patients with diabetic foot ulcers using conventional cultures, biochemical identification, and antimicrobial susceptibility tests. The phytochemical and proximate contents of the extracts were assessed by standard technique. The in vitro antimicrobial activities of the extracts were determined at a concentration of 200mg/ml of the extract using the agar well diffusion technique. The minimum inhibitory and bactericidal concentrations were determined by serial doubling dilution. The methanol extract time-kill assay was performed to determine the time of kill of the bactericidal concentration. Results: The phytochemical analysis showed that M. lucida root contains essential secondary metabolites such as flavonoids, alkaloids, tannin, saponin, glucosides, anthraquinone and quinine. The methanol and aqueous extracts showed higher in vitro antibacterial activity, producing the highest zone of inhibition of 27mm against S. aureus but a lower activity of 18mm with n-hexane extract against all isolates except S. aureus. The MIC ranges from 3.125mg/ ml and 25mg/ml. The time-kill assay of methanol extract at 2x and 3x MIC showed that bactericidal activity occurred within 0-8 hours of incubation, indicating high activity. Conclusion: The antibacterial potency of M. lucida root extract and the phytochemical components from this study shows that it can serve as a source of alternative antimicrobial agent that may be effective in the treatment MDR bacterial infections.

РАЗРАБОТКА ЭКСПЕРИМЕНТАЛЬНОЙ ЛЕКАРСТВЕННОЙ ФОРМЫ ДЛЯ МЕСТНОГО ПРИМЕНЕНИЯ С ПРОТИВОГРИБКОВЫМ ДЕЙСТВИЕМ

Currently, mycoses are one of the urgent health problems. According to WHO, up to 1/3 of the world's population suffers from fungal pathologies. Currently, fungal infections are becoming increasingly difficult to treat, which is associated both with the identification of new species of fungi that were previously considered non-pathogenic, and with the development of resistance of pathogens to available medicines. The problem of mycoses is becoming more urgent due to the variety of pathways of transmission of pathogens and their high resistance in the external environment, changes in the immune response of the human body. Therefore, it is so important to expand the range and constantly improve the compositions and forms of antifungal agents. The substance of the silver salt pyrazole-3-carboxamide (SSP) synthesized in PGFA showed antifungal activity, which allowed it to be included in the experimental mild dosage form. The purpose of this study is to develop an experimental mild dosage form based on the silver salt of pyrazole–3-carboxamide for topical use in candidiasis dermatomycosis and to evaluate its antifungal activity. Materials and methods. The pharmacologically active component is the pharmaceutical substance of the silver salt N-{4-[(1,3-thiazole-2-yl)sulfamoyl]phenyl}-1-phenyl-5-(4-ethoxyphenyl)pyrazole-3-carboxamide. The antifungal activity of the compound was studied by double serial dilutions in a liquid nutrient medium in relation to reference strains of fungi of the genus Candida: Candida albicans NCTC 885-653, Candida krusei RCPGU-1472/310, Candida glabrata RCPGU-1485/47. To determine the antifungal activity of an experimental mild dosage form in relation to a typical strain of fungi of the genus Candida, a three-nesting variant of the agar diffusion method was used. Statistical processing of the results of chemical studies was carried out according to generally accepted methods using standard EXCEL computer programs. Results and discussion. As a result of the conducted research, the composition of an experimental mild dosage form based on the silver salt of pyrazole-3-carboxamide has been substantiated and developed. A technology for the manufacture of ointment has been developed to ensure the quality of the dosage form, its therapeutic activity and stability. Conclusion. Based on technological and microbiological studies, an experimental mild dosage form based on the silver salt of pyrazole-3-carboxamide has been developed with the aim of further introduction into medical practice for the local treatment of dermatomycosis after appropriate studies.

The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility

Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2 µg/ml) in "TALP-Sperm” and “TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.

Quantitative drug susceptibility testing for Mycobacterium tuberculosis using unassembled sequencing data and machine learning.

There remains a clinical need for better approaches to rapid drug susceptibility testing in view of the increasing burden of multidrug resistant tuberculosis. Binary susceptibility phenotypes only capture changes in minimum inhibitory concentration when these cross the critical concentration, even though other changes may be clinically relevant. We developed a machine learning system to predict minimum inhibitory concentration from unassembled whole-genome sequencing data for 13 anti-tuberculosis drugs. We trained, validated and tested the system on 10,859 isolates from the CRyPTIC dataset. Essential agreement rates (predicted MIC within one doubling dilution of observed MIC) were above 92% for first-line drugs, 91% for fluoroquinolones and aminoglycosides, and 90% for new and repurposed drugs, albeit with a significant drop in performance for the very few phenotypically resistant isolates in the latter group. To further validate the model in the absence of external MIC datasets, we predicted MIC and converted values to binary for an external set of 15,239 isolates with binary phenotypes, and compare their performance against a previously validated mutation catalogue, the expected performance of existing molecular assays, and World Health Organization Target Product Profiles. The sensitivity of the model on the external dataset was greater than 90% for all drugs except ethionamide, clofazimine and linezolid. Specificity was greater than 95% for all drugs except ethambutol, ethionamide, bedaquiline, delamanid and clofazimine. The proposed system can provide quantitative susceptibility phenotyping to help guide antimicrobial therapy, although further data collection and validation are required before machine learning can be used clinically for all drugs.

Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumonia

PurposeTo assess performance of Etest®, Vitek®2 and BD Phoenix™ to determine the susceptibility of Streptococcus pneumoniae strains to penicillin, ampicillin and cefotaxime.MethodsSixty unique S. pneumoniae challenge strains were selected to cover a wide range of penicillin, ampicillin and cefotaxime minimal inhibitory concentrations (MICs). Strains were analyzed in four different Belgian laboratories. Etest® benzylpenicillin (BEN), ampicillin/amoxicillin (AMP) and cefotaxime (CTA) (bioMérieux), Vitek®2 AST-ST03 (bioMérieux) and BD Phoenix™ SMIC/ID-11 testing were each performed in two different labs. Results were compared to Sensititre® broth microdilution (BMD) (Thermo Fisher Scientific) results. MIC results were interpreted using EUCAST non-meningitis breakpoints (v 13.0).ResultsEssential agreement (EA) was ≥ 90% for all methods compared to BMD, except for Etest® BEN on Oxoid plate (58.3%), Etest® AMP (both on Oxoid (65.8%) and BD BBL plate (84.2%)). Categorical agreement (CA) for penicillin was only ≥ 90% for Vitek®2, for other methods CA ranged between 74 and 84%. CA for AMP was for all methods < 90% (range 75.8–88.3%) and CA for CTA was between 87 and 90% for all methods except for Etest on Oxoid plate (79.2%).ConclusionsOur study indicates that Vitek®2 and BD Phoenix™ are reliable for providing accurate pneumococcal susceptibility results for BEN, AMP and CTA. Using Etest BEN or AMP on Oxoid plate carries a risk of underestimating the MIC and should be interpreted with caution, especially when the obtained MIC is 1 or 2 doubling dilutions below the S or R clinical breakpoint.

Antifungal Potency of Some Antifungal Chemotherapeutics and Antidandruff Solutions against Dermatophytic Flora from Local Hairdressers’ Tools in Awka, Nigeria

Dermatophytes produce spores that can survive for a long time on fomites such as hairdressing tools (combs, scissors, clippers and hairpins). These fomites intend serve in the transmission of dermatophytosis. This study was aimed at evaluating and comparing the antifungal efficacy of some antifungal chemotherapeutics and antidandruff solutions against dermatophytic flora of hairdressers’ tools in Eke-Awka Market in Awka, Anambra State, Nigeria. A total of forty-five (45) samples were collected by swabbing the hairdressing tools of the local hair dressers using sterile swab sticks. The antifungal drugs (Ketoconazole, Miconazole and Griseofulvin) and the antidandruff solutions (Shampoo and Conditioner) were also bought from Eke-Awka Market. The samples where inoculated into sterile Sabouraud Dextrose Broth base in test tubes and incubated at 25-27ºC for 5-14 days. After incubation, the test tubes with visually observed growth (turbidity) were subsequently plated out by streaking on freshly prepared Sabouraud Dextrose Agar supplemented with Chloramphenicol (50µg/ml) and incubated at 25°C for up to 7-14 days. Identification was done using standard methods. Agar well diffusion method was employed for the in vitro antifungal susceptibility testing using different concentrations of both the antifungal drugs and antidandruff solutions obtained using double fold serial dilutions. Twenty Four (24) dermatophytes were isolated and identified. The isolates included Microsporum ferrugineum 6 (25%), Microsporum gypseum 3 (12.5%), Micosporum audouinii 6 (25%), Trichophyton schoenleinii 5 (20.8%) and Trichophyton mentagrophytes 4 (16.7%). The percentage susceptibility pattern of the isolates showed that 60℅ of the isolates were susceptible to Ketoconazole, 40℅ respectively susceptible and susceptible doze dependent to Miconazole and 100℅ resistant to Griseofulvin. For the antidandruff solutions, 20℅ of the isolates were susceptible to hair shampoo while 100℅ were resistant to hair conditioner. The results of this study showed that dermatophytes are prevalent on the tools used by local hair dressers and also that the antifungal drugs had better activity on the isolates than the antidandruff solutions used. Also, based on this study, ketoconazole is recommended for dermatophytosis. There is therefore need to educate the populace on the health hazards associated with sharing local hair making tools and the need for clients to have their personal hair making tools.

PROBLEMS OF DETERMINING THE MEDICINAL HERBAL REMEDIES ANTIMICROBIAL ACTIVITY AND THEIR SOLUTION METHODS

Intriduction. Currently, the antibiotic resistance development in pathogenic microorganisms remains a global problem. To solve this problem, an active search and development of new antimicrobial drugs, including those of plant origin, is underway. In the many years course of experience in this area, methods used to determine antimicrobial activity are being developed and improved, however, not all of them are suitable for determining the activity of biologically active substances of plant origin. The purpose of the study. To summarize information about existing methods for determining antimicrobial activity under in vitro experimental conditions. Material and methods. The article is based on publication materials from the PubMed and E-library databases, search.rsl. The literary search was carried out by keywords: antimicrobial activity, antibacterial effect, antibiotic, herbal preparations. The review includes articles that are relevant to the research topic and have been published over the past 10 years. Results and discussion. The article describes the main methods for determining antimicrobial activity in vitro and determines the possibility of using these methods in relation to herbal remedies. Conclusions. The findings indicated that the choice of the research method should depend primarily on the object under study and the type of microorganism in respect of which the activity is determined. Most of the developed and widely used methods for determining antimicrobial activity do not allow an adequate assessment of the activity of herbal remedies. Thus, when working with herbal remedies, the methods of double serial dilutions in liquid nutrient media with the use of indicators and the developing direction of activity research on biofilms can be considered the most accurate.

CHALCONE ANALOGUES AS POTENTIAL MEDICINES OF PATHOGENETIC THERAPY OF ALZHEIMER'S DISEASE: IN VITRO SCREENING

Introduction. Alzheimer's disease is one of the most common forms of dementia, the pathogenesis of which is based on the accumulation of β–amyloid plaques in brain structures and the development of cholinergic deficiency. The aim of the study was to evaluate the effect of chalcone analogues on the change in acetylcholinesterase activity and the process of β-amyloid aggregation in vitro. Material and methods. The tested compounds were six bis-substituted chalcone analogues, which were dissolved in dimethylsulfoxide during the analysis to obtain double dilutions. The effect of the studied substances on the activity of acetylcholinesterase was evaluated by the modified Ellman method. The change in the aggregation process of β-amyloid particles was determined in reaction with congo red after 3, 6 and 9 days of incubation. Based on the obtained results of the «% inhibition- concentration» relationships, the IC50 index was calculated, which was expressed in mmol/ml. Results. In the course of the study, it was shown that the analyzed chalcone analogues inhibit the aggregation of β-amyloid particles starting from the 6th day of incubation with a maximum on the 9th day. At the same time, the compounds that showed the highest level of activity were trimethoxy-substituted substances under the codes AZBAX4 and AZBAX6, whose IC50 on the 9th day of the study was 21.4±0.928 mmol/ml and 32.4±0.456 mmol/ml, respectively. Also, a high level of anticholinesterase properties was established for these compounds with IC50 of 35.9±0.991mmol/ml and 25.1±0.261 mmol/ml, respectively. The rest of the tested compounds showed a lower level of activity. Conclusion. The study showed that chalcone analogues under the codes AZBAX4 and AZBAX6 inhibit the activity of acetylcholinesterase and the pro-cess of aggregation of β-amyloid in vitro, which makes these compounds promising for further study in order to develop medicines for the pathogenet-ic treatment of Alzheimer's disease.

Heterophilic Interference of Rheumatoid Factor in TSH Immunometric Assay: A Cross-Sectional Observational Study.

Considering the inherent vulnerability of immunoassays for heterophilic interference and the potential of Rheumatoid Factor (RF) to act as a heterophile-like antibody, we conducted this study to investigate if RF leads to any such heterophilic interference in seropositive rheumatoid arthritis (RA) patients. The study was done on the TSH assay as it is a noncompetitive, double antibody sandwich assay, which is known to be vulnerable to heterophilic interference. In this cross-sectional observational study, eighty-four consecutive newly diagnosed RF-positive RA patients underwent TSH, Free T4, and anti-TPO estimation using the chemiluminescence technique (CLIA) on Siemens Immulite 1000 platform. The samples were screened for TSH interference using four methods: 1) analysis on a different platform, 2) assessment of linearity using doubling dilutions, 3) polyethylene glycol (PEG) precipitation, and 4) addition of a commercial blocker. Ten samples had a loss of linearity on serial dilution, indicating potential interference. After heterophile blocker treatment, five cases exhibited interference. One patient had diagnostic interpretation discordance on the second platform. No sample on PEG precipitation suggested the influence of antibodies. It is worth noting that even in cases where interference was suspected, the clinical interpretation was largely unaffected by the correction of TSH values based on mean dilution or measurement after heterophile blocker treatment. RF can cause heterophilic interference in TSH immunoassays used commercially. However, in most cases, this interference does not affect clinical decision-making.

Antimicrobial Activity of New &lt;i&gt;N&lt;/i&gt;-substituted Amides of Aroylpyruvic Acids and Their Silver Salts

Introduction. An important direction of modern pharmacy is the production of new domestic substances with anti-infectious activity, within the framework of ensuring the technological sovereignty of the state. Optimization of methods for determining antimicrobial activity is an integral part of these studies. Previously obtained silver salts of pyrazoles and pyrazolecarboxamideshad high antimicrobial activity. This work presents the results of determining the antimicrobial activity of new biologically active compounds synthesized by a salt formation reaction with silver ions at the carboxyl group of aroylpyruvic acid amides containing anthranilic acid in the amide fragment.Aim. Obtaining previously unknown amides of aroylpyruvic acids and their silver salts and studying their antimicrobial activity (AMA).Materials and methods. New N-substituted amides of anthranilic acids and their silver salts were obtained by organic synthesis methods. To study their antimicrobial activity, a micromethod of double serial dilutions was used on screening strains of S. aureus ATCC 6538P, E. coli ATCC 25922, as well as E. faecalis ATCC 29212, S. aureus Wood 46, S. abony No. 103/39 for highly active compounds. Additionally, a method of accounting for the results was carried out using the phenomenon of fluorescence in interaction with rezazurin, indicating the presence of living microbial cells.Results and discussion. The presence of antibacterial activity of silver salts 2a and 2b in relation to the studied strains was established: the minimum suppressive concentration (MPC) of compound 2a is 23.4–31.2 micrograms/ml, 2b is 3.9–5.8 micrograms/ml. When taking into account the results of the study of AMA using the phenomenon of fluorescence, it was found that they either coincide with the values of MPC determined visually, or indicate less antibacterial activity. Comparison of these results and seeding of the contents of the wells on a dense nutrient medium with respect to screening strains indicates that the suppression of fluorescence in experimental wells in the range of 99–82 % compared with the control is accompanied by the absence of bacterial growth (bactericidal effect), with fluorescence inhibition of less than 60 %, a pronounced growth of microorganisms is observed. In the intermediate range, the growth of single colonies is observed (bacteriostatic effect).Conclusion. The results of the study showed that the new silver salts of N-substituted amides of aroylpyruvic acids have high antimicrobial activity. The correspondence of the type of antibacterial activity to the degree of fluorescence inhibition was established.

Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a β-lactam/β-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane β-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine β-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of β-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

Evaluation of the LifeScale system for rapid phenotypic antimicrobial susceptibility testing from positive blood cultures

Abstract Objective The spread of antimicrobial resistance is a growing threat to public health, thus there is a critical need for prompt and reliable antimicrobial susceptibility testing (AST). Rapid AST may improve patient outcomes by allowing for quicker start of optimal therapy and reducing hospital length of stay, therefore decreasing overall healthcare costs. However, rapid results may not improve patient care if they are not considered equivalent to standard AST. Several platforms are under development that can provide rapid AST results; the objective of this work is to assess the performance of the new LifeScale rapid AST platform (Affinity Biosensors) that uses resonant mass measurements for calculation of antimicrobial susceptibility results. Methods Positive BACTEC blood-cultures collected from patients were included if the gram stain revealed only Gram-negative rods. Standard of care (SOC) AST results, as determined by a combination of BD Phoenix automated, Sensititre broth microdilution, and/or standard disk diffusion methods, were compared to results from direct positive blood culture testing on the LifeScale system. Minimum inhibitory concentrations (MICs) and categorical interpretations based on CLSI and/or FDA AST breakpoints were compared by microorganism and drug to calculate essential and categorical agreement for five species with pending claims on the LifeScale system: Escherichia coli (N=33), Klebsiella pneumoniae (N=10), K. oxytoca (N=5), Pseudomonas aeruginosa (N=11) and Acinetobacter sp. (N=1); tested against 13 relevant antibiotics: Amikacin, ampicillin, aztreonam, cefazolin, cefepime, ceftazidime, ceftazidime/avibactam ertapenem, gentamicin, levofloxacin, meropenem, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole. Results LifeScale had 95.3% essential agreement with the SOC results, and a 93.1% categorical agreement based on CLSI M52 criteria. Of the 665 organism-drug combinations evaluated, agreement was high, with only 1 very major discrepancy, 8 major discrepancies and 37 minor discrepancies, of which 22 (47.8%) were within ±1 doubling dilution. Among these discrepancies, LifeScale MICs averaged 1 dilution higher than SOC MICs, which may reduce the risk of false-susceptible interpretations. Conclusion s: The accuracy and reliability of results from rapid AST is of utmost importance. The LifeScale technology is unique among AST platforms as it measures bacterial cell mass and counts to determine MICs for up to 14 antibiotics in ~4 h. These results suggest this platform is able to deliver rapid, actionable results to guide antimicrobial therapy for patients with bacteremia.

Susceptibility profile and β-lactamase content of global Pseudomonas aeruginosa isolates resistant to ceftolozane/tazobactam and/or imipenem/relebactam-SMART 2016-21.

To determine susceptibility profiles and β-lactamase content for ceftolozane/tazobactam-resistant and imipenem/relebactam-resistant Pseudomonas aeruginosa isolates collected in eight global regions during 2016-21. Broth microdilution MICs were interpreted using CLSI breakpoints. PCR to identify β-lactamase genes or WGS was performed on selected isolate subsets. Ceftolozane/tazobactam-resistant [from 0.6% (Australia/New Zealand) to 16.7% (Eastern Europe)] and imipenem/relebactam-resistant [from 1.3% (Australia/New Zealand) to 13.6% (Latin America)] P. aeruginosa varied by geographical region. Globally, 5.9% of isolates were both ceftolozane/tazobactam resistant and imipenem/relebactam resistant; 76% of these isolates carried MBLs. Most ceftolozane/tazobactam-resistant/imipenem/relebactam-susceptible isolates carried ESBLs (44%) or did not carry non-intrinsic (acquired) β-lactamases (49%); 95% of imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible isolates did not carry non-intrinsic β-lactamases. Isolates that carried indicators of strong PDC (Pseudomonas-derived cephalosporinase) up-regulation without a mutation known to expand the spectrum of PDC, or non-intrinsic β-lactamases, showed an 8-fold increase in ceftolozane/tazobactam modal MIC; however, this rarely (3%) resulted in ceftolozane/tazobactam resistance. Isolates with a PDC mutation and an indicator for PDC upregulation were ceftolozane/tazobactam non-susceptible (MIC, ≥ 8 mg/L). MICs ranged widely (1 to >32 mg/L) for isolates with a PDC mutation and no positively identified indicator for PDC up-regulation. Imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible isolates without non-intrinsic β-lactamases frequently (91%) harboured genetic lesions implying OprD loss of function; however, this finding alone did not account for this phenotype. Among imipenem-non-susceptible isolates without non-intrinsic β-lactamases, implied OprD loss only shifted the distribution of imipenem/relebactam MICs up by 1-2 doubling dilutions, resulting in ∼10% imipenem/relebactam-resistant isolates. P. aeruginosa with ceftolozane/tazobactam-resistant/imipenem/relebactam-susceptible and imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible phenotypes were uncommon and harboured diverse resistance determinants.

Sensitivity to antibacterial drugs of biofilms formed by strains of vibrio cholerae of various serogroups.

The ability of Vibrio cholerae to form biofilms may cause ineffectiveness of cholera treatment and prevention. The aim of the study was to determine the antibiotic sensitivity of V.cholerae in biofilms. Material and methods. Sensitivity to 11 antibacterial agents was determined for biofilms of 10 strains of V.cholerae of different serogroups isolated from humans and from water obtained for 48 hours at 37°C on plastic plates in vials with autoclaved tap water infected with a suspension of 104 V.cholerae microbial cells. For this purpose the plates were washed three times in physiological solution and transferred into penicillin vials with double dilutions of antibacterial agents in liquid nutrient medium (Martin's broth, pH 7.7). After 24 hours of cultivation in the thermostat (37°C), the biofilms were imprinted and 0.1 ml of plankton culture was seeded onto plates with Marten's agar (pH 7.7). After 24 hours in the thermostat (37°C), the biofilms were imprinted and 0.1 ml of plankton culture was sown on plates with Marten's agar (pH 7.7). The result was counted after 24 h, determining the minimum suppressive concentrations of preparations by the presence or absence of V.cholerae growth. Results. The strains studied in biofilm communities, in contrast to the planktonic form, were highly resistant to all antibacterial drugs taken in the study, except for ciprofloxacin and ceftazidime. To improve the effectiveness of treatment of cholera and other infections whose causative agents are cholera vibrio, it is necessary to determine the antibiotic sensitivity of V.cholerae biofilms.

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

O04 A novel, fast, accurate, cost-effective antimicrobial susceptibility test for urine samples

Abstract Objectives To show that iFAST (impedance-based fast antimicrobial susceptibility test) can give accurate susceptibility results for urinary tract infection (UTI) samples in less than 5 h. Background UTIs are one of the most common forms of infection and reasons for antibiotic prescribing. Empirical prescribing without reference to antimicrobial susceptibility tests (ASTs) is common due to the high incidence, burden of symptoms and the length of time needed for an AST (24–48 h). However, untreated UTIs can lead to complications and in severe cases to bacteraemia and death. In addition, resistance to frontline antibiotics is increasing, leading to increased treatment failure. There is, therefore, a need for a fast, accurate and cost-effective AST for urine samples. Bacterial impedance cytometry can measure the opacity and electrical size of individual cells by measuring the change in the electrical current as single cells flow between micro-electrodes. Bacteria treated with antibiotics change their electrical size and opacity compared with untreated cells. This change is visible on the iFAST within 2 h of exposure to above MIC of antibiotics, thereby enabling detection of cells susceptible to breakpoint concentrations of antibiotics. The iFAST technology and workflow can fit into current diagnostic laboratory practice. Methods Fifty-eight Escherichia coli and Klebsiella pneumoniae strains were streaked onto standard agar plates and incubated at 37°C. After 2 h a streak of cells was inoculated into 0.9% saline. Cell count was measured using the iFAST and adjusted to 5 × 105 cfu/mL. 100 μL samples were exposed to EUCAST susceptibility breakpoint concentrations of frontline UTI antibiotics in MH2: co-amoxiclav (2/8 mg/L), amoxicillin (8 mg/L), ceftazidime (1mg/L), cefalexin (16mg/L), ciprofloxacin (0.25mg/L), gentamicin (2 mg/L), nitrofurantoin (64mg/L), trimethoprim (4mg/L) or a no antibiotic control and incubated at 37°C. After 2 h, 60 μL of each culture were measured in the iFAST for 2 min and compared with the no antibiotic control, using percentage cell count and a 50% population contour. In parallel, a gold standard microbroth dilution MIC was carried out, according to EUCAST guidelines, on the cultures. Results For the 441 tests conducted (58 strains in 8 antibiotics, nitrofurantoin is only recommended for E. coli treatment and was therefore disregarded for K. pneumoniae), overall concordance with the gold standard microbroth dilution MIC method was 95.2% for total cell count metric and 94.9% for cells in the population contour. The highest concordance was seen for ceftazidime (100% for both metrics) and amoxicillin (100 and 99% respectively), the lowest for trimethoprim (92% and 89% respectively). All non-concordances, except one strain with trimethoprim, showed an MIC of ±1 log2 (doubling dilution) different to the breakpoint concentration, which was used for iFAST. This is the normal technical variability of the gold standard method. Conclusions iFAST has up to 100% concordance with current gold standard MIC methods and delivers the results in less than 5 h compared with 24–48 h for current ASTs. This novel, fast, accurate and cost-effective AST could increase evidence-based antibiotic prescribing, thereby improving patient treatment, decreasing risk of UTI complications and overall decreasing antimicrobial resistance development.

Genotypic resistance determined by whole genome sequencing versus phenotypic resistance in 234 Escherichia coli isolates

Whole genome sequencing (WGS) enables detailed characterization of bacteria at single nucleotide resolution. It provides data about acquired resistance genes and mutations leading to resistance. Although WGS is becoming an essential tool to predict resistance patterns accurately, comparing genotype to phenotype with WGS is still in its infancy. Additional data and validation are needed. In this retrospective study, we analysed 234 E. coli isolates from positive blood cultures using WGS as well as microdilution for 11 clinically relevant antibiotics, to compare the two techniques. We performed whole genome sequencing analyses on 234 blood culture isolates (genotype) to detect acquired antibiotic resistance. Minimal inhibitory concentrations (MIC) for E. coli were performed for amoxicillin, cefepime, cefotaxime, ceftazidime, meropenem, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, gentamicin, tobramycin, and ciprofloxacin, using the ISO 20776-1 standard broth microdilution method as recommended by EUCAST (phenotype). We then compared the two methods for statistical ‘agreement’. A perfect (100%) categorical agreement between genotype and phenotype was observed for gentamicin and meropenem. However, no resistance to meropenem was observed. A high categorical agreement (> 95%) was observed for amoxicillin, cefepime, cefotaxime, ceftazidime, amikacin, and tobramycin. A categorical agreement lower than 95% was observed for amoxicillin/clavulanic acid, piperacillin/tazobactam, and ciprofloxacin. Most discrepancies occurred in isolates with MICs within ± 1 doubling dilution of the breakpoint and 22.73% of the major errors were samples that tested phenotypically susceptible at higher antibiotic exposure and were therefore considered as ‘not resistant’. This study shows that WGS can be used as a valuable tool to predict phenotypic resistance against most of the clinically relevant antibiotics used for the treatment of E. coli bloodstream infections.

Distribution of MIC values of antibacterial drugs for Flavobacteriales isolated from respiratory samples in Russian patients with cystic fibrosis

Objective. Analysis of the distribution of the values of the minimum inhibitory concentrations (MIC) of a number of antibacterial drugs in relation to representatives of the order Flavobacteriales isolated from respiratory samples from patients with cystic fibrosis of the Russian Federation by the method of double serial dilutions. Materials and Methods. The distribution of the values of MIC of a number of antibacterial drugs was evaluated in relation to 100 strains of bacteria, representatives of the order Flavobacteriales, isolated from respiratory samples from patients with cystic fibrosis from 60 regions of the Russian Federation as part of a routine microbiological examination. Of these, 75 representatives of Chryseobacterium spp., among which C. arthrospherae – 28, C. formosense – 1, C. gambrini – 3, C. gleum – 10, C. indologenes – 20, C. joostei – 1, C. oraniemense – 10, C. shandongense – 2 strains, 4 strains of Elizabethkingia spp., among which 2 – E. miricola, and one strain of E. meningoseptica and E. anopheles, respectively, as well as 21 strains of E. falsenii. Identification of all isolated cultures was carried out using MALDI-ToF mass spectrometry (Bruker, Germany). The MIC values were determined for 17 antimicrobial drugs: amikacin, amoxicillin/clavulanate, aztreonam, cefotaxime, ceftazidime, ceftazidime/avibactam, ceftolozane/ tazobactam, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, piperacillin/ tazobactam, tigecycline, tobramycin and trimethoprim/sulfamethoxazole using Sensititre kits DKMGN. Results. The MIC values of colistin, cefotaxime and tobramycin more than 8 µg/ml, as well as more than 2 µg/ml with respect to ertapenem was demonstrated for 100% of isolates. For most strains, the MIC values of imipenem and meropenem were more than 4 µg/ml. MIC of ceftazidime against 20% of Chryseobacterium spp. strains was up to 2 µg/ml. Indicators of MIC of amikacin in relation to Elizabethkingia spp. strains were 32 µg/ml or more. For 38% of the strains of Chryseobacterium spp. and E.falsenii, this value did not exceed 8 µg/ml. As in the case of amikacin, all Elizabethkingia spp. strains demonstrated high levels of gentamicin MIC – 8 µg/ml (25% of strains) and more (75% of strains). For 20% of all tested strains, the MIC value was in the range of ≤ 2 µg/ml. In relation to half of the tested isolates, the MIC values for ceftazidime/avibactam, as well as ceftolozane/tazobactam were at the level of up to 2⁄4 µg/ml inclusive. More than a third of the strains had a level of MIC ciprofloxacin up to 0.25 µg/ml inclusive. For 25% of strains, the level of MIC of tigecycline was up to 0.5 µg/ml, inclusive, the lowest MIC indicators for the tested group of strains were demonstrated for trimethoprim/sulfamethoxazole: for 88% of strains, the MIC value was ≤ 1⁄19 µg/ml, for another 9% of strains, this indicator was 2⁄38 µg/ml. Conclusions. Representatives of the order Flavobacteriales are a group of microorganisms characterized by multiple antibiotic resistance. Most strains isolated from patients with cystic fibrosis in the Russian Federation retain low MIC values for trimethoprim/sulfamethoxazole.

520. Development of Cefepime-taniborbactam MIC Antimicrobial Susceptibility Test (AST) for Enterobacterales and Pseudomonas aeruginosa on MicroScan Dried Gram-negative MIC Panels

Abstract Background Automated AST devices are critical to inform appropriate care of patients with bacterial infections. Cefepime-taniborbactam is an investigational agent under development to treat infections due to multidrug-resistant bacteria, including carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa. Development of a cefepime-taniborbactam antimicrobial susceptibility test was completed for the MicroScan Dried Gram-negative MIC (MSDGN) Panel when compared to CLSI broth microdilution reference panels. Methods Development was conducted by comparing MICs obtained using the MSDGN panel to MICs using a CLSI broth microdilution reference panel. The concentration range of cefepime-taniborbactam evaluated was 0.12/4-64/4 µg/mL. A total of 1376 isolates (1256 Enterobacterales and 120 P. aeruginosa) were tested at 16, 18, and 20 hours incubation times (for visual read and autoSCAN-4). A total of 917 isolates (837 Enterobacterales and 80 P. aeruginosa) were tested at 16 and 18 hours incubation time for the WalkAway System. All isolates were tested using turbidity and Prompt® methods of inoculation. MSDGN panels were incubated at 35 ± 1°C. Reference panels, prepared according to ISO methodology, were inoculated using the turbidity inoculation method. All frozen reference panels were incubated at 35 ± 2°C and read visually. Rates of essential agreement (MicroScan MIC within ± 1 doubling dilution of reference MIC) were determined for each combination of inoculation method and read method. Results Essential agreement rates ranged from 93.0% to 98.8% for all isolates tested during development. Essential agreement rates for each inoculation and read method are presented in Table 1. Table 1- Results Conclusion Cefepime-taniborbactam MIC results obtained with the MSDGN panel correlated well with MIC results obtained using reference panels. Rates of essential agreement were ≥93.0% for all inoculation and read methods. These development data support the continued evaluation of MSDGN panel with cefepime-taniborbactam in a multicenter trial. Pending submission and clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use in the US. For Investigational Use Only. The performance characteristics of this product have not been established. Disclosures Enrique J. Fernandez, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Robert L. Williams, PhD, Beckman Coulter, Inc.: Employee of Beckman Coulter Vasna Carr, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Vasna Carr, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter Miller Renae, BS, Beckman Coulter, Inc.: Employee of Beckman Coulter.

SENSITIVITY OF GRAM-NEGATIVE CAUSATIVE AGENTS OF INFECTIOUS INFLAMMATORY DISEASES OF SOFT TISSUES OF THE FACIAL REGION TO ANTIBIOTICS

Today timely monitoring of the sensitivity of gram-negative non-fermenting bacteria taken from various sites of the human body to antibiotics is extremely important in the way of stopping and overcoming significant spread of antibiotic resistance. The aim of this study is to determine the sensitivity of gram-negative causative agents of infectious inflammatory diseases of soft tissues of the face region to antibiotics. Materials and methods. The study included 25 clinical isolates of the genus Acinetobacter and 22 clinical isolates of the genus Pseudomonas. Determination of the sensitivity of microorganisms was carried out using the disc diffusion method and the method of double serial dilutions in a liquid nutrient medium in accordance with the recommendations by EUCAST. Results. The level of sensitivity of representatives of genus Acinetobacter to imipenem and meropenem did not exceed 29.6%. The lowest sensitivity was found to fluoroquinolones and the highest sensitivity was revealed to aminoglycosides. Pseudomonas spp. showed low sensitivity to penicillins and cephalosporins. The similarity of the sensitivity results of Pseudomonas spp. to carbapenems imipenem and meropenem was detected. Along with this, testing the sensitivity of pseudomonads to protected carbapenems showed a better result. Moreover, clinical isolates of Pseudomonas spp. showed high resistance to fluoroquinolones and aminoglycosides. Conclusion. Acinetobacter spp. and Pseudomonas spp., isolated from patients with infectious and inflammatory diseases of the soft facial tissues have a rather low sensitivity to antibacterial drugs of various groups. Acinetobacter spp showed the highest sensitivity to gentamicin and amikacin. Pseudomonas possesses the highest sensitivity to cefiderocol, imipenem relabactam and meropenem verabactam.