Determination Of Antibiotic Susceptibility Research Articles

Significant reduction of the culturing time required for bacterial identification and antibiotic susceptibility determination by infrared spectroscopy.

Rapid testing of bacteria for antibiotic susceptibility is essential for effective treatment and curbing the emergence of multidrug-resistant bacteria. The misuse of antibiotics, coupled with the time-consuming classical testing methods, intensifies the threat of antibiotic resistance, a major global health concern. In this study, employing infrared spectroscopy-based machine learning techniques, we significantly shortened the time required for susceptibility testing to 10 hours, a significant improvement from the 24 hours in our previous studies as well as the conventional methods that typically take at least 48 hours. This remarkable reduction in turnaround time (from 48 hours to 10 hours), achieved by minimizing the culturing period, offers a game-changing advantage for clinical applications. Our study involves a dataset comprising 400 bacterial samples (200 E. coli, 100 Klebsiella pneumoniae, and 100 Pseudomonas aeruginosa) with an impressive 96% accuracy in the taxonomic classification at the species level and up to 82% accuracy in bacterial susceptibility to various antibiotics.

In vitro antimicrobial activity of silver nanoparticles against selected Gram-negative and Gram-positive pathogens

Background and aim. Infections caused by pathogenic bacteria increase patient morbidity and mortality and significantly raise treatment costs. The use of silver nanoparticles as an alternative treatment for S aureus, E coli, MRSA, E faecalis, K pneumoniae and P aeruginosa indicates their antibacterial effect and prompts medical research to consider the next generation of antibacterial drugs that could change antibiotic therapy. By combining silver nanoparticles with different classes of antibiotics, the antibacterial effect is evidenced by increased values of the inhibition zone compared to the values obtained for some antibiotics commonly used in the treatment of bacterial infections. This study focuses on comparing the antibacterial activity of antibiotics versus antibiotics combined with silver nanoparticles against various bacteria, by comparing inhibition zones obtained for both. We aim to prove that the size of the inhibition zone for antibiotics combined with silver nanoparticles is greater, thus confirming the improved antibacterial effect. Metods. In this study we tested the antibacterial activity of solutions of silver nanoparticles alone or in combination with different antibiotics. We used standard bacterial strains, ATCC, both Gram positive bacteria Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, as well as Gram negative bacteria Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, but also on clinical isolates: a strain MRSA (Methicillin Resistant Staphylococcus aureus) and a PDR strain (pan drug resistant) of Klebsiella pneumoniae. Bacterial identification was performed using Vitek MS analyzer (bioMerieux). Antibiotic susceptibility determination was performed with VITEK2 COMPACT SYSTEM (bio Merieux, Inc Durham NC) with ready to use VITEK AST cards. The interpretation of the results was done in compliance with EUCAST 2023-2024 standards. Testing was performed for several classes of antibiotics, silver nanoparticle solutions in 2 concentrations (10 μg/mL and 100 μg/mL) and for combinations of antibiotics with silver nanoparticle solutions. The diameter of the inhibition zone (ZOI) for silver nanoparticles, antibiotics and silver nanoparticles combined with antibiotic against each bacterium was expressed in millimeters. The Kirby-Bauer disk-diffusion method, in accordance with current EUCAST standards, was used to analyze the antibacterial effect of antibiotics, silver nanoparticles, and antibiotics combined with silver nanoparticles at biocompatible doses of 10 and 100 μg/mL. The experiments were conducted in triplicate, and the results were almost identical. Results. The results of this study show that the silver nanoparticles displayed antibacterial activity, proven by the appearance of the inhibition zone, in various sizes, for all bacteria studied. The antibiotic classes tested were beta-lactamins, first, second, third and fourth generation cephalosporins, macrolides, fluoroquinolones, lincosamides, aminoglycosides, glycopeptides, tetracyclines, oxazolidinones, sulfonamides, rifamycins, amphenicols. Testing S aureus ATCC 29213, the highest zone of inhibition was demonstrated for cephalosporins (32.6667 ± 0.701 mm), macrolides (31.6667 ± 0.701 mm, and lincosamides (29.6667 ± 0.701 mm). Testing MRSA (internal code GR0333), the highest zone of inhibition for combination of silver nanoparticles and antibiotics was demonstrated for fluoroquinolones (36.3333 ± 0.701 mm), lincosamides (32.3333 ± 0.701 mm), Fusid acid (32.3333 ± 0.701 mm) and aminoglicosides (31.3333 ± 0.701 mm). Testing E coli ATCC 25922 the highest zone of inhibition was for Fosfomycine, 39 mm and for E faecalis ATCC 29212 for aminoglicosides was 19 mm. For K pneumoniae (internal code GQ8575) the inhibition zone for silver nanoparticles 100 μg/mL was 12.3333 ± 0.701 mm and for P aeruginosa ATCC 27253 was 16 ± 1.214 mm. Conclusions. The use of metallic nanoparticles, especially silver ones, as antimicrobial agents with definite bactericidal activity has led medical specialists to consider this new treatment which may change antibacterial therapy. Studies of in vitro combinations between silver nanoparticles and different classes of antibiotics represent a highly efficient and effective new antibacterial treatment against multidrug-resistant bacteria. To avoid the problem of antimicrobial resistance associated with conventional antibiotics, it is necessary to understand the adaptive mechanisms of bacteria under the action of metal nanoparticles, which could be exploited in future studies. Further in vitro and in vivo studies that would assess specify the biocompatibility and toxicity of silver nanoparticles will make these super nanomaterials the medicines of the future.

Detection of resistance to macrolides and fluoroquinolones in Mycoplasma genitalium by targeted next-generation sequencing.

Mycoplasma genitalium is fastidious to culture, and its detection in human clinical specimens relies mainly on molecular methods. Phenotypic determination of antibiotic susceptibility for this bacterium is not a timely or feasible option for most clinical laboratories. This study sought to determine whether next-generation sequencing technologies can effectively be employed in determining genetic mutations associated with drug resistance in M. genitalium samples collected in Aptima Hologic tubes and possibly integrating them into viable workflows in public health laboratories. Following analysis by a custom-designed bioinformatics pipeline, at least one mutation/sample has been identified in 94/98 specimens in at least one of seven loci (macrolides: rrl, rplD, rplV; fluoroquinolones: parC, parE, gyrA, gyrB) described previously to be connected to antibiotic resistance. This method identified a total of 469 single nucleotide polymorphisms (SNPs) (452 mutations): 134 of 23S rRNA SNPs and 318 amino acid mutations: 114 substitutions and 204 synonymous; the turnaround time (sample to analyzed sequence) was typically 3 days. The assays and workflows described in this work demonstrated that the determination of a drug resistance profile for macrolides and fluoroquinolones of M. genitalium samples by using next-generation sequencing in clinical samples is a feasible approach that can be implemented in clinical laboratories, following thorough and extensive validation studies.IMPORTANCEThe mechanisms of drug resistance in Mycoplasma genitalium are complex and involve several genetic loci. The molecular methods for accurately characterizing resistance to fluoroquinolones and macrolides in this organism are often not available or approved for patient use and do not cover all genetic determinants. To this end, we propose a next-generation sequencing-based method with a turnaround time of 3 days that includes the investigation of all drug resistance loci of M. genitalium. Following adaptation, validation, and verification for routine clinical use, assays based on this method may yield molecular results that can be used to guide proper treatment regimens and for surveillance of drug resistance in the general population.

Predicting Salmonella MIC and Deciphering Genomic Determinants of Antibiotic Resistance and Susceptibility.

Salmonella spp., a leading cause of foodborne illness, is a formidable global menace due to escalating antimicrobial resistance (AMR). The evaluation of minimum inhibitory concentration (MIC) for antimicrobials is critical for characterizing AMR. The current whole genome sequencing (WGS)-based approaches for predicting MIC are hindered by both computational and feature identification constraints. We propose an innovative methodology called the "Genome Feature Extractor Pipeline" that integrates traditional machine learning (random forest, RF) with deep learning models (multilayer perceptron (MLP) and DeepLift) for WGS-based MIC prediction. We used a dataset from the National Antimicrobial Resistance Monitoring System (NARMS), comprising 4500 assembled genomes of nontyphoidal Salmonella, each annotated with MIC metadata for 15 antibiotics. Our pipeline involves the batch downloading of annotated genomes, the determination of feature importance using RF, Gini-index-based selection of crucial 10-mers, and their expansion to 20-mers. This is followed by an MLP network, with four hidden layers of 1024 neurons each, to predict MIC values. Using DeepLift, key 20-mers and associated genes influencing MIC are identified. The 10 most significant 20-mers for each antibiotic are listed, showcasing our ability to discern genomic features affecting Salmonella MIC prediction with enhanced precision. The methodology replaces binary indicators with k-mer counts, offering a more nuanced analysis. The combination of RF and MLP addresses the limitations of the existing WGS approach, providing a robust and efficient method for predicting MIC values in Salmonella that could potentially be applied to other pathogens.

Comparative study of oral microbiota in the experimental long-term opioid exposure, after its withdrawal and the use of complex drug correction.

Aim: To study changes of dental biofilm microbiota composition during experimental opioid exposure, after its withdrawal and when using of complex drug correction.. Materials and Methods: Microbiological studies (48 rats) included microscopic and bacteriological methods, as well as determination of antibiotic susceptibility of microbial isolates. Ceftriaxone and pentoxifylline were used to correction the changes. Results: The action of opioid for 10 weeks caused considerable changes in the microbiocenosis, which was illustrated by a significant increasing of the opportunistic pathogens quantitative indicators and the emergence of pathogenic microbiota. Changes in the microbiocenosis at 6 weeks of opioid exposure and after its withdrawal for 4 weeks were expressed in the appearance of pathogenic microbiota and the absence of significant differences in quantitative indicators of saprophytic and opportunistic microflora compared to similar indicators in animals with 10 weeks opioid exposure. This indicated a slow progression of dysbiotic changes and the inflammatory process in the oral cavity of rats. Conclusions: After 10 weeks of experiment with opioid administration for 6 weeks and the use of ceftriaxone and pentoxifylline on the background of 4-week opioid withdrawal, a significant reduction of quantitative indicators of opportunistic bacteria and elimination of pathogenic species of microorganisms was determined. The use of complex drug correction on the background of 10 weeks of opioid exposure led to a significant reduction in the quantitative indicators of opportunistic pathogens and contributed to the elimination of most pathogenic species of microbiota under the action of ceftriaxone.

Susceptibility trends of swine respiratory pathogens from 2019 to 2022 to antimicrobials commonly used in Spain

BackgroundAntimicrobial resistance is one of the most important health challenges in humans and animals. Antibiotic susceptibility determination is used to select the most suitable drug to treat animals according to its success probability following the European legislation in force for these drugs. We have studied the antibiotic susceptibility pattern (ASP) of Actinobacillus pleuropneumoniae (APP) and Pasteurella multocida (PM) isolates, collected during the period 2019–2022 in Spain. ASP was measured by determining minimum inhibitory concentration using standardized laboratory methods and its temporal trend was determined by logistic regression analysis of non-susceptible/susceptible isolates using clinical breakpoints.ResultsIt was not observed any significant temporal trends for susceptibility of Actinobacillus pleuropneumoniae to ceftiofur, florfenicol, sulfamethoxazole/trimethoprim, tulathromycin and tildipirosin during the study period (p > 0.05). Contrarily, a significant temporal trend (p < 0.05) was observed for quinolones (enrofloxacin and marbofloxacin), tetracyclines (doxycycline and oxyteracycline), amoxicillin, tiamulin and tilmicosin. On the other hand, it was not observed any significant temporal trends for susceptibility of Pasteurella multocida to quinolones (enrofloxacin and marbofloxacin), amoxicillin, ceftiofur, florfenicol and macrolides (tildipirosin, tulathromycin and tilmicosin) during the study period (p > 0.05). Contrarily, a significant temporal trend (p < 0.05) was observed for tetracyclines (oxyteracycline), tiamulin and sulfamethoxazole/trimethoprim.ConclusionsIn general terms, pig pathogens (APP and PM) involved in respiratory diseases analysed herein appeared to remain susceptible or tended to increase susceptibility to antimicrobials over the study period (2019–2022), but our data clearly showed a different pattern in the evolution of antimicrobial susceptibility for each combination of drug and microorganism. Our results highlight that the evolution of antimicrobial susceptibility must be studied in a case-by-case situation where generalization for drug families and bacteria is not possible even for bacteria located in the same ecological niche.

Quick Detection of Proteus and Pseudomonas in Patients' Urine and Assessing Their Antibiotic Susceptibility Using Infrared Spectroscopy and Machine Learning.

Bacterial resistance to antibiotics is a primary global healthcare concern as it hampers the effectiveness of commonly used antibiotics used to treat infectious diseases. The development of bacterial resistance continues to escalate over time. Rapid identification of the infecting bacterium and determination of its antibiotic susceptibility are crucial for optimal treatment and can save lives in many cases. Classical methods for determining bacterial susceptibility take at least 48 h, leading physicians to resort to empirical antibiotic treatment based on their experience. This random and excessive use of antibiotics is one of the most significant drivers of the development of multidrug-resistant (MDR) bacteria, posing a severe threat to global healthcare. To address these challenges, considerable efforts are underway to reduce the testing time of taxonomic classification of the infecting bacterium at the species level and its antibiotic susceptibility determination. Infrared spectroscopy is considered a rapid and reliable method for detecting minor molecular changes in cells. Thus, the main goal of this study was the use of infrared spectroscopy to shorten the identification and the susceptibility testing time of Proteus mirabilis and Pseudomonas aeruginosa from 48 h to approximately 40 min, directly from patients' urine samples. It was possible to identify the Proteus mirabilis and Pseudomonas aeruginosa species with 99% accuracy and, simultaneously, to determine their susceptibility to different antibiotics with an accuracy exceeding 80%.

A novel inductively coupled capacitor wireless sensor system for rapid antibiotic susceptibility testing

BackgroundThe increasing prevalence and severity of antimicrobial resistance (AMR) present a major challenge to our healthcare system. Rapid detection of AMR is essential for lifesaving under emergent conditions such as sepsis. The current gold standard phenotypic antibiotic susceptibility testing (AST) takes more than a day to obtain results. Genotypic ASTs are faster (hours) in detecting the presence of resistance genes but require specific probes/knowledge of each AMR gene and do not provide specific information at the phenotype level. To address this unmet challenge, we developed a new rapid phenotypic AST.ResultWe designed a new electrochemical biosensor based on the concept of magnetically coupled LC sensors. The engineered LC sensors can be placed in 96-well plates and communicate the reading remotely with a receiver coil for signal analysis. The sensors were validated by monitoring the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in the presence and absence of different antibiotics. Drug-resistant strains were used as controls. Bacterial growth was detected within 30 min after inoculation, allowing rapid determination of antibiotic susceptibility at the phenotype level. The sensor also functions in the presence of host proteins when tested with 2% FBS in growth media.ConclusionsWith the compatibility with 96-well plates, this label-free rapid 30-min AST has the potential for low-cost applications with simple integration into the existing workflow in clinical settings.

Evaluation of surgical site infections along with the study of antibiogram and drug-resistant isolates from exudate samples in a tertiary care hospital

Abstract Background: Infections of the skin and soft tissue caused by pyogenic bacteria, are a major cause of morbidity, prolonged hospital stay and post-surgical complications. Present study was designed to determine the rate of surgical site infection (SSI), burden of multi drug resistant (MDR) isolates in exudate samples and to study the antimicrobial resistance and susceptibility pattern among these isolates. Methods: One thousand five hundred and seven pus samples from pyogenic infections and SSI received over 1 year in a microbiology laboratory, which yielded aerobic growth, were identified by standard culture methods and biochemical reactions. Antibiotic susceptibility test and determination of extended-spectrum beta-lactamase (ESBL) and methicillin-resistant staphylococci [methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococci (MRCoNS)] rates was done according to the Clinical and Laboratory Standards Institute guidelines. SSI rates of the hospital were determined. Results: Staphylococcus aureus, Klebsiella, Pseudomonas and methicillin-resistant coagulase-negative staphylococci were the most common organisms isolated from pyogenic lesions. SSI rate was 5.9%. Susceptibility of Gram-negative bacteria to imipenem was 88%, amikacin 86%, gentamicin 83% and piperacillin/tazobactam 80%. Susceptibility was least for cephalosporins 54%, ciprofloxacin 39% and ampicillin 28%. ESBL rate was 33% and MRSA was 38%. In SSI, ESBL and MRSA rates were 35% and 43%, respectively. Among Gram positives, susceptibility to linezolid was 100% and clindamycin 71% susceptible. It was least to ciprofloxacin 19% and co-trimoxazole 24.6%. Conclusions: Developing antibiogram helps in framing local antibiotic policy and adhering to this contributes to controlling drug-resistant strains and also aids in reducing SSI rates.

Phenotypic and Genotypic Identification of Carbapenem-Resistant Klebsiella Pneumoniae and Determination of Antibiotic Susceptibility

Amaç: K. pneumoniae izolatlarında karbapenemaz genlerinin tanımlanması amaçlanmıştır. Hastalar ve Yöntem: Bu çalışmaya Temmuz 2016-Aralık 2017 tarihleri arasında örneklerden izole edilen çok ilaca dirençli 95 K. pneumoniae suşu dahil edilmiştir. Meropenem, tigesiklin ve kolistinin MIC değerleri Vitek 2, Etest ve Sıvı Mikrodilüsyon Yöntemleri (SMD) ile belirlenmiştir. Kategorik uyum (KU), Çok Büyük Hata (ÇBH) ve Büyük Hata (BH) oranları hesaplanarak değerlendirilmiştir. blaOXA-48, blaOXA-181, blaNDM-1, blaVIM, blaIMP ve mcr-1 kolistin direnç geni araştırılmıştır. Bulgular: SMD, Vitek 2 ve Etest ile izolatlarda belirlenen meropenem direnci sırasıyla; %70,5, %87,4, %81,1 olarak tespit edilmiştir. SMD yöntemine göre KU, ÇBH ve BH oranları Vitek 2 ve Etest için sırasıyla %69,5, %4,8 ve %20; %70,5, %1,5 ve %0 olarak belirlenmiştir. İzolatlarımızda en yüksek duyarlılık tigesikline karşı tespit edilmiştir. Tigesiklin için KU oranları Vitek 2 ve Etest ile %70,5 ve %95,8 olarak bulunmuştur. Vitek 2 tarafından belirlenen BH oranı, kabul edilebilir %7,6 (&lt;%3) sınırının üzerinde olarak saptanmıştır. Kolistin direnci SMD ile %48,4 olarak tespit edilmiştir. Kolistinin belirlenen KU; ÇBH; BH oranları Vitek 2/Etest için sırasıyla %86,3/%72,6; %17,4/%50; %10,2/%6,1 olarak bulunmuştur. Çalışmamıza dâhil ettiğimiz izolatlarda ağırlıklı olarak blaOXA-48 (%93,7) saptanmıştır. blaOXA-48, izolatların 56'sında (%59) tek başına, 33 izolatta (%34,74) ise blaOXA-181 genleriyle birlikte tespit edilmiştir. İzole OXA-48 pozitif suşlarda, OXA-181 ile birliktelik gösteren izolatlara göre daha düşük kolistin MİK seviyelerine rastlanmıştır. İzolatların %3,2 ’sinde blaNDM-1 geni tespit edilmiştir. PZR ile karbapenemaz kombine disk sonuçları izolatların %91,3’ünde uyumlu olarak bulunmuştur. Sonuç: OXA-48 direnç geni bölgemizde yaygınlığını korumaktadır. Bunun yanında blaOXA-48-benzeri gen bölgelerinin gerçek prevalansının ve direnç dinamiklerinin kapsamlı olarak ortaya konması, ulusal karbapenemaz sürveyans politikalarının gelişimine katkı sağlayacaktır.

Bacterial ecology of sepsis in febrile neutropenics on hematology-oncology services

Introduction: Febrile neutropenia is a hematologic emergency that develops following treatment of hematologic malignancies. It exposes patients to the risk of sepsis, which is a major cause of morbidity and mortality. Currently, coagulase-negative staphylococci are the most common bloodborne isolates while enterobacteria are isolated less frequently. The objective of our study is to determine the epidemiological, bacteriological and antibiotic susceptibility profile of strains isolated from blood cultures collected from febrile neutropenic patients at Hassan II University Hospital, Fez. Materials and methods: This is a prospective study, over a period of 4 years (January 2019 - December 2022) including 83 patients with bacteremia hospitalized in the department of hemato-oncology. Identification and detection of antibiotic resistance was performed according to conventional bacteriology methods and CA-SFM/EUCAST recommendations. Results: Among 524 blood cultures received, we diagnosed 83 cases of bacteremia. The average age of the patients was 55 years. The average age of the patients was 55 years. The predominance of women was noted, with a sex ratio of 0.88. The majority of the episodes, 94.87%, occurred in patients followed for a neoplastic pathology. Hematological malignancies were the most responsible with a rate of 61.54% against 33.33% of solid tumors. Gram-positive bacteria represent 56.6%, and 43.4% of Gram-negative bacteria. The proportions of the different bacterial classes (BGN / CG+) have remained stable over the last 4 years, Staphylococcus epidermidis was the most frequent isolated Gram-positive bacteria 24.1℅(n=29) then Staphylococcus aureus 24.6% (n=18), followed by Gram-negative bacteria of enterobacteria type especially Escherichia coli which was the most frequently isolated pathogen (16.86%), followed by Klebsiella pneumoniae (10.84℅) and non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa (8.4) and Acinetobacter baumanii (6℅). Bacteria of clinical interest have shown increasingly worrisome levels of beta-lactam resistance, with extended-spectrum beta-lactamase (ESBL)-producing strains of Klebsiella pneumoniae accounting for 4, 8% with only one case of carbapenemase, meticillin-resistant Staphylococcus aureus was 3.61% while no imipenem-resistant Pseudomonas aeruginosa or vancomycin-resistant enterococci were found. While Acinetobacter baumanii isolates (n=5) showed increased resistance to the majority of antibiotics tested, especially to imipenem which was 100%. Discussion and conclusion: Gram-positive bacteremia of the genus coagulase-negative staphylococcus is the most common type of infection in febrile neutropenias showing an increasingly resistant profile to first-line antibiotics. However, Gram-negative infections have shown decreased susceptibility compared to previous years. A regular study of the bacterial ecology of blood culture isolates and a determination of antibiotic susceptibility are necessary to optimize the probabilistic antibiotic therapy of bacteremia.

Determination of pilus-islands profile and antibiotic susceptibility of Streptococcus agalactiae isolated from urine of pregnant women.

Group B Streptococcus (GBS) is one of the most important causes of neonatal diseases and postpartum fever. GBS infection can be transmitted from the infected mother to her baby during delivery. This bacterium is also involved in causing urinary tract infections and asymptomatic bacteriuria, pyelonephritis, cystitis and urethritis. In addition to capsule, Pilus is known as a virulence factor of GBS. The aim of this study was to evaluate the frequency of pilus islands and antibiotic resistance in GBS isolated from urine of pregnant women in Yazd, Iran. In this cross-sectional study, 33 GBS samples isolated from the urine of pregnant women were studied by the multiplex polymerase chain reaction (PCR) method for the presence of pilus islands PI-1, PI-2a and PI-2b. Antibiotic resistance phenotype of tetracycline, penicillin, gentamicin, erythromycin, levofloxacin and clindamycin was determined by disk diffusion method. Data were analyzed using SPSS, version 16. PI-1+PI-2a was the most frequent pilus island in the GBS isolates 28 (84.8%) and the frequency of PI-2b was 5 (15.2%). The frequency of PI-1+PI-2a was 50% in serotype III and 25%, 14.3%, 7.1% and 3.6% in serotypes Ia, II, Ib and V respectively (P=0.492). The sensitivity of all GBS isolates to penicillin was 93.9% and highest resistance to tetracycline (97%), clindamycin (24.2%) and erythromycin (21.2%). Most of the GBS urine isolates examined carried the PI-1+PI-2a gene, which increases bacterial potency in colonization and resistance to the immune system. Penicillin was best choice for prevention.

Identification of a novel carbapenem-hydrolysing class D β-lactamase RAD-1 in Riemerella anatipestifer.

To elucidate the role of a novel carbapenem-hydrolysing class D β-lactamase (RAD-1) from Riemerella anatipestifer. We applied WGS and bioinformatic analysis to screen putative β-lactamase genes in R. anatipestifer SCVM0004. A putative class D β-lactamase gene was cloned into pET24a and transferred into Escherichia coli BL21 (DE3) for antibiotic susceptibility determination and protein purification. Meanwhile, the purified native protein was used to determine the enzymatic activities. A class D β-lactamase, RAD-1, was identified in the genome of R. anatipestifer SCVM0004. It was distinct from all characterized class D β-lactamases (≤42% amino acid sequence identity). Searching in GenBank showed that blaRAD-1 was widely disseminated among R. anatipestifer. Genomic environment analysis indicated that the chromosomal structures of blaRAD-1-located regions were relatively conserved. Expression of RAD-1 in E. coli results in elevated MICs for various β-lactam antibiotics, including penicillins, extended-spectrum cephalosporins, a monobactam and carbapenems. Moreover, kinetic analysis of purified RAD-1 revealed: (i) high-level activity against penicillins; (ii) highest affinity for carbapenems; (iii) moderate hydrolysis of extended-spectrum cephalosporins and a monobactam; and (iv) no activity for oxacillin and cefoxitin. This study identified a novel chromosomally located class D carbapenemase RAD-1 (Bush-Jacoby functional group 2def) in R. anatipestifer SCVM0004. Moreover, bioinformatic analysis confirmed that the RAD-1 was widely prevalent and conserved in R. anatipestifer.

Molecular diagnostics for genotypic detection of antibiotic resistance: current landscape and future directions.

Antimicrobial resistance (AMR) among bacteria is an escalating public health emergency that has worsened during the COVID-19 pandemic. When making antibiotic treatment decisions, clinicians rely heavily on determination of antibiotic susceptibility or resistance by the microbiology laboratory, but conventional methods often take several days to identify AMR. There are now several commercially available molecular methods that detect antibiotic resistance genes within hours rather than days. While these methods have limitations, they offer promise for optimizing treatment and patient outcomes, and reducing further emergence of AMR. This review provides an overview of commercially available genotypic assays that detect individual resistance genes and/or resistance-associated mutations in a variety of specimen types and discusses how clinical outcomes studies may be used to demonstrate clinical utility of such diagnostics.

Determination of Antibiotic Susceptibility of Avian Pathogenic Escherichia coli by Phenotypic and Genotypic Methods

tEscherichia coli infection is one of the most important infections, which causes heavy economic losses in the poultry industry. It is caused by avian pathogenic E.coli (APEC), which belongs to the group Extra-intestinal Pathogenic E. coli (ExPEC). In this study, a total of 60 APEC isolates from various poultry farms located in and around Anand were evaluated for antibiotics susceptibility by disc diffusion (phenotypic) method for 21 different antibiotics. The E. coli isolates showed 100% resistance against pefloxacin, moxifloxacin and tetracycline followed by ampicillin (86.67%), levofloxacin (81.67%), amoxiclav and ciprofloxacin (71.67% each), co-trimoxazole (56.67%), sulphadiazine (53.34%) and erythromycin (33.33%). The study also revealed that E. coli isolates were highly susceptible to colistin (100.00%), followed by ceftriaxone and spectinomycin (85.00% each), cefixime (81.67%), amikacin (80.00%) and gentamicin (76.67%). All these isolates were also screened for the presence of 20 different antibiotic-resistant genes (ARGs) by genotypic method, i.e., polymerase chain reaction. PCR revealed presence of cmlA gene responsible for chloramphenicol resistance in cent percent isolates. The distribution of other ARGs in the E. coli isolates were qnrS (85.00%), aac(3)-IV (56.66%), strB (53.33%), aadA1 (51.66%), dhfrI (50.00%), tetB (41.66%) sulI and tetA (33.33% each), blaOXA (31.66%), cat1 and blaCMY (21.66% each), strA (6.67%), blaSHV (3.33%) and dhfrV (1.66%), while all the isolates were negative for blaTEM, ere(A), qnrA, qnrB and mcr-1 genes.

16s rRNA-based phylogenetic analyses of Elizabethkingia anophelis: Detection of Elizabethkingia anophelis, a rare infectious agent from blood and determination of antibiotic susceptibility in Turkey

PurposeElizabethkingia anophelis was firstly isolated from the midgut of the Anopheles gambiae mosquito in 2011. After this year, it was isolated in some intensive care cases in Africa and Asia. This study, it was aimed to confirm the identification of E. anophelis in the blood of a pediatric patient. MethodsAfter the suspicious bacteria were grown on blood agar, MALDI-TOF MS and 16s rRNA gene sequencing methods were used to identify and an antibiotic susceptibility test was carried out by Vitek 2 Compact system according to the EUCAST. Finally, a phylogenetic tree was created based on the 16s rRNA gene region. ResultsThe isolate was identified as E. anophelis by both methods. It was found to be resistant to all beta-lactam antibiotics and also susceptible to ciprofloxacin and levofloxacin. According to the 16S rRNA-based phylogenetic tree, our isolate clustered within a branch containing other E. anophelis. ConclusionThese findings will guide clinicians in choosing which antibiotic to choose if they encounter this agent. Also, the clinicians should be vigilant against this agent, as it is a newly emerging infectious agent in Turkey.

Determination of antibiotic susceptibility of bacteria by flow cytometric method

In this study, it was aimed to determine the antibiotic susceptibility of bacterial strains by using flow cytometry method by comparing them with current standardized methods.Eleven clinical isolates and 6 standard bacterial strains were included in the study. MIC values were determined by broth microdilution method (BMD), VITEK 2® automated system and flow cytometric method (FCM). FCM was performed with the Accuri C6 flow cytometer.For all strains except P. aeuruginosa ATCC 27853 [BMD-FCM:r = 0.557(p = 0.048); VITEK 2-FCM:r = 0.529(p = 0.063)], E. faecalis ATCC 29212 [BMD-FCM:r = 0.393(p = 0.295); BMD-VITEK 2:r = 0.393(p = 0.295)], and vancomycin-resistant E. faecium clinical isolate [BMD-FCM:r = 0.452(p = 0.063)] r values were in the range of 0.802-0.969 for BMD-FCM (p < 0.001), 0.655-0.941 for BMD-VITEK 2 (p < 0.005) and 0.667-0.953 for FCM-VITEK 2 (p < 0.005).Correlation values of antibiotic susceptibility test results between three methods for Gram-negative bacteria were found as follows; r = 0.927(p < 0.001) for BMD-FCM, r = 0.851(p < 0.001) for BMD-VITEK 2, r = 0.807(p < 0.001) for VITEK 2-FCM. Correlation values were found as follows for Gram positive bacteria; r = 0.848(p < 0.001) for BMD-FCM, r = 0.877(p < 0.001) for BMD-VITEK 2, r = 0.800(p < 0.001) for VITEK 2-FCM. When all bacteria included in the study were evaluated as a total; it was r = 0.911(p < 0.001) for BMD-FCM, r = 0.888(p < 0.001) for BMD-VITEK 2, r = 0.835(p < 0.001) for VITEK 2-FCM. The methicillin resistance of the clinical methicillin resistant S. aureus isolate could not be detected by FCM.It was determined that there was a high level of correlation between methods. FCM shortens the duration of antibiotic susceptibility tests by 12-14h and gives results within the same day. However, it has not been standardized to be widely used in microbiology laboratories and experienced personnel are needed for its implementation.

Roles of Bacterial Mechanosensitive Channels in Infection and Antibiotic Susceptibility

Bacteria accumulate osmolytes to prevent cell dehydration during hyperosmotic stress. A sudden change to a hypotonic environment leads to a rapid water influx, causing swelling of the protoplast. To prevent cell lysis through osmotic bursting, mechanosensitive channels detect changes in turgor pressure and act as emergency-release valves for the ions and osmolytes, restoring the osmotic balance. This adaptation mechanism is well-characterized with respect to the osmotic challenges bacteria face in environments such as soil or an aquatic habitat. However, mechanosensitive channels also play a role during infection, e.g., during host colonization or release into environmental reservoirs. Moreover, recent studies have proposed roles for mechanosensitive channels as determinants of antibiotic susceptibility. Interestingly, some studies suggest that they serve as entry gates for antimicrobials into cells, enhancing antibiotic efficiency, while others propose that they play a role in antibiotic-stress adaptation, reducing susceptibility to certain antimicrobials. These findings suggest different facets regarding the relevance of mechanosensitive channels during infection and antibiotic exposure as well as illustrate that they may be interesting targets for antibacterial chemotherapy. Here, we summarize the recent findings on the relevance of mechanosensitive channels for bacterial infections, including transitioning between host and environment, virulence, and susceptibility to antimicrobials, and discuss their potential as antibacterial drug targets.

Isolation and Characterization of Microbes of Different Kinds of Street Food and Determination of Antibiotic Susceptibility of the Isolates

Aim: Improper personal hygiene can facilitate the transmission of the pathogenic microorganisms found in environment and on people’s hands via food to humans. The present study was undertaken to investigate the microbiological quality of different street food and determining the antibiotic susceptibility of the isolated microorganisms.&#x0D; Study Design: Collection of food samples for isolation of Pathogenic Microorganisms, to identify them by using Biochemical test, molecular test (16sr RNA typing), Antibiotic susceptibility was done by using different antibiotics against the isolates.&#x0D; Place and Duration of Study: Food samples were collected from street vendors of Sainkpuri area, work was done from December 2020 to April 2021 at Microbiology Department, St. Francis College for Women, Hyderabad.&#x0D; Methodology: Five samples of street food were collected in sterilized bottles, tested for the presence of microorganisms by following standard microbiological method used for isolation of microorganisms. The organisms were identified by carrying out various biochemical test according to Bergey’s Manual of Systematic Bacteriology. The Molecular characterization was done based on 16sr RNA typing. Determining the sensitivity of the isolates against different Antibiotics by employing Kirby Bauer technique.&#x0D; Results: The organisms isolated from Manchuria and Ragada samples: Lactobacillus delbrueckii; Lactobacillus casei, Klebsiella pneumonia, Enterococcus faecalis and Vibrio spp respectively; Samosa and bonda: Staphylococcus aureus, Lactobacillus delbrueckii and Pantoea dispersa; Pani puri water: Staphylococcus aureus and Providencia vermicola. Antibiotic susceptibility tests showed that most of the isolated microorganisms were sensitive to Ciproflaxin.&#x0D; Conclusion: Hence the quality of street food is found to be low due to following factor: lack hygiene conditions in the food preparation, ingredients may also affect quality of the food, and presence of air borne microorganisms in the surrounding areas of service points.

Phenotypical resistance profile of Stenotrophomonas maltophilia insolated from farm amimals

Stenotrophomonas maltophilia is a multidrug‐resistant opportunistic pathogen. Infections caused by resistant strains of bacteria in animals are characterized by a more severe manifestation, increased duration of treatment and often leads to recurrence. Isolation, identification and determination of pathogens and interpretation of their antibioticograms are the basis of effective and rational antibiotic therapy. The aim of the study was to analyze the antibiotic resistance of Stenotrophomonas maltophilia cultures isolated in cases of respiratory diseases in livestock. The materials for the study were nose scrapings collected from 50 postpartum period cows and 69 newborn calves, and 30 horses up to 3 years old. Primary cultures of tracheal and nasal swabs from horses, cows, and calves were incubated on meat‐peptone broth with 1% glucose and horse serum at 37℃ for 24 hours. After that the culture was inoculated on nutrient media such as columbia ram blood agar, chocolate agar, Endo medium and then incubated at 37℃ for 24 hours. Identification of the isolated cultures and study of their biochemical properties were performed using Nefermtest 24 (Erba Lachema, Czech Republic) test systems. Antibiotics resistance measurement was carried out by the MIC method using MicroScan WalkAway 40 plus (Siemens, Germany) automatic analyzer and GN3F, RUSTEF, NF Sensititre (Trek Diagnostic Systems, Great Britain) plates. Disk diffusion test with Mueller‐Hinton agar was performed according to the recommendations of the European Committee for the determination of antibiotic susceptibility (EUCAST). Pathogenicity of cultures was studied by in vivo testing on white mice. The total number of multidrug resistant S. maltophilia isolates made up less than 5% of 149 clinical samples. Following groups of antibiotics were selected to test the antibiotic sensitivity of this bacterium based on the study’s results: penicillins combined with β‐lactamase inhibitors (co‐amoxiclav, ampicillin/sulbactam, ticarcillin/clavulanic acid), quinolone antibiotics (levofloxacin, moxifloxacin, ciprofloxacin, enrofloxamethacin (trimethoxoprimolamines), sulfanilamylamines (TMP/SMX), nitrofurans (nitrofurantoin), tetracyclines (doxycycline), and macrolides (erythromycin). S. maltophilia cultures isolated from livestock were sensitive only to levofloxacin and moxifloxacin. Interpretation of antibioticograms is one of the main factors that determine the effectiveness of antibiotic therapy.