Antibiotic Susceptibility Testing Research Articles

Microbroth dilution method for antibiotic susceptibility testing of fastidious and anaerobic bacteria of the urinary microbiome.

Antibiotic susceptibilities of fastidious and anaerobic bacteria of the human urinary microbiome are largely underreported due to difficulty in growing them in the lab environment. The current standard medium, Muller-Hinton broth, has difficulty supporting the growth of many of these species, leaving microbiologists without a standardized method. To address this need, this study offers a methodology to survey susceptibilities in a high-throughput manner of these understudied microbes with a proposed harmonized medium, NYCIII, which is capable of supporting the growth of both fastidious and non-fastidious urinary microbes. Broader standardization of this method can allow for the development of antibiotic-resistant breakpoints of the many uncharacterized urinary microbes.

Antibiotic resistance of Bacillus species isolated in foodstuff samples that were collected in Babylon (Iraq)

A total of 164 food samples were collected from various supermarkets and farmers in various areas of Babylon, were placed in plastic containers, and were transported to the laboratory. The isolation of Bacillus species was performed by culture in blood agar, chromogenic agar, and the absence of growth on McConkey agar, by staining with Gram stain, and through molecular identification of the species’ 16SrRNA gene and sequencing. The antibiotic susceptibility test used eight types of antibiotics. The undertaken sequencing identified the Bacillus isolates to belong to the follow¬ing species: B. subtilis (2), B. cereus (6), B. thuringiensis (1), B. anthracis (1), and B. spizizenii (1). The susceptibility test of the six B. cereus isolates revealed that 5 (83.3%), 4 (66.6%), 3 (50%), 2 (33.3%), 2 (33.3%), 2 (33.3%), 1 (16.6%), and 0 (0%) were resistant to rifampicin, clindamycin, erythromycin, tetracycline, trimethoprim, nitrofurantoin, gentamicin, and ciprofloxacin, while the respective resistance numbers for the two isolates of B. subtilis were 2 (100%), 2 (100%), 2 (100%), 0 (0%), 1 (50%), 0 (0%), 0 (0%), and 0 (0%). One isolate of B. thuringiensis presented resistance to erythromycin, erythromycin, trimethoprim, and rifampicin, while one isolate of B. anthracis was found to be resistant to gentamicin, erythromycin, nitrofurantoin, and rifampicin. The B. spizizenii isolate was resistant to all antibiotics except gentamicin and trimethoprim.

Identification of Acinetobacter schindleri isolated from Chinese giant salamanders (Andrias davidianus)

At a particular aquaculture facility in Zhangjiajie in China, the Chinese giant salamander (Andrias davidianus) exhibited analogous clinical manifestations, culminating in sequential mortalities. This study used rigorous aseptic sampling methods to isolate bacteria from the affected liver of salamanders to determine the causative agent behind the decline in amphibians. A bacterial pathogen was isolated from diseased A. davidianus, and the strain was named DN-3. This isolate was subjected to bacterial identification, antibiotic susceptibility assays, reinfection experiments, and biochemical profiling. The isolated bacterial strain was definitively identified as Acinetobacter schindleri using 16S rRNA sequence analysis and biochemical identification. Antibiotic susceptibility testing revealed that this isolate was susceptible to neomycin, macrolides, doxycycline, piperacillin, nitrofurantoin, and carbenicillin. Subsequent reinfection assays, in which varying concentrations of the bacterial inoculum were administered to healthy salamanders, confirmed that the pathogen elicited varying degrees of morbidity or mortality within an eight-day observation period. The median lethal dose (LD50) of A. schindleri DN-3 for A. davidianus was calculated to be 6.25×104 CFU/mL. This result supports the significant pathogenicity of the strain for A. davidianus. The findings of this study provide empirical insights into the clinical management and epidemiological control of diseases affecting A. davidianus.

A Study of Genotypic Characterization of ESBL and MBL Genes of β-Lactamase Producing Pseudomonas aeruginosa in Various Clinical Samples

Pseudomonas aeruginosa, a Gram-negative bacterium, presents a substantial challenge in healthcare due to its adaptability and resistance. This study delves into its genotypic characteristics, focusing on ESBL and MBL genes. The prevalence of P. aeruginosa in nosocomial infections, the research aims to decipher resistance mechanisms crucial for tailored interventions. The study includes 170 non-repetitive clinical samples with protocols. Antibiotic susceptibility testing reveals diverse resistance patterns. Molecular detection of ESBL and MBL genes involves DNA isolation, PCR amplification, and gel electrophoresis. The study examined 170 P. aeruginosa samples, revealing gender-specific variations with 65.91% male and 34.09% female isolates. Antimicrobial testing displayed resistance in Ceftazidime (59%) and Ciprofloxacin (48%), while Ticarcillin-clavulanic acid showed promising sensitivity (58%). Molecular identification unveiled diverse resistance genes across sample types, emphasizing genetic complexity. The study underscores the urgency for targeted therapeutic interventions and novel antimicrobial strategies against P. aeruginosa infections. As antimicrobial resistance complexities persist, this research guides efforts toward a profound understanding of clinical interventions and strategic antimicrobial management.

MICROBIOLOGICAL ASSESSMENT OF INDOOR AIR QUALITY OF STUDENT HOSTELS AND CANTEENS IN A NIGERIAN UNIVERSITY CAMPUS: RISKS ON STUDENTS’ HEALTH

Microbial air contaminants affect the quality of air we breathe and these microbial air contaminants can settle on body parts, clothes, food etc. which poses a serious threat to human health. Therefore, the level of bacterial contamination in various in-door air of the students’ hostels and canteens of Kaduna State University (KASU) and their antibiotics susceptibility patterns was studied. A total of 44 samples were collected from the rooms (18), toilets (18) of the students hostels and canteen (8) of KASU; using the settle plate method. Gram staining and standard biochemical methods were used to identify the bacterial isolates and antibiotic susceptibility testing was determined by disc diffusion method. The bacteria load was between 1.2 x 103 to 6.7 x 103 cfu/ml3. A total of 30 bacteria isolates were identified, Staphylococcus aureus had the highest (9, 30%) occurrence, followed by Streptococcus spp (6, 20%), Bacillus subtilis and Clostridium spp had similar (4, 13.2%) occurrence, The antibiotic susceptibility testing carried out revealed that the Gram negative and Gram positive bacteria isolates were more susceptible to vancomycin (75%, 43.5%), tetracycline (75%, 47.8%) and gentamicin (50%, 43.5%) while high resistance was observed with chloramphenicol (100%, 73.9%), ceftriaxone (100%, 100%), cefoxitin (100%, 73.9%), amoxicillin/clavulanic acid (100%, 87%), and trimethoprim/sulphamethoxazole (100%, 78%).  All the indoor samples were contaminated with pathogenic bacteria and there was high level of multidrug resistance.  This is very dangerous to the students’ health because it can be a source of transmission infections and antibiotic resistant bacterial strains.

PHENOTYPIC DETECTION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF EXTENDED SPECTRUM BETA LACTAMASE PRODUCING Escherichia coli FROM SUSPECTED CASES OF URINARY TRACT INFECTION IN KANO METROPOLIS, NIGERIA

Urinary tract infections (UTIs) caused by antimicrobial-resistant bacteria, especially ESBL-producing Escherichia coli can be life-threatening as therapeutic options available to treat infected patients are limited. Resistance due to ESBL-producing bacteria poses a peculiar challenge in treating infections because of its association with multidrug resistance. The aim of this study was thus to determine the susceptibility pattern and phenotypic detection of ESBL-producing E. coli from UTI patients. Two hundred and forty-six (246) E.coli isolates obtained from patients with suspected urinary tract infections were studied. The identity of the isolates was confirmed using standard biochemical tests. Antibiotic susceptibility testing was carried out using the Kirby-Bauer Disc Diffusion Technique. Screening for ESBL production was done using the Clinical Laboratory Standards Institute breakpoint. Suspected ESBL producers were subjected to confirmation using the Double Disc Synergy Test. Standard Discs of Augmentin (AMC 30µG Oxoid England), Ceftazidime (CAZ 30µG, Oxoid England) and Cefotaxime (CTX 30µG, Oxoid England) were used for the screening and confirmation. Multidrug-resistant E.coli were found to be 65.4%. Screening for ESBL production showed 67.1% suspected ESBLs producing E.coli. The Double Disc Synergy Test showed 22.4% confirmed ESBLs producing E.coli. Antimicrobial sensitivity of the ESBLs producing organisms showed 100% resistance to augmentin, ceftriaxone, ceftazidime, ciprofloxacin and cefotaxime while resistance to gentamicin was 91.1%, chloramphenicol 89.2%, nitrofurantoin 78.4%, and cotrimoxazole 94.6%. A 100% sensitivity to imipenem was also observed. ESBL-producing E.coli are present in Kano metropolis and are resistant to commonly prescribed antibiotics. We, therefore, suggest screening and confirmation for ESBL, to prevent treatment failure.

Antibiotics resistance and phenotypic virulence factors of Vibrio species isolated from poultry litters at Obafemi Awolowo University, research farm.

The study investigates the antimicrobial resistance and phenotypic virulence factors of Vibrio isolates in poultry litter. Samples were enriched in 5 mL alkaline peptone water 1:10 (w/v) for 6 hours and cultured on Thiosulphate Citrate Bile salt (TCBS) agar. Vibrio isolates were identified through biochemical tests assay and were subjected to antibiotic susceptibility tests using standards disc diffusion technique. The phenotypic virulence factors, including hemolytic and proteolytic activity were determined. A total of 100 Vibrio isolates were identified as Vibrio parahaemolyticus (49%), V. cholerae (26%), V. vulnificus (11%), V. alginolyticus (9%), and V. damsela (5%). The Vibrio isolates showed a significant resistance to the selected antibiotics tested with 99% resistant to Gentamycin, 95% Cefixime, 96% Ofloxacin, 98% Augmentin, 96% Nitrofurantoin, 100% Ciprofloxacin, and 100% Cefuroxime. The Vibrio isolates showed varying levels of haemolysis with 17% alpha haemolysis, 61% beta haemolysis, 22% gamma haemolysis, and 29% proteolytic activity. The study reveals that poultry litter contains Vibrio isolates with antibiotic resistance and virulence factors, increasing their risk of severe infections. The high beta haemolysis prevalence highlights the interconnected relationship between animal, environmental, and human health, underscoring the need for effective management.

Incidence of drug resistance bacterial isolates from different Pyoderma cases attending at tertiary care hospital of Central Nepal

Background & Objectives: Pyoderma is defined as any purulent skin disease and represents infections in epidermis and dermis including hair follicles. Thus, this study was conducted to find out incidence and bacteriological profile of pyodermas as well as to determine their antibiotic susceptibility test of isolated bacteria. Materials and Methods: The samples were collected in dermatological OPD and sent the collected samples immediately to bacteriology laboratory for culture and sensitivity tests from December 2018 to May 2019 A.D. Antimicrobial susceptibility testing was done using Kirby–Bauer disc diffusion method and the results were interpreted according to the CLSI guidelines. Quality control of the test was done by standard ATCC strains of S.aureus (ATCC 25923), E.coli (ATCC 25922). Results: The incidence of pyoderma was 3.24%. Maximum cases were detected in the age group below 13 years of age. Primary pyoderma (61.8%) was more frequent when compared to secondary pyoderma (38.2%). The highest number of organism isolated was S. aureus (24.7%) from different cases of pyoderma followed by E. coli (8.2%), CONS (6.1%) and Klebsiella pneumoniae (5.1%) whereas other organisms were found in less number. Gram positive bacteria mainly S. aureus and other isolates for the cause of pyoderma were mostly resistant to penicillin (100%), ciprofloxacillin (75.0%) and clindamycin (79.2%). Gram negative E. coli (8.2%) was second most bacterial isolates and others GNB isolates were (14/63) where resistance pattern were seen more (75.0%) in each ceftrixone and imipenem. Conclusion: Incidence of pyoderma was low among the patients visiting to the Dermatology Department. Antibiotic resistance is increasing day by day; therefore, routine culture and sensitivity tests are essential to guide proper treatment procedure.

Genetic Analyses of Rare ESBL ST628 Klebsiella pneumoniae Detected during a Protracted Nosocomial Outbreak in the United Kingdom

Klebsiella pneumoniae (K. pneumoniae) cultures from a hospital-wide outbreak in the UK, which lasted for over 12 months, were sequenced. We sought to sequence and genetically characterise the outbreak strain. Antibiotic Susceptibility Testing (AST) was performed on 65 K. pneumoniae isolates saved from the outbreak. All isolates were sequenced using the Oxford Nanopore Technologies (ONT) MinION flowcell: 10 isolates, including the isolate with the earliest collection date in 2017, were additionally sequenced on the NovaSeq 6000 platform to build high-accuracy nanopore-illumina assemblies. Among the sequenced strains, 60 were typed as ST628. 96.6% (n = 58/60) ST628 strains harboured a large ~247-kb FIB(K) plasmid carrying up to 11 antimicrobial resistance genes, including the extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15. Clonality between the outbreak isolates was confirmed using single nucleotide polymorphism (SNP) typing. The outbreak strains were phylogenetically related to clinical ST628 strains identified in 2012, 6 years prior to the outbreak. A rare ESBL K. pneumoniae K2 ST628 strain harbouring a multi-drug resistant (MDR) plasmid encoding the ESBL gene blaCTX-M-15 was detected across multiple independent wards during the protracted nosocomial outbreak. Surveillance of this strain is recommended to prevent future nosocomial outbreaks.

Physical quality and antibiotic susceptibility profile of <i>Escherichia coli</i> isolates from roof-harvested rainwater: A microcosm study

Owing to the inadequate supply of potable water in rural settlements in developing countries, roof-harvested rainwater (RHRW) has been  favoured as an alternative source of water supply. The present study investigated the physical characteristics and microbial quality  of 10 RHRW samples collected from storage tanks in Ikwo community of Ebonyi State, Nigeria. Triplicate water sampling was performed  over a 3-month sampling regime. Onsite determination of odour, colour, pH and temperature, and isolation and enumeration of  Escherichia coli were done using standard procedures. The identity of the isolates was confirmed using molecular techniques, and the  susceptibility of the isolates to 11 antibiotics was determined following the Kirby-Bauer disc diffusion assay. Multiple antibiotic resistance  phenotypes and indices (MARPs and MARI) were equally determined. Our findings revealed that the RHRWs had unobjectionable odour  and were colourless. Across the 3-month o sampling, the pH ranged from 6.9 to 7.8 while the temperature was between 24 °C and 29 C.  While E. coli was absent in samples E (September), H (August and October) and J (August, September, and October), the highest E. coli  count in August, September, and October was 37, 32, and 38 cfu/100 mL, respectively. The antibiotic susceptibility test on 40 E. coli  isolates showed high resistance to ampicillin (100%), aztreonam (98%), cefotetan (97%), nitrofurantoin (90%), imipenem (83%),  streptomycin (72%), and ertapenem (69%). Interestingly, the  ighest MARP, 11 (AK/S/ETP/IMI/OFX/NOR/AMP/CTT/ATM/F/TE) (sample B),  and the least, 4 (IMI/AMP/CTT/ATM) were recorded in samples B and D, respectively in September. MARI was from 0.42 to 1.00. The  physical parameters conform with WHO standards, on the other hand, detection of E. coli poses risks of water-borne illness(es) and  transmission of antimicrobial resistance. Hence stringent measures including proper treatment, sanitation and hygiene are advocated to  safeguard the health of dwellers who depend on this water source for daily life activities. 

Identification and florfenicol-treatment of pseudomonas putida infection in gilthead seabream (Sparus aurata) fed on tilapia-trash-feed

The present study aimed to determine the major cause of the high mortality affecting farmed gilthead seabream (Sparus aurata) and controlling this disease condition. Fifteen diseased S. aurata were sampled from a private fish farm located at Eldeba Triangle, Damietta, fish showed external skin hemorrhages, and ulceration. Bacterial isolates retrieved from the diseased fish were identified biochemically as Pseudomonas putida and then confirmed by phylogenetic analysis of the 16 S rRNA gene sequence. P. putida was also isolated from three batches of tilapia-trash feed given to S. aurata. Biofilm and hemolytic assay indicated that all P. putida isolates produced biofilm, but 61.11% can haemolyse red blood cells. Based on the antibiotic susceptibility test results, P. putida was sensitive to florfenicol with minimum inhibitory concentrations ranging between 0.25 and 1.0 µg mL− 1, but all isolates were resistant to ampicillin and sulfamethoxazole-trimethoprim. Pathogenicity test revealed that P. putida isolate (recovered from the tilapia-trash feed) was virulent for S. aurata with LD50 equal to 4.67 × 107 colony forming unit (CFU) fish− 1. After intraperitoneal (IP) challenge, fish treated with 10 mg kg− 1 of florfenicol showed 16.7% mortality, while no mortality was recorded for the fish group that received 20 mg kg− 1. The non-treated fish group showed 46.7% mortality after bacterial challenge. HPLC analysis of serum florfenicol levels reached 1.07 and 2.52 µg mL− 1 at the 5th -day post-drug administration in the fish groups received 10 and 20 mg kg− 1, respectively. In conclusion, P. putida was responsible for the high mortality affecting cultured S. aurata, in-feed administration of florfenicol (20 mg kg− 1) effectively protected the challenged fish.

Rapid diagnosis of bloodstream infections using a culture-free phenotypic platform

BackgroundBloodstream infections (BSIs) are a life-threatening acute medical condition and current diagnostics for BSIs suffer from long turnaround time (TAT). Here we show the validation of a rapid detection-analysis platform (RDAP) for the diagnosis of BSIs performed on clinical blood samplesMethodsThe validation was performed on a cohort of 59 clinical blood samples, including positive culture samples, which indicated confirmed bloodstream infections, and negative culture samples. The bacteria in the positive culture samples included Gram-positive and Gram-negative pathogenic species. RDAP is based on an electrochemical sandwich immunoassay with voltage-controlled signal amplification, which provides an ultra-low limit of detection (4 CFU/mL), allowing the platform to detect and identify bacteria without requiring culture and perform phenotypic antibiotic susceptibility testing (AST) with only 1–2 h of antibiotic exposure. The preliminary diagnostic performance of RDAP was compared with that of standard commercial diagnostic technologies.ResultsUsing a typical clinical microbiology laboratory diagnostic workflow that involved sample culture, agar plating, bacteria identification using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry, and AST using MicroScan as a clinical diagnostic reference, RDAP showed diagnostic accuracy of 93.3% and 95.4% for detection-identification and AST, respectively. However, RDAP provided results at least 15 h faster.ConclusionsThis study shows the preliminary feasibility of using RDAP to rapidly diagnose BSIs, including AST. Limitations and potential mitigation strategies for clinical translation of the present RDAP prototype are discussed. The results of this clinical feasibility study indicate an approach to provide near real-time diagnostic information for clinicians to significantly enhance the treatment outcome of BSIs.

Assessing Clinician Utilization of Next-Generation Antibiotics Against Resistant Gram-Negative Infections in U.S. Hospitals : A Retrospective Cohort Study.

The U.S. antibiotic market failure has threatened future innovation and supply. Understanding when and why clinicians underutilize recently approved gram-negative antibiotics might help prioritize the patient in future antibiotic development and potential market entry rewards. To determine use patterns of recently U.S. Food and Drug Administration (FDA)-approved gram-negative antibiotics (ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, plazomicin, eravacycline, imipenem-relebactam-cilastatin, and cefiderocol) and identify factors associated with their preferential use (over traditional generic agents) in patients with gram-negative infections due to pathogens displaying difficult-to-treat resistance (DTR; that is, resistance to all first-line antibiotics). Retrospective cohort. 619 U.S. hospitals. Adult inpatients. Quarterly percentage change in antibiotic use was calculated using weighted linear regression. Machine learning selected candidate variables, and mixed models identified factors associated with new (vs. traditional) antibiotic use in DTR infections. Between quarter 1 of 2016 and quarter 2 of 2021, ceftolozane-tazobactam (approved 2014) and ceftazidime-avibactam (2015) predominated new antibiotic usage whereas subsequently approved gram-negative antibiotics saw relatively sluggish uptake. Among gram-negative infection hospitalizations, 0.7% (2551 [2631 episodes] of 362 142) displayed DTR pathogens. Patients were treated exclusively using traditional agents in 1091 of 2631 DTR episodes (41.5%), including "reserve" antibiotics such as polymyxins, aminoglycosides, and tigecycline in 865 of 1091 episodes (79.3%). Patients with bacteremia and chronic diseases had greater adjusted probabilities and those with do-not-resuscitate status, acute liver failure, and Acinetobacter baumannii complex and other nonpseudomonal nonfermenter pathogens had lower adjusted probabilities of receiving newer (vs. traditional) antibiotics for DTR infections, respectively. Availability of susceptibility testing for new antibiotics increased probability of usage. Residual confounding. Despite FDA approval of 7 next-generation gram-negative antibiotics between 2014 and 2019, clinicians still frequently treat resistant gram-negative infections with older, generic antibiotics with suboptimal safety-efficacy profiles. Future antibiotics with innovative mechanisms targeting untapped pathogen niches, widely available susceptibility testing, and evidence demonstrating improved outcomes in resistant infections might enhance utilization. U.S. Food and Drug Administration; NIH Intramural Research Program.

Fourteen-day vonoprazan-based bismuth quadruple therapy for H. pylori eradication in an area with high clarithromycin and levofloxacin resistance: a prospective randomized study (VQ-HP trial)

Potassium-competitive acid blockers (P-CABs) provide potent acid inhibition, yet studies on P-CAB-based quadruple therapy for H. pylori eradication are limited. We theorized that integrating bismuth subsalicylate into a quadruple therapy regimen could enhance eradication rates. However, data on the efficacy of vonoprazan bismuth quadruple therapy are notably scarce. Therefore, the aim of this study was to evaluate the efficacy of vonoprazan-based bismuth quadruple therapy in areas with high clarithromycin and levofloxacin resistance. This was a prospective, single-center, randomized trial conducted to compare the efficacy of 7-day and 14-day vonoprazan-based bismuth quadruple therapy for H. pylori eradication between June 1, 2021, and March 31, 2022. Qualified patients were randomly assigned to the 7-day or 14-day regimen (1:1 ratio by computer-generated randomized list as follows: 51 patients for the 7-day regimen and 50 patients for the 14-day regimen). The regimens consisted of vonoprazan (20 mg) twice daily, bismuth subsalicylate (1024 mg) twice daily, metronidazole (400 mg) three times daily, and tetracycline (500 mg) four times daily. CYP3A4/5 genotyping and antibiotic susceptibility tests were also performed. Successful eradication was defined as 13negative C-UBTs 4 weeks after treatment. The primary endpoint was to compare the efficacy of 7-day and 14-day regimens as first-line treatments, which were assessed by intention-to-treat (ITT) and per-protocol (PP) analyses. The secondary endpoints included adverse effects. A total of 337 dyspeptic patients who underwent gastroscopy were included; 105 patients (31.1%) were diagnosed with H. pylori infection, and 101 patients were randomly assigned to each regimen. No dropouts were detected. The antibiotic resistance rate was 33.3% for clarithromycin, 29.4% for metronidazole, and 27.7% for levofloxacin. The CYP3A4 genotype was associated with 100% rapid metabolism. The H. pylori eradication rates for the 7-day and 14-day regimens were 84.4%, 95% CI 74.3–94.2 and 94%, 95% CI 87.4–100, respectively (RR difference 0.25, 95% CI 0.03–0.53, p value = 0.11). Interestingly, the 14-day regimen led to 100% eradication in the clarithromycin-resistant group. Among the patients in the 7-day regimen group, only two exhibited resistance to clarithromycin; unfortunately, neither of them achieved a cure from H. pylori infection. The incidence of adverse events was similar in both treatment groups, occurring in 29.4% (15/51) and 28% (14/50) of patients in the 7-day and 14-day regimens, respectively. No serious adverse reactions were reported. In conclusion, 14 days of vonoprazan-based bismuth quadruple therapy is highly effective for H. pylori eradication in areas with high levels of dual clarithromycin and levofloxacin resistance.

Co-production of metallo-β-lactamase and OXA-type β-lactamases in carbapenem-resistant Acinetobacter baumannii clinical isolates in North East India.

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to public health globally, especially due to its ability to produce multiple carbapenemases, leading to treatment challenges. This study aimed to investigate the antibiotic resistance pattern of carbapenem-resistant A. baumannii isolates collected from different clinical settings in North East India, focusing on their genotypic and phenotypic resistance profiles. A total of 172 multidrug-resistant A. baumannii isolates were collected and subjected to antibiotic susceptibility test using the Kirby-Bauer disk diffusion method. Various phenotypic tests were performed to detect extended-spectrum β-lactamase (ESBL), metallo-β-lactamase (MBL), class C AmpC β-lactamase (AmpC), and carbapenem hydrolyzing class D β-lactamase (CHDL) production among the isolates. Overexpression of carbapenemase and cephalosporinase genes was detected among the isolates through both phenotypic and genotypic investigation. The antibiotic resistance profile of the isolates revealed that all were multidrug-resistant; 25% were extensively drug-resistant, 9.30% were pan-drug-resistant, whereas 91.27% were resistant to carbapenems. In the genotypic investigation, 80.81% of isolates were reported harbouring at least one metallo-β-lactamase encoding gene, with blaNDM being the most prevalent at 70.34%, followed by blaIMP at 51.16% of isolates. Regarding class D carbapenemases, blaOXA-51 and blaOXA-23 genes were detected in all the tested isolates, while blaOXA-24, blaOXA-48, and blaOXA-58 were found in 15.11%, 6.97%, and 1.74% isolates respectively. Further analysis showed that 31.97% of isolates co-harboured ESBL, MBL, AmpC, and CHDL genes, while 31.39% of isolates co-harboured ESBL, MBL, and CHDL genes with or without ISAba1 leading to extensively drug-resistant or pan drug-resistant phenotypes. This study highlights the complex genetic profile and antimicrobial-resistant pattern of the isolates circulating in North East India, emphasizing the urgent need for effective infection control measures and the development of alternative treatment strategies to combat these challenging pathogens.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Comparative Analysis of Antibiotic Resistance and Biofilm Characteristics of Two Major Enterococcus Species from Poultry Slaughterhouses in South Korea.

The spread of antibiotic-resistant Enterococcus in the poultry industry poses significant public health challenges due to multidrug resistance and biofilm formation. We investigated the antibiotic resistance profiles and biofilm characteristics of E. faecalis and E. faecium isolates from chicken meat in poultry slaughterhouses in South Korea. Ninety-six isolates (forty-eight each of E. faecalis and E. faecium) were collected between March and September 2022. Both species were analyzed using MALDI-TOF, PCR, antibiotic susceptibility testing, and biofilm assays. A high level of multidrug resistance was observed in E. faecalis (95.8%) and E. faecium (93.8%), with E. faecium exhibiting a broader range of resistance, particularly to linezolid (52.1%) and rifampicin (47.9%). All E. faecalis isolates formed biofilm in vitro, showing stronger biofilm formation than E. faecium with a significant difference (p < 0.001) in biofilm strength. Specific genes (cob, ccf, and sprE) were found to be correlated with biofilm strength. In E. faecium isolates, biofilm strength was correlated with resistance to linezolid and rifampicin, while a general correlation between antibiotic resistance and biofilm strength was not established. Through analysis, correlations were noted between antibiotics within the same class, while no general trends were evident in other analyzed factors. This study highlights the public health risks posed by multidrug-resistant enterococci collected from poultry slaughterhouses, emphasizing the complexity of the biofilm-resistance relationship and the need for enhanced control measures.

Inhibition Zones: A Window into Antibiotic Resistance

Antibiotic resistance poses a significant global health threat, necessitating innovative approaches to combat it effectively. This survey paper presents a comprehensive overview of the challenges associated with antibiotic resistance and the critical role of antibiotic susceptibility testing (AST) in guiding treatment decisions. We propose leveraging advancements in image processing and machine learning to develop an automated algorithm for measuring the Zone of Inhibition in AST, addressing the limitations of manual methods. Additionally, we discuss the potential of mobile applications and expert systems to simplify AST, particularly in resource-limited settings. Standardization efforts and future trends in AST are also explored to enhance testing strategies and preserve antibiotic efficacy. Through this research, we aim to contribute to the ongoing efforts to mitigate antibiotic resistance and improve patient care worldwide.

Durlobactam, a Diazabicyclooctane β-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum β-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb β-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KIapp 9.2 ± 0.9 μM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KIapp 3.3 ± 0.6 μM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KIapp values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 μg/mL, similar to the ranges for meropenem (1-32 μg/mL) and imipenem (0.5-64 μg/mL). In β-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel β-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Characterization of genes related to the efflux pump and porin in multidrug-resistant Escherichia coli strains isolated from patients with COVID-19 after secondary infection

BackgroundEscherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin.MethodsA total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated.ResultsThe E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin.ConclusionsThe antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.