Antimicrobial Susceptibility Testing Research Articles

Exploring the Role of Artificial Intelligence in Revolutionizing Microbial Diagnostics

Identifying the microorganisms responsible for illnesses and infections is crucial for accurate diagnosis, which is an essential aspect of providing medical care. Artificial intelligence (AI) systems can enhance drug discovery, epidemiological monitoring, prediction of antibiotic resistance, and disease management in the field of microbiological diagnosis. Artificial intelligence (AI) is a branch of science and technology that uses computers to simulate human intelligence. AI has recently shown tremendous promise as a powerful computational tool for the detection and management of bacterial infections. These machines can imitate human thought processes and cognitive capacities. Today, pathogen identification and Antimicrobial Susceptibility Testing (AST) in clinical laboratories often rely on culturing and isolating pathogens. With the rapid advancement of technology, Artificial Intelligence (AI) has become a vital tool for bacterial AST, providing numerous fast and efficient methods for testing drug susceptibility. AI systems offer enhanced diagnostic methods and early detection of antibiotic resistance. Moreover, they can rapidly and accurately identify diseases, including those that are novel or drug-resistant. In addition, AI can be applied to identify cells infected with malaria, detect drug resistance in pathogens like tuberculosis (TB) and HIV, understand and tackle antimicrobial resistance (AMR), and predict outbreaks. The primary goals of applying AI to bacterial diagnosis are the rapid and precise identification of pathogens and the prediction of drug resistance.

Machine Learning Approaches for Microorganism Identification, Virulence Assessment, and Antimicrobial Susceptibility Evaluation Using DNA Sequencing Methods: A Systematic Review.

Microbial infections pose a substantial global health challenge, particularly impacting immunocompromised individuals and exacerbating the issue of antimicrobial resistance (AMR). High virulence of pathogens can lead to severe infections and prolonged antimicrobial treatment, increasing the risk of developing resistant strains. Integrating machine-learning (ML) with DNA sequencing technologies offers potential solutions by enhancing microbial identification, virulence assessment, and antimicrobial susceptibility evaluation. This review explores recent advancements in these integrated approaches, addressing current limitations and identifying gaps in the literature. A comprehensive literature search was conducted across databases including PubMed, Scopus, Web of Science, and IEEE Xplore, covering publications from January 2014 to June 2024. Using a detailed Boolean search string, relevant studies focusing on ML applications in microorganism identification, antimicrobial susceptibility testing, and microbial virulence were included. The screening process involved a two-stage review of titles, abstracts, and full texts, with data extraction and critical appraisal performed using the QIAO tool. Data were analyzed through narrative synthesis to identify common themes and innovations. Out of 1,650 initially identified records, 19 studies met the inclusion criteria. These studies primarily focused on AMR, with additional research on microbial virulence and identification. Machine learning algorithms such as Random Forest, Support Vector Machines, and Convolutional Neural Networks, combined with DNA sequencing techniques like Whole Genome Sequencing and Metagenomic Sequencing, demonstrated significant advancements in predictive accuracy and efficiency. High-quality studies achieved impressive performance metrics, including F1-scores up to 0.88 and AUC scores up to 0.96. The integration of ML and DNA sequencing technologies has significantly enhanced microbial analysis, improving the identification of pathogens, assessment of virulence, and evaluation of antimicrobial susceptibility. Despite advancements, challenges such as data quality, high costs, and model interpretability persist. This review highlights the need for continued innovation and provides recommendations for future research to address these limitations and improve disease management and public health strategies. The systematic review is registered with PROSPERO (CRD42024571347).

Evaluation of anti-acne activity of human whole saliva against acne vulgaris: An invitro study.

Acne vulgaris (AV) is a multifactorial inflammatory skin disorder, affecting 9.45% of the world's population. AV can be painful, discomforting, and disfiguring due to scarring, leading to physiological distress, and economic burden. AV pathogenies can be due to various factors, key ones include follicular plugging, colonization by the microorganism, endocrinological factors, and inflammation. Gram-positive bacteria Cutibacterium acnes and Staphylococcus epidermidis are the most common pathogens isolated from patients with AV. The present study aimed to investigate the anti-acne activity of whole human saliva against bacteria that cause AV. The saliva sample from four different individuals was collected at three different intervals. These samples were used to perform antimicrobial susceptibility test (AST) against the pathogens. Kirby-Bauer disk diffusion susceptibility test, and Broth dilution method using Resazurin were performed. The human saliva was effective in inhibiting Cutibacterium acnes and Staphylococcus epidermidis those causing AV. The Minimum inhibitory activity and disc diffusion assay depicted potency of saliva. The pH of the saliva samples was found to be more acidic in the morning samples when compared with the evening samples. The afternoon sample was found to be more effective when compared with the other two intervals. Saliva shows effective anti-acne activity by inhibiting the growth and proliferation of acne-causing bacteria.

Investigating the Prevalence of Bacterial Infections in Patients with Coronavirus Disease 2019 Hospitalized in Intensive Care Unit and Determining their Antibiotic Resistance Patterns.

COVID-19 patient hospitalization, particularly in intensive care units, exposes them to bacterial and fungi co-infections, which can have very serious consequences, including increased mortality. In addition, antibiotic resistance among pathogens is a hidden threat behind COVID-19. In the period from 2020 September to 2021 August, bacterial isolates from COVID-19 patients admitted to the ICU of Sina Hospital in Hamadan, Iran, were collected and identified based on standard biochemical tests. COVID-19 cases were confirmed based on clinical symptoms, computed tomography, and polymerase chain reaction. Antimicrobial susceptibility tests were conducted using disc diffusion and broth microdilution methods. In total, 207 bacterial isolates were collected, with Klebsiella pneumoniae account-ing for 69 (33.33%) and Acinetobacter baumannii accounting for 59 (28.15%). The frequency and percentage of isolated bacteria were as follows: Alcaligenes species 28 (13.59%), Staphylococcus aureus 18 (8.73%), Pseudomonas aeruginosa 15 (7.28%), Escherichia coli 11 (5.33%), Stenotrophomonas maltophilia 3 (1.45%), Enterococcus species 3 (1.45%), and Serratia species 1 (0.48%). About 95.38% resistance to ceftazidime and cefotaxime and 92.31% resistance to ciprofloxacin and cefepime were found in K. pneumoniae isolates. A. baumannii isolates were 100% resistant to cefotaxime, ceftriaxone, and cefepime. About 22.22% resistance to vancomycin and 66.67% resistance to clindamycin, erythromycin, and cefoxitin were seen in S. aureus isolates. Knowledge of bacterial co-infections and their antibiotic resistance pattern in COVID-19 patients can help in choosing effective antibiotics for the treatment and prevention of antibiotic resistance.

Colistin, Meropenem–Vaborbactam, Imipenem–Relebactam, and Eravacycline Testing in Carbapenem-Resistant Gram-Negative Rods: A Comparative Evaluation of Broth Microdilution, Gradient Test, and VITEK 2

Objectives. This study aimed to evaluate and compare the performance of different assays for antimicrobial susceptibility testing (AST) and minimum inhibitory concentration (MIC) determination for reserve antibiotics in carbapenem-resistant Enterobacterales (CREs), Pseudomonas aeruginosa (CRPAs), and Acinetobacter baumannii (CRABs). Methods. An analysis was conducted on 100 consecutive isolates: 50 CREs, 35 CRPAs, and 15 CRABs. Sensititre broth microdilution was used as a reference standard to evaluate the performance of VITEK 2 card AST-XN24 (bioMérieux), the respective gradient tests (bioMérieux), and UMIC colistin broth microdilution test strips (Bruker Daltonics). Errors, essential agreement (EA), and categorical agreement of MICs for colistin (COL), meropenem–vaborbactam (MVB), imipenem–relebactam (IRL), and eravacycline (ERV) were assessed. Results. The agreement between both of the COL broth microdilution (BMD) methods was perfect (100/100). The gradient test and VITEK 2 analysis yielded comparable EA rates (92/100 and 72/79, respectively), with the latter not registering any very major errors (VMEs). The MVB gradient test achieved EA in 66 of 85 isolates and VITEK 2 in 70/85. For IRL, EA was reached in 69 and 64 of 85 cases by gradient test and VITEK 2 analysis, respectively. The ERV gradient test yielded false results in nearly all (12/15) CRABs but achieved EA in 46 of 50 CREs. The VITEK system recorded EA for ERV in 60 of 65 isolates. Conclusions. We observed substantial variability in the measured MICs between BMD and the alternative methods. In only a few constellations, VITEK 2 or gradient tests could substitute the reference method. BMD is the method of choice for COL analysis, with VITEK 2 representing an alternative method for CRPA testing. Alternative methods for MVB did not provide reliable results, except for Enterobacterales, when tested with the gradient test. However, resistant results need to be confirmed by BMD. Only BMD can be used for IRL MIC determination. VITEK 2 was mostly accurate in measuring ERV MICs, while the corresponding gradient test yielded reliable results exclusively in CREs. It is essential that laboratories are aware of which testing method provides reliable results for each combination of microorganisms and reserve antibiotics.

Antibiotic susceptibility testing using minimum inhibitory concentration (MIC) assays

Antimicrobial resistance is due to genetic changes that allow bacteria to evade antibiotic treatment. Antimicrobial susceptibility testing is critical for the detection of antibiotic-resistant strains, the selection of effective therapeutic strategies against bacterial infections, and the evaluation of the efficacy of novel antimicrobials. Among the variety of clinical microbiology methods used for antibiotic susceptibility testing, minimum inhibitory concentration (MIC) assays have become the gold standard in clinical practice. MIC assays determine the lowest concentration of an antimicrobial agent that is required to inhibit visible bacterial growth in vitro. Here, we outline MIC assay protocols, in strict accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines that aim to assess the susceptibility of non-fastidious organisms to antimicrobial agents. The protocols described in this methods paper are intended to aid the performance of reliable and informative MIC assays for research purposes that are in line with clinical microbiology practices.

Non-invasive Streptococcus pneumoniae infections are associated with different serotypes than invasive infections, Belgium, 2020 to 2023.

BackgroundDespite widely implemented pneumococcal vaccination programmes, Streptococcus pneumoniae remains a global risk for human health. Streptococcus pneumoniae can cause invasive (IPD) or non-invasive pneumococcal disease (NIPD). Surveillance is mainly focusing on IPD, assessing the full impact of pneumococcal vaccination programmes on pneumococcal disease is challenging.AimWe aimed to prospectively investigate serotype distribution and antimicrobial resistance (AMR) of S. pneumoniae isolates from patients with NIPD and compare with data on IPD isolates and with a 2007-2008 dataset on NIPD.MethodsBetween September 2020 and April 2023, we collected isolates and patient data from patients with NIPD from 23 clinical laboratories in Belgium. Capsular typing was performed by a validated Fourier-Transform Infrared spectroscopic method, and AMR was assessed with broth microdilution, using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints.ResultsWe received S. pneumoniae isolates from 1,008 patients with lower respiratory tract infections (n = 760), otitis media (n = 190) and sinusitis (n = 58). Serotype 3 was the most prevalent serotype among the NIPD isolates. Serotypes not included in the 20-valent pneumococcal conjugate vaccine (PCV20) were significantly more common among the NIPD than among the IPD isolates. Antimicrobial resistance levels were significantly higher among the NIPD isolates (n = 539; 2020-2022) compared with the IPD isolates (n = 2,344; 2021-2022). Resistance to several β-lactam antimicrobials had increased significantly compared with 15 years before.ConclusionsThe NIPD isolates were strongly associated with non-vaccine serotypes and with increased AMR levels. This underlines the importance of continued NIPD surveillance for informed policy making on vaccination programmes.

Evaluation of Antibacterial Activity of Bismuth Ferrites Nanoparticles in the Inhibition of E. Coli and S. Aureus Bacteria.

In this work, bismuth ferrites (BFO) nanoparticles were produced in the form of using sol-gel technique, followed by annealing in a tube furnace in temperatures from 400 °C to 650 °C. X-ray diffraction (XRD) results showed the formation of small sizes nanoparticles (NPs) with high purity. Structural analysis displayed that annealing at 600 °C could make BFO NPs be fitted to rhombohedral space group (R3c), with small quantity of spurious phases. The sizes of the BFO nanoparticles determined by transmission electron microscopy (HRTEM) are between 50 to 100 nm. To evaluate the efficiency of BFO in antimicrobial susceptibility tests, the nanoparticles were dispersed through cand tested agar diffusion method and dilution in a 96 well plate using a Gram-positive strains (Staphylococcus aureus) and Gram negative strain (Escherichia coli). The antibacterial activity of the BFO NPs was tested at concentrations of 2 mg/mL with MIC greater than 60 μg/mL for both bacteria.

BACTERIOLOGICAL PROFILE OF SURGICAL SITE INFECTIONS AND THEIR ANTIBIOGRAM: A STUDY IN A TERTIARY CARE HOSPITAL

Objective: To ascertain the frequency of surgical site infection (SSI) occurrences and the prevalence of aerobic bacterial infections associated with their antibiogram. Methods: For 1 year, a tertiary care hospital affiliated with Government Medical College, Amritsar, served as the site of this cross-sectional study. Pus aspirates were aseptically taken from 86 consecutive individuals suspected of having SSI using a sterile surgical syringe with a needle and sterile cotton surgical swabs. The specimens were cultivated and placed in an incubator at 37°C for 24 h. The individual strains were then identified using traditional techniques. The Kirby–Bauer’s Disc Diffusion Method was employed to conduct antimicrobial susceptibility testing. Results: Among 1021 patients, 86 were clinically suspected of SSI, and 64 were culture-positive. The incidence of SSI was 8.42%. The most common organisms were Klebsiella pneumoniae 34% (17/50) and Escherichia coli 32% (16/50), whereas Staphylococcus aureus 100% (14/14) was the only organism found among Gram-positive cocci. The antimicrobial Profile of Gram-positive isolates showed maximum sensitivity to linezolid (100%), followed by erythromycin (85.7%). The antimicrobial profile of lactose fermenters revealed maximum sensitivity to polymyxin (100%). Antibiogram of Proteus species showed maximum sensitivity to meropenem (100%) and ceftazidime tazobactam (100%). Non-fermenters showed 100% polymyxin and 80% sensitivity to aztreonam. Conclusion: Our study has revealed significant findings. The incidence of SSI was found to vary between 8% and 12% in our tertiary care center. K. pneumoniae 26.5% (17/64) and E. coli 25% (16/64) were the most common organisms isolated. S. aureus 21.2% (14/64) was the only organism found among Gram-positive cocci. SSI below waist surgery was far more 75% (48/64) than above waist surgery of 25% (16/64).

Avian Pathogenic Escherichia coli Isolated in Poultry Farms in Bangladesh that Use Antibiotics Extensively.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is causing economic losses to the global poultry industry. Increased prevalence of antibiotic resistance in APEC is the leading cause for increased indiscriminate use of various antimicrobial compounds in farms. The study aimed to investigate the presence of phenotypic and genotypic markers for antibiotic resistance, metals, and biocides in APEC from Bangladeshi poultry and details about the antimicrobials used in poultry farms. In total, 55 APEC were isolated from hearts or liver samples of 86 sick or dead chickens using culture on agar plate and biochemical testing. APEC isolates were tested for antibiotic susceptibility to 14 antimicrobial agents according to the European Committee on Antimicrobial Susceptibility Testing. A series of PCRs was performed to screen the presence of genes for quinolones, colistin, aminoglycosides, ESBL, metals, and biocides. Detailed information regarding antibiotic use was collected from farmers during clinical investigations. Resistance was found to 10 antibiotics and prevalence was as follows: ampicillin (86%), ciprofloxacin (86%), trimethoprim-sulphamethoxazole (73%), chloramphenicol (33%), mecillinam (13%), gentamicin (11%), cefoxitin (11%), cefotaxime (9%), tigecycline (2%), and nitrofurantoin (2%). The most common multiresistance phenotype was CIP-AMP-SXT, and 35% of isolates were multidrug resistant. Genotypic analysis confirmed the presence of quinolone resistance genes [qnrS1 and aac-(6')-lb-cr], silver-resistant genes (silE), and mercury-resistant genes (merA) but not others. In total, 88% farmers were using different antimicrobial compounds, and, of them, 56% were using antimicrobials without prescriptions from veterinarians. Ciprofloxacin was most extensively used followed by oxytetracycline. Critically important antibiotics like ciprofloxacin, colistin, and gentamicin are extensively used in the farms. This study confirmed the presence of antibiotics, metals, and biocide-resistant APEC in poultry farms in Bangladesh. Increased resistance to quinolones is a serious ongoing problem. The indiscriminate use of antibiotics in poultry farms is alarming and should be stopped.

Ultrastructural polymicrobial Staphylococcus aureus-Pseudomonas aeruginosa interactions and antimicrobial resistance in ex vivo cornea model.

Aim: To investigate antagonistic interactions among pathogens, in ex vivo donor corneas infected with monomicrobial or polymicrobial combinations of antibiotic susceptible and resistant clinical isolates of Staphylococcus aureus (MSSA, MRSA) and Pseudomonas aeruginosa (S-PA, MDR-PA).Materials & methods: Scanning electron microscopy and antimicrobial susceptibility testing (AST, broth microdilution for minimum inhibitory and bactericidal concentrations [MIC/MBC]) pre-and post-polymicrobial interactions, in infected donor corneas.Results: MSSA lost viability with S-PA/MDR-PA, while MRSA formed larger cells, biofilm and lower MIC (teicoplanin) with S-PA, but lost viability with MDR-PA. S-PA had lower MIC (ceftazidime, meropenem, chloramphenicol) with MSSA, and lower MBC (cefoperazone, ciprofloxacin) and fewer cells with MRSA. MDR-PA had abundant cells and no change in AST with MSSA or MRSA.Conclusion: Significant antagonistic interactions occur in ocular polymicrobial infections, affecting antibiotic susceptible isolates more than resistant ones.

Emergence of Salmonella enterica Serovar Heidelberg Producing OXA 48 Carbapenemase in Eastern Algeria.

Salmonella infections have become increasingly resistant to antibiotics, including fluoroquinolones, third-generation cephalosporins (C3G), and even carbapenems. This report describes the emergence of a strain of Salmonella enterica serovar Heidelberg that produces the carbapenemase OXA 48. The strain was isolated from a stool sample taken from a newborn. Antimicrobial susceptibility testing was carried out following the recommendations of the Clinical and Laboratory Standard Institute. Whole genome sequencing was performed on MiSeq Illumina™. The strain was resistant to ertapenem (minimal inhibitory concentration [MIC] = 12 µg/mL), intermediate to imipenem (MIC = 1.5 µg/mL), resistant to nalidixic acid, and intermediate to fluoroquinolones but was susceptible to C3G, cotrimoxazole, chloramphenicol, and colistin (MIC = 0.064 µg/mL). The strain was identified as ST-15. The strain of Salmonella Heidelberg ST-15 was found to have antimicrobial resistance genes, specifically blaOXA-48, aac(6')-Iaa and fosA7, which mediate resistance to carbapenems, aminoglycosides and fosfomycin, respectively. Additionally, mutations were detected in the gyrA, parC. Three plasmid replicon type IncL, IncX1, and Col156 have been identified. The strain has the potential to cause an epidemic. The genomic analysis of the strain allowed us to understand the mechanisms of resistance. Preventing the spread of Salmonella carbapenemase-producing strains is crucial, particularly in hospital settings. Epidemiological measures are necessary to achieve this goal.

Resistance profiles of carbapenemase-producing Enterobacterales in a large centre in England: are we already losing cefiderocol?

Carbapenemase-producing Enterobacterales (CPE) pose difficult therapeutic challenges. We aimed to characterize antimicrobial resistance profiles of CPE in our centre. All non-duplicate CPE isolates between 1 August 2020 and 31 August 2023 in a large teaching trust in England were retrospectively studied. Cefiderocol antimicrobial susceptibility testing (AST) was performed using disc diffusion, ceftazidime/avibactam using disc diffusion and gradient diffusion, and ceftazidime/avibactam aztreonam synergy using the double disc diffusion method. EUCAST version 14.0 breakpoints were used. A total of 158 CPE from 136 patients were isolated. Most patients were colonized with CPE, but only 16.9% had active infections. Thirty-day all-cause mortality was 10.3%, increasing to 13% for patients with infections and to 18.2% for bacteraemias. OXA-48 was the most prevalent carbapenemase (48.1%), followed by NDM (38%). All isolates exhibited MDR profiles, with high levels of resistance to meropenem (41.1%). Resistance to cefiderocol was found in 69.7% of NDM-producing isolates, with a further 18.2% in the area of technical uncertainty. Ceftazidime/avibactam and aztreonam synergy was seen in 87.5% of isolates, whereas colistin and fosfomycin susceptibility remained high (98.1% and 97.2%, respectively). All OXA-48-producing isolates were susceptible to ceftazidime/avibactam, and 15.3% were resistant to cefiderocol. No patients had been exposed to cefiderocol beforehand, whereas three had been exposed to ceftazidime/avibactam. The most common risk factor for CPE isolation was travel and receiving healthcare abroad, especially in Asia. We found high rates of resistance to cefiderocol in CPE isolates without prior cefiderocol exposure. Our results prohibit empirical use of cefiderocol for the treatment of CPE infections in our setting.

Fosfomycin—Overcoming Problematic In Vitro Susceptibility Testing and Tricky Result Interpretation: Comparison of Three Fosfomycin Susceptibility Testing Methods

Background: Fosfomycin (FOS) is an older antimicrobial agent newly rediscovered as a possible treatment for infections with limited therapeutic options (e.g., Gram-negative bacteria with difficult-to-treat resistance, DTR), especially in intravenous form. However, for correct usage of FOS, it is necessary to have a reliable susceptibility testing method suitable for routine practice and robust interpretation criteria. Results: The results were interpreted according to 2023 interpretation criteria provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). DTR Gram-negatives were more likely to be resistant to FOS (45% in Enterobacterales and 20% in P. aeruginosa) than non-DTR (10% and 6.7%, resp.). All isolates of S. aureus were susceptible to FOS. In Gram-negatives, all agreement values were unacceptable. Etest® performed better in the DTR cohort (categorical agreement, CA, 80%) than in the non-DTR cohort (CA 45.7%). There were no very major errors (VREs) observed in P. aeruginosa. S. aureus had surprisingly low essential agreement (EA) rates (53% for MRSA and 47% for MSSA) for Etest®, but categorical agreement was 100%. Methods: A total of 130 bacterial isolates were tested and compared using the disc diffusion method (DD) and gradient strip method (Etest®) with the reference method (agar dilution, AD). The spectrum of isolates tested was as follows: 40 Enterobacterales (20 DTR vs. 20 non-DTR), 30 Pseudomonas aeruginosa (15 DTR vs. 15 non-DTR), and 60 Staphylococcus aureus (30 methicillin-susceptible, MSSA, vs. 30 methicillin-resistant, MRSA). Conclusions: Neither one of the tested methods was identified as a suitable alternative to AD. It would be beneficial to define more interpretation criteria, at least in some instances.

First documentation of a clinical multidrug-resistant Enterobacter chuandaensis ST2493 isolate co-harboring blaNDM-1 and two blaKPC-2 bearing plasmids.

The increasing prevalence of carbapenem-resistant Enterobacter cloacae complex (CREC) poses great challenges to infection treatment in the clinical setting. In this study, we reported the emergence of carbapenemase in a rare species, Enterobacter chuandaensis, belonging to the Enterobacter cloacae complex (ECC). We elucidated the genetic characteristics of carbapenem-resistant isolate FAHZZU5885, co-harboring blaNDM-1 and blaKPC-2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and average nucleotide identity (ANI) analysis were used to identify E. chuandaensis. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were used to clarify the number and size of the plasmids in FAHZZU5885. Antimicrobial phenotypes were identified by antimicrobial susceptibility testing (AST), and the characteristics of the strain were examined with whole-genome sequencing (WGS). The conjugation experiment and stability assay were conducted to verify the transferability and stability of the plasmid carrying carbapenemase-encoding genes. E. chuandaensis FAHZZU5885 was isolated from a perianal swab of a patient admitted to the ICU. This strain simultaneously carried blaNDM-1 and two blaKPC-2 genes. FAHZZU5885 was resistant to most of the tested antibiotics except for amikacin, tigecycline, and colistin. Two blaKPC-2 were located separately on two different plasmids, the ~ 120kb IncFIA-IncFII plasmid and the ~ 80kb IncR plasmid. Both plasmids shared the conserved sequence klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. The blaNDM-1-bearing plasmid had the potential to transfer and can remain stable after successive passages. In addition, the blaNDM-1 was carried on a ~ 80kb IncFII plasmid with the conserved sequence ISAba125-blaNDM-1-ble-trpF-dsbD-cutA-groS-groL. In summary, this study marks the first report of the multidrug-resistant E. chuandaensis strain FAHZZU5885 harboring two blaKPC-2-bearing plasmids, indicating the potential for the further dissemination of carbapenemase-encoding genes in novel species. The findings contribute to enhancing our understanding of CREC strains, emphasizing the need for continued and comprehensive surveillance of this species.

Whole-Genome Sequencing Reveals the Population Structure and Genetic Diversity of Salmonella Typhimurium ST34 and ST19 Lineages.

Salmonella Typhimurium is an invasive gastrointestinal pathogen for both humans and animals. To investigate the genetic framework and diversity of S. Typhimurium, a total of 194 S. Typhimurium isolates were collected from patients in a tertiary hospital between 2020 and 2021. Antimicrobial susceptibility testing was used to confirm the resistance phenotype. Whole-genome sequencing and bioinformatics analysis were performed to determine the sequence type, phylogenetic relationships, resistance gene profiles, Salmonella pathogenicity island (SPI) and the diversity of the core and pan genome. The result showed that 57.22% of S. Typhimurium isolates were multidrug resistant and resistance of total isolates to the first-line drug ciprofloxacin was identified in 60.82%. The population structure of S. Typhimurium was categorized into three lineages: ST19 (20.10%, 39/194), ST34-1 (47.42%, 92/194) and ST34-2 (40.65%, 63/194), with the population size exhibiting increasing trends. All lineages harbored variety of fimbrial operons, prophages, SPIs and effectors that contributed to the virulence and long-term infections of S. Typhimurium. Importantly, ST34-1 lineage might potentially be more invasive due to the possession of SPI1-effector gene sopE which was essential for the proliferation, internalization and intracellular presence of S. Typhimurium in hosts. Multiple antimicrobial resistance genes were characteristically distributed across three lineages, especially carbapenem genes only detected in ST34-1&2 lineages. The distinct functional categories of pan genome among three lineages were observed in metabolism, signaling and gene information processing. This study provides a theoretical foundation for the evolved adaptation and genetic diversity of S. Typhimurium ST19 and ST34, among which ST34 lineages with multidrug resistance and potential hypervirulence need to pay more attention to epidemiological surveillance.

Analysis of molecular mechanisms of delafloxacin resistance in Escherichia coli

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

Microbial Spectrum, Antimicrobial Resistance, and Prevalence of MDR/XDR Pathogens in Medical Device-Associated Infections

Background: Medical device-associated infections (MDAIs) are a major healthcare concern due to their high morbidity, mortality, and rising prevalence of MDR and XDR bacteria. This study examined the microbiological composition, antimicrobial susceptibility, and prevalence of MDR/XDR infections in MDAI patients at Dr. D.Y. Patil Medical College, Pune. Methods: One-year retrospective research included 100 MDAI patients. Antimicrobial susceptibility testing was done on microbial isolates from blood, urine, and wound swabs according to CLSI standards. The prevalence of MDR and XDR pathogens was determined. Results: Escherichia coli (30%) and Klebsiella pneumoniae (20%) caused most infections, while Staphylococcus aureus (25%) was the most common Gram-positive bacteria. Antimicrobial susceptibility testing showed that 40% of the isolates were MDR and 15% were XDR. Klebsiella pneumoniae had the highest resistance rate (70%), followed by Pseudomonas aeruginosa. Conclusion: The study shows that MDAIs are dominated by MDR and XDR infections, mainly Gram-negative bacteria, making therapy difficult. These findings highlight the critical need for improved infection control, antimicrobial stewardship, and regular surveillance to address medical device-associated resistance infections. Keywords: Medical device infections, multidrug-resistant, extensively drug-resistant organisms, antimicrobial susceptibility, microbiological profile, healthcare-associated infections.

Selection and Evaluation of Suitable Quality Control Strains for Meropenem Antimicrobial Susceptibility Testing through Preliminary External Quality Assessment

BackgroundQuality control (QC) of carbapenem susceptibility testing for Gram-negative bacteria faces challenges due to limited measuring ranges and the lack of suitable QC strains. This study aimed to select and evaluate QC strains for meropenem antimicrobial susceptibility testing (AST) through a pilot external quality assessment (EQA). MethodsCandidate QC strains for meropenem AST were selected based on primary AST data and genomic information from the Japan Antimicrobial Resistant Bacterial Bank. Phenotype stability was verified through serial passaging and MIC comparison with original strains. The validated broth microdilution method was used to determine the target MIC value in a pilot EQA involving 47 clinical laboratories in Japan using ten different AST methods. ResultsTwo strains, Citrobacter freundii JBBDAJB-19-0032 (Strain-A) and Enterobacter hormaechei subsp. steigerwaltii JBEBAAB-19-0102 (Strain-B), both carrying blaIMP-1, were selected as candidate QC strains. The meropenem MICs for Strain-A and Strain-B were 4 mg/L and 2 mg/L, respectively. In the pilot EQA, 43 laboratories reported appropriate results for Strain-A and 40 laboratories reported appropriate results for Strain-B. The MIC range was 2-8 mg/L for Strain-A and 1-4 mg/L for Strain-B. However, 19 and 12 laboratories, respectively, reported out-of-range MICs using AST plates on the MicroScan WalkAway (Beckman Coulter). Inappropriate results were reported by four and seven laboratories, respectively, using common methods for Strain-A and Strain-B, respectively. ConclusionsThe candidate QC strains selected for this study are suitable for meropenem AST EQA, except when the measuring range of certain methods does not match their QC range.

Identification and antimicrobial susceptibility profiles of Campylobacter isolated from camel at municipal abattoirs in eastern Ethiopia

Campylobacteriosis is an infectious zoonotic disease caused by the genus Campylobacter. The disease is transmitted from animals to humans predominantly through the consumption of contaminated food and water. However, the lack of information on the status of Campylobacter makes it difficult to quantify the role of camel meat in the dissemination of the pathogen. A cross-sectional study was conducted from June 2022 to August 2023 to investigate the occurrence of Campylobacter and associated risk factors and to determine the antimicrobial susceptibility profiles of Campylobacter species from camels slaughtered at municipal abattoirs in the towns of Harar, Babile, Jigjiga and Dire Dawa in eastern Ethiopia. A total of 324 (146 carcass swabs, 146 camel feces and 32 abattoir environmental swab samples) were collected and analyzed using TaqMan real-time PCR and culture techniques following standard procedures. In addition, antimicrobial susceptibility tests were performed using the disk diffusion method for eight antimicrobial agents according to the Clinical Laboratory Standards Institute. The overall prevalence of Campylobacter was 7.7%. Campylobacter was more frequently detected from carcasses and surface contact environmental swabs. We isolated Campylobacter at the genus level from approximately half of the PCR-positive samples, representing 54.2% (13 out of 24) of the total. The isolation levels of C. jejuni and C. coli were also 5.56% and 2.2%, respectively, which varied significantly (p = 0.037) based on sample type and site. The odds of occurrence of C. jejuni in samples collected from abattoir environments was 7.52 times greater than those in carcass and fecal samples. We detected resistance to chloramphenicol (78.6%), followed by amoxicillin (71.4%). However, 93%, 78.6%, and 71.4% of the isolates were susceptible to ceftriaxone, ciprofloxacin, and nalidixic acid, respectively. Multidrug resistance (MDR) was detected in 60% of the isolates. Of these MDR isolates, 9 (75%) were C. jejuni and 3 (25%) were C. coli. This study revealed that a considerable proportion of multidrug-resistant Campylobacter species circulate in both camel meat and abattoir environments. This indicates possible carcass cross-contamination by Campylobacter during slaughtering that can pose a threat to humans and limit therapeutic options, which could be prevented by applying good hygienic practices at abattoirs. Therefore, abattoir workers need to be aware of abattoir hygienic standard operating procedures. Regular coordinated actions should be implemented for the rational use of veterinary and medical drugs at the national level, together with training and awareness of hygienic practices.