Detection Of Antimicrobial Resistance Research Articles

Suppression PCR-Based Selective Enrichment Sequencing for Pathogen and Antimicrobial Resistance Detection on Cell-Free DNA in Sepsis-A Targeted, Blood Culture-Independent Approach for Rapid Pathogen and Resistance Diagnostics in Septic Patients.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. The rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcomes in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we targeted eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested in an experimental research study on the first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be additionally detected. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time-to-results to 11-13 h and suggesting diagnostic potential in sepsis.

Antimicrobial Resistance and ARGs Detection in Treated Final Effluent from STPs: An Upcoming Challenge to the Environment

Aim: The present study analysed the presence of antimicrobial resistance organisms and genes in the final effluent from STPs of hospitals. Study Area and Sampling: Samples for microbiological analysis were collected from two different hospitals in Trivandrum City and carried out further microbiological analysis. Methodology: In this study, MDRB (Multi Drug Resiatant Bacteria) were detected from the hospital effluent samples collected from two hospitals. Antibiotic susceptibility analysis showed that the 90% screened organisms was resistant to different antibiotics -Tetracycline (30µg), Amikacin (30µg), Gentamycin (10µg), Ciprofloxacin (5µg), Colistin (10µg) and Amoxicillin (30µg)). Metagenomic surveillance of effluent helped to assess the efficacy of STPs, at the same time assessing the local clinical antibiotic resistance condition by detection of the presence of antimicrobial resistance towards antibiotics and their genes (ARGs) in the hospital effluent. Results: In this study, a total of 3 antibiotic resistant bacterial strains obtained from hospital STP effluent were identified by16S rRNA sequence analysis. The sequences of E.coli, Klebsiella and Enterobacter were submitted in Genbank with accession number MT784125, OM978270 andMN437586 respectively.The final effluent from Hospital 1 showed100% resistance to Tetracycline and 86% resistance to Amoxicillin followed by sensitivity 28%, 22%, 18% and 10% respectively to Ciprofloxacin, Gentamycin, Amikacin and Colistin. The bacterial strains isolated from final effluent of Hospital 2 showed highest resistance to Amikacin and Colistin which is 100% and 86% and 82% resistance to Gentamycin and Ciprofloxacin. The gene primers used for the respective genes above have been amplified in the sample with a higher efficiency of 16 SrRNA, ermB and ampC primers showing a lower Cq value. Thus, these three genes were detected in the samples at high amount which showed the prominent use of the consecutive antibiotics in the clinical field

Pharmacovigilance Strategies to Address Resistance to Antibiotics and Inappropriate Use-A Narrative Review.

The spread of antimicrobial resistance (AMR) is a global challenge. Close and continuous surveillance for quick detection of AMR can be difficult, especially in remote places. This narrative review focuses on the contributions of pharmacovigilance (PV) as an auxiliary tool for identifying and monitoring the ineffectiveness, resistance, and inappropriate use of antibiotics (ABs). The terms "drug ineffective", "therapeutic failure", "drug resistance", "pathogen resistance", and "multidrug resistance" were found in PV databases and dictionaries, denoting ineffectiveness. These terms cover a range of problems that should be better investigated because they are useful in warning about possible causes of AMR. "Medication errors", especially those related to dose and indication, and "Off-label use" are highlighted in the literature, suggesting inappropriate use of ABs. Hence, the included studies show that the terms of interest related to AMR and use are not only present but frequent in PV surveillance programs. This review illustrates the feasibility of using PV as a complementary tool for antimicrobial stewardship activities, especially in scenarios where other resources are scarce.

Exploring the causes of the prevalence of vancomycin-resistant Enterococcus faecalis

BackgroundBacterial drug resistance represents a significant global concern, with vancomycin-resistant Enterococcus faecalis posing a particularly grave threat to contemporary healthcare systems. This study aims to reveal the reasons for the prevalence of VRE in China.MethodsThis study collected data from the China Antimicrobial Resistance Detection System, China Statistical Yearbook, and China Meteorological Network. The collected data are meticulously organized and subjected to both single-factor and multi-factor analyses. An accurate multiple linear regression model was developed by utilizing this comprehensive dataset.ResultsSingle-factor analysis revealed significant regional variations in the resistance rate of vancomycin-resistant Enterococcus faecalis (P = 0.003). Specifically, there were noteworthy disparities observed between regions experiencing temperate and monsoon climates (P = 0.029; P = 0.005). Furthermore, multi-factor regression analysis demonstrated a negative correlation between the drug resistance rate and both rainfall and rGDP, while a positive correlation was observed with nPI.ConclusionWe successfully established a prediction model for the VRE and found that the resistance rate was low in areas with high rainfall and high per capita economic income, but high in areas with many specialized public health institutions. This is critical for public health strategies and helps policymakers and healthcare practitioners tailor antibiotic resistance approaches to local geography, meteorology, economic conditions.

Flow Cytometry as a Rapid and Valuable Method in Investigation of Colistin Resistance in Carbapenem-Resistant Klebsiella pneumoniae Isolates.

Rapid detection of antimicrobial resistance is crucial for early initiation of appropriate therapy. The aim of this study was to investigate whether resistance to colistin, the last-resort antibiotic, in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates can be detected accurately and rapidly by flow cytometry (FCM). The VITEK 2 automated system was used to identify 85 K. pneumoniae strains and to determine their resistance to carbapenems. The minimum inhibitory concentration (MIC) values for colistin in 85 CRKP strains were determined by broth microdilution (BMD), which is the reference method. In addition, FCM was used, combined with DiBAC4(3) fluorescent stain, to determine colistin susceptibility. The MIC₅₀ value of the strains, 80% of which were resistant to colistin by the BMD method, was 16 mg/L, and the MIC₉₀ value was 32 mg/L. When FCM was compared with the reference method, it was determined that the specificity was 94.1%, sensitivity was 100% of FCM, and Cohen's kappa value was 0.96. Colistin susceptibility results with FCM were obtained within an average of 2 h. These findings suggest that FCM holds great promise as a rapid and reliable alternative method for detecting colistin resistance in CRKP strains.

Genotypic and phenotypic antimicrobial resistance of Irish Clostridioides difficile isolates, 2022

ObjectivesInfection with Clostridioides difficile (C. difficile) usually occurs after antibiotic treatment for other infections and can cause gastro-intestinal disorders of variable severity. C. difficile can be resistant to a wide spectrum of antimicrobials. Detection of antimicrobial resistance (AMR) is important to direct optimal treatment and surveillance of AMR patterns in the overall population. Correlation between genotypic markers and phenotypic AMR is not yet well defined. The aim for this study is to assess whether and to what extent genotypic determinants of AMR correlate with phenotypic resistance. Methods99 C. difficile isolates were phenotypically characterized for resistance to eight antibiotics using Sensititre plates or E-tests. Their genomes were screened for genetic markers of resistance. Accuracy, sensitivity, specificity, positive and negative predictive values were calculated. ResultsWe found high rates of resistance (>50%) to cefoxitin and clindamycin, intermediate rates of resistance (10% - 50%) to moxifloxacin and tetracycline and low rates of resistance (<10%) to imipenem, metronidazole, vancomycin, and rifampicin. For moxifloxacin, tetracycline, and clindamycin, we found a good correlation between genotypic and phenotypic AMR, with an overall accuracy of 96% (95% CI 90%-100%), 78% (95% CI 68%-86%) and 86% (95% CI 77%-92%) respectively. For the other five antibiotics, accurate estimates on the correlation could not be made. ConclusionOur results suggest that for moxifloxacin, tetracycline and clindamycin, phenotypic resistance in C. difficile can be predicted by genetic indicators and used for public health purposes. However, for the other five antibiotics, the model is not accurate and further development is necessary.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Antibiotic Resistance Detection in Pseudomonas aeruginosa: Recent Strategies, Advances, and Challenges

AbstractAntibiotic resistance presents a worldwide public health emergency, impeding the effective management of infectious diseases. Pseudomonas aeruginosa plays a substantial role as a bacterial pathogen, particularly in infections among hospitalized individuals, and those with weakened immune systems. Timely and accurate detection of antimicrobial resistance in P. aeruginosa is crucial for initiating tailored antibiotic therapy promptly, thus improving patient outcomes. Nevertheless, this endeavor encounters challenges due to the intricate and varied nature of antibiotic‐resistant strains. Extensive efforts have been invested in developing sensors and instrumentations for assessing antibiotic resistance or susceptibility (AST), aiming to enable personalized patient treatment with appropriate medications. This paper focuses on recent advancements in these methodologies, utilized for evaluating the degree of antibiotic resistance or susceptibility in pathogenic bacteria. Flow cytometry, dual molecular recognition and mass spectrometry are presented as newer phenotypic AST techniques. Genomic methods and the pyocyanin detection are listed as methodologies for the detection of resistance indicators.

Laboratory tools for the direct detection of bacterial respiratory infections and antimicrobial resistance: a scoping review.

Rapid laboratory tests are urgently required to inform antimicrobial use in food animals. Our objective was to synthesize knowledge on the direct application of long-read metagenomic sequencing to respiratory samples to detect bacterial pathogens and antimicrobial resistance genes (ARGs) compared to PCR, loop-mediated isothermal amplification, and recombinase polymerase amplification. Our scoping review protocol followed the Joanna Briggs Institute and PRISMA Scoping Review reporting guidelines. Included studies reported on the direct application of these methods to respiratory samples from animals or humans to detect bacterial pathogens ±ARGs and included turnaround time (TAT) and analytical sensitivity. We excluded studies not reporting these or that were focused exclusively on bioinformatics. We identified 5,636 unique articles from 5 databases. Two-reviewer screening excluded 3,964, 788, and 784 articles at 3 levels, leaving 100 articles (19 animal and 81 human), of which only 7 studied long-read sequencing (only 1 in animals). Thirty-two studies investigated ARGs (only one in animals). Reported TATs ranged from minutes to 2 d; steps did not always include sample collection to results, and analytical sensitivity varied by study. Our review reveals a knowledge gap in research for the direct detection of bacterial respiratory pathogens and ARGs in animals using long-read metagenomic sequencing. There is an opportunity to harness the rapid development in this space to detect multiple pathogens and ARGs on a single sequencing run. Long-read metagenomic sequencing tools show potential to address the urgent need for research into rapid tests to support antimicrobial stewardship in food animal production.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Rapid determination of antimicrobial susceptibility of Gram-negative bacteria from clinical blood cultures using a scattered light-integrated collection device.

Background. A bloodstream infection (BSI) presents a complex and serious health problem, a problem that is being exacerbated by increasing antimicrobial resistance (AMR).Gap Statement. The current turnaround times (TATs) for most antimicrobial susceptibility testing (AST) methods offer results retrospective of treatment decisions, and this limits the impact AST can have on antibiotic prescribing and patient care. Progress must be made towards rapid BSI diagnosis and AST to improve antimicrobial stewardship and reduce preventable deaths from BSIs. To support the successful implementation of rapid AST (rAST) in hospital settings, a rAST method that is affordable, is sustainable and offers comprehensive AMR detection is needed.Aim. To evaluate a scattered light-integrated collection (SLIC) device against standard of care (SOC) to determine whether SLIC could accelerate the current TATs with actionable, accurate rAST results for Gram-negative BSIs.Methods. Positive blood cultures from a tertiary referral hospital were studied prospectively. Flagged positive Gram-negative blood cultures were confirmed by Gram staining and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Vitek 2, disc diffusion (ceftriaxone susceptibility only) and an SLIC device. Susceptibility to a panel of five antibiotics, as defined by European Committee on Antimicrobial Susceptibility Testing breakpoints, was examined using SLIC.Results. A total of 505 bacterial-antimicrobial combinations were analysed. A categorical agreement of 95.5 % (482/505) was achieved between SLIC and SOC. The 23 discrepancies that occurred were further investigated by the broth microdilution method, with 10 AST results in agreement with SLIC and 13 in agreement with SOC. The mean time for AST was 10.53±0.46 h and 1.94±0.02 h for Vitek 2 and SLIC, respectively. SLIC saved 23.96±1.47 h from positive blood culture to AST result.Conclusion. SLIC has the capacity to provide accurate AST 1 day earlier from flagged positive blood cultures than SOC. This significant time saving could accelerate time to optimal antimicrobial therapy, improving antimicrobial stewardship and management of BSIs.

Antimicrobial Resistance in Selected Bacteria from Food Animals in New Zealand 2018–2022

Antimicrobial resistance (AMR) presents a significant threat to human health worldwide. One important source of antimicrobial-resistant infections in humans is exposure to animals or animal products. In a phased survey, we investigated AMR in 300 Escherichia coli isolates and 300 enterococci (Enterococcus faecalis and E. faecium) isolates each from the carcasses of poultry, pigs, very young calves, and dairy cattle (food animals); all Salmonella isolates from poultry, very young calves, and dairy cattle; and 300 Campylobacter (Campylobacter jejuni and C. coli) isolates from poultry. The highest resistance levels in E. coli were found for sulfamethoxazole, tetracycline, and streptomycin, for all food animals. Cefotaxime-resistant E. coli were not found and low resistance to ciprofloxacin, colistin, and gentamicin was observed. The majority of enterococci isolates from all food animals were bacitracin-resistant. Erythromycin- and/or tetracycline-resistant enterococci isolates were found in varying proportions from all food animals. Ampicillin- or vancomycin-resistant enterococci isolates were not identified, and ciprofloxacin-resistant E. faecalis were not found. Salmonella isolates were only recovered from very young calves and all eight isolates were susceptible to all tested antimicrobials. Most Campylobacter isolates were susceptible to all tested antimicrobials, although 16.6% of C. jejuni were resistant to quinolones and tetracycline. Results suggest that AMR in E. coli, enterococci, Salmonella, and Campylobacter isolates from food animals in New Zealand is low, and currently, AMR in food animals poses a limited public health risk. Despite the low prevalence of AMR in this survey, ongoing monitoring of antimicrobial susceptibility in bacteria from food animals is recommended, to ensure timely detection of AMR with potential impacts on animal and human health.

AntimicrobialResistance and Virulence of Shiga-Toxin-Producing Escherichia coli from Milk Samples of Some Cattle Farms Al-Buḥayrah Governorate Egypt

Objective: Contamination of milk with Escherichia coli (E. coli) can pose significant public health and economic concerns. The current study was conducted to explore the prevalence and antibiotic resistance profiles of Shiga-toxin-producing Escherichia coli in milk samples of dairy cows in Egypt. Study Design: Twenty milk samples were gathered from dairy cattle (ten from healthy cows and ten from mastitic cows) and examined for the presence of Shiga-toxin-producing E. coli using Eosin Methylene Blue agar. Molecular detection of virulence genes and phenotypic antimicrobial resistance were performed. Results: E. coli was isolated from 65% (13/20) of the total milk samples (90% from mastitic cows and 40% from normal healthy cows). The virulence gene profiling of E.coli by polymerase chain reaction revealed that 84.62% (11/13) were Stx2a, 69.23% (9/13) were Stx2f, and 61.54% (8/13) were positive for Stx1 and Stx2 similarly. All the isolates showed resistance to Amikacin, Ciprofloxacin, Erythromycin, Linezolid, Penicillin G, and Trimethoprim/Sulfamethoxazole, while 92.3% expressed resistance to Amoxicillin/ Clavulanic acid, and Tylosin, and 84.62% of the isolates were resistant to Oxytetracycline indicating multidrug-resistant profiles. Conclusion: The present work highlights the presence of various E. coli strains, including potential pathogens and multidrug-resistant isolates, in milk samples from dairy cows. Regular milk testing and improved mastitis control measures are the key to reduce the public health risks and to safeguarde dairy farm productivity.

Detection of antimicrobial resistance in

Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

MALDI-TOF MS: A Reliable Tool in the Real Life of the Clinical Microbiology Laboratory.

Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in the last decade has revealed itself as a valid support in the workflow in the clinical microbiology laboratory for the identification of bacteria and fungi, demonstrating high reliability and effectiveness in this application. Its use has reduced, by 24 h, the time to obtain a microbiological diagnosis compared to conventional biochemical automatic systems. MALDI-TOF MS application to the detection of pathogens directly in clinical samples was proposed but requires a deeper investigation, whereas its application to positive blood cultures for the identification of microorganisms and the detection of antimicrobial resistance are now the most useful applications. Thanks to its rapidity, accuracy, and low price in reagents and consumables, MALDI-TOF MS has also been applied to different fields of clinical microbiology, such as the detection of antibiotic susceptibility/resistance biomarkers, the identification of aminoacidic sequences and the chemical structure of protein terminal groups, and as an emerging method in microbial typing. Some of these applications are waiting for an extensive evaluation before confirming a transfer to the routine. MALDI-TOF MS has not yet been used for the routine identification of parasites; nevertheless, studies have been reported in the last few years on its use in the identification of intestinal protozoa, Plasmodium falciparum, or ectoparasites. Innovative applications of MALDI-TOF MS to viruses' identification were also reported, seeking further studies before adapting this tool to the virus's diagnostic. This mini-review is focused on the MALDI-TOF MS application in the real life of the diagnostic microbiology laboratory.

A One Health perspective on the use of genotypic methods for antimicrobial resistance prediction.

Antimicrobial resistance is a global One Health concern with critical implications for the health of humans, animals, and the environment. Phenotypic methods of bacterial culture and antimicrobial susceptibility testing remain the gold standards for the detection of antimicrobial resistance and appropriate patient care; however, genotypic-based methods, such as PCR, whole genome sequencing, and metagenomic sequencing, for detection of genes conferring antimicrobial resistance are increasingly available without inclusion of appropriate standards for quality or interpretation. Misleading test results may lead to inappropriate antimicrobial treatment and, in turn, poor patient outcomes and the potential for increased incidence of antimicrobial resistance. This article explores the current landscape of clinical and methodological aspects of antimicrobial susceptibility testing and genotypic antimicrobial resistance test methods. Additionally, it describes the limitations associated with employing genotypic-based test methods in the management of veterinary patients from a One Health perspective. The companion Currents in One Health by Maddock et al, AJVR, March 2024, addresses current and future needs for veterinary antimicrobial resistance research.

Detection of Antimicrobial Resistance by Phenotypic and Genotypic Methods in E. coli Isolated from Diarrhea of Cattle Calves in Mhow, Indore

Background: Neonatal calf diarrhoea, a complex illness, continues to be the leading cause of neonatal calf deaths. The complex pathophysiology of infectious calf diarrhoea results in severe losses to the dairy sector, either directly via mortality or indirectly through the costs of prophylaxis and slowed growth in infected calves. Neonatal calf diarrhoea during the first month of life causes between 80 to 85 per cent of all deaths in India. Methods: A total 137 cattle calve diarrhea samples were collected from calves that show clinical signs of diarrhea. Swabs of the rectum were taken directly from diarrhoeic calves and collected in sterile test tubes. They were then immediately transported to the laboratory for the isolation of E. coli. Result: A total 64 E. coli isolates were detected and identified from samples via biochemical and molecular assays employing PCR targeting the E. coli specific 16s rRNA gene. The result of antibiotic sensitivity of 64 isolated strains of E. coli to 15 antimicrobial drugs shows highest sensitivity towards antibiotic gentamycin (68.75%) followed by sulfadiazine and co-trimoxazole (39.06), ciprofloxacin (32.81%) and highest resistance against ampicillin (87.5%) followed by ceftriaxone (82.813%), ceftazidime plus clav (81.25%). There is the variation in the expression of the AMR genes in the isolated E. coli strains. The detection rate of AMR gene blaTEM was highest 90% and that of aadA was the lowest (0%). The other AMR genes blaCTX-M, blaSHV, sul1 and tetA showing 50%, 20%, 50% and 30%.

Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water

BackgroundWater is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites.ResultsAll strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic.ConclusionsThe study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

A rapid and specific antimicrobial resistance detection of Escherichia coli via magnetic nanoclusters.

Drinking water contamination, often caused by bacteria, leads to substantial numbers of diarrhea deaths each year, especially in developing regions. Human urine as a source of fertilizer, when handled improperly, can contaminate drinking water. One dominant bacterial pathogen in urine is Escherichia coli, which can trigger serious waterborne/foodborne diseases. Considering the prevalence of the multi-drug resistant extended-spectrum beta-lactamase (ESBL) producing E. coli, a rapid detection method for resistance is highly desired. In this work, we developed a method for quick identification of E. coli and, at the same time, capable of removal of general bacterial pathogens from human urine. A specific peptide GRHIFWRRGGGHKVAPR, reported to have a strong affinity to E. coli, was utilized to modify the PEGylated magnetic nanoclusters, resulting in a specific capture and enrichment of E. coli from the bacteria-spiked artificial urine. Subsequently, a novel luminescent probe was applied to rapidly identify the antimicrobial resistance of the collected E. coli within 30 min. These functionalized magnetic nanoclusters demonstrate a promising prospect to rapidly detect ESBL E. coli in urine and contribute to reducing drinking water contamination.

The impact of the BIOFIRE® Blood Culture Identification 2 Panel on antimicrobial treatment of children with suspected systemic inflammatory response syndrome and sepsis.

This study aimed to assess the therapeutic effects of implementing the BioFire® Blood Culture Identification 2 (BCID2) Panel (bioMérieux, Marcy l'Etoile, France) in the clinical practice of children with sepsis.This retrospective cross-sectional study included children from 15days of age to 18years old with sepsis and of whom the BCID2 Panel was studied from the positive blood culture. If the antimicrobial treatment was changed according to the results of BCID2 Panel, it was recorded and re-grouped as targeted antimicrobial therapy, de-escalation of the antimicrobial treatment and shifting to another antimicrobial drug if any antimicrobial resistance was detected. Seven-days and thirty-days mortality rate was recorded.Thirty-two patients with 36 septic episodes with positive BCID2 Panel results were included. The median age was 10months 15days (ranging from 15days to 16.5years). The mean difference between having positive results by the BCID2 Panel and conventional culture methods was 82.2 ± 45.4h (ranging from 12.3 to 207h). Effect of the BCID2 Panel on the antimicrobial treatment was detected in 69.4% of the episodes (n = 25). Glycopeptides were ceased at 6 patients, piperacillin/tazobactam was ceased at 6 patients, and cefotaxime was ceased at one patient and de-escalation was achieved in 13 episodes which formed the 36.1% of the initial antimicrobial treatment. Conclusion: The BCID2 Panel had an important impact on the patients care and optimization according to the principles of antimicrobial stewardship. The BCID2 Panel may be one of the key items for rapid and accurate diagnosis in children with sepsis. Blood culture is still the mainstay for especially detection of antimicrobial drug resistance, while BCID2 tests had several advantages such as speed and diagnostic accuracy as good as blood culture. What is Known: • The BioFire®Blood Culture Identification 2 (BCID2) Panel (bioMérieux, Marcy l'Etoile, France) is a well-validated assay that allows for the simultaneous identification of 43 nucleic acid targets associated with bloodstream infections within about 1h. What is New: • BCID2 Panel had a direct impact on the patient's treatment decision at the 69.4% of the sepsis episodes. • The clinicians received the microbiological results 82.2h earlier with BCID2 panel compared to blood culture methods with antimicrobial resistanceandde-escalation of the antimicrobial drugs was achieved at 13 episodes which formed the 36.1% of the initial treatment.