Conventional Antimicrobial Susceptibility Testing Research Articles

Molecular Characterization of Escherichia coli Causing Urinary Tract Infections Through Next-Generation Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes.

Introduction An enormous increase in antimicrobial resistance (AMR) among bacteria isolated from human clinical specimens contributed to treatment failures. Increased surveillance through next-generation sequencing (NGS) or whole genome sequencing (WGS) could facilitate the study of the epidemiology of drug-resistant bacterial strains, resistance genes, and other virulence determinants they are potentially carrying. Methods This study included 30 Escherichia coli (E. coli) isolates obtained from patients suffering from urinary tract infections (UTIs) attending Prathima Institute of Medical Sciences, Karimnagar, India. All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS to identify genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes carried by the isolates. Results The conventional antimicrobial susceptibility testing revealed that 15 (50%) isolates were resistant to imipenem (IPM), 10 (33.33%) were resistant to amikacin (AK), 13 (43.33%) were resistant to piperacillin-tazobactam (PTZ), 17 (56.66%) were resistant to cephalosporins, and 14 (46.66%) were resistant to nitrofurantoin (NIT). Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaCTX-M(19/30, 63.33%) genes, followed by blaTEMand blaOXA-1. The blaNDM-5 gene was found in three isolates (3/30, 10%). The virulence genes identified in the present study wereiutA, sat, iss, and papC, among others. The E. coli serotype found predominantly belonged to O25:H4 (5, 16.66%), followed by O102:H6 (4, 13.33%). A total of 16 MLST variants were identified among the examined samples. Of the MLST-based sequence types (STs) identified, ST-131 (7, 23.33%) was the predominant one, followed by ST-167 (3, 10%)and ST-12 (3, 10%). Conclusions The study results demonstrated that the E. coli strains isolated from patients suffering from UTIs potentially carried antimicrobial resistanceand virulence genes and belonged to different strain types based on MLST. Careful evaluation of bacterial strains using molecular analyses such as NGS could facilitate an improved understanding of bacterial antibiotic resistance and its virulence potential. This could enable physicians to choose appropriate antimicrobial agents and contribute to better patient management, thereby preventing the emergence and spread of drug-resistant bacteria.

Predictive Modeling of Phenotypic Antimicrobial Susceptibility of Selected Beta-Lactam Antimicrobials from Beta-Lactamase Resistance Genes.

The outcome of bacterial infection management relies on prompt diagnosis and effective treatment, but conventional antimicrobial susceptibility testing can be slow and labor-intensive. Therefore, this study aims to predict phenotypic antimicrobial susceptibility of selected beta-lactam antimicrobials in the bacteria of the family Enterobacteriaceae from different beta-lactamase resistance genotypes. Using human datasets extracted from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program conducted by Pfizer and retail meat datasets from the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), we used a robust or weighted least square multivariable linear regression modeling framework to explore the relationship between antimicrobial susceptibility data of beta-lactam antimicrobials and different types of beta-lactamase resistance genes. In humans, in the presence of the blaCTX-M-1, blaCTX-M-2, blaCTX-M-8/25, and blaCTX-M-9 groups, MICs of cephalosporins significantly increased by values between 0.34-3.07 μg/mL, however, the MICs of carbapenem significantly decreased by values between 0.81-0.87 μg/mL. In the presence of carbapenemase genes (blaKPC, blaNDM, blaIMP, and blaVIM), the MICs of cephalosporin antimicrobials significantly increased by values between 1.06-5.77 μg/mL, while the MICs of carbapenem antimicrobials significantly increased by values between 5.39-67.38 μg/mL. In retail meat, MIC of ceftriaxone increased significantly in the presence of blaCMY-2, blaCTX-M-1, blaCTX-M-55, blaCTX-M-65, and blaSHV-2 by 55.16 μg/mL, 222.70 μg/mL, 250.81 μg/mL, 204.89 μg/mL, and 31.51 μg/mL respectively. MIC of cefoxitin increased significantly in the presence of blaCTX-M-65 and blaTEM-1 by 1.57 μg/mL and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of cefoxitin increased by an average of 8.66 μg/mL over 17 years. Compared to E. coli isolates, MIC of cefoxitin in Salmonella enterica isolates decreased significantly by 0.67 μg/mL. On the other hand, MIC of ceftiofur increased in the presence of blaCTX-M-1, blaCTX-M-65, blaSHV-2, and blaTEM-1 by 8.82 μg/mL, 9.11 μg/mL, 8.18 μg/mL, and 1.04 μg/mL respectively. In the presence of blaCMY-2, MIC of ceftiofur increased by an average of 10.20 μg/mL over 14 years. The ability to predict antimicrobial susceptibility of beta-lactam antimicrobials directly from beta-lactamase resistance genes may help reduce the reliance on routine phenotypic testing with higher turnaround times in diagnostic, therapeutic, and surveillance of antimicrobial-resistant bacteria of the family Enterobacteriaceae.

Performance of targeted next-generation sequencing in the detection of respiratory pathogens and antimicrobial resistance genes for children.

Introduction. Respiratory tract infection, which is associated with high morbidity and mortality, occurs frequently in children. At present, the main diagnostic method is culture. However, the low pathogen detection rate of the culture approach prevents timely and accurate diagnosis. Fortunately, next-generation sequencing (NGS) can compensate for the deficiency of culture, and its application in clinical diagnostics has become increasingly available.Gap Statement. Targeted NGS (tNGS) is a platform that can select and enrich specific regions before data enter the NGS pipeline. However, the performance of tNGS in the detection of respiratory pathogens and antimicrobial resistance genes (ARGs) in infections in children is unclear.Aim and methodology. In this study, we estimated the performance of tNGS in the detection of respiratory pathogens and ARGs in 47 bronchoalveolar lavage fluid (BALF) specimens from children using conventional culture and antimicrobial susceptibility testing (AST) as the gold standard.Results. RPIP (Respiratory Pathogen ID/AMR enrichment) sequencing generated almost 500 000 reads for each specimen. In the detection of pathogens, RPIP sequencing showed targeted superiority in detecting difficult-to-culture bacteria, including Mycoplasma pneumoniae. Compared with the results of culture, the sensitivity and specificity of RPIP were 84.4 % (confidence interval 70.5-93.5 %) and 97.7 % (95.9 -98.8%), respectively. Moreover, RPIP results showed that a single infection was detected in 10 of the 47 BALF specimens, and multiple infections were detected in 34, with the largest number of bacterial/viral coinfections. Nevertheless, there were also three specimens where no pathogen was detected. Furthermore, we analysed the drug resistance genes of specimens containing Streptococcus pneumoniae, which was detected in 25 out of 47 specimens in the study. A total of 58 ARGs associated with tetracycline, macrolide-lincosamide-streptogramin, beta-lactams, sulfonamide and aminoglycosides were identified by RPIP in 19 of 25 patients. Using the results of AST as a standard, the coincidence rates of erythromycin, tetracycline, penicillin and sulfonamides were 89.5, 79.0, 36.8 and 42.1 %, respectively.Conclusion. These results demonstrated the superiority of RPIP in pathogen detection, particularly for multiple and difficult-to-culture pathogens, as well as in predicting resistance to erythromycin and tetracycline, which has significance for the accurate diagnosis of pathogenic infection and in the guidance of clinical treatment.

Portable Colorimetric Hydrogel Beads for Point-of-Care Antimicrobial Susceptibility Testing.

Sepsis is a life-threatening condition with systemic inflammatory responses caused by bacterial infections. Considering the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), sepsis is a great threat to public health. The gold standard methods for antimicrobial susceptibility testing (AST), however, take at least approximately 3 days to implement the entire blood culture, pure culture, and AST processes. To overcome the time-consuming nature of conventional AST, a method employing a chromatic biosensor composed of poly(diacetylene), alginate, and LB broth (PAL) is introduced in this study. Compared to the gold standards, AST with PAL biosensors can be completed within a time frame as short as 16 h. Such a significant reduction in time is possible because the consecutive cultures and AST are carried out simultaneously by encapsulating the bacterial nutrients and detection molecules into a single component. The bead-like hydrogel sensors were used in their freeze-dried form, which endows them with portability and stability, thus making them adequate for point-of-care testing. The PAL biosensor yields minimum inhibitory concentrations comparable to those from the Clinical and Laboratory Standards Institute, and the applicability of the biosensor is further shown in MRSA-infected mice.

Detection of Salmonella DNA and drug-resistance mutation by PCR-based CRISPR-lbCas12a system

Salmonella is an important foodborne pathogen, which can cause serious public health problems. Rapid and accurate detection of Salmonella infection and drug resistance mutations in patients will provide timely guidance for clinical treatment and avoid disease progression and other related clinical problems. Here, we established a highly sensitive and quick method for Salmonella and drug resistance mutation detection based on polymerase chain reaction (PCR) and CRISPR-lbCas12a system and evaluated its practicability with clinical samples.Specific CRISPR RNAs (crRNAs) and primers are designed for Salmonella DNA and parC gene S80I mutation diagnosis. CrRNAs with and without phosphorylated modification and different crRNA preparation methods are used to assess the effect on the detection system. After optimization, we detected as low as one copy of Salmonella DNA and drug resistance mutation parC S80I with the Salmonella DNA standard. For 94 clinical samples, this method also showed high sensitivity (100%, 95% CI: 84.98–100%) and specificity (98.48%, 95% CI: 90.73–99.92%) with less time (3 h) than plate culture (16 h) and conventional antimicrobial susceptibility testing (over 16 h). Besides, one parC S80I mutant strain was detected, which is consistent with the result of DNA sequencing. Taken together, we established a highly sensitive and specific method for Salmonella infection and parC S80I drug resistance mutation detection with fewer reagents and ordinary instruments. This assay has wide application prospects for fast detection of pathogen (bacterium and virus) infection, drug resistance determination, and proper treatment guidance.

A prospective study evaluating the effect of a ‘Diagnostic Stewardship Care-Bundle’ for automated blood culture diagnostics

We prospectively implemented a diagnostic stewardship care-bundle checklist, 'Sepsis-48 DSB', with the aim of reducing intervening duration of key steps of automated blood culture diagnostics (aBCD). Sepsis-48 DSB was implemented for automated blood culture bottles (BCBs) received from adult intensive care units (AICUs) during the intervention period (P2; July 2020-June 2021) and intervening durations were compared with those during the retrospective, pre-intervention period (P1; March-June 2020). During both periods, provisional blood culture reports (pBCR) were issued wherein direct microbial identification (dID) was performed in BCBs with Gram-negatives by directly inoculating conventional biochemical tests and direct antimicrobial susceptibility testing (dAST) using EUCAST RAST method. The results were compared with the standard of care (SoC) method (i.e. full incubation followed by identification and AST by VITEKⓇ-2 Compact). During P2, significant reductions in loading time (LT) [median: 63.5 vs. 32 minutes, P < 0.001], time to dID+dAST performance (TTD) [186 vs. 115 minutes, P=0.0018] and an increase in compliance to bundle targets [LT ≤45: 44% vs. 66%, P=0.006 and TTD ≤120: 34% vs. 51.7%, P=0.03] were observed. Using dID+dAST method, results were read 694 minutes earlier than SoC method. Of 176 pBCR, 165 (94%) were concordant with SoC in microbial identification of species. Categorical agreement for any drug-bug combination was 92.7% (1079/1164) and corresponding major, very major, and minor error rates were 8.8% (19/216), 4.9% (45/921), and 1.8% (21/1164), respectively. The 'diagnostic stewardship care-bundle' strategy was successfully implemented with considerable diagnostic accuracy leading to significant reductions in duration of targeted steps of aBCD.

Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan.

The study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producing Klebsiella pneumoniae clinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out. Plasmids were isolated from 10 clinical isolates of Klebsiella pneumoniae, and from a corresponding set of Escherichia coli transconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. For E. coli transconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results. All carbapenemase-producing K. pneumoniae isolates (carrying either blaNDM-1, or/and blaOXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of the blaNDM-1 and blaESBL-plasmids identified in this study were previously described. Most blaNDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange between K. pneumoniae isolates of this collection. The majority of NGS-based AMR profiles from the E. coli transconjugants correlated well with both NGS-based predicted and conventional AST results. This study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.

A COMPARATIVE STUDY TO DETERMINE THE ACCURACY OF DIRECT SUSCEPTIBILITY TESTING WITH CONVENTIONAL ANTIMICROBIAL SUSCEPTIBILITY TESTING

Introduction: Clinically significant bacteria are identified in the laboratory and the accurate information isprovided with antimicrobial susceptibility testing (AST) which is essential for an accuratemanagementofpatientssufferingwith abacterial infection orbacterialdisease[1].As the process of AST istime consuming and Results of AST are provided with in a timespan of 48–72 h after sampling.Disk diffusion has many benefits, which includes low expenditure, time duration reduction ofresults and few more basic benefits. One of the benefits is the probability or chances ofexecuting direct susceptibility testing (DST).when is potentially very useful in the management of critically used selectively and interpreted carefully, DST on clinical samples ill patients, as the time to results is shortened by approximately 24 h. Aim:To do a comparative study for determining the accuracy of direct susceptibility testing with conventional antimicrobial susceptibility testing. Material And Method:This cross- sectional study was conducted in the Department of Microbiology, peoples College of MedicalSciences and Research Centre, over a period of 1 year-from February 2021 to February 2022. Result:A total of 311 samples of urine, pus and body fluid were collected and positive sample processed from 124males and 187 females from age groups 1 to 90 years.From total of 311 samples Out of all the samples 132 were of pus, 175 were of urine specimen 4 were offluidssamples.Comparison of the bacterial response to 14 antimicrobial agents using directantimicrobial sensitivity testing (DST) versus standard antimicrobial sensitivity testing(AST) for Gram positive bacteriaOut of 91 gram positive bacteria we found that on performing AST and DST on norfloxacin probability valuewas 1, which shows that there is no significant difference in results by these two methods. Similarly,Ciprofloxacin, Cefoxitin, Doxycycline, Amoxy-clavulanic acid, Erythromycin, Clindamycin, Linezolid,Chloramphenicol, Cefuroxime all have a p-value as 1 on performing both the different types of antimicrobialsusceptibility testing method which indicates a similarity in their results. Conclusion:After 1 year of systematic study even though we found that AST and DST both gives almost similar result, but keeping in mind major and minor differences in antibiotic sensitivity test . We should consider it only foremergencypurposes.In emergency it is observed that DST proved to be more successful because DST provides results with in 24to 32 hours, this providing results faster but it should always be followed by AST for confirmation of results.With DST, we have to apply more no. of antibiotics disk to coues all bacteria increasingcost of ABST.

Test Performance and Potential Clinical Utility of the GenMark Dx ePlex Blood Culture Identification Gram-Negative Panel.

The test performance and potential clinical utility of the ePlex blood culture identification Gram-negative (BCID-GN) panel was evaluated relative to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry on bacterial isolates and conventional antimicrobial susceptibility testing. The majority (106/108, 98.1%) of GN bacteria identified by MALDI were on the BCID-GN panel, and valid tests (107/108, 99.1%) yielded results on average 26.7 h earlier. For all valid tests with on-panel organisms, the positive percent agreement was 102/105 (97.2%) with 3 false negatives and the negative percent agreement was 105/105. Chart review (n = 98) showed that in conjunction with Gram stain results, negative pan-Gram-positive (GP) markers provided the opportunity to discontinue GP antibiotic coverage in 63/98 (64.3%) cases on average 26.2 h earlier. Only 8/12 (66.7%) Enterobacterales isolates with resistance to third-generation cephalosporins harbored the CTX-M gene. In contrast, 8/8 CTX-M+ samples yielded a resistant isolate. Detection of 1 Stenotrophomonas maltophilia (18 h), 1 OXA23/48+ Acinetobacter baumannii (52.4 h), and 3 CTX-M+ Enterobacterales isolates on ineffective treatment (47.1 h) and 1 on suboptimal therapy (72.6 h) would have additionally enabled early antimicrobial optimization in 6/98 (6.1%) patients. IMPORTANCE The GenMark Dx ePlex rapid blood culture diagnostic system enables earlier time to identification of antimicrobial-resistant Gram-negative bacteria causing bloodstream infections. Its ability to rule out Gram-positive bacteria enabled early discontinuation of unnecessary antibiotics in 63/98 (64.3%) cases on average 26.2 h earlier. Detection of bacteria harboring the CTX-M gene as well as early identification of highly resistant bacteria such as Stenotrophomonas maltophilia and Acinetobacter baumannii enabled optimization of ineffective therapy in 6/98 (6.1%) patients. Its implementation in clinical microbiology laboratories optimizes therapy and improves patient care.

Comparative analysis of conventional and direct antimicrobial susceptibility testing of blood cultures in a tertiary care cancer hospital: the way forward

ABSTRACT Bloodstream infections (BSI) are associated with high mortality rates, especially in immunocompromised patients. Identifying pathogens early and selecting appropriate antimicrobials to treat BSI is integral in reducing the mortality rate. There is a need to reduce the turnaround time (TAT) of pathogen identification as well as to accelerate the antimicrobial susceptibility testing (AST) of blood cultures, which can be achieved by following relevant identification methods and performing the direct AST (DAST) by the disk diffusion method. In this study, blood samples were collected from patients with suspected bacteremia/septicemia, and aseptic precautions were taken to prevent contamination. Samples containing gram-negative bacilli (GNB) were then analyzed by DAST and conventional AST (CAST). We tested 118 GNB-positive isolates in total to compare the results of DAST and CAST. DAST and CAST showed good categorical agreement (CA) for various groups of microorganisms: 98.9% and 99.6% for Enterobacterales and Pseudomonas spp., respectively. Early detection of pathogens in blood along with the determination of their antibiotic susceptibility patterns is a need of the hour. By performing DAST on positive blood culture broth, clinical teams can obtain the information necessary for switching from empirical therapy to definitive treatment one day faster. This rapid identification of the pathogen, along with corresponding AST results, will help clinicians to accelerate targeted antimicrobial therapy for critical patients and, thus, reduce mortality and morbidity rates in patients with bloodstream infections.

Direct single-cell antimicrobial susceptibility testing of Escherichia coli in urine using a ready-to-use 3D microwell array chip.

Empirical antibiotic therapies are prescribed for treating uncomplicated urinary tract infections (UTIs) due to the long turnaround time of conventional antimicrobial susceptibility testing (AST), leading to the prevalence of multi-drug resistant pathogens. We present a ready-to-use 3D microwell array chip to directly conduct comprehensive AST of pathogenic agents in urine at the single-cell level. The developed device features a highly integrated 3D microwell array, offering a dynamic range from 102 to 107 CFU mL-1, and a capillary valve-based flow distributor for flow equidistribution in dispensing channels and uniform sample distribution. The chip with pre-loaded reagents and negative pressure inside only requires the user to initiate AST by loading samples (∼3 s) and can work independently. We demonstrate an accessible sample-to-result workflow, including syringe filter-based bacteria separation and rapid single-cell AST on chip, which enables us to bypass the time-consuming bacteria isolation and pre-culture, speeding up the AST in ∼3 h from 2 days of conventional methods. Moreover, the bacterial concentration and AST with minimum inhibitory concentrations can be assessed simultaneously to provide comprehensive information on infections. With further development for multiple antibiotic conditions, the Dsc-AST assay could contribute to timely prescription of targeted drugs for better patient outcomes and mitigation of the threat of drug-resistant bacteria.

182. Impact of Rapid Identification and Phenotypic Results on Clinical, Economic, Antimicrobial Stewardship Outcomes in Patients with Gram-Negative Bacteremia

Abstract Background Timely and effective antibiotic therapy is a critical determinant of clinical outcome in Gram-negative bacilli (GNB) bloodstream infections (BSI). Conventional antimicrobial susceptibility testing (AST) remains a rate-limiting step in determining targeted therapy. Accelerate Pheno™ (AP) is a novel system providing both identification (ID) and AST results within 8 hours. The primary objective of this study was to determine the impact of rapid ID and AST with AP on time to achieving targeted therapy (TTT). Methods This single center, quasi-experimental study compared outcomes with AP (POST; November 2020 to July 2021) versus a historical standard (PRE; November 2019 to July 2020) of rapid diagnostic testing (BioFire® FilmArray®BCID) and conventional AST (VITEK®2) with pharmacist-driven antimicrobial stewardship (AMS) intervention. PRE and POST BSI were retrospectively identified by positive blood culture (BCx) with an on-panel GNB. Inpatients were excluded if less than 18 years of age, pregnant, polymicrobial BCx, BCx with the same organism and source within 7 days, or if died or transitioned to palliative or hospice care before final AST results. Secondary outcomes included inpatient all-cause mortality, hospital and ICU length of stay, hospital-acquired Clostridioides difficile infection, antibiotic and microbiologic costs, days of anti-pseudomonal therapy, antibiotic intensity score, and time to first intervention following Gram stain, ID, and AST results. Results Overall, 373 BSI were screened and 294 were included [161 PRE vs 133 POST]. Baseline demographics and characteristics were similar between groups. Median TTT was shorter POST [50 hours (IQR 44–92) vs 28 hours (IQR 14–55), p &amp;lt; 0.05]. Median microbiology testing costs per episode were higher POST [Δ$40.33 (32%), p &amp;lt; 0.05]. Other secondary outcomes did not differ significantly between groups. Pharmacists intervened more POST [69 (43%) vs 77 (58%), p &amp;lt; 0.05]. AP completed ID and AST in 155 of 205 (76%) BSI. Off-panel GNB occurred in 26 (13%) BSI POST. Conclusion Implementing the AP system together with an established AMS program resulted in a shorter TTT and a higher cost to test. Clinical, AMS, and other economic outcomes were not impacted by the addition of AP. Disclosures All Authors: No reported disclosures.

Clinically undetected polyclonal heteroresistance among Pseudomonas aeruginosa isolated from cystic fibrosis respiratory specimens.

Pseudomonas aeruginosa infection is the leading cause of death among patients with cystic fibrosis (CF) and a common cause of difficult-to-treat hospital-acquired infections. P. aeruginosa uses several mechanisms to resist different antibiotic classes and an individual CF patient can harbour multiple resistance phenotypes. To determine the rates and distribution of polyclonal heteroresistance (PHR) in P. aeruginosa by random, prospective evaluation of respiratory cultures from CF patients at a large referral centre over a 1 year period. We obtained 28 unique sputum samples from 19 CF patients and took multiple isolates from each, even when morphologically similar, yielding 280 unique isolates. We performed antimicrobial susceptibility testing (AST) on all isolates and calculated PHR on the basis of variability in AST in a given sample. We then performed whole-genome sequencing on 134 isolates and used a machine-learning association model to interrogate phenotypic PHR from genomic data. PHR was identified in most sampled patients (n = 15/19; 79%). Importantly, resistant phenotypes were not detected by routine AST in 26% of patients (n = 5/19). The machine-learning model, using the extended sampling, identified at least one genetic variant associated with phenotypic resistance in 94.3% of isolates (n = 1392/1476). PHR is common among P. aeruginosa in the CF lung. While traditional microbiological methods often fail to detect resistant subpopulations, extended sampling of isolates and conventional AST identified PHR in most patients. A machine-learning tool successfully identified at least one resistance variant in almost all resistant isolates by leveraging this extended sampling and conventional AST.

An antibiotic concentration gradient microfluidic device integrating surface-enhanced Raman spectroscopy for multiplex antimicrobial susceptibility testing.

Antimicrobial susceptibility testing (AST) is a key measure in clinical microbiology laboratories to enable appropriate antimicrobial administration. During an AST, the determination of the minimum inhibitory concentration (MIC) is an important step in which the bacterial responses to an antibiotic at a series of concentrations obtained in separate bacterial growth chambers or sites are compared. However, the preparation of different antibiotic concentrations is time-consuming and labor-intensive. In this paper, we present a microfluidic device that generates a concentration gradient for antibiotics that is produced by diffusion in the laminar flow regime along a series of lateral microwells to encapsulate bacteria for antibiotic treatment. All the AST preparation steps (including bacterium loading, antibiotic concentration generation, buffer washing, and isolated bacterial growth with an antibiotic) can be performed in a single chip. The viable bacterial cells in each microwell after the antibiotic treatment are then quantified by their surface-enhanced Raman scattering (SERS) signals that are acquired after placing a uniform SERS-active substrate in contact with all the microwells. For proof-of-concept, we demonstrated the AST performance of this system on ampicillin (AMP)-susceptible and -resistant E. coli strains. Compared with the parameters for conventional AST methods, the AST procedure based on this chip requires only 20 μL of bacteria solution and 5 h of operation time. This result indicates that this integrated system can greatly shorten and simplify the tedious and labor-intensive procedures required for current standard AST methods.

LB16. Surveillance Rates of Cefiderocol Heteroresistance Correlate With All-cause Mortality in the APEKS-NP and CREDIBLE-CR Trials

BackgroundCefiderocol is a recently FDA approved, novel siderophore beta-lactam antibiotic. Conventional antimicrobial susceptibility testing (AST) suggests that a variety of Gram-negative pathogens, including carbapenem-resistant (CR) isolates, are overwhelmingly susceptible to cefiderocol.. This antibiotic performed well in the APEKS-NP trial for the treatment of nosocomial pneumonia caused largely by carbapenem susceptible isolates. However, in the CREDIBLE-CR trial involving exclusively CR Gram-negative bacteria, cefiderocol was associated with a higher rate of all-cause mortality. We hypothesized one explanation for these discrepant data might be undetected cefiderocol heteroresistance (HR).HR is a form of antibiotic resistance in which an isolate harbors a minority resistant subpopulation of cells co-existing with a majority susceptible population, and is often undetected by standard AST. Isolates exhibiting undetected HR, with as low as 1 in 1 million resistant cells, can cause treatment failure in in vivo models.MethodsWe quantified HR to cefiderocol by population analysis profile (PAP) of 161 Acinetobacter, 180 Klebsiella, and 108 Pseudomonas isolates collected in Georgia, USA.ResultsWe observed CR isolates exhibited a high frequency of HR, which was largely undetected by standard AST, and correlated with all-cause mortality in the CREDIBLE-CR study (Table). Carbapenem-susceptible isolates exhibited no or low rates of cefiderocol HR (Table). Cephalosporin-resistant bacteria mostly exhibited increased rates of cefiderocol HR, but below those of CR strains. These differences in rates of cefiderocol HR correlated with the mortality data from the APEKS-NP and CREDIBLE-CR trials, across the bacterial species tested (Table).Table: Surveillance rates of cefiderocol heteroresistance correlate with all-cause mortality in the APEKS-NP and CREDIBLE-CR trials ConclusionThese data suggest that the lower rates of cefiderocol HR in carbapenem-susceptible isolates that predominated the APEKS-NP trial may explain the enhanced efficacy of the drug in that study as compared to the CREDIBLE-CR trial. Importantly, the widespread, undetected cefiderocol HR observed among CR pathogens may explain the discordance between this drug’s excellent in vitro susceptibility profile and increased patient mortality in the CREDIBLE-CR trial.Disclosures All Authors: No reported disclosures

Direct versus conventional antimicrobial susceptibility testing: An important antimicrobial stewardship tool in ICU care

Direct versus conventional antimicrobial susceptibility testing: An important antimicrobial stewardship tool in ICU care

Evaluation of the InTray and Compact Dry culture systems for the diagnosis of urinary tract infections in patients presenting to primary health clinics in Harare, Zimbabwe

Antimicrobial resistance surveillance data is lacking from many resource-limited settings mainly due to limited laboratory testing. Novel culture systems may address some of the limitations of conventional culture media and expand the availability of microbiology services. The aims of this study were to evaluate the performance of InTray COLOREX Screen/ESBL and Compact Dry for the detection of uropathogens and of extended-spectrum beta-lactamase (ESBL)-producing organisms from urine samples. Urines samples were collected from patients presenting with symptoms of urinary tract infection to primary care clinics in Harare. Performance of the InTray COLOREX Screen, ESBL and Compact Dry chromogenic media were compared to the reference of culture using Brilliance UTI agar and conventional antimicrobial susceptibility testing. A total of 414 samples were included in the analysis. Of the included samples, 98 were positive on Brilliance UTI agar and 83 grew Enterobacterales. The sensitivities and specificities for Enterobacterales were 89.2% (95% CI 80.4–94.9) and 98.2% (95% CI 96.1–99.3) for InTray Screen and 95.2% (95% CI 88.1–98.7) and 99.7% (95% CI 98.3–100) for Compact Dry. Extended-spectrum beta-lactamases were present in 22 isolates from the Brilliance UTI agar. The sensitivity of the InTray COLOREX ESBL culture plates for the detection of ESBL-producing organisms was 95.5% (95% CI 77.2–99.9) and specificity was 99.5% (95% CI 98.2–99.9%). Our findings show good performance of the novel culture systems for the detection of uropathogens and ESBL-producing organisms. Both systems have several advantages over conventional media and have the potential to expand and decentralize laboratory testing.

Novel electronic biosensor for automated inoculum preparation to accelerate antimicrobial susceptibility testing

A key predictor of morbidity and mortality for patients with a bloodstream infection is time to appropriate antimicrobial therapy. Accelerating antimicrobial susceptibility testing from positive blood cultures is therefore key to improving patient outcomes, yet traditional laboratory approaches can require 2–4 days for actionable results. The eQUANT—a novel instrument utilizing electrical biosensors—produces a standardized inoculum equivalent to a 0.5 McFarland directly from positive blood cultures. This proof-of-concept study demonstrates that eQUANT inocula prepared from clinically significant species of Enterobacterales were comparable to 0.5 McF inocula generated from bacterial colonies in both CFU/ml concentration and performance in antimicrobial susceptibility testing, with ≥ 95% essential and categorical agreement for VITEK2 and disk diffusion. The eQUANT, combined with a rapid, direct from positive blood culture identification technique, can allow the clinical laboratory to begin antimicrobial susceptibility testing using a standardized inoculum approximately 2–3 h after a blood culture flags positive. This has the potential to improve clinical practice by accelerating conventional antimicrobial susceptibility testing and the resulting targeted antibiotic therapy.

Rapid versus standard antimicrobial susceptibility testing to guide treatment of bloodstream infection.

BackgroundRapid antimicrobial susceptibility tests are expected to reduce the time to clinically important results of a blood culture. This might enable clinicians to better target therapy to a person's needs, and thereby, improve health outcomes (mortality, length of hospital stay), and reduce unnecessary prescribing of broad‐spectrum antibiotics; thereby reducing antimicrobial resistance rates.ObjectivesTo assess the effects of rapid susceptibility testing versus standard susceptibility testing for bloodstream infections (BSIs).Search methodsTo identify studies with selected outcomes, we searched the Cochrane Infectious Diseases Group Specialised Register, CENTRAL, MEDLINE, LILACS, and two trials registries, between 1987 and October 2020. We used 'bloodstream infection' and 'antimicrobial susceptibility tests' as search terms. We had no language or publication status limitations.Selection criteriaRandomized controlled trials (RCTs) comparing rapid antimicrobial susceptibility testing (with a time‐to‐result of ≤ 8 hours) versus conventional antimicrobial susceptibility testing in people with a BSI caused by any bacteria, as identified by a positive blood culture.Data collection and analysisTwo review authors independently screened references, full‐text reports of potentially relevant studies, extracted data from the studies, and assessed risk of bias. Any disagreement was discussed and resolved with a third review author. For mortality, a dichotomous outcome, we extracted the number of events in each arm, and presented a risk ratio (RR) with 95% confidence interval (CI) to compare rapid susceptibility testing to conventional methods. We used Review Manager 5.4 to meta‐analyse the data. For other outcomes, which are time‐to‐event outcomes (time‐to‐discharge from hospital, time‐to‐first appropriate antibiotic change), we conducted qualitative narrative synthesis, due to heterogeneity of outcome measures. Main resultsWe included six trials, with 1638 participants. For rapid antimicrobial susceptibility testing compared to conventional methods, there was little or no difference in mortality between groups (RR 1.10, 95% CI 0.82 to 1.46; 6 RCTs, 1638 participants; low‐certainty evidence). In subgroup analysis, for rapid genotypic or molecular antimicrobial susceptibility testing compared to conventional methods, there was little or no difference in mortality between groups (RR 1.02, 95% CI 0.69 to 1.49; 4 RCTs, 1074 participants; low‐certainty evidence). For phenotypic rapid susceptibility testing compared to conventional methods, there was little or no difference in mortality between groups (RR 1.37, 95% CI 0.80 to 2.35; 2 RCTs, 564 participants; low‐certainty evidence).In qualitative analysis, rapid susceptibility testing may make little or no difference in time‐to‐discharge (4 RCTs, 1165 participants; low‐certainty evidence). In qualitative analysis, rapid genotypic susceptibility testing compared to conventional testing may make little or no difference in time‐to‐appropriate antibiotic (3 RCTs, 929 participants; low‐certainty evidence). In subgroup analysis, rapid phenotypic susceptibility testing compared to conventional testing may improve time‐to‐appropriate antibiotic (RR ‐17.29, CI ‐45.05 to 10.47; 2 RCTs, 564 participants; low‐certainty evidence). Authors' conclusionsThe theoretical benefits of rapid susceptibility testing have not been demonstrated to directly improve mortality, time‐to‐discharge, or time‐to‐appropriate antibiotic in these randomized studies. Future large prospective studies should be designed to focus on the most clinically meaningful outcomes, and aim to optimize blood culture pathways.

Antimicrobial Resistance Profile of Bacterial Isolates Identified from Surgical Site Infections at a Referral Hospital, Northwest Ethiopia.

Surgical wound infections (SWI) remain as a major source of postoperative illness that increases the time of hospital stay and health care related costs globally. On top of this, the emergence and spread of drug-resistant pathogens continue to challenge the proper management of surgical wound infections. A hospital based cross-sectional study was conducted at Felege Hiwot Referral Hospital (FHRH). A total of 165 study participants were included. Socio-demographic data were collected using a pre-tested structured questionnaire. Isolates were identified by conventional bacteriological technique and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. Among 165 study participants, 98 (59.4%) were males. The overall prevalence of culture confirmed surgical wound infection was 115 (69.7%). A total of 125 bacteria isolates were identified among which, Staphylococcus aureus was predominant followed by Pseudomonas aeruginosa and Klebsiella species with a proportion of 31 (24.8%), 26 (20.8%) and 17 (13.6%), respectively. Majority (80.8%) of the isolates were found multidrug resistant (MDR). Dirty wound and duration of hospital stay were found significantly associated with culture confirmed surgical wound infections. S. aureus, P. aeruginosa and Klebsiella species were the most common isolates identified from surgical wound sites. Most of these pathogens were found MDR. Therefore, regular surveillance on the types of bacterial isolates and their drug resistance pattern should be considered.