Antimicrobial Susceptibility Testing Results Research Articles

Investigation of virulence factors, phylogenetic grouping, multiple-locus variable number tandem repeat analysis, and antimicrobial susceptibility of E. coli isolated from aborted bovine fetal tissue.

Escherichia coli is an important microorganism for cattle breeding. The aim of this study was to investigate the presence of phylogenetic groups, virulence factors, genotyping with multi-locus variable tandem repeat analysis (MLVA), and susceptibility to commonly used antimicrobial agents in E. coli strains isolated from aborted bovine fetal samples. In this study, phylogrouping and various virulence genes were analyzed by PCR in E. coli strains isolated from 637 bovine fetal tissue samples. Consequently, E. coli was isolated and identified in 24 samples in culture. Of the 24 isolates identified as positive, 12.5% were defined as group A, 83.3% as B1, and 4.2% as group B2. Of the E. coli isolates, virulence factor fimH was identified in eight (33.3%), traT in 15 (62.5%), ompT in five (20.8%), CNF1 in one (4.16%), and CNF2 in six (25%). Seven genotypic groups were determined as a result of the analysis with the MLVA 10 method. According to the antimicrobial susceptibility test results, high resistance was determined against amoxicillin/clavulanic acid and oxytetracycline. In conclusion, strains of E. coli containing CNF1, CNF2, fimH, traT, and ompT virulence factors can be associated with bovine abortions. It is noteworthy that the dominant phylogenetic group B1 has been observed in cases of cattle abortions.

Antimicrobial Drug Resistance Trends of Bacteria Causing Bloodstream Infections in a Diagnostic Centre in Lahore

Bacteraemia due to multidrug-resistant (MDR) bacteria, particularly those producing carbapenemase or extended-spectrum beta-lactamase (ESBL), causes a significant threat to patients and associated morbidity and mortality. The global rise in the incidence of bacteremia necessitates the rapid and accurate identification of pathogens to ensure effective patient health management. Objective: To investigate antimicrobial drug resistance trends among bacteria causing bloodstream infections from a diagnostic centre in Lahore. Methods: This research was conducted at the Institute of Microbiology and Molecular Genetics, University of the Punjab, Lahore and Citilab and Research Centre, Lahore, from January 2020 to December 2022. A total of 2919 blood samples were cultured to screen the bacteremia patients. Following standard protocols, four hundred twenty isolates proceeded for gram-staining, biochemical characterization, and antimicrobial susceptibility testing (AST). The AST results of each strain calculated multiple antibiotic resistance (MAR). Results: Of 420 bacterial isolates, Gram-negative and Gram-positive isolates accounted for 48.57% and 51.43%, respectively. The predominant pathogens were Staphylococcus epidermidis (48.10%) and Salmonella typhi (27.14%), with other significant pathogens including Klebsiella spp., Pseudomonas spp., Enterobacter, Acinetobacter spp., Escherichia coli, Staphylococcus aureus, Enterococcus spp., Citrobacter, Morganella morganii, and Proteus mirabilis. AST revealed high resistance to Cephalosporins, Nitrofurantoin, Fosfomycin, and Quinolones. In contrast, Carbapenems demonstrated notable sensitivity. Salmonella typhi and Staphylococcus epidermidis exhibit the highest MAR values. Conclusions: The study highlights the prevalence of multidrug resistance bacteremia-causing pathogens, with a concerning trend towards decreasing antibiotic efficacy.

Lateral ear canal resection and bulla osteotomy with marsupialization to treat otitis media in rabbits: forty-eight procedures.

Describe lateral ear canal resection and bulla osteotomy with marsupialization (LECARBOM) in rabbits with otitis media (OM), and report outcomes, complications, bacteria cultured from middle ears, and their antimicrobial susceptibility testing (AST) results. Retrospective clinical case series; single referral hospital. Forty-two rabbits with naturally occurring OM. Medical records (2011-2021) of rabbits with CT-confirmed OM undergoing surgery were reviewed for outcomes, complications, bacteria cultured from middle ears, and AST. Surgery was performed on 48 ears, and outcomes determined 21 days postoperatively. All rabbits survived the procedure. Otitis externa resolved in all lop-eared rabbits. Of 29 ears with OM-associated head tilt, eight (28%) resolved, nine (31%) improved, seven (24%) remained unchanged and five (17%) worsened. Outcome was not associated with head tilt duration nor OM severity. Postoperative complications occurred in 12 (25%) cases, seven of which had wound margin dehiscence that healed by 21 days. The most frequent bacteria isolated were Pasteurella multocida (16%), Bordetella bronchiseptica (14%) and Staphylococcus aureus (14%). Bacteria were sensitive to azithromycin, marbofloxacin or enrofloxacin and resistant to penicillin. LECARBOM is a well-tolerated surgical procedure to treat OM. It also improved or stabilized 83% of cases with head tilt and resolved all cases with otitis externa. LECARBOM with administration of an AST-sensitive antibiotic, or azithromycin when no AST is performed, should be effective in rabbits with OM. Postsurgically, it resolves or alleviates most secondary conditions such as otitis externa, head tilt and facial nerve paralysis.

Potential impact of real-time processing and rapid susceptibility testing of blood samples in Gram-negative bloodstream infections in intensive care patients

IntroductionTimely and appropriate therapy is critical in patients with Gram-negative bloodstream infections (GNBSI). Most bacteriology laboratories process blood specimen in the daytime, during laboratory operating hours, and use conventional culture for antimicrobial susceptibility testing (AST). We simulated the potential impact of real-time processing and rapid AST (7 hours) on early adaptation of the antibiotic regimen in intensive care unit (ICU) patients with GNBSI. MethodsAll GNBSI episodes occurring in the ICUs of 2 hospitals in Paris were included. Data were collected. For each episode of bacteremia, we simulated the impact of three strategies: (1) Real-time processing coupled with conventional techniques (Gram stain and standard AST); (2) Standard processing coupled with rapid AST; and (3) Real-time processing coupled with rapid AST. ResultsWe included 109 episodes in 98 patients. Forty-two patients (48%) died during ICU stay. AST results led to a change of the antibiotic regimen in 66 (61%) episodes, mainly de-escalation (54/109, 55%). In standard care, median time from sample collection to definitive AST result was 65.9 hours (±26.7). The three strategies would have reduced time-to-result by 9.2 hours (±7.1), 30.8 hours (±19.7) and 40.0 hours (±20.6) respectively. Compared to standard care, strategies 1, 2 and 3 would have avoided 20, 69 and 90 patient-days of broad-spectrum antibiotics respectively. ConclusionIn addition to real-time processing of blood samples, rapid AST would be the most effective strategy to shorten time-to-result in critical patients with GNBSI.

Bacterial ventriculoperitoneal shunt infections: changing trends in antimicrobial susceptibility, a 7-year retrospective study from Pakistan

BackgroundVentriculoperitoneal (VP) shunt infections in adults represent a severe complication and make treatment more challenging. Therefore, drug susceptibility patterns are crucial for therapeutic decisions and infection control in neurosurgical centers. This 7-year retrospective study aimed to identify the bacteria responsible for adult VP shunt infections and determine their drug susceptibility patterns.MethodsThis single-center study was performed from 2015 to 2021 in Lahore, Pakistan, and included CSF cultures from VP shunt infections. Demographic data, causative organisms, and antimicrobial susceptibility testing results were collected. Multivariate analysis of variance (MANOVA) and two-sample t-tests were used to analyze and compare the antibiotic sensitivity trends over the study period.Results14,473 isolates recovered from 13,937 CSF samples of VP shunt infections were identified and analyzed for their susceptibility patterns to antimicrobials. The proportion of Gram-negative and Gram-positive bacteria were 11,030 (76%) and 3443 (24)%, respectively. The predominant bacteria were Acinetobacter species (n = 5898, 41%), followed by Pseudomonas species (n = 2368, 16%) and coagulase-negative Staphylococcus (CoNS) (n = 1880, 13%). 100% of Staphylococcus aureus (S.aureus) and CoNS were sensitive to vancomycin and linezolid (n = 2580). However, 52% of S. aureus (719/1,343) were methicillin-resistant Staphylococcus aureus (MRSA). Acinetobacter showed maximum sensitivity to meropenem at 69% (2759/4768). Pseudomonas was 80% (1385/1863 sensitive to piperacillin-tazobactam, Escherichia coli (E. coli) showed 72% to amikacin (748/1055), while Klebsiella spp. was 57% (574/1170) sensitive to piperacillin-tazobactam. The sensitivity of piperacillin-tazobactam and meropenem for Gram-negative bacteria decreased significantly (p < 0.05) over 7 years, with 92.2% and 88.91% sensitive in 2015 and 66.7% and 62.8% sensitive in 2021, respectively.ConclusionThe significant decrease in the effectiveness of carbapenem and beta-lactam/beta-lactamase inhibitor combination drugs for the common Gram-negative causative agents of VP shunt infections suggests that alternative antibiotics such as colistin, fosfomycin, ceftazidime/avibactam, ceftolozane/tazobactam, and tigecycline should be considered and in consequence included in testing panels. Additionally, it is recommended to adopt care bundles for the prevention of VP shunt infection.

P27 Rethinking UTI diagnosis: how gold is the ‘gold standard’ and how much does antimicrobial susceptibility testing really matter?

Abstract Objectives To analyse the results provided by culture methods for UTI diagnosis and estimate its potential effectiveness for clinical decision making. Methods The results of all 80981 urine samples processed by the Public Health Wales laboratory in Cardiff in 2022 were collated and analysed. Prevalence and resistance data were collected and analysed using Datastore. Specific aims were as follows: (i) determine the prevalence of ‘no growth’ and ‘mixed growth’ samples, and of each individual uropathogen; (ii) determine resistance rates for each pathogen; and (iii) estimate the potential effectiveness of these culture results for making clinical decisions. Results Of the 80981 urine samples analysed, 22775 (28%) were negative following flow cytometry analysis and were not cultured. Of the 58206 urine samples cultured, 18656 (32%) showed no growth and 20516 (35%) showed mixed growth and were not analysed further. A total of 19034 (33%) showed growth of one or more specific pathogens. The most common uropathogen was Escherichia coli (58%), followed by Klebsiella pneumoniae (8.6%), Enterococcus spp. (7.3%) and Proteus mirabilis (4.2%). Resistance rates varied by pathogen. Resistance to nitrofurantoin in E. coli was low (2.4%) but resistance to trimethoprim amongst E. coli isolates was higher (30.5%). Klebsiella, Pseudomonas and Proteus spp. were not routinely tested for resistance to nitrofurantoin, presumably due to high levels of inherent resistance. Of the 22 P. mirabilis isolates tested, all were resistant to nitrofurantoin. Trimethoprim resistance was high for all species routinely tested. Only 24% of all samples analysed returned an antimicrobial susceptibility test (AST) result. AST results relevant to the first-choice antibiotics of nitrofurantoin or trimethoprim were obtained for only 16% and 19% of all urines tested, respectively. Overall resistance to nitrofurantoin was 2.8% and to trimethoprim 32%, representing 0.44% and 6% of total samples tested, respectively. Conclusions These results highlight issues with laboratory culturing. Over one-third of all samples cultured did not return a usable clinical result due to mixed growth and AST results were returned for less than one-quarter of all samples tested. The clinical utility of AST depends on the specific antibiotic and species being tested. With nitrofurantoin resistance currently so low in all species routinely tested, knowing whether there is an infection and which bacteria is causing it will have more impact than performing susceptibility testing for this antibiotic. However, for trimethoprim, resistance rates are higher and more variable, so AST has more potential clinical impact.

P14 Optimization of real-time sequencing for rapid detection of bacterial species and antimicrobial resistance in blood and urine infections

Abstract Objectives To optimize the process to obtain bacterial DNA directly from human urine and blood samples to produce high-quality genomic data using long-read Nanopore sequencer. Methods A total of 22 blood cultures and 24 urine samples positive for Escherichia coli or Klebsiella pneumoniae and corresponding isolates were collected from the microbiology diagnostics laboratory. DNA isolation optimization was performed testing three different sample volumes and seven DNA extraction kits. DNA concentration was quantified with Qubit fluorometer. Nanopore sequencing was performed on a MinION Mk1C device in R9.4.1 flow cells using the Rapid Barcoding Kit. Bacterial species and antimicrobial resistance (AMR) genes were determined with EPI2ME ARMA pipeline. Genomes were assembled with Flye, polished with Medaka and remaining human contigs removed with Krakentools. Bacterial isolates from the same samples were also sequenced using Illumina technology. Illumina and Nanopore assemblies were quality checked with Quast, Kraken was used to identify bacterial species, MLST for multilocus sequence typing and ABRicate with ResFinder database to detect AMR genes. Results A total of 72 DNA extractions from 24 urine and 22 blood samples with different kits were performed to find the optimal extraction protocol for each sample type. DNA from urine samples was isolated from 5 mL, while 1 mL was best for blood cultures. We found that the host depletion step prior to DNA extraction using MolYsis basic 5 Kit (VH-Bio) was essential. DNA from urine samples was extracted with GenElute Bacterial Genomic DNA Kit (Merck) and QIAamp Biostic bacteremia Kit (Qiagen) was best to allow successful bacterial genome sequencing for blood samples. Bacterial isolates from 11 urine and 1 blood sample (out of 54 sequenced) were successfully sequenced. In all 12 cases, the highest percentage of bacterial reads matched with the causative organism found during routine microbiology diagnostics (E. coli). A variety of clinically relevant AMR genes were detected, such as dfrA and bla CTX-M genes, which matched the phenotypic antimicrobial susceptibility testing results. Comparison of genomic assemblies obtained through Nanopore sequencing of 10 urine samples and Illumina sequencing of the corresponding clinical isolates showed a high degree of matching, validating Nanopore sequencing as an alternative option to support diagnosis and treatment choice. Conclusions We have successfully optimized the protocols to extract and sequence pathogenic bacterial DNA directly from blood and urine samples. Short-read sequencing confirmed results acquired by Nanopore sequencer, endorsing long-read sequencing in clinical settings to support timely diagnosis and treatment.

Characteristics of Clostridioides difficile isolates circulating in the Slovak hospitals

Clostridioides difficile is a frequent cause of hospital-acquired diarrhea associated with antibiotics. Hypervirulent strains of Clostridioides difficile are associated with a severe course of Clostridioides difficile infection and a higher mortality. We have confirmed 322 non-duplicate Clostridioides difficile isolates in stool samples of patients hospitalized in three Slovak hospitals providing primary health care between January and December 2021 and determined genes encoding toxins, ribotypes and susceptibility to antimicrobials. 93.7% of them were toxigenic and co-occurrence of genes tcdA, tcdB, cdtA, cdtB, which encode toxins, was detected in 71.4% isolates. Nosocomially associated ribotypes 176, 001, and 027 were confirmed in 61.1%, 10.2%, and 5.2% of isolates, respectively, and found to be the most frequent. Although ribotype 176 predominated in all three hospitals, the highest incidence was recorded in Košice (73.5%). Results of antimicrobial susceptibility testing revealed 65.8% resistance rate for rifampicin, 5.2% for vancomycin, 1.5% for metronidazole, 1.2% for teicoplanin and 0.6% for doxycycline. The correlation of frequent ribotypes and results of antimicrobial susceptibility testing points to the predominance of rifampicin resistance in isolates belonging to ribotypes 176 and 027. The above results reflect the problematic situation in our hospitals during the COVID-19 pandemic, that was related to the misuse of broad-spectrum antibiotics and impossibility to follow epidemiological measures to prevent the spread of hypervilulent isolates.

BlaPSZ-1, a novel AmpC gene identified from a Pantoea isolate.

Pantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance. A rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The β-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance gene-related sequences were analyzed by bioinformatic methods. The isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for third-generation cephalosporins or aztreonam. In this study, a novel AmpC β-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance.

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.

Molecular Analysis of Fosfomycin Resistance Among Escherichia coli Isolates from Urinary Tract Infections in Kidney Transplant Patients During 2019 - 2020

Background: This study aimed to estimate the prevalence of fosfomycin resistance and the frequency of extended-spectrum beta-lactamase (ESBL) production in Escherichia coli isolates from three kidney transplant patients (KTPs) in Tehran. Methods: Sixty clinical isolates of uropathogenic E. coli were collected from three kidney transplant centers in Tehran between April and May 2019. Antimicrobial susceptibility testing (AST), minimum inhibitory concentration (MIC) of fosfomycin, and screening for ESBL production were conducted following the protocols established by the Clinical and Laboratory Standards Institute (CLSI). The presence of the blaTEM, blaSHV, blaCTX-M, fosA3, and fosC2 genes was evaluated using polymerase chain reaction (PCR) and sequencing. Additionally, mutations in the murA, glpT, uhpT, and cya genes were assessed. The activity of the carbohydrate phosphate transporter was measured using the real-time PCR assay. Results: According to the AST results, ampicillin showed the highest resistance rate (86%), while ertapenem and doripenem exhibited complete susceptibility (100%). According to the E-test, 1.6% of E. coli isolates were resistant to fosfomycin. Furthermore, 33.4% of E. coli isolates in KTPs were ESBL producers, with the most frequent occurrence of the blaTEM gene (55%). Additionally, mutations were identified in the murA, uhpT, and glpT genes of resistant samples. No plasmid genes for fosA3 and fosC2 were detected. The expression of the uhpT gene increased 32-fold in a susceptible isolate, as determined by qPCR. Conclusions: The high resistance of E. coli isolates from urinary tract infections (UTIs) of KTPs to β-lactam antibiotics remains a significant clinical challenge. However, no correlation was found between ESBL production and resistance to fosfomycin. The resistance rate to fosfomycin was low, and the primary cause of resistance was mutations in chromosomal genes.

Ten simple rules for the sharing of bacterial genotype-Phenotype data on antimicrobial resistance.

The increasing availability of high-throughput sequencing (frequently termed next-generation sequencing (NGS)) data has created opportunities to gain deeper insights into the mechanisms of a number of diseases and is already impacting many areas of medicine and public health. The area of infectious diseases stands somewhat apart from other human diseases insofar as the relevant genomic data comes from the microbes rather than their human hosts. A particular concern about the threat of antimicrobial resistance (AMR) has driven the collection and reporting of large-scale datasets containing information from microbial genomes together with antimicrobial susceptibility test (AST) results. Unfortunately, the lack of clear standards or guiding principles for the reporting of such data is hampering the field's advancement. We therefore present our recommendations for the publication and sharing of genotype and phenotype data on AMR, in the form of 10 simple rules. The adoption of these recommendations will enhance AMR data interoperability and help enable its large-scale analyses using computational biology tools, including mathematical modelling and machine learning. We hope that these rules can shed light on often overlooked but nonetheless very necessary aspects of AMR data sharing and enhance the field's ability to address the problems of understanding AMR mechanisms, tracking their emergence and spread in populations, and predicting microbial susceptibility to antimicrobials for diagnostic purposes.

P07 Use of the Reveal rapid antimicrobial susceptibility testing system to shorten time to appropriate antibiotic use: assessment of clinical utility in Sheffield Teaching Hospitals NHS Trust

Abstract Objectives Gram-negative bacteraemia accounts for significant morbidity and mortality,1 while increasing the prevalence of resistant organisms over the recent years2 highlights the importance of obtaining rapid antimicrobial susceptibility testing (AST) results. Antimicrobial stewardship (AMS) is part of the WHO global strategy on antimicrobial resistance,3 and shortening the length of time a patient is treated with unnecessarily broad-spectrum agents is paramount. Our aim was to review whether the Reveal rapid AST system (bioMérieux) shortened time-to-best-fit antibiotic, and whether this was a spectrum escalation or de-escalation, in Gram-negative bacteraemias in Sheffield Teaching Hospitals NHS Trust. Methods The Reveal rapid AST system was used, in conjunction with standard laboratory methodology, to ascertain antimicrobial susceptibilities for Gram-negative bacilli in blood cultures processed at the start of the day between June and August 2022. Data was gathered regarding Medical Microbiology advice issued to clinical teams and the impact the Reveal AST results had on this. For comparison, we retrospectively reviewed microbiology advice given for Gram-negative bacteraemias between April and May 2022, with our routine microbiology testing methods of MALDI-TOF MS and breakpoint agar dilutions AST, to see whether Reveal would have been predicted to have an impact had it been available. Results 66 bacteraemias were reviewed between June and August 2022, compared with 82 in April and May 2022. Reveal results shortened time-to-best-fit antibiotic in 35/66 (53%) cases. Of these, 8/66 (12%) resulted in antibiotic spectrum escalation and 21/66 (32%) in de-escalation, with 6/66 (9%) resulting in shorter time to an alternative susceptible antibiotic of similar spectrum. In the retrospective cohort, earlier susceptibility results would have been predicted to shorten time-to-best-fit antibiotic in 53/82 (65%) cases [escalation 16/82 (20%), de-escalation 37/82 (45%)], consistent with the study period findings. Extrapolating this to cultures received throughout 2022 in our trust (n = 1722), with a mean AST turnaround time of 491 min compared with 1553 min for standard AST testing, the Reveal system could save 4877 hours in time to escalation and 11 887 hours in time to de-escalation to appropriate antibiotic. Conclusions In our experience, Reveal resulted in a shortening of time-to-best-fit antibiotic in the majority of patients with Gram-negative bacteraemia. The impact on escalation may impact on mortality and morbidity, while de-escalation could have significant benefit in terms of AMS, with possible implications for shortened time to oral switch and shortened length of stay, with associated financial, operational and quality of care benefits.

Metagenomic next-generation sequencing for the identification of infections caused by Gram-negative pathogens and the prediction of antimicrobial resistance.

The aim of this study was to evaluate the efficacy of metagenomic next-generation sequencing (mNGS) for the identification of Gram-negative bacteria (GNB) infections and the prediction of antimicrobial resistance. A retrospective analysis was conducted on 182 patients with diagnosis of GNB infections who underwent mNGS and conventional microbiological tests (CMTs). The detection rate of mNGS was 96.15%, higher than CMTs (45.05%) with a significant difference (χ 2=114.46, P < .01). The pathogen spectrum identified by mNGS was significantly wider than CMTs. Interestingly, the detection rate of mNGS was substantially higher than that of CMTs (70.33% vs 23.08%, P<.01) in patients with but not without antibiotic exposure. There was a significant positive correlation between mapped reads and pro-inflammatory cytokines (interleukin-6 and interleukin-8). However, mNGS failed to predict antimicrobial resistance in 5 of 12 patients compared to phenotype antimicrobial susceptibility testing results. Metagenomic next-generation sequencing has a higher detection rate, a wider pathogen spectrum, and is less affected by prior antibiotic exposure than CMTs in identifying Gram-negative pathogens. The mapped reads may reflect a pro-inflammatory state in GNB-infected patients. Inferring actual resistance phenotypes from metagenomic data remains a great challenge.

Evaluation of the Liquid Colony™ Produced by the FAST System for Shortening the Time of Bacterial Identification and Phenotypic Antimicrobial Susceptibility Testing and Detection of Resistance Mechanisms from Positive Blood Cultures.

the aim of this study was to evaluate the performance of the Liquid Colony™ (LC) generated directly from positive blood cultures (PBCs) by the FAST System (Qvella, Richmond Hill, ON, Canada) for rapid identification (ID) and antimicrobial susceptibility testing (AST) compared with the standard of care (SOC) workflow. Anonymized PBCs were processed in parallel by the FAST System and FAST PBC Prep cartridge (35 min runtime) and SOC. ID was performed by MALDI-ToF mass spectrometry (Bruker, Billerica, MA, USA). AST was performed by reference broth microdilution (Merlin Diagnostika, Bornheim, Germany). Carbapenemase detection was carried out with the lateral flow immunochromatographic assay (LFIA) RESIST-5 O.O.K.N.V. (Coris, Gembloux, Belgium). Polymicrobial PBCs and samples containing yeast were excluded. 241 PBCs were evaluated. ID results showed 100% genus-level concordance and 97.8% species-level concordance between LC and SOC. The AST results for Gram-negative bacteria showed a categorical agreement (CA) of 99.1% (1578/1593), with minor error (mE), major error (ME), and very major error (VME) rates of 0.6% (10/1593), 0.3% (3/1122), and 0.4% (2/471), respectively. The results from Gram-positive bacteria showed a CA of 99.6% (1655/1662), with mE, ME, and VME rates of 0.3% (5/1662), 0.2% (2/1279), and 0.0% (0/378), respectively. Bias evaluation revealed acceptable results for both Gram-negatives and Gram-positives (-12.4% and -6.5%, respectively). The LC yielded the detection of 14/18 carbapenemase producers by LFIA. In terms of turnaround time, the ID, AST, and carbapenemase detection results were generally obtained one day earlier with the FAST System compared with the SOC workflow. The ID, AST, and carbapenemase detection results generated with the FAST System LC were highly concordant with the conventional workflow. The LC allowed species ID and carbapenemase detection within around 1 h after blood culture positivity and AST results within approximately 24 h, which is a significant reduction in the turnaround time of the PBC workflow.

Epidemiological characteristics of New Delhi Metallo-β-Lactamase-producing Enterobacteriaceae in the Fourth hospital of Hebei Medical University

The epidemiological characteristics of New Delhi Metallo-β-Lactamase-Producing (NDM) Enterobacteriaceae were analyzed to provide theoretical support for clarifying the distribution characteristics of carbapenem-resistant Enterobacteriaceae (CRE) in the hospital environment and early identification of susceptible patients. From January 2017 to December 2021,42 strains of NDM-producing Enterobacteriaceae were gathered from the Fourth Hospital of Hebei Medical University, primarily Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. The micro broth dilution method combined with the Kirby-Bauer method was used to determine the minimal inhibitory concentrations (MICs) of antibiotics. The carbapenem phenotype was detected by the modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM). Carbapenem genotypes were detected by colloidal gold immunochromatography and real-time fluorescence PCR. The results of antimicrobial susceptibility testing showed that all NDM-producing Enterobacteriaceae were multiple antibiotic resistant, but the sensitivity rate to amikacin was high. Invasive surgery prior to culture, the use of excessive amounts of different antibiotics, the use of glucocorticoids, and ICU hospitalization were clinical characteristics of NDM-producing Enterobacteriaceae infection. Molecular typing of NDM-producing Escherichia coli and Klebsiella pneumoniae was carried out by Multilocus Sequence Typing (MLST), and the phylogenetic trees were constructed. Eight sequence types (STs) and two NDM variants were detected in 11 strains of Klebsiella pneumoniae, primarily ST17, and NDM-1. A total of 8 STs and 4 NDM variants were detected in 16 strains of Escherichia coli, mainly ST410, ST167, and NDM-5. For high-risk patients who have CRE infection, CRE screening should be done as soon as feasible to adopt prompt and efficient intervention measures to prevent outbreaks in the hospital.

Phenotypic and genetic extended spectrum beta lactamase profiles of bacterial isolates from ICU in tertiary level hospital in Kenya

Background: Bacterial infections in the ICUs are a threat to the lives of critically ill patients. Moreover, their vulnerable immunity predisposes them to developing bacteria-associated sepsis, further deteriorating their already fragile health. In the face of increasing drug resistance menace, the problem of bacterial infection in ICU is likely to worsen. This study sought to assess bacterial infections in ICU setting by identifying prevalent gram-negative bacterial species and characterizing their antibiotic susceptibility patterns. Phenotypic and genetic resistance were determined among gram-negative isolates from ICU patients. Methods: Cross-sectional samples collected from Kenyatta National Hospital ICU between January and June 2021 were cultured for subsequent analysis. Phenotypic identification of culture-positive samples was performed using VITEK 2 while Antibiotic susceptibility patterns were determined based on Antimicrobial Susceptibility Testing (AST) results. Cephalosporin-resistant gram-negative bacteria were assessed by PCR to detect the presence of ESBL genes. Results and discussion: Out of the 168 gram-negative isolates, Acinetobacter baumanii was the most abundant (35%). Other isolates that were present at frequencies more than 15% are Klebsiella pneumoniae and Escherichia. coli. A. baumaniii is known to be a notorious bacterium in ICU due to its multidrug resistance nature. Indeed, A. baumanii isolates from Kenyatta National Hospital showed significantly high level of phenotypic resistance. Concordant with the high level of phenotypic resistance, we found high carriage of the ESBL genes among the isolates analysed in this study. Moreover, majority of isolates harboured all the four ESBL genes. Conclusion: A high rate of phenotypic and genetic resistance was detected among the tested isolates. Resistance to cephalosporins was primarily driven by acquisition of the ESBL genes. The high-rate multidrug resistance in ICU bacterial isolates in this study has a crucial implication for ICU patient management and general antibiotics use.

Rapid antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales: an implementation study.

The goal of this descriptive study was to assess the performance as well as the extent of the clinical impact of rapid automated antimicrobial susceptibility testing in patients with bacteraemia due to Enterobacterales. We also aimed to analyse how rapid automated antimicrobial susceptibility testing influences clinical decision-making. This single-centre study conducted at the University Hospital of Zurich included data from all consecutive patients with Enterobacterales bacteraemia from November 2019 to October 2020. There was no control group. The primary outcome was the effect of rapid automated antimicrobial susceptibility testing on antibiotic therapy (no adjustment, escalation to a broader-spectrum antibiotic or de-escalation to a narrower-spectrum antibiotic). Rapid automated antimicrobial susceptibility testing results were further compared to susceptibility tests using European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard methods and erroneous results were noted. Additionally, we investigated turnaround times for rapid automated antimicrobial susceptibility testing and routine diagnostic testing. We analysed 106 patients with 116 episodes of bacteraemia due to Enterobacterales, withEscherichia coliandKlebsiella pneumoniaebeing the most frequent isolates. Almost 8% of pathogens were multidrug resistant. Rapid automated antimicrobial susceptibility testing showed category agreement in 98.4% of all interpretable cases. A significant reduction of more than 20 h in turnaround times could be achieved with rapid automated antimicrobial susceptibility testing compared to the routine diagnostic workflow. In the majority of cases, rapid automated antimicrobial susceptibility testing had no effect, given that the empirical therapy was already correct or circumstances did not allow for de-escalation. In 38.8% of cases, antimicrobial therapy was adjusted, whereas eight cases were de-escalated based on rapid automated antimicrobial susceptibility testing alone. Rapid automated antimicrobial susceptibility testing may be a valuable and safe way to accelerate diagnosis. In particular, time to suitable therapy can be shortened in cases of incorrect therapy. However, physicians are reluctant to de-escalate antibiotic therapy based on rapid automated antimicrobial susceptibility testing alone, limiting its impact in everyday clinics. To further explore the potential of rapid automated antimicrobial susceptibility testing, a stringent/compulsory antibiotic stewardship programme would be a valuable next step.

DIRECT ANTIMICROBIAL SUSCEPTIBILITY TESTING OF GRAM NEGATIVE BACILLI FROM POSITIVE BLOOD CULTURES

Conventional blood culture and susceptibility pattern takes around 48 hours after blood culture bottles turns positive on Bact/ALERT system. Rapid initiation of antimicrobial therapy is required in management of sepsis patients to reduce its adverse effects on mortality and morbidity. Objective of this study was to execute rapid and accurate direct antimicrobial susceptibility testing (DST) of gram negative bacilli on positive blood culture bottles by the disk diffusion method and compare the results with those of standard antimicrobial susceptibility testing (AST). This prospective study was performed at the Saveetha Institute of Medical and Technical Science, Sriperumbudur, with mono-bacterial gram negative bacilli. 100-gram negative bacilli (GNB) samples were agged from positive blood cultures with duration of 3 months. Both DST and AST results were interpreted, and the isolates were identied by vitek2. On 100 GNB isolates, the most common organism was Escherichia coli, followed by Klebsiella pneumoniae (27%), Pseudomonas aeruginosa (11%), Acinetobacter baumanii (8%), Salmonella typhi (7%), Enterobacter cloacae (3%), Acinetobacter lwof(3%) and Serratia marsacens. The DST and AST correlations were found to be in 99% categorical agreement, with 1% minor error, 0% major error, and 0% very major error. The concentration of bacterial inoculums impacts the results between DST and AST. DST reduces the crucial time from 24 to 48 hours for patients with bloodstream infections treated with empirical antibiotic therapy, which may or may not be resistant. DST initiates early antibiotic therapy that is susceptible to treating a bloodstream infection or sepsis. Aims and Objective: To evaluate categorical agreement of antimicrobials between DST and AST from positive blood culture bottles by Kirby-Bauer disk diffusion method. Materials and methods: A prospective study was conducted in tertiary care hospital, with sample size of 100 gram negative bacilli from positive blood culture bottles in patients suspected with blood stream infections during the period from October 2022 to December 2022. RESULTS: Out of 100 gram negative bacilli , Escherichia coli (37%) was the most common isolate, followed by Klebsiella pneumonia (27%), Pseudomonass aeroginosa (11%), Acinetobacter baumanii (8%), Salmonella typhi (7%), Enterobacter clocae (3%), Acinetobacter lw of(3%) and Serratia marsacens (2%). Conclusion: During this study, there was 99% categorical agreement between DST and AST. Earlier treatment with antimicrobials can reduce the mortality and morbidity in bacteraemia patients

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.