Novel Organism Verification and Analysis (NOVA) study: identification of 35 clinical isolates representing potentially novel bacterial taxa using a pipeline based on whole genome sequencing

BACKGROUND Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA - Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS). RESULTS We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently. CONCLUSION Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods

Probability of pharmacological target attainment with flucloxacillin in Staphylococcus aureus bloodstream infection: a prospective cohort study of unbound plasma and individual MICs.

OBJECTIVES MSSA bloodstream infections (BSIs) are associated with considerable mortality. Data regarding therapeutic drug monitoring (TDM) and pharmacological target attainment of the β-lactam flucloxacillin are scarce. PATIENTS AND METHODS We determined the achievement of pharmacokinetic/pharmacodynamic targets and its association with clinical outcome and potential toxicity in a prospective cohort of 50 patients with MSSA-BSI. Strain-specific MICs and unbound plasma flucloxacillin concentrations (at five different timepoints) were determined by broth microdilution and HPLC-MS, respectively. RESULTS In our study population, 48% were critically ill and the 30 day mortality rate was 16%. The median flucloxacillin MIC was 0.125 mg/L. The median unbound trough concentration was 1.7 (IQR 0.4-9.3), 1.9 (IQR 0.4-6.2) and 1.0 (IQR 0.6-3.4) mg/L on study day 1, 3 and 7, respectively. Optimal (100% fT>MIC) and maximum (100% fT>4×MIC) target attainment was achieved in 45 (90%) and 34 (68%) patients, respectively, throughout the study period. Conversely, when using the EUCAST epidemiological cut-off value instead of strain-specific MICs, target attainment was achieved in only 13 (26%) patients. The mean unbound flucloxacillin trough concentration per patient was associated with neurotoxicity (OR 1.12 per 1 mg/L increase, P = 0.02) and significantly higher in deceased patients (median 14.8 versus 1.7 mg/L, P = 0.01). CONCLUSIONS Flucloxacillin pharmacological target attainment in MSSA-BSI patients is frequently achieved when unbound flucloxacillin concentrations and strain-specific MICs are considered. However, currently recommended dosing regimens may expose patients to excessive flucloxacillin concentrations, potentially resulting in drug-related organ damage

Unexpectedly High False-Positive Rates for Haemophilus influenzae Using a Meningoencephalitis Syndromic PCR Panel in Two Tertiary Centers

False-positive results in the diagnostic of meningitis and encephalitis pose important challenges. This study aimed to determine false-positive rates for Haemophilus influenzae in cerebrospinal fluids evaluated by the BioFire FilmArray® Meningitis/Encephalitis Panel. We conducted a retrospective study of all H. influenzae-positive FilmArray®. Meningitis/Encephalitis Panel results from June 2016 to October 2019 in two Swiss university hospitals. Cases were classified as true positive, likely true-positive, and likely false-positive results according to cerebrospinal fluid culture, H. influenzae-specific quantitative real-time PCR (qPCR), and Gram staining, as well as culture of other materials. We performed 3,082 panels corresponding to 2,895 patients: 0.6% of the samples (18/3,082) were positive for H. influenzae. Culture and H. influenzae-specific qPCR were performed on 17/18 (94.4%) and 3/18 (16.7%) cerebrospinal fluid samples, respectively; qPCR was negative in all cases. Among 17 samples sent for culture, 10 concerned patients were not treated with antibiotics prior to lumbar puncture. Only 1/17 revealed growth of H. influenzae and was classified as a true positive. We further classified 3/18 (16.7%) cases with the identification of Gram-negative rods in the cerebrospinal fluid or positive blood cultures for H. influenzae as likely true-positive and 14/18 (77.8%) cases as likely false-positive. Diagnostic results should always be interpreted together with the clinical presentation, cerebrospinal fluid analysis, and other available microbiological results. All H. influenzae-positive results should be viewed with special caution and a H. influenzae-specific qPCR should be systematically considered

High prevalence of ESBL-Producing E. coli in private and shared latrines in an informal urban settlement in Dar es Salaam, Tanzania

Abstract Background Data about the burden of extended-spectrum beta-lactamase (ESBL)-producing microorganisms in Africa are limited. Our study aimed to estimate the prevalence of human faecal ESBL carriage in the community of an informal urban settlement in Dar es Salaam (Tanzania, East Africa) by using environmental contamination of household latrines with ESBL as a surrogate marker. Methods Within the context of a large survey in February 2014 assessing 636 randomly selected household latrines for faecal contamination by the detection of growth of E. coli and total faecal coliform bacteria, a randomly selected subset of the samples were screened for ESBL. Results Seventy latrines were screened for ESBL. An average of 11.4 persons (SD ±6.5) were sharing one latrine. Only three (4.3%) latrines had hand-washing facilities and 50 showed faeces on the floor. ESBL-producing Enterobacteriaceae were confirmed in 17 (24.3%) of the 70 latrine samples: 16 E. coli and 1 Klebsiella pneumoniae. Five ESBL E. coli strains were detected on door handles. The most prevalent ESBL type was CTX-M-1 group (76.5%). Pulsed-field gel electrophoresis typing of a subset of ESBL-producing E. coli isolates revealed both diverse singular types and a cluster of 3 identical isolates. There was no significant difference of the latrine and household characteristics between the group with ESBL (n = 17) and the group with non-ESBL E. coli (n = 53) (p > 0.05). Conclusions Almost a quarter of private and shared latrines in an informal urban settlement in Tanzania are contaminated with ESBL-producing microorganisms, suggesting a high prevalence of human ESBL faecal carriage in the community. Shared latrines may serve as a reservoir for transmission in urban community settings in Tanzania

Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms

Background Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. Results Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. Conclusions The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance

Cefiderocol for Extensively Drug-Resistant Gram-Negative Bacterial Infections: Real-world Experience From a Case Series and Review of the Literature

Cefiderocol is a new siderophore cephalosporin with activity against carbapenem-resistant gram-negative bacteria. Data on its clinical efficacy are limited to complicated urinary tract infections. We present a series of 3 patients successfully treated with cefiderocol for complicated health care-associated infections and review published case reports

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile