Introduction and benchmarking of pyMLST: open-source software for assessing bacterial clonality using core genome MLST.

Core genome multilocus sequence typing (cgMLST) has gained in popularity for bacterial typing since whole-genome sequencing (WGS) has become affordable. We introduce here pyMLST, a new complete, stand-alone, free and open source pipeline for cgMLST analysis. pyMLST can create or import a core genome database. For each gene, the first allele is aligned against the bacterial genome of interest using BLAT. Incomplete genes are aligned using MAFT. All data are stored in a SQLite database. pyMLST accepts assembly genomes or raw data (with the option pyMLST-KMA) as input. To evaluate our new tool, we selected three genome collections of major bacterial pathogens (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and compared them with pyMLST, pyMLST-KMA, ChewBBACA, SeqSphere and the variant calling approach. We compared the sensitivity, precision and false-positive rate for each method with those of the variant calling approach. Minimal spanning trees were generated with each type of software to evaluate their interest in the context of a bacterial outbreak. We found that pyMLST-KMA is a convenient screening method to avoid assembling large bacterial collections. Our data showed that pyMLST (free, open source, available in Galaxy and pipeline ready) performed similarly to the commercial SeqSphere and performed better than ChewBBACA and pyMLST-KMA.

Population structure of community-acquired extended-spectrum beta-lactamase producing Escherichia coli and methicillin resistant Staphylococcus aureus in a French region showed no difference between urban and rural areas.

Antimicrobial resistance is a global health issue and extended-spectrum ß-lactamase producing Escherichia coli (ESBL-Ec) and methicillin-resistant Staphylococcus aureus (MRSA) are of particular concern. Whole genome sequencing analysis of isolates from the community is essential to understand the circulation of those multidrug-resistant bacteria. Our main objective was to determine the population structure of clinical ESBL-Ec and MRSA isolated in the community setting of a French region. For this purpose, isolates were collected from 23 sites belonging to 6 private medical biology laboratories in the Bourgogne-Franche-Comté region. One hundred ninety ESBL-Ec and 67 MRSA were sequenced using the Illumina technology. Genomic analyses were performed to determine the bacterial typing, presence of antibiotic resistance genes, metal resistance genes as well as virulence genes. Analysis showed that ST131 was the major ESBL-Ec clone circulating in the region, representing 42.1% of the ESBL-Ec isolates. The blaCTX-M genes represented 98% of blaESBL with the majority being blaCTX-M-15 (53.9%). MRSA population consisted of mainly of CC8 (50.7%) and CC5 (38.8%) clonal complexes. Interestingly, we found a prevalence of 40% of the zinc resistance gene czrC in our MRSA population. We observed no differences in our ESBL-Ec or MRSA populations between urban and rural areas in our French region, suggesting no impact of population density or rural environment.

Effect of a karst system (France) on extended spectrum beta-lactamase (ESBL)-producing Escherichia coli.

Karst aquifers are an important water resource worldwide particularly exposed to anthropogenic pollution, including antibiotic-resistance. The release of antibiotic-resistant bacterial pathogens in the environment is a major public health challenge worldwide. In this One Health study, we aimed to determine the effect of karst on antibiotic-resistant bacteria. For this purpose, we determined the concentrations of extended-spectrum ß-lactamases-producing Escherichia coli (ESBL-Ec) for 92 weeks in a rural karst hydrosystem providing drinking water. ESBL-Ec isolates (n = 130) were sequenced by whole genome sequencing. We analysed the isolates at different levels of granularity, i.e., phylogroup, sequence type, presence of antibiotic-resistance genes, mutations conferring antibiotic-resistance, and virulence genes. The ESBL-Ec concentrations were spatially and temporally heterogeneous in the studied karst hydrosystem. ESBL-Ec isolates survived in the karst and their concentrations were mostly explained by the hydrodynamic of the hydrosystem. We demonstrate that the studied karst has no filtration effect on ESBL-Ec, either quantitatively (i.e., in the ESBL-Ec concentrations) or qualitatively (i.e., in the genetic characteristics of ESBL-Ec isolates).

Performance of discs, sensititre EUMDROXF microplates and MTS gradient strips for the determination of the susceptibility of multidrug-resistant Pseudomonas aeruginosa to cefiderocol.

OBJECTIVES: To evaluate the performance of commercially available tests to determine the susceptibility of multidrug-resistant (MDR) clinical Pseudomonas aeruginosa strains to cefiderocol. METHODS: A collection of 150 clinical strains of P. aeruginosa resistant to ceftazidime, (MIC, Minimal Inhibitory Concentration, MIC > 8 mg/L) imipenem (MIC> 4 mg/L) and ceftolozane/tazobactam (MIC> 4/4 mg/L), isolated from 2015 to 2022 was selected. Cefiderocol susceptibility was determined in parallel (a) by disc diffusion using Mast, Oxoid and Liofilchem discs deposited on Mueller-Hinton agar batches from Bio-Rad, BioMérieux, Mast, Becton Dickinson, I2A and Oxoid; (b) by MIC gradient test strips (MTS) (Liofilchem); and (c) by EUMDROXF Sensititre microplates. MICs and inhibition zones were compared with the broth microdilution reference method (BMD) MICs. RESULTS: The MIC50 and MIC90 of cefiderocol were 1 mg/L and 8 mg/L by BMD, respectively, including 21.3% (32/150) resistant strains. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ± 30%). Although the Sensititre EUMDROXF microplates overestimated MIC values (categorical agreement [CA] = 86.7% [130/150, 95% CI 80.3-91.2]; EA = 69.3% [104/150, 95% CI 61.6-76.2]; bias = 68.2%), MTS strips underestimated the MIC values for many strains (CA = 86.7%, 130/150, 95% CI 80.3-91.2; EA = 69.3%, 104/150, 95% CI 61.6-76.2; bias = -30.4%), classifying properly only 50% (16/32) of resistant strains. Finally, many cefiderocol-resistant strains were not identified by the disc method, although the CA ranged from 78.0% (117/150, 95% CI 70.7-83.0) to 89.3% (134/150, 95% IC 83.4-93.3) according to Mueller-Hinton agar batches. CONCLUSION: Determination of cefiderocol susceptibility in MDR P. aeruginosa clinical strains by Sensititre EUMDROXF microplates is an alternative to the reference BMD method. However, MIC values ± 1 dilution apart from the breakpoint (2 mg/L) should be controlled by BMD whereas the use of MTS gradient strips is discouraged. Disc diffusion might be useful for screening, unfortunately many cefiderocol-resistant strains are not detected.

SARS-CoV-2 respiratory viral loads and association with clinical and biological features.

To determine the distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) respiratory viral loads (VL) during the acute phase of infection and their correlation with clinical presentation and inflammation-related biomarkers. Nasopharyngeal swabs from 453 adult SARS-CoV-2-infected patients from the Department of Infectious Diseases, Besançon, France, were collected at the time of admission or consultation for reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Clinical information and concentrations of biological parameters (C-reactive protein [CRP], fibrinogen, lactate dehydrogenase [LDH], prealbumin) were noticed. Mean respiratory VL homogeneously decreased from 7.2 log10 copies/ml (95% confidence interval [CI]: 6.6-7.8) on the first day of symptoms until 4.6 log10 copies/ml (95% CI: 3.8-5.4) at day 10 (slope = -0.24; R2 = .95). VL were poorly correlated with COVID-19 symptoms and outcome, excepted for dyspnea and anosmia, which were significantly associated with lower VL (p < .05). CRP, fibrinogen, and LDH concentrations significantly increased over the first 10 days (median CRP concentrations from 36.8 mg/L at days 0-1 to 99.5 mg/L at days 8-10; p < .01), whereas prealbumin concentrations tended to decrease. Since SARS-CoV-2 respiratory VL regularly decrease in the acute phase of infection, determining the level of VL may help predicting the onset of virus shedding in a specific patient. However, the role of SARS-CoV-2 VL as a biomarker of severity is limited.