Linezolid resistance: detection of the cfr(B) gene in French clinical MRSA strains.

OBJECTIVES: To describe two linezolid-resistant MRSA strains carrying the cfr(B) gene detected in the French National Reference Centre for staphylococci. METHODS: Two linezolid-resistant MRSA strains isolated from cystic fibrosis patients in two different French hospitals in 2017 and 2019 were examined to explore the mechanisms of linezolid resistance. Antimicrobial susceptibility was tested using broth microdilution and gradient strips. The genetic determinants of linezolid resistance were assessed by a multiplex PCR targeting cfr/cfr(B), optrA and poxtA genes, by amplification and sequencing of individual 23S rRNA genes and by WGS using both Illumina and Nanopore technologies. RESULTS: The two MRSA strains were resistant to linezolid but susceptible to tedizolid, and PCR-positive for cfr/cfr(B). The WGS analysis indicated that they belonged to two different STs (ST8-MRSA-IV and ST5382-MRSA-IV) and that they both harboured the cfr(B) gene on the same 9.7 kb Tn6218-like chromosomal transposon, a finding only previously reported in Enterococcus sp. and Clostridioides difficile. CONCLUSIONS: To the best of our knowledge, this is the first description of the presence of cfr(B) in staphylococci, more specifically in linezolid-resistant MRSA strains. This finding illustrates the risk of horizontal intergenus transfer of oxazolidinone resistance genes in Staphylococcus aureus and highlights the need to monitor such emergence in this species.

In vitro study to evaluate the antimicrobial activity of various multifunctional cosmetic ingredients and chlorphenesin on bacterial species at risk in the cosmetic industry.

AIMS: We evaluated the activity of the preservative chlorphenesin and of four antimicrobial cosmetic multifunctional ingredients against various strains of gram-negative and gram-positive human opportunistic pathogens. METHODS AND RESULTS: Growth kinetics, modelling growth parameters and statistical analyses enabled comparing bacterial behaviour in the presence and in the absence of the compound. Whatever compound tested (i.e. chlorphenesin, phenylpropanol, hexanediol, ethylhexylglycerin, hydroxyacetophenone) and strain origin (i.e. clinical versus industrial), the growth of 42 strains belonging to Acinetobacter spp., Burkholderia cepacia complex and Stenotrophomonas maltophilia, was totally inhibited. On the opposite all of the P. aeruginosa strains (n = 13) as well as 4 and 6 out of 10 strains of Pluralibacter gergoviae grew in the presence of chlorphenesin and ethylhexylglycerin, respectively. Some P. gergoviae and Staphylococcus hominis strains withstand hydroxyacetophenone. Within a species, the different strains show variable latency phase, growth rate (r) and carrying capacity (K). They can be similar, lower or higher than those measured in control conditions. CONCLUSIONS: Data showed differences in the antimicrobial activity of compounds. Upon exposure, strains differed in their behaviour between and within species. Whatever species and strains, compound sensitivity could not be related to antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Most multifunctional ingredients showed significant antimicrobial properties against the wide panel of species and strains evaluated. This will help adjusting preservation strategies in the cosmetic industry.

Runoff microbiome quality assessment of a city center rainwater harvesting zone shows a differentiation of pathogen loads according to human mobility patterns.

The hygienic quality of urban surfaces can be impaired by multiple sources of microbiological contaminants. These surfaces can trigger the development of multiple bacterial taxa and favor their spread during rain events through the circulation of runoff waters. These runoff waters are commonly directed toward sewer networks, stormwater infiltration systems or detention tanks prior a release into natural water ways. With water scarcity becoming a major worldwide issue, these runoffs are representing an alternative supply for some usage like street cleaning and plant watering. Microbiological hazards associated with these urban runoffs, and surveillance guidelines must be defined to favor these uses. Runoff microbiological quality from a recently implemented city center rainwater harvesting zone was evaluated through classical fecal indicator bacteria (FIB) assays, quantitative PCR and DNA meta-barcoding analyses. The incidence of socio-urbanistic patterns on the organization of these urban microbiomes were investigated. FIB and DNA from Human-specific Bacteroidales and pathogens such as Staphylococcus aureus were detected from most runoffs and showed broad distribution patterns. 16S rRNA DNA meta-barcoding profilings further identified core recurrent taxa of health concerns like Acinetobacter, Mycobacterium, Aeromonas and Pseudomonas, and divided these communities according to two main groups of socio-urbanistic patterns. One of these was highly impacted by heavy traffic, and showed recurrent correlation networks involving bacterial hydrocarbon degraders harboring significant virulence properties. The tpm-based meta-barcoding approach identified some of these taxa at the species level for more than 30 genera. Among these, recurrent pathogens were recorded such as P. aeruginosa, P. paraeruginosa, and Aeromonas caviae. P. aeruginosa and A. caviae tpm reads were found evenly distributed over the study site but those of P. paraeruginosa were higher among sub-catchments impacted by heavy traffic. Health risks associated with these runoff P. paraeruginosa emerging pathogens were high and associated with strong cytotoxicity on A549 lung cells. Recurrent detections of pathogens in runoff waters highlight the need of a microbiological surveillance prior allowing their use. Good microbiological quality can be obtained for certain typologies of sub-catchments with good hygienic practices but not all. A reorganization of Human mobility and behaviors would likely trigger changes in these bacterial diversity patterns and reduce the occurrences of the most hazardous groups.

Targeted proteomics links virulence factor expression with clinical severity in staphylococcal pneumonia.

Introduction: The bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins. Methods: We present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival. Results: We found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models. Discussion: These findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens.

Draft Genome Sequences of Stenotrophomonas maltophilia Strains Sm32COP, Sm41DVV, Sm46PAILV, SmF3, SmF22, SmSOFb1, and SmCVFa1, Isolated from Different Manures in France.

Stenotrophomonas maltophilia is a major opportunistic human pathogen responsible for nosocomial infections. Here, we report the draft genome sequences of Sm32COP, Sm41DVV, Sm46PAILV, SmF3, SmF22, SmSOFb1, and SmCVFa1, isolated from different manures in France, which provide insights into the genetic determinism of intrinsic or acquired antibiotic resistance in this species.

Impact of untreated urban waste on the prevalence and antibiotic resistance profiles of human opportunistic pathogens in agricultural soils from Burkina Faso.

This study examined the long-term effects of the landfill disposal of untreated urban waste for soil fertilization on the prevalence and antibiotic resistance profiles of various human opportunistic pathogens in soils from Burkina Faso. Samples were collected at three sites in the periphery of Ouagadougou during two campaigns in 2008 and 2011. At each site, amendment led to changes in physico-chemical characteristics as shown by the increase in pH, CEC, total C, total N, and metal contents. Similarly, the numbers of total heterotrophic bacteria were higher in the amended fields than in the control ones. No sanitation indicators, i.e., coliforms, Staphylococci, and Enterococci, were detected. Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) were detected at a low level in one amended field. Stenotrophomonas maltophilia was detected from both campaigns at the three sites in the amended fields and only once in an unamended field. Diversity analysis showed some opportunistic pathogen isolates to be closely related to reference clinical strains responsible for nosocomial- or community-acquired infections in Northern countries. Antibiotic resistance tests showed that P. aeruginosa and Bcc isolates had a wild-type phenotype and that most S. maltophilia isolates had a multi-drug resistance profile with resistance to 7 to 15 antibiotics. Then we were able to show that amendment led to an increase of some human opportunistic pathogens including multi-drug resistant isolates. Although the application of untreated urban waste increases both soil organic matter content and therefore soil fertility, the consequences of this practice on human health should be considered.

Comparative Genomics of Environmental and Clinical Stenotrophomonas maltophilia Strains with Different Antibiotic Resistance Profiles.

Stenotrophomonas maltophilia, a ubiquitous Gram-negative γ-proteobacterium, has emerged as an important opportunistic pathogen responsible for nosocomial infections. A major characteristic of clinical isolates is their high intrinsic or acquired antibiotic resistance level. The aim of this study was to decipher the genetic determinism of antibiotic resistance among strains from different origins (i.e., natural environment and clinical origin) showing various antibiotic resistance profiles. To this purpose, we selected three strains isolated from soil collected in France or Burkina Faso that showed contrasting antibiotic resistance profiles. After whole-genome sequencing, the phylogenetic relationships of these 3 strains and 11 strains with available genome sequences were determined. Results showed that a strain's phylogeny did not match their origin or antibiotic resistance profiles. Numerous antibiotic resistance coding genes and efflux pump operons were revealed by the genome analysis, with 57% of the identified genes not previously described. No major variation in the antibiotic resistance gene content was observed between strains irrespective of their origin and antibiotic resistance profiles. Although environmental strains generally carry as many multidrug resistant (MDR) efflux pumps as clinical strains, the absence of resistance-nodulation-division (RND) pumps (i.e., SmeABC) previously described to be specific to S. maltophilia was revealed in two environmental strains (BurA1 and PierC1). Furthermore the genome analysis of the environmental MDR strain BurA1 showed the absence of SmeABC but the presence of another putative MDR RND efflux pump, named EbyCAB on a genomic island probably acquired through horizontal gene transfer.

Diversity among strains of Pseudomonas aeruginosa from manure and soil, evaluated by multiple locus variable number tandem repeat analysis and antibiotic resistance profiles.

The results of a multiple locus variable number of tandem repeat (VNTR) analysis (MLVA)-based study designed to understand the genetic diversity of soil and manure-borne Pseudomonas aeruginosa isolates, and the relationship between these isolates and a set of clinical and environmental isolates, are hereby reported. Fifteen described VNTR markers were first selected, and 62 isolates recovered from agricultural and industrial soils in France and Burkina Faso, and from cattle and horse manure, along with 26 snake-related isolates and 17 environmental and clinical isolates from international collections, were genotyped. Following a comparison with previously published 9-marker MLVA schemes, an optimal 13-marker MLVA scheme (MLVA13-Lyon) was identified that was found to be the most efficient, as it showed high typability (90%) and high discriminatory power (0.987). A comparison of MLVA with PFGE for typing of the snake-related isolates confirmed the MLVA13-Lyon scheme to be a robust method for quickly discriminating and inferring genetic relatedness among environmental isolates. The 62 isolates displayed wide diversity, since 41 MLVA types (i.e. MTs) were observed, with 26 MTs clustered in 10 MLVA clonal complexes (MCs). Three and eight MCs were found among soil and manure isolates, respectively. Only one MC contained both soil and manure-borne isolates. No common MC was observed between soil and manure-borne isolates and the snake-related or environmental and clinical isolates. Antibiotic resistance profiles were performed to determine a potential link between resistance properties and the selective pressure that might be present in the various habitats. Except for four soil and manure isolates resistant to ticarcillin and ticarcillin/clavulanic acid and one isolate from a hydrocarbon-contaminated soil resistant to imipenem, all environmental isolates showed wild-type antibiotic profiles.

Low occurrence of Pseudomonas aeruginosa in agricultural soils with and without organic amendment.

The occurrence of Pseudomonas aeruginosa was monitored at a broad spatial scale in French agricultural soils, from various soil types and under various land uses to evaluate the ability of soil to be a natural habitat for that species. To appreciate the impact of agricultural practices on the potential dispersion of P. aeruginosa, we further investigated the impact of organic amendment at experimental sites in France and Burkina Faso. A real-time quantitative PCR (qPCR) approach was used to analyze a set of 380 samples selected within the French RMQS ("Réseau de Mesures de la Qualité des Sols") soil library. In parallel, a culture-dependent approach was tested on a subset of samples. The results showed that P. aeruginosa was very rarely detected suggesting a sporadic presence of this bacterium in soils from France and Burkina Faso, whatever the structural and physico-chemical characteristics or climate. When we analyzed the impact of organic amendment on the prevalence of P. aeruginosa, we found that even if it was detectable in various manures (at levels from 10(3) to 10(5) CFU or DNA targets (g drywt)(-1) of sample), it was hardly ever detected in the corresponding soils, which raises questions about its survival. The only case reports were from a vineyard soil amended with a compost of mushroom manure in Burgundy, and a few samples from two fields amended with raw urban wastes in the sub-urban area of Ouagadougou, Burkina Faso. In these soils the levels of culturable cells were below 10 CFU (g drywt)(-1).