Biology subjects

Piveteau, P.

Publications and source records attributed to Piveteau, P..

5 recordsLinked to original sources

bioRxiv2025

Pangenome structure and ecological adaptation in the Klebsiella pneumoniae species complex: insights from a geographically and time limited multi-habitat study

Klebsiella pneumoniae species complex (KpSC) members inhabit distinct ecological habitats, yet the diversity and adaptation of environmental KpSC populations remain underexplored. We investigated KpSC transmission, pangenome structure and diversity, and functional gene enrichment across four distinct habitats in Burgundy, France over one year. In total, 664 environmental samples were collected from an organic vegetable farm (n=329), an organic cattle farm (n=304) and wastewater treatment plants (WWTP; n=31), alongside 47 clinical isolates. KpSC was detected in 22.4% of environmental samples, most commonly in WWTP (83.9%), followed by vegetable farm (27.1%) and cattle farm (11.2%). A total of 336 isolates were collected and whole-genome sequenced. K. pneumoniae sensu stricto (phylogroup Kp1) was predominant (76%), followed by K. variicola subsp. variicola (phylogroup Kp3) (21%). Genomic analyses revealed substantial novel diversity, especially in environmental habitats (67 novel STs), with limited cross transmission but clear local persistence. Over 90% of environmental isolates lacked acquired antimicrobial resistance genes, whereas half of the clinical isolates exhibited a multi-drug resistance profile. Comparative pangenome analyses showed Kp1 possessed a larger and more diverse pangenome than Kp3. Human-associated populations (clinical and WWTP) shared similar pangenome structures, although clinical isolates exhibited an expanded accessory genome. Cattle farm isolates had the most restricted and distinct pangenome while vegetable farm isolates displayed the largest total genomic repertoire. Functional enrichment analysis highlighted Kp3s environmental adaptation via conserved functions linked to the phylogroup genetic background, such as metabolic and regulatory pathways, including nitrogen fixation genes. Contrarily, human-associated populations, especially Kp1 members, were enriched in acquired functions linked to the ecological context, including antimicrobial and metal resistance determinants and mobile genetic elements. These findings emphasize phylogroup- and ecological niche-driven pangenome contrasts in KpSC, contributing to explain the successful adaptation of Kp1 to multiple habitats, including human-related settings.

genomics↗

bioRxiv2024

Triplex real-time PCR ZKIR-T assay for simultaneous detection of the Klebsiella pneumoniae species complex and identification of K. pneumoniae sensu stricto

Klebsiella pneumoniae species complex (KpSC) members, including the most important species K. pneumoniae (phylogroup Kp1 of the KpSC), are important opportunistic pathogens which display increasing rates of antimicrobial resistance worldwide. As they are widespread in food and the environment, there is a need for fast, sensitive and reliable methods to detect KpSC members in complex matrices. Previously, the ZKIR real-time PCR assay was developed to detect all KpSC members without distinction. Given that Kp1 is the clinically most significant phylogroup of the KpSC, here we aimed to simultaneously identify Kp1 while detecting all KpSC members. Three TaqMan probes were developed and used: the zkir P1 probe to detect phylogroups Kp1 to Kp5 and Kp7; the zkir P2 probe to detect phylogroup Kp6; and the Kp1 probe to specifically identify this phylogroup. The new assay was tested on a total of 95 KpSC and 19 non-KpSC strains from various sources, representing the different phylogroups as defined by whole genome sequencing. The results showed almost complete specificity, as the expected PCR results were obtained for 112 (98%) strains. The new triplex real-time PCR assay, called ZKIR-T, enables detection of all KpSC taxa while discriminating Kp1, which will be useful for rapid screening and to focus downstream analyses on chosen phylogroups of the KpSC. ImportanceThe pathogens of the Klebsiella pneumoniae species complex are widespread in food and animals and are amongst the main pathogens responsible for multidrug resistant infections in humans. In this study, we developed a highly sensitive detection assay that enables detection of this group of bacteria, with the simultaneous identification of the most common and clinically important species. This triplex one-reaction assay was shown to be highly sensitive and precise, enabling fast screening of varied samples for the presence of KpSC and K. pneumoniae sensu stricto.

microbiology↗

bioRxiv2022

In vitro evolution of Listeria monocytogenes reveals selective pressure for loss of SigB and AgrA function at different incubation temperatures.

The alternative sigma factor B ({sigma}B) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the General Stress Response. We previously showed that {sigma}B loss-of-function mutations arise frequently in strains of L. monocytogenes, and suggested that mild stresses might favour the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42{degrees}C) or lower (30{degrees}C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42{degrees}C. However, at 30{degrees}C independent alleles in the agr locus arose, resulting in the inactivation of the Agr quorum sensing. Colonies of both sigB- and agr- strains exhibited a greyer colouration on 7-days-old agar plates compared with the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB- strains outcompeted the parental strain at 42{degrees}C, but not at 30{degrees}C, whilst agr- strains showed a small increase in competitive fitness at 30{degrees}C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of {sigma}B or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. ImportanceTo withstand environmental aggressions L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B ({sigma}B). However, {sigma}B becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to {sigma}B loss-of-function alleles. Temperatures of 42{degrees}C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of {sigma}B loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30{degrees}C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of {sigma}B in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

molecular biology↗

bioRxiv2021

High prevalence of Klebsiella pneumoniae in European food products: a multicentric study comparing culture and molecular detection methods

Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices, and to characterize food isolates phenotypically and genotypically. Chicken meat (n=160) and salad (n=145) samples were collected in five European countries and screened for KpSC presence using culture-based and ZKIR qPCR methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44{degrees}C/48h) was defined as the most suitable selective culture method for KpSC recovery. High prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples: same food type and same country (15 cases); different food types and same country (1); same food type and two countries (1), suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. ImportanceBacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous and K. pneumoniae (Kp) is a leading cause of antibiotic-resistant infections in humans and animals. Despite the urgent public health threat represented by Kp, there is a lack of knowledge on the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing KpSC presence in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low-levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

microbiology↗

bioRxiv2019

The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

Klebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 4222 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using SCAI agar medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 x 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 x 103 to 1.5 x 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCEThe Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

microbiology↗