Biology subjects

Janecko, N.

Publications and source records attributed to Janecko, N..

4 recordsLinked to original sources

bioRxiv2026

A low-cost rpoB-based multiplex MAMA PCR for differentiation of the Klebsiella pneumoniae species complex

The Klebsiella pneumoniae species complex (KpSC) is a clinically important group of closely related pathogens associated with invasive infections. The complex comprises seven closely related members, which are often reported as K. pneumoniae, particularly in resource-limited settings. Accurate differentiation of KpSC members remains challenging because routine laboratory methods lack sufficient resolution, and approaches like mass spectrometry and whole genome sequencing (WGS) are not widely available. Consequently, the epidemiology and clinical significance of non-K. pneumoniae members of the KpSC remain underrecognized. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR targeting species- and subspecies-specific single-nucleotide polymorphisms in the housekeeping gene rpoB, with six primer sets for differentiation of common KpSC members. The assay was validated against 49 genomically characterized clinical isolates, after which 179 wastewater-derived isolates provisionally identified as Klebsiella spp. by standard microbiological methods were tested. Of these, 174 were assigned to specific KpSC members by the assay, while 5 produced inconclusive amplification patterns. A subset of 16 environmental isolates was selected for WGS, including four of the five inconclusive isolates. All environmental isolates with interpretable MAMA PCR patterns were concordant with WGS. The four inconclusive environmental isolates were identified as Enterobacter spp. Overall, comparison of MAMA PCR with WGS showed 100% sensitivity and 100% specificity for all tested targets, and the total cost was approximately US$1. This rpoB-based multiplex MAMA PCR provides a simple, accurate, and low-cost approach for differentiation of KpSC members in routine laboratories and may support improved identification and surveillance in resource-limited settings. ImportanceThe Klebsiella pneumoniae species complex (KpSC) has seven members but is often reported as a single organism in routine laboratories, masking clinically and epidemiologically important diversity. As a result, the contribution of non-K. pneumoniae KpSC members to human and environmental microbiology remains poorly defined, especially in low-resource settings. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR based on discriminatory rpoB single nucleotide polymorphisms for differentiation of common KpSC members using standard PCR and agarose gel electrophoresis. The assay demonstrated 100% sensitivity and 100% specificity against whole-genome sequencing and excluded non-Klebsiella environmental isolates initially identified as Klebsiella pneumoniae using standard microbiological procedures. With an estimated per-test cost of about US$1, this method offers an affordable and scalable option for laboratories seeking more accurate KpSC identification and improved surveillance.

microbiology↗

bioRxiv2025

Case study: Genomic characteristics of the gut microbiome, Campylobacter and Salmonella genotypes in three cases of gastroenteritis co-infections

Campylobacter jejuni and Salmonella enterica co-infections in gastroenteritis cases are underexplored, particularly in relation to microbiome dysbiosis. This study uses metagenomic and culture-based approaches to investigate pathogen genotypes and stool microbiome composition. Stool samples from three patients were analysed using culture, qPCR and whole genome sequencing (WGS) of genomes and metagenomes. Strain typing and antimicrobial resistance (AMR) profiling were conducted for both pathogens. The stool microbiome structure was compared between the three patients and across three time points in one patient. C. jejuni and Salmonella enterica were of different strains and different proportions in the cases. C. jejuni sequence types were identified as ST-794, ST-50 and ST-10025, while S. enterica serovars Enteritidis ST-183 and S. Derby ST-39 were identified. C. jejuni AMR genotypes included blaOXA-447, gyrA (T86I), and tet(O). S. Enteritidis contained AMR gene mdsaA-B, and S. Derby contained mdsaA-B and fosA7 genes. Despite similarities in Bristol scales for stool composition, the three cases exhibited distinct microbiome population structures, suggesting there is no one signature microbial profile of microbiomes with a Campylobacter-Salmonella co-infection. A temporal trend towards microbial diversity was observed within a patient, shifting from Proteobacteria dominance (93.3%) during acute infection towards Bacteroidetes (89.5%) abundance post-infection, suggesting that moving towards diverse communities may play a key role in patient recovery. These findings reveal a case study of microbiome disruption for patients infected with C. jejuni and S. enterica and highlight the value of direct stool sequencing in understanding pathogen abundance in stool and microbiome dysbiosis.

microbiology↗

bioRxiv2025

Genomic diversity of non-typhoidal Salmonella found in patients suffering from gastroenteritis in Norfolk, UK

Salmonella is a significant public health pathogen responsible for a wide spectrum of diseases, ranging from gastroenteritis to invasive non-typhoidal salmonellosis (iNTS) and enteric fever. Although advancements in whole genome sequencing (WGS) have improved surveillance and outbreak investigations, traditional single-colony sequencing methods overlook within-host diversity, potentially underestimating the complexity of infections. This study explores the genome-wide diversity of Salmonella strains recovered from stool samples of eight patients, with up to 20 isolates analysed per sample. A total of 156 Salmonella enterica isolates were recovered. All isolates from individual patients displayed consistent serovars and sequence types. Despite the serotype consistency, microevolution was observed in S. Java ST149 and S. Java ST43, with SNP analyses revealing higher diversity (13 and 5 SNP differences, respectively) compared to the clonal populations of other serovars. Phylogenetic analysis of S. Java ST149 isolates from Patient 2 revealed distinct branching, driven by mutations in genes such as secY and cnoX, while S. Java ST43 isolates from Patient 1 displayed multiple clades with notable SNPs affecting transcriptional regulators. Genome structure (GS) analyses using hybrid assemblies identified uniform GS1.0 across all isolates. Antimicrobial resistance (AMR) profiling revealed the presence of multidrug efflux pump genes (mdsA and mdsB) in all isolates. However, S. Typhimurium isolates from Patient 4 exhibited additional AMR genes, including sul2, aph(3)-Ib, and blaTEM-1, associated with an 8.7 kb resistance region. A single isolate from Patient 4 lacked these additional genes due to the deletion of a [~]19 kb genomic region, highlighting structural variation as a driver of phenotypic differences. These findings emphasise the genetic diversity of Salmonella within hosts, particularly in serovars such as S. Java, and underscore the limitations of single-colony sequencing in capturing this complexity. The study highlights the utility of hybrid sequencing strategies for comprehensive analysis of genome variation, offering valuable insights into transmission dynamics, antimicrobial resistance, and evolutionary processes in Salmonella. Impact statementThis study provides a transformative perspective on Salmonella genomics, uncovering the significant within-host diversity of Salmonella populations through the analysis of multiple single-colony isolates. By leveraging hybrid sequencing technologies, we reveal critical insights into the microevolution of Salmonella during infection, capturing genome-wide variation at both single nucleotide and chromosomal scales. Our findings highlight the inherent limitations of traditional single-colony sequencing in detecting within-host diversity, particularly in genetically diverse serovars such as S. Java ST149. The identification of antimicrobial resistance (AMR) determinants and structural genomic variation demonstrates the dynamic nature of Salmonella genomes, offering crucial implications for clinical management and public health surveillance. Importantly, we show that within-host diversity may influence epidemiological interpretations, particularly in outbreak investigations and source attribution, where genetic differences between isolates can mask deeper transmission links. Data SummaryAll relevant supporting data are available in the accompanying supplementary data files. The online version of this article contains four supplementary table. All Salmonella isolate genome sequences are available in the National Centre for Biotechnology Information (NCBI), Sequence Read Archive (SRA) under the Bioproject accession numbers PRJNA1230128. SRA accession numbers and associated metadata for isolate genomes are included in the supplementary table S2-S4.

genomics↗

bioRxiv2021

Genome-wide insights into population structure and host specificity of Campylobacter jejuni

The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.

genomics↗