Statistical evidence that honeybees competitively reduced wild bee abundance in the Munich Botanic Garden in 2020 compared to 2019

In a commentary on our paper (Renner et al., Oecologia 195:825-831, 2021), Harder and Miksha lay out why they think that our finding of higher honeybee abundances reducing wild bee abundances in an urban botanical garden is not statistically supported. Here, we explain the statistical test provided in our paper, which took advantage of a natural experiment offered by 2019 being a poorer year for bee keeping than 2020

Non-O1/Non-O139 Vibrio cholerae - An Underestimated Foodborne Pathogen? An Overview of Its Virulence Genes and Regulatory Systems Involved in Pathogenesis

In recent years, the number of foodborne infections with non-O1 and non-O139 Vibrio cholerae (NOVC) has increased worldwide. These have ranged from sporadic infection cases to localized outbreaks. The majority of case reports describe self-limiting gastroenteritis. However, severe gastroenteritis and even cholera-like symptoms have also been described. All reported diarrheal cases can be traced back to the consumption of contaminated seafood. As climate change alters the habitats and distribution patterns of aquatic bacteria, there is a possibility that the number of infections and outbreaks caused by Vibrio spp. will further increase, especially in countries where raw or undercooked seafood is consumed or clean drinking water is lacking. Against this background, this review article focuses on a possible infection pathway and how NOVC can survive in the human host after oral ingestion, colonize intestinal epithelial cells, express virulence factors causing diarrhea, and is excreted by the human host to return to the environment

How to Evaluate Non-Growing Cells - Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria

Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches

German coasts harbor non-O1/non-O139 Vibrio cholerae with clinical virulence gene profiles

Non-O1/non-O139 Vibrio cholerae (NOVC) are ubiquitous in aquatic ecosystems. In rare cases, they can cause intestinal and extra-intestinal infections in human. This ability is associated with various virulence factors. The presence of NOVC in German North Sea and Baltic Sea was observed in previous studies. However, data on virulence characteristics are still scarce. Therefore, this work aimed to investigating the virulence potential of NOVC isolated in these two regions. In total, 31 NOVC strains were collected and subjected to whole genome sequencing. In silico analysis of the pathogenic potential was performed based on the detection of genes involved in colonization and virulence. Phenotypic assays, including biofilm formation, mobility and human serum resistance assays were applied for validation. Associated toxin genes (hlyA, rtxA, chxA and stn), pathogenicity islands (Vibrio pathogenicity island 2 (VPI-II) and Vibrio seventh pathogenicity island 2 (VSP-II)) and secretion systems (Type II, III and VI secretion system) were observed. A maximum likelihood analysis from shared core genes revealed a close relationship between clinical NOVCs published in NCBI and environmental strains from this study. NOVC strains are more mobile at 37 °C than at 25 °C, and 68% of the NOVC strains could form strong biofilms at both temperatures. All tested strains were able to lyse erythrocytes from both human and sheep blood. Additionally, one strain could survive up to 60% and seven strains up to 40% human serum at 37 °C. Overall, the genetic virulence profile as well as the phenotypic virulence characteristics of the investigated NOVC from the German North Sea and Baltic Sea suggest potential human pathogenicity

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring

Genomic Characterization of Arcobacter butzleri Strains Isolated from Various Sources in Lithuania

Arcobacter (A.) butzleri, the most widespread species within the genus Arcobacter, is considered as an emerging pathogen causing gastroenteritis in humans. Here, we performed a comparative genome-wide analysis of 40 A. butzleri strains from Lithuania to determine the genetic relationship, pangenome structure, putative virulence, and potential antimicrobial- and heavy-metal-resistance genes. Core genome single nucleotide polymorphism (cgSNP) analysis revealed low within-group variability (≤4 SNPs) between three milk strains (RCM42, RCM65, RCM80) and one human strain (H19). Regardless of the type of input (i.e., cgSNPs, accessory genome, virulome, resistome), these strains showed a recurrent phylogenetic and hierarchical grouping pattern. A. butzleri demonstrated a relatively large and highly variable accessory genome (comprising of 6284 genes with around 50% of them identified as singletons) that only partially correlated to the isolation source. Downstream analysis of the genomes resulted in the detection of 115 putative antimicrobial- and heavy-metal-resistance genes and 136 potential virulence factors that are associated with the induction of infection in host (e.g., cadF, degP, iamA), survival and environmental adaptation (e.g., flagellar genes, CheA-CheY chemotaxis system, urease cluster). This study provides additional knowledge for a better A. butzleri-related risk assessment and highlights the need for further genomic epidemiology studies in Lithuania and other countries

Enzymatic synthesis and formation kinetics of mono- and di-hydroxylated chlorinated paraffins with the bacterial dehalogenase LinB from Sphingobium indicum

Transformation studies of chlorinated paraffins (CPs) and the effects of CP transformation products on humans, biota and environment are rare. The focus here is on hydroxylation reactions. As for polyhalogenated persistent organic pollutants (POPs) in general, hydroxylation reactions convert lipophilic material to more polar compounds with increased mobility. We investigated the in-vitro transformation of single-chain CP-mixtures to hydroxylated products with the dehalogenase LinB from Sphingobium indicum. C11-, C12- and C13-single-chain CP-homologues were exposed to LinB and mono-hydroxylated (CP-ols) and di-hydroxylated (CP-diols) transformation products were formed. Liquid-chromatography coupled to mass-spectrometry (LC-MS) was used to detect hydroxylated products and to separate them from the starting material. The presented data can be used to identify these CP-ol and CP-diol homologues in other samples. Hydroxylated products had lower chlorination degrees (nCl) than respective CP-starting-materials. Reactive and persistent CP-material was found in each homologue group. Reactive material is converted within hours by LinB, while more persistent CPs are transformed within days. Homologue-specific kinetic models were established to simulate the stepwise hydroxylation of persistent CPs to mono- and di-hydroxylated products. First-order rate constants for the formation of CP-ols (k1) and CP-diols (k2) were deduced for different homologues. Lower-chlorinated CP-ols did not accumulate to large extent and were transformed quickly to CP-diols, while higher-chlorinated CP-ols and -diols both accumulated. By enzymatic transformation of single-chain CPs with LinB, we synthesized unique sets of mono- and di-hydroxylated materials, which can be used as analytical standards and as starting materials for metabolic, toxicity and environmental fate studies

High Resolution Discrimination of Clinical Mycobacterium tuberculosis Complex Strains Based on Single Nucleotide Polymorphisms

Recently, the diversity of the Mycobacterium tuberculosis complex (MTBC) population structure has been described in detail. Based on geographical separation and specific host pathogen co-evolution shaping MTBC virulence traits, at least 20 major lineages/genotypes have evolved finally leading to a clear influence of strain genetic background on transmissibility, clinical presentation/outcome, and resistance development. Therefore, high resolution genotyping for characterization of strains in larger studies is mandatory for understanding mechanisms of host-pathogen-interaction and to improve tuberculosis (TB) control. Single nucleotide polymorphisms (SNPs) represent the most reliable markers for lineage classification of clinical isolates due to the low levels of homoplasy, however their use is hampered either by low discriminatory power or by the need to analyze a large number of genes to achieve higher resolution. Therefore, we carried out de novo sequencing of 26 genes (approx. 20000 bp per strain) in a reference collection of MTBC strains including all major genotypes to define a highly discriminatory gene set. Overall, 161 polymorphisms were detected of which 59 are genotype-specific, while 13 define deeper branches such as the Euro-American lineage. Unbiased investigation of the most variable set of 11 genes in a population based strain collection (one year, city of Hamburg, Germany) confirmed the validity of SNP analysis as all strains were classified with high accuracy. Taken together, we defined a diagnostic algorithm which allows the identification of 17 MTBC phylogenetic lineages with high confidence for the first time by sequencing analysis of just five genes. In conclusion, the diagnostic algorithm developed in our study is likely to open the door for a low cost high resolution sequence/SNP based differentiation of the MTBC with a very high specificity. High throughput assays can be established which will be needed for large association studies that are mandatory for detailed investigation of host-pathogen-interaction during TB infection