Objectives WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. Methods The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. Results Genotype–phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype–phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. Conclusions WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

The Gram-negative gastric pathogen Helicobacter pylori depends on natural transformation for genomic plasticity, which leads to host adaptation and spread of resistances. Here, we show that H. pylori takes up covalently labeled fluorescent DNA preferentially at the cell poles and that uptake is dependent on the type IV secretion system ComB. By titration of external pH and detection of accessibility of the fluorophor by protons, we localized imported fluorescent DNA in the periplasm. Single molecule analysis revealed that outer membrane DNA transport occurred at a velocity of 1.3 kbp·sand that previously imported DNA was reversibly extracted from the bacterium at pulling forces exceeding 23 pN. Thus, transport velocities were 10-fold higher than in Bacillus subtilis, and stalling forces were substantially lower. dsDNA stained with the intercalator YOYO-1 was transiently detected in the periplasm in wild-type H. pylori but was periplasmatically trapped in a mutant lacking the B. subtilis membrane-channel homolog ComEC. We conclude that H. pylori uses a two-step DNA uptake mechanism in which ComB transports dsDNA across the outer membrane at low force and poor specificity for DNA structure. Subsequently, Hp-ComEC mediates transport into the cytoplasm, leading to the release of the noncovalently bound DNA dye. Our findings fill the gap to propose a model for composite DNA uptake machineries in competent bacteria, all comprising the conserved ComEC channel for cytoplasmic membrane transport in combination with various transporters for access of external DNA to the cytoplasmic membrane.genome plasticity | horizontal gene transfer | molecular motor | natural transformation | single-cell analysis

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Campylobacter infection is the most commonly notified bacterial enteritis in Germany. We performed a large combined case-control and source attribution study (Nov 2011-Feb 2014) to identify risk factors for sporadic intestinal Campylobacter infections and to determine the relative importance of various animal sources for human infections in Germany. We conducted multivariable logistic regression analysis to identify risk factors. Source attribution analysis was performed using the asymmetric island model based on MLST data of human and animal/food isolates. As animal sources we considered chicken, pig, pet dog or cat, cattle, and poultry other than chicken. Consumption of chicken meat and eating out were the most important risk factors for Campylobacter infections. Additional risk factors were preparation of poultry meat in the household; preparation of uncooked food and raw meat at the same time; contact with poultry animals; and the use of gastric acid inhibitors. The mean probability of human C. jejuni isolates to originate from chickens was highest (74%), whereas pigs were a negligible source for C. jejuni infections. Human C. coli isolates were likely to originate from chickens (56%) or from pigs (32%). Efforts need to be intensified along the food chain to reduce Campylobacter load, especially on chicken meat.

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni 2؉ incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Protein-protein interactions are operative at the majority of cellular processes; thus, their study gives valuable insight into proteomic and functional associations. Helicobacter pylori is a Gram-negative bacterium that is infecting the stomach of about half of the human population. It is responsible for the development of gastric pathologies such as gastritis, gastroduodenal ulcer, and adenocarcinoma (1). One remarkable feature of H. pylori is its capacity to persist (often during decades) and multiply in the hostile environment of the stomach. In H. pylori, urease is a major virulence factor and is essential for the resistance to acidity because of its capacity to hydrolyze urea into bicarbonate and ammonia (2), which results in pH homeostasis of the bacterium. The protein is an extremely active and highly regulated member of the nickel metalloenzyme family. Activation of urease, i.e. incorporation of nickel into the active site, requires urease-specific accessory proteins whose homologues have been extensively studied genetically and biochemically in Klebsiella pneumoniae (3)(4)(5)(6)(7)(8). It is still enigmatic why vast amounts of urease, up to 10% of the total protein, are produced, whereas only a minor active portion is sufficient for acid resistance (9). Furthermore the observation that urease is even indispensable for the colonization under neutral conditions (10) suggested additional essential functions for this central enzyme that remained unidentified.H. pylori possesses another nickel-containing enzyme, a [NiFe] hydrogenase, allowing this organism to utilize hydrogen as an energy source. Accordingly hydrogenase has been shown to be essential for H. pylori colonization in the mouse model (11). The assembly of the [NiFe] hydrogenase metal center located in the large hydrogenase subunit is partially understood and requires several proteins involved in the sequential delivery of iron before nickel (for a review, see Ref. 12). In addition, the [NiFe] hydrogenase possesses a small subunit containing [Fe-S] clusters. For the assembly of the latter no distinct biosynthetic pathway has been attributed so far, suggesting that the housekeeping [Fe-S] cluster assembly system might be involved (12). H. pylori is unique in that the maturation events of these two nickel-containing essential proteins are interconnected as H. pylori mutants deficient in one of the accessory proteins, HypA or HypB, involved in nickel insertion into hydrogenase, lack significant activation of urease (13).