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In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E coli (EAEC)–specific virulence factors. Here, we identified an E coli O104:H4 isolate that carried a single-nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS , encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E coli O104:H4 Δ stx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and Δ rpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR , encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E coli O104:H4 virulence, primarily through an AggR-dependent mechanism. [ABSTRACT FROM AUTHOR]

Neutralising antibodies against the SARS-CoV-2 spike (S) protein are major determinants of protective immunity, though insufficient antibody responses may cause the emergence of escape mutants. We studied the humoral immune response causing intra-host evolution in a B-cell depleted, haemato-oncologic patient experiencing clinically severe, prolonged SARS-CoV-2 infection with a virus of lineage B.1.177.81. Following bamlanivimab treatment at an early stage of infection, the patient developed a bamlanivimab-resistant mutation, S:S494P. After five weeks of apparent genetic stability, the emergence of additional substitutions and deletions within the N-terminal domain (NTD) and the receptor binding domain (RBD) of S was observed. Notably, the composition and frequency of escape mutations changed in a short period with an unprecedented dynamic. The triple mutant S:Delta141-4 E484K S494P became dominant until virus elimination. Routine serology revealed no evidence of an antibody response in the patient. A detailed analysis of the variant-specific immune response by pseudotyped virus neutralisation test, surrogate virus neutralisation test, and immunoglobulin-capture enzyme immunoassay showed that the onset of an IgM-dominated antibody response coincided with the appearance of escape mutations. The formation of neutralising antibodies against S:Delta141-4 E484K S494P correlated with virus elimination. One year later, the patient experienced clinically mild re-infection with Omicron BA.1.18, which was treated with sotrovimab and resulted in an increase in Omicron-reactive antibodies. In conclusion, the onset of an IgM-dominated endogenous immune response in an immunocompromised patient coincided with the appearance of additional mutations in the NTD and RBD of S in a bamlanivimab-resistant virus. Although virus elimination was ultimately achieved, this humoral immune response escaped detection by routine diagnosis and created a situation temporarily favouring the rapid emergence of various antibody escape mutants with known epidemiological relevance. Author summary: In the immunocompromised host, prolonged infection and insufficient antibody responses favour the emergence of highly mutated SARS-CoV-2 variants. To better understand the selective pressure exerted by antiviral antibodies, we characterised the variant-specific humoral immune response and resulting intra-host evolution of SARS-CoV-2 in a B-lymphocyte-depleted patient experiencing severe infection. Treatment with a monoclonal antibody early in infection led to the selection and outgrowth of resistant virus. As their B-cell function recovered, the patient mounted an IgM-dominated antiviral antibody response, the onset of which closely correlated with the appearance of additional escape mutations within the spike protein. The strength and breadth of this antibody response gradually increased, resulting in the selection of a variant carrying several escape mutations within spike. The formation of neutralising antibodies against this variant resulted in virus elimination. Our study provides new insights into the selection process of viral escape mutants in immunocompromised patients. The resuming humoral immunity against SARS-CoV-2 can be dominated by IgM. This immune response is poorly detectable with routine diagnostic procedures yet may trigger and drive substantial viral intra-host evolution. Determination of antiviral antibody responses and correlating them with intra-host evolution may provide a better picture of the adaptability of currently circulating SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

Methicillin‐resistant Staphylococcus aureus (MRSA) can be transmitted between pigs and humans on farms. Hence, the reduction of MRSA carriage in pigs could decrease the risk of zoonotic transmission. Recently, straw bedding has been found to significantly reduce MRSA carriage in pigs. The mechanisms behind this effect remain unclear but changes in the nasal microbiome may play a role. In this exploratory study, the nasal microbiota of pigs kept on straw was examined using V1/V2 16S rRNA gene sequencing. Nasal swabs were collected from 13 pigs at six different time points during the course of a full fattening cycle resulting in 74 porcine samples. In addition, straw samples were collected at each time point. Eleven out of 13 pigs were MRSA positive at housing‐in. We found a strong temporal pattern in the microbial communities. Both microbial diversity and abundance of Staphylococcus species peaked in week 5 after introduction to the straw stable decreased in week 10, when all pigs turned MRSA‐negative, and increased again toward the end of the fattening period. These findings show that the introduction of pigs into a new environment has a huge impact on their nasal microbiota, which might lead to unfavorable conditions for MRSA. Moreover, other Staphylococcus species may play a role in eliminating MRSA carriage. We designed a follow‐up study including two different husbandry systems to further assess these effects. [ABSTRACT FROM AUTHOR]

Plasma-activated water (PAW) generated from tap water has gained attention as a disinfectant when used directly in its pure form. Little is known about the application of PAW for bacterial inactivation in aqueous environments because its use in fluids results in dilutions. We investigated the effect of PAW in aqueous suspensions simulating such dilutions, and we focused on the minimal addition of PAW volumes to bacterial aqueous suspensions still resulting in high inactivation rates. The antimicrobial effect was highly dependent on the activation of PAW. An increase in activation power from 90 to 100 W resulted in a greater microbial reduction with an identical 10 min activation time. The susceptibility to PAW dilutions was analyzed in detail regarding nine Gram-negative species out of Enterobacterales and other waterborne microorganisms as well as four Gram-positive species present in two different matrices, in saline and in tap water, at high concentrations simulating massive contamination situations. For this purpose, the PAW activation setting of 90 W and 30 min was defined in order to be able to differentiate the limitations of inactivation in individual bacterial species. The Gram-negatives in saline demonstrated susceptibility when one volume unit of PAW was added. However, twice the PAW volume was necessary for inactivation when bacteria were present in tap water. Gram-positive microorganisms were more robust, indicated by prolonged contact times before inactivation. Our results indicate that PAW can be used for bacterial decontamination processes in aqueous environments when added in surplus. Optimized activation settings such as electric power to generate PAW and the contact times to the samples increase the effect of the inactivation a wide range of bacteria, regardless of their resistance profiles. [ABSTRACT FROM AUTHOR]

Purpose: Carbapenemase-producing Enterobacterales (CPE) pose a serious threat for healthcare facilities worldwide, yet the mode of transmission is often unclear. Recently, we recorded an increase of blaOXA-48-harboring isolates at our hospital associated with patients with previous medical treatment in the Ukraine. We used long-read whole genome sequencing (lrWGS) to characterize these isolates including their plasmids. Methods: Samples were collected as part of clinical routine diagnostic or screening of multi-drug resistance bacteria (MDRB). Antimicrobial susceptibility testing was performed and all MDRB (n = 10) were sequenced by lrWGS for genotyping, identification of antimicrobial resistance (AMR) genes, and characterization of plasmids. Results: While routine analysis of core genome multilocus sequence typing (cgMLST) did not show any genetic similarities between isolates, we found an unexpected high similarity in the plasmid diversity of different Enterobacterales in patients with previous medical treatment in the Ukraine. This included an IncL/M plasmid carrying blaOXA-48 and additional small non-AMR-coding plasmids. Conclusion: Our results show that lrWGS can be used in the routine setting to uncover similarities in plasmids and may give further information about potential epidemiologic associations. In the future, analysis of both AMR and non-AMR plasmids may provide an additional layer of information for molecular surveillance of CPE. [ABSTRACT FROM AUTHOR]

Introduction: The emergence of extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an ISmediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids. [ABSTRACT FROM AUTHOR]