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Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1 ) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV . We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K . pneumoniae isolates carrying one or more bla SHV alleles but no other acquired β-lactamases to assess genotype-phenotype relationships for bla SHV . This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae ), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Background: Nosocomial infections pose a considerable risk to patients who are susceptible, and this is particularly acute in intensive care units when hospital-associated bacteria are endemic. During the first wave of the COVID-19 pandemic, the surge of patients presented a significant obstacle to the effectiveness of infection control measures. We aimed to assess the risks and extent of nosocomial pathogen transmission under a high patient burden by designing a novel bacterial pan-pathogen deep-sequencing approach that could be integrated with standard clinical surveillance and diagnostics workflows., Methods: We did a prospective cohort study in a region of northern Italy that was severely affected by the first wave of the COVID-19 pandemic. Inpatients on both ordinary and intensive care unit (ICU) wards at the San Matteo hospital, Pavia were sampled on multiple occasions to identify bacterial pathogens from respiratory, nasal, and rectal samples. Diagnostic samples collected between April 7 and May 10, 2020 were cultured on six different selective media designed to enrich for Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, and DNA from each plate with positive growth was deep sequenced en masse. We used mSWEEP and mGEMS to bin sequencing reads by sequence cluster for each species, followed by mapping with snippy to generate high quality alignments. Antimicrobial resistance genes were detected by use of ARIBA and CARD. Estimates of hospital transmission were obtained from pairwise bacterial single nucleotide polymorphism distances, partitioned by within-patient and between-patient samples. Finally, we compared the accuracy of our binned Acinetobacter baumannii genomes with those obtained by single colony whole-genome sequencing of isolates from the same hospital., Findings: We recruited patients from March 1 to May 7, 2020. The pathogen population among the patients was large and diverse, with 2148 species detections overall among the 2418 sequenced samples from the 256 patients. In total, 55 sequence clusters from key pathogen species were detected at least five times. The antimicrobial resistance gene prevalence was correspondingly high, with key carbapenemase and extended spectrum ß-lactamase genes detected in at least 50 (40%) of 125 patients in ICUs. Using high-resolution mapping to infer transmission, we established that hospital transmission was likely to be a significant mode of acquisition for each of the pathogen species. Finally, comparison with single colony Acinetobacter baumannii genomes showed that the resolution offered by deep sequencing was equivalent to single-colony sequencing, with the additional benefit of detection of co-colonisation of highly similar strains., Interpretation: Our study shows that a culture-based deep-sequencing approach is a possible route towards improving future pathogen surveillance and infection control at hospitals. Future studies should be designed to directly compare the accuracy, cost, and feasibility of culture-based deep sequencing with single colony whole-genome sequencing on a range of bacterial species., Funding: Wellcome Trust, European Research Council, Academy of Finland Flagship program, Trond Mohn Foundation, and Research Council of Norway., Competing Interests: Declaration of interests SWL received Robert Austrian Research Award sponsored by Pfizer, outside of the scope of this study. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Several enzymes from the metallo-β-lactamase-like family of lactonases (MLLs) degrade N- acyl L-homoserine lactones (AHLs). They play a role in a microbial communication system known as quorum sensing, which contributes to pathogenicity and biofilm formation. Designing quorum quenching ( QQ ) enzymes that can interfere with this communication allows them to be used in a range of industrial and biomedical applications. However, tailoring these enzymes for specific communication signals requires a thorough understanding of their mechanisms and the physicochemical properties that determine their substrate specificities. We present here a detailed biochemical, computational, and structural study of GcL, which is a highly proficient and thermostable MLL with broad substrate specificity. We show that GcL not only accepts a broad range of substrates but also hydrolyzes these substrates through at least two different mechanisms. Further, the preferred mechanism appears to depend on both the substrate structure and/or the nature of the residues lining the active site. We demonstrate that other lactonases, such as AiiA and AaL, show similar mechanistic promiscuity, suggesting that this is a shared feature among MLLs. Mechanistic promiscuity has been seen previously in the lactonase/paraoxonase PON1, as well as with protein tyrosine phosphatases that operate via a dual general acid mechanism. The apparent prevalence of this phenomenon is significant from both a biochemical and protein engineering perspective: in addition to optimizing for specific substrates, it may be possible to optimize for specific mechanisms, opening new doors not just for the design of novel quorum quenching enzymes but also of other mechanistically promiscuous enzymes., Competing Interests: The authors declare the following competing financial interest(s): MHE has patents WO2020185861A1, WO2015014971A1. MHE is a co-founder, a former CEO and an equity holder of Gene&Green TK, a company that holds the license to WO2014167140A1, FR3132715A1, FR3068989A1, EP3941206 for which MHE is an inventor. These interests have been reviewed and managed by the University of Minnesota in accordance with its Conflict-of-Interest policies. CB is an inventor of WO2020185861A1., (© 2024 The Authors. Published by American Chemical Society.)

The protein tyrosine phosphatase (PTP) SHP-1 plays an important role in both immune regulation and oncogenesis. This enzyme is part of a broader family of PTPs that all play important regulatory roles in vivo. Common to these enzymes is a highly conserved aspartic acid (D421 in SHP-1) that acts as an acid/base catalyst during the PTP-catalyzed reaction. This residue is located on a mobile loop, the WPD-loop, the dynamic behavior of which is intimately connected to the catalytic activity. The SHP-1 WPD-loop variants H422Q, E427A, and S418A have been kinetically characterized and compared to those of the wild-type (WT) enzyme. These variants exhibit limiting magnitudes of k cat ranging from 43 to 77% of the WT enzyme. However, their pH profiles are significantly broadened in the basic pH range. As a result, above pH 6, the E427A and S418A variants have turnover numbers notably higher than those of WT SHP-1. Molecular modeling results indicate that the shifted pH dependencies result primarily from changes in solvation and hydrogen-bonding networks that affect the p K a of the D421 residue, explaining the changes in pH-rate profiles for k cat on the basic side. In contrast, a previous study of a noncatalytic residue variant of the PTP YopH, which also exhibited changes in pH dependency, showed that the catalytic change arose from mutation-induced changes in conformational equilibria of the WPD-loop. This finding and the present study show the existence of distinct strategies for nature to tune the activity of PTPs in particular environments through controlling the pH dependency of catalysis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Purpose: To compare the outcomes and complications of two perioperative protocols for the management of patients who underwent medial unicompartmental knee arthroplasty (UKA): 24 h (1-day surgery [OS]) versus 72 h (enhanced recovery after surgery [ERAS]) of the length of hospital stay (LOS). In our hypothesis, the reduction of the LOS from 3 to 1 day did not influence the outcomes and complications., Methods: A total of 42 patients (21 in each group) with isolated anteromedial knee osteoarthritis and meeting specific criteria were prospectively included in the study. Clinical outcomes included Knee Society Score (KSS) and Forgotten joint score while pain evaluation was performed using a Visual Analogue Scale (VAS). Functional outcomes were assessed measuring the knee range of motion (ROM) while radiographic outcomes were evaluated measuring the amelioration of the varus deformity through the hip-knee-ankle angle (HKA)., Results: Clinical and functional outcomes did not significantly differ between the two groups. Complications occurred in 9.5% of OS and 4.7% of ERAS group patients. Significant improvements in knee ROM, VAS pain, KSS and HKA angle were observed postsurgery, with no significant differences between groups except in KSS expectations and function trends., Conclusion: The OS protocol is safe and effective and LOS, in a well-defined fast-track protocol, did not significantly impact clinical and functional outcomes. OS may lead to reduced hospitalisation costs and potential reductions in complications associated with prolonged stays, benefiting both patients and healthcare facilities. However, further research with larger sample sizes and longer follow-up periods is needed to confirm these findings. Early mobilisation and rehabilitation protocols are key components of successful patient recovery following UKA procedures., Level of Evidence: Level II., (© 2024 The Author(s). Knee Surgery, Sports Traumatology, Arthroscopy published by John Wiley & Sons Ltd on behalf of European Society of Sports Traumatology, Knee Surgery and Arthroscopy.)

Background: Renin-angiotensin-aldosterone system inhibitors (RAASIs) play a crucial role in the treatment of several chronic cardiovascular conditions. Nonetheless, hyperkalemia, a frequent side effect, often leads to the discontinuation of RAASIs. The implications of hyperkalemia-driven changes in RAASI medications are poorly understood., Methods: Population-based, observational, retrospective cohort study. Two large healthcare databases were utilized to identify 77,089 individuals aged 55 years and older with chronic conditions who were prescribed RAASIs between 2015 and 2017 in Southern Barcelona, Spain. We assessed the interplay between serum potassium abnormalities, RAASI management, and their associations with clinical outcomes, adjusting for potential confounders including socioeconomic factors, medical conditions, and potassium levels., Results: The one-year prevalence of hyperkalemia (defined as serum potassium, K+ >5.0 mmol/L) was 17.8 %. RAASI were down-titrated in 16.1 % of these 13,673 patients with K+ levels. Factors linked to a higher likelihood of reducing/discontinuing RAASI after developing hyperkalemia included older age, impaired kidney function, higher potassium levels, and previous hospitalizations. Dose reduction/discontinuation of RAASI after developing hyperkalemia was associated with an increased risk of hospitalization (adjusted hazard ratio [HR] 1.16, 95 % confidence interval [CI] 1.10-1.21) and with increased mortality (HR 1.60, 95 % CI 1.56-1.84)., Conclusion: In this large, observational study, hyperkalemia was linked to a greater likelihood of discontinuing RAASIs. Down-titration of RAASI was independently associated with unfavorable clinical outcomes such as hospitalization and specially mortality. Although the observational nature of the study, these findings underscore the importance of preventing circumstances that may lead to RAASI down-titration, such as hyperkalemia, as well as preventing hospitalizations and mortality, to ensure RAASI benefits., Competing Interests: Declaration of competing interest Josep Comin-Colet has received speaker fees from Vifor Pharma. Josep Comin-Colet and Miguel Cainzos-Achirica have participated in other research projects funded by unrestricted grants from CLS Vifor., (Copyright © 2024. Published by Elsevier B.V.)

Since October 2022, alerts have spread from several countries about the increase in invasive group A streptococcal (iGAS) and scarlet fever cases affecting young children. We aim to analyze the epidemiology of GAS infections in the last 12 years in our hospital and identify the clinical features of invasive cases observed in 2023. We conducted a retrospective study enrolling children and adolescents hospitalized at our pediatric clinic from January to December 2023 for a definitive diagnosis of iGAS infection. Clinical, laboratory, and imaging data were collected and analyzed. Comparing 2016 and 2023, we observed a similar number of GAS infections (65 vs. 60 cases). Five children with iGAS infection were hospitalized between March and April 2023. The median age was five years. At admission, all patients showed tachycardia disproportionate to their body temperature. Vomiting was a recurrent symptom (80%). Laboratory tests mostly showed lymphopenia, hyponatremia, and high inflammatory markers. The number of pediatric iGAS cases significantly increased in 2023. Clinical (pre-school-aged children with high fever, unexplained tachycardia, and vomiting) and laboratory parameters (high procalcitonin levels, hyponatremia, and lymphopenia) could help identify and suspect a potential iGAS infection.

Newborns admitted to neonatal intensive care units (NICU) are at increased risk of health care-associated infections. Serratia marcescens represent the third most common pathogen in NICU outbreaks. Here we present an outbreak investigation performed using Whole Genome Sequencing (WGS) analyses and the control measures implemented to limit the spread of S. marcescens in the NICU of an Italian hospital. In February 2023 S. marcescens was isolated from six newborns, when in 2022 this pathogen was isolated only from two samples in the same ward. Measures for infection prevention were adopted. Routinary surveillance screening, performed with rectal swabs collected at admission and weekly thereafter, was implemented to search for S. marcescens presence. Environmental samples were collected. All the isolates, obtained from the conjunctival swab of six newborns, from rectal swab of two newborns who did not develop infections, as well as from the aerators of two faucets, were sequenced. WGS analyses showed no correlation between the isolates from newborns and environmental isolates. The implementation of the measures for infection prevention and control had enabled us to successfully control the outbreak within a short period. WGS analyses proved to be crucial in outbreak investigation to limit the spreading of the pathogens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigate the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses reveal a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets reveals only a partial habitat separation of the clusters. Globally, two clusters only show a separate gene composition coherent with ecological adaptations. These results suggest that genetic isolation has preceded ecological adaptations in the shaping of the species diversity, an evolutionary scenario coherent with the Evolutionary Extended Synthesis., (© 2024. The Author(s).)

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens , with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Minimum Inhibitory Concentrations (MICs) are the gold standard for quantitatively measuring antibiotic resistance. However, lab-based MIC determination can be time-consuming and suffers from low reproducibility, and interpretation as sensitive or resistant relies on guidelines which change over time. Genome sequencing and machine learning promise to allow in silico MIC prediction as an alternative approach which overcomes some of these difficulties, albeit the interpretation of MIC is still needed. Nevertheless, precisely how we should handle MIC data when dealing with predictive models remains unclear, since they are measured semi-quantitatively, with varying resolution, and are typically also left- and right-censored within varying ranges. We therefore investigated genome-based prediction of MICs in the pathogen Klebsiella pneumoniae using 4367 genomes with both simulated semi-quantitative traits and real MICs. As we were focused on clinical interpretation, we used interpretable rather than black-box machine learning models, namely, Elastic Net, Random Forests, and linear mixed models. Simulated traits were generated accounting for oligogenic, polygenic, and homoplastic genetic effects with different levels of heritability. Then we assessed how model prediction accuracy was affected when MICs were framed as regression and classification. Our results showed that treating the MICs differently depending on the number of concentration levels of antibiotic available was the most promising learning strategy. Specifically, to optimise both prediction accuracy and inference of the correct causal variants, we recommend considering the MICs as continuous and framing the learning problem as a regression when the number of observed antibiotic concentration levels is large, whereas with a smaller number of concentration levels they should be treated as a categorical variable and the learning problem should be framed as a classification. Our findings also underline how predictive models can be improved when prior biological knowledge is taken into account, due to the varying genetic architecture of each antibiotic resistance trait. Finally, we emphasise that incrementing the population database is pivotal for the future clinical implementation of these models to support routine machine-learning based diagnostics.

The dissemination of carbapenemase-producing Escherichia coli, although still at low level, should be continuously monitored. OXA-244 is emerging in Europe, mainly in E. coli . In Italy, this carbapenemase was reported from an environmental river sample in 2019. We report clinical isolates of OXA-244-producing ST131 E. coli in four patients admitted to an acute care hospital in Pavia, Italy. The association of this difficult-to-detect determinant with a globally circulating high-risk clone, ST131 E. coli, is of clinical relevance.

Protein tyrosine phosphatases (PTPs) are crucial regulators of cellular signaling. Their activity is regulated by the motion of a conserved loop, the WPD-loop, from a catalytically inactive open to a catalytically active closed conformation. WPD-loop motion optimally positions a catalytically critical residue into the active site, and is directly linked to the turnover number of these enzymes. Crystal structures of chimeric PTPs constructed by grafting parts of the WPD-loop sequence of PTP1B onto the scaffold of YopH showed WPD-loops in a wide-open conformation never previously observed in either parent enzyme. This wide-open conformation has, however, been observed upon binding of small molecule inhibitors to other PTPs, suggesting the potential of targeting it for drug discovery efforts. Here, we have performed simulations of both enzymes and show that there are negligible energetic differences in the chemical step of catalysis, but significant differences in the dynamical properties of the WPD-loop. Detailed interaction network analysis provides insight into the molecular basis for this population shift to a wide-open conformation. Taken together, our study provides insight into the links between loop dynamics and chemistry in these YopH variants specifically, and how WPD-loop dynamic can be engineered through modification of the internal protein interaction network., Competing Interests: The authors declare none., (© The Author(s) 2024.)

Coagulase-negative staphylococci (CoNS) are commensal on human body surfaces and, for years, they were not considered a cause of bloodstream infection and were often regarded as contamination. However, the involvement of CoNS in nosocomial infection is increasingly being recognized. The insertion of cannulas and intravascular catheters represents the primary source of CoNS entry into the bloodstream, causing bacteremia and sepsis. They owe their pathogenic role to their ability to produce biofilms on surfaces, such as medical devices. In this study, we evaluate the adhesive capacity of CoNS isolated from blood cultures by comparing a spectrophotometric phenotypic assay with genotypic analysis based on the evidence of the ica operon. We retrospectively reviewed the database of CoNS isolated from blood cultures from January to December 2021 that were considered responsible for 361 bloodstream infections. Eighty-nine CoNS were selected among these. Our data show that Staphylococcus epidermidis was the predominant species isolated, expressing greater adhesive capacities, especially those with the complete operon. Knowledge of the adhesive capabilities of a microorganism responsible for sepsis can be useful in implementing appropriate corrective and preventive measures, since conventional antibiotic therapy cannot effectively eradicate biofilms.

In this study, we present two cases of Klebsiella pneumoniae , one KPC-33- and one NDM-1-producing, showing resistance to cefiderocol and ceftazidime/avibactam, collected in the intensive care unit of a hospital in Northern Italy from two patients who had recently undergone lung transplantation. Whole-genome sequencing was performed to investigate the molecular features of these strains.