BackgroundBacteriophages (phages), viruses ubiquitous in the environment infecting bacteria, are an increasingly attractive option for adjunct therapy with conventional antibiotics in complicated, refractory multidrug-resistant infections. Bacteria (“hypermutators”) with defects in methyl-directed mismatch repair (MMR) have been described in Pseudomonas aeruginosa, with the resultant defect in DNA repair responsible for rapid development of antimicrobial resistance.Case SummaryA 72-year-old male with mild chronic obstructive pulmonary disease, diagnosed with resectable, advanced right frontoethmoid adenoid cystic carcinoma, underwent endoscopic resection to remove the affected bone and tissue. The subsequent large craniofacial defect was reconstructed with non-vascularized calvarial bone grafts, rigidly fixated with titanium hardware and externally covered with a vascularized pericranial flap. He developed complete dehiscence of the skull base reconstruction, and wound cultures grew multiple susceptible P. aeruginosa isolates with distinct morphologies that became progressively drug resistant on treatment. Whole-genome sequencing of isolates showed the presence of a non-synonymous mutation in the mutS gene impacting the MMR pathway, consistent with a hypermutator. Phage therapy was pursued due to ongoing infection with retained hardware, lack of clinical response to antibiotic therapy alone, extreme antibiotic resistance, and possible surgical cure of his cancer. He received twice-daily intravenous phage therapy in combination with cefiderocol for 2 weeks with clinical and microbiological resolution of his infection.ConclusionThis case suggests that phage therapy may be useful in the treatment of deep-seated hypermutator P. aeruginosa infections with retained hardware.

BACKGROUND:Human conflicts are associated with increased prevalence of multi-drug-resistant organisms (MDROs). Here, we investigate a collection of extensively drug resistant (XDR) bacteria recovered from foreign combatants wounded in Ukraine and evacuated to a U.S. military treatment facility in Germany between November 2022 and March 2024. METHODS:As part of routine surveillance, perirectal swabs were collected upon admission from all patients arriving from outside Germany. Whole genome sequencing, antimicrobial resistance gene (AMR) carriage, and antimicrobial susceptibility testing (AST) was determined for all isolates. Genetic relatedness to a set of isolates from soldiers treated at Ukrainian hospitals was assessed. RESULTS:Eighty-six deduplicated clinically significant MDROs were cultured from 32 combatants from 10 countries injured in Ukraine and subsequently treated in Germany. Twenty-three (72%) patients carried isolates from well-established global lineages of Enterococcus faecium (ST117), Klebsiella pneumoniae (ST147, 395), Acinetobacter baumannii (ST78) and Pseudomonas aeruginosa (ST235, ST773) that harbored carbapenemase (IMP, NDM, OXA), a 16S rRNA methyltransferase (ArmA, RmtB, RmtC), and/or the van operon, with 14 patients having >1 of these organisms. Sixteen combatants carried isolates genetically related to those from Ukrainian patients, suggesting acquisition occurred in Ukraine. AST revealed multiple isolates non-susceptible to cefiderocol (n = 8) or aztreonam-avibactam (n = 6), further complicating treatment options for carbapenemase-producing organisms. CONCLUSIONS:This is the first study describing the epidemiology of MDROs collected from a population of foreign combatants injured in Ukraine. The findings forecast challenges to clinicians and infection prevention and control both in Ukraine and foreign healthcare systems as combatants are repatriated.

The multiply antibiotic-resistant lineage 1 of Acinetobacter baumannii global clone 1 (GC1) emerged in the 1970s, and subsequently more extensively resistant sublineages have emerged. Here, we examined the evolution of the extensively resistant MRSN56 sublineage and showed it is characterised by insertions carrying resistance genes at specific chromosomal positions. An evolved form of the sublineage carries KL17 replacing KL1 at the capsule locus and includes an additional integrative element Aci-IE1 carrying further resistance genes including blaNDM. Further members of the modified sublineage (isolated 2014–2021) identified among publicly available genomes were from several countries and appear to have replaced the original form (2007–2010). Some KL17 type isolates had acquired even more resistance genes including blaPER. The blaNDM and blaPER genes contribute to reduced susceptibility to cefiderocol and/or sulbactam/durlobactam. The phylogeny indicated that separation of the sublineage into KL1 and KL17 groups coincided with the KL switch and Aci-IE1 was acquired later.

Extensive drug resistance (XDR) in Acinetobacter baumannii and other pathogens has revitalized bacteriophage as a therapeutic consideration. Six phages (AB1I1L, AB1I1M, AB1I1P, AB1I1T, AB2I2, and AB2I3) targeting A. baumannii were isolated from wastewater. These represent a previously undescribed phage genus with rapid adsorption and potent lysis. 18/40 A. baumannii clinical isolates, including 11/27 carbapenem-resistant isolates, were susceptible to one of the isolated phages. Importantly, in vitro-derived, phage resistant bacteria were killed in human ascites, demonstrating decreased biofitness. In contrast to most described phages that target A. baumannii, the bacterial capsule is not the primary receptor. Capsule impedes phage activity in vitro. The treatment of an XDR isolate using phage monotherapy in a rat subcutaneous abscess model showed dose-dependent efficacy, though a higher sustained concentration of phage was needed when compared with in vitro conditions. These phages are potential candidates for phage therapy, warranting additional preclinical evaluation as adjunctive treatment for A. baumannii infections.

The Russian invasion of Ukraine in 2022 has placed extraordinary pressure on hospitals there. One consequence of this has been the alarming increase in infections caused by multi-drug resistant organisms (MDROs), both within Ukraine and among the Ukrainian diaspora. The original source of these MDROs remains obscure although nosocomial origin is suspected. Here, we analyzed a collection of Acinetobacter baumannii and Pseudomonas aeruginosa collected from Ukraine before and after the invasion to glean a greater understanding of their relationship and origins. Genomic analysis was conducted on 167 A. baumannii and 93 P. aeruginosa cultured from 223 Ukrainian patients hospitalized in Ukraine or other European countries. Fifty-three isolates were cultured between 2014 and 2021, prior to the invasion, and the remaining 207 after. Highly genetically related extensively-drug resistant (XDR) clones were identified that spanned the pre- and post-invasion periods. For A. baumannii, isolates encompassed three sequence types (STs), including carbapenemase-producing strains from ST-2 (blaOXA-23) and ST-78 (blaOXA-72), as well as ST-400 carrying the ESBL blaGES-11. For P. aeruginosa, isolates encompassed three STs: ST-773 carrying blaNDM-1, ST-1047 carrying blaIMP-1, and ST-244. For all, the mobile genetic elements associated with carbapenemase carriage were fully characterized. Notably, post-invasion ST-773 and ST-1047 P. aeruginosa had a signature of host adaptation with multiple loss-of-function mutations in the quorum-sensing regulator LasR, known to modulate immune responses and provide survival advantages in animal models of infection. XDR epidemic clones circulating in Ukraine and across Europe since 2022 share a close genetic relationship to historical strains from Ukraine. In some cases, direct links to medical facilities within Ukraine can be inferred. These data suggest that surveillance efforts should focus on tracking nosocomial transmission within Ukrainian hospitals while infection control efforts are being disrupted by the ongoing Russian invasion.

In this work, we present an optimized nanopore long-read only sequencing workflow for epidemiologic analysis of clonal outbreaks built with open-source tools. A set of unrelated clinical Pseudomonas aeruginosa isolates (n = 10) was chosen for workflow optimization, and sequencing libraries were prepared using a modified rapid barcoding strategy that incorporates temperature ramps to improve performance for high-GC content genomes. Sequencing data were used to benchmark the performance of the dorado suite (v0.9.1), including its basecaller, pre-assembly read error correction, and post-assembly polishing algorithms. All long-read assemblies and core genome multilocus sequence typing (cgMLST) were performed with Flye and pyMLST, respectively. Results were compared with a standard reference Illumina short-read approach, and discordant positions were determined at the core and whole-genome levels. Optimal performance was found with dorado sup@v5.0.0 basecalling with the inclusion of dorado error correction and dorado polish with its bacterial model. This workflow was then validated with four retrospective hospital outbreak isolate sets, including Klebsiella pneumoniae (n = 12), P. aeruginosa (n = 11), Enterococcus faecium (n = 10), and Staphylococcus aureus (n = 10). The nanopore-only assemblies obtained from the optimized pipeline demonstrated fully concordant cgMLST-based minimum spanning trees compared to the Illumina short-read reference. At the whole-genome level, high concordance was also observed, with as few as two discordant positions per genome compared to short-read assemblies. This optimized library preparation and open-source computational workflow enables nanopore-only clonality and outbreak analysis with performance comparable to that of Illumina short-read sequencing and will contribute critically to hospital infection control. IMPORTANCE:For the past decade, bacterial whole-genome sequencing has been performed using high-accuracy short-read sequencing. More recently, long-read sequencing with Oxford Nanopore Technologies (ONT) instruments has emerged as a potential alternative based on multiple advantages, including lower costs, portability, and speed. However, this platform has suffered from basecall error rates that were too high for many applications in clinical microbiology, including outbreak tracing. With the release of new flow cell chemistries and basecall algorithms, the accuracy has improved dramatically, making this approach feasible for outbreak investigations. In this work, we optimize a streamlined nanopore-only workflow for epidemiologic analysis of bacterial pathogens. The workflow was validated with isolates from four previously identified clinical outbreaks with varying GC content and demonstrated fully concordant cgMLST clustering as compared to short-read references. This workflow will facilitate the broader implementation of ONT-only genomes and cgMLST analysis to assist in hospital outbreaks worldwide.

Background Acinetobacter baumannii is an important nosocomial pathogen worldwide. During the current invasion of Ukraine, reports of infections caused by this organism have proliferated. Here, we provide a phenotypic and genotypic analysis of A. baumannii associated with the conflict. Methods Between March 2022 and September 2023, 68 A. baumannii strains were cultured from wounded Ukrainian service members in three hospitals in west-central Ukraine. Antibiotic susceptibility and WGS were performed on all isolates. Results Strains encompassed eight different STs, including the emerging ST78 (and its single locus variant ST1077) and globally distributed ST2 lineages, with ST19 being the most common (25%). Fifty strains carried at least one acquired carbapenemase (blaOXA-23 or blaOXA-72), with seven strains carrying both. Overall, susceptibility ranged from 0% (fluoroquinolones) to 100% (SUL/durlobactam) and all strains had CST MICs <1 mg/mL. Notably, all but one ST2 isolates were resistant to FDC, and this correlated with the presence of the blaPER-1 or blaPER-7 ESBL genes. In contrast, 8 of 13 ST78 were FDC non-susceptible, but non-susceptibility was correlated with the disruption of the pirA siderophore receptor gene by ISAba35. Finally, passage in MEM of one strain for 8 days resulted in a mutation of the blaGES-11 ESBL to the blaGES-14 carbapenemase. Conclusions Sampling of A. baumannii strains infecting injured Ukrainian soldiers revealed the predominance of known (ST2) and emerging (ST19, ST78) lineages carrying carbapenemases. Antibiotic resistance was broad, including the recently approved catechol-substituted siderophore cephalosporin, FDC, highlighting the immense treatment challenges faced by medical personnel during this ongoing conflict.

Aortitis due to hypervirulent Klebsiella pneumoniae (hvKp) in a Belarusian male from central New York is described. Isolates at the time of diagnosis (Kp031824-1, Kp031824-2) and after 12 weeks of antimicrobial therapy (Kp070124) were characterized. Kp070124 was genomically and phenotypically unchanged retaining its capsular polysaccharide and mucoviscosity. In vitro studies established that capsule minus derivatives of Kp031824-1 and Kp031824-2 can occur due to mutations in wcaJ. But this genotype/phenotype was not selected for in Kp070124 after ≥15 weeks in the systemic compartment. Compared to capsule minus derivatives, the capsule positive phenotype demonstrated resistance to phagocytosis, but not to complement mediated bactericidal activity, suggesting resistance to phagocytosis is a more important defense mechanism at this site of infection. These data also support that a capsule positive, mucoviscous phenotype is selected for during infections in the systemic compartment. The surprising result that capsule positive strains have increased sensitivity to complement mediated bactericidal activity compared to capsule minus strains requires further investigation. The duration of therapy for this syndrome remains unclear but should be prolonged; adjunctive therapies (e.g. phage therapy, passive immunization, augmentation of cell mediated bactericidal activity) may be needed to overcome the protection endowed by the mucoviscous capsule of hvKp.

Pseudomonas aeruginosa sequence type (ST)-1047 is emerging globally as a carbapenemase-rich lineage, yet its evolutionary history and population structure are not known. Here, we performed a comprehensive genomic and epidemiological investigation of 141 ST-1047 isolates from 15 countries, integrating short- and long-read sequencing data with Bayesian phylogenetics and mobile genetic element analyses. Two clonal subpopulations were identified. Subclone 1, defined by blaVIM-11 carriage and loss of exoU, is proposed to have been imported to the United States following the medical evacuation of wounded service members from Afghanistan in 2005 and later seeded a nosocomial outbreak in Washington state. Subclone 2, carrying blaIMP-1, is undergoing rapid clonal expansion due to nosocomial outbreaks in Ukraine hospitals where infection control is impaired by the war with Russia. Genomic islands resembling P. aeruginosa genomic island-97B mediated blaIMP-1 duplication and integration at multiple chromosomal sites, including between iron-regulated small RNAs PrrF1 and PrrF2. Outside these subclones, independent acquisitions of blaNDM-1 and/or blaDIM-1 occurred via diverse resistance islands. While plasmids were detected in some ST-1047 isolates, chromosomal integration of carbapenemase genes has promoted stability and driven the population structure. This global study reveals that, since its emergence in the late 19th century, the ST-1047 lineage showed an exceptional ability to acquire diverse carbapenemases, and that geopolitical conflicts influenced its global spread on at least two occasions. These findings underscore the need for sustained global surveillance and high-resolution genomic analyses to prevent further spread of this high-risk pathogen.IMPORTANCECarbapenemase-producing Pseudomonas aeruginosa is a major cause of healthcare-associated infections worldwide and is associated with high mortality due to limited treatment options. In this study, we characterize the emergence and international spread of a previously underrecognized lineage of P. aeruginosa that has independently acquired and stabilized multiple resistance genes, including those encoding VIM, IMP, NDM, and Dutch imipenemase carbapenemases. Using genomic sequencing and evolutionary analyses, we show how this lineage emerged in the late 19th century and has since adapted by integrating resistance genes directly into its chromosome, promoting long-term stability and outbreak potential. Strikingly, we link its global expansion to population movements, soldier evacuations, and healthcare disruptions during armed conflicts in Afghanistan and Ukraine. This work reveals how political instability can drive the spread of multidrug-resistant bacteria and underscores the value of high-resolution surveillance to detect and contain emerging threats before they become dominant in clinical settings.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the MultidrugResistant Organism Repository and Surveillance Network, 97 were ST19 PAS , 34 of which carbapenemresistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23 , bla OXA-72 , and/or bla NDM-5 . Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub -lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Abstract Increasing antimicrobial resistance in Acinetobacter baumannii has resulted in limited to no treatment options. Bacteriophage therapy promises to assist in filling this treatment void. Six related phages (AB1I1L, AB1I1M, AB1I1P, AB1I1T, AB2I2, AB2I3) that target A. baumannii were isolated from wastewater and possessed an icosahedral structure and contractile tail consistent with myoviridae. Purified phage of high quality and low endotoxin levels (< 0.5EU/mL) demonstrated rapid adsorption and potent lysis. Encouragingly 18/40 (45%) tested clinical isolates were susceptible to one or more members of this phage species, including 11 of 27 (41%) carbapenem resistant A. baumannii isolates. Importantly, pools of phage tolerant/resistant A. baumannii that developed after exposure to each of the six phages were highly susceptible to human ascites mediated bactericidal activity ex vivo. An unusual and potentially important feature of this phage species is that the bacterial capsule is not their receptor; in fact, capsule impedes phage activity. Whole genome sequencing did not identify bacterial virulence or resistance genes or phage lysogenic genes. Taken together, these phages are potential candidates for phage therapy and warrant additional preclinical evaluation as a treatment modality for XDR-A. baumannnii.

Background Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed. Methods Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log10 10 LD50 50 (range 1.5-2.9)) - 2.9)) were created to measure the effects on LD50 50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, p rmpA, p rmpA2 (truncated), irp2, and clbBC. Findings Curing pVir had the greatest increase in survival (mean LD50 50 to 7.6 (range 7.0-9.0, - 9.0, p <= 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD50s 50 s for O p rmpA (5.9, p <= 0.0001), O iucA (3.6, p <= 0.0001), O irp2 (3.4), O rmpA O iucA (6.3, p <= 0.0001), and O pVir O irp2 (8.7, p <= 0.0001). Notably O pVir had an additional mean LD50 50 increase of 1.3 compared to the pVir-encoded O p rmpA O iucA (p <= 0.01), suggesting presence of additional pVir-virulence genes. Truncated p RmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, p RmpA/aerobactin, p RmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively. Interpretation These data can guide countermeasure development. Funding This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance. Copyright (c) 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Carriage of CTX-M-type extended-spectrum β-lactamase (ESBL) is rare in Pseudomonas aeruginosa. During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla CTX-M-15. A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla CTX-M-15. The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla CTX-M-15. Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla CTX-M-15. This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn4661-mediated transposition. ESBL bla CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa, likely initiated by plasmid transfer from a K. michiganensis strain.

Genomic surveillance detected clonal Escherichia coli sequence type-361 isolates carrying blaNDM-5, blaKPC-3, blaCTX-M-15, and rmtB1 from a patient in Ukraine and four wounded foreign soldiers evacuated to Germany. Isolates were non-susceptible to carbapenems, aminoglycosides, and cefiderocol and aztreonam/avibactam due to a PBP3 YRIN insertion and the blaCMY-145 AmpC β-lactamase. Coordinated surveillance efforts across civilian, military, and veteran healthcare systems are essential to prevent further spread as international volunteers return home after medical evacuation from Ukraine.

In 2003−2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19 PAS , 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23 , bla OXA-72 , and/or bla NDM-5 . Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Objectives:To better understand the source and potential transmission routes of antibiotic-resistant bacteria infecting injured service members in Ukraine.Methods:Phenotypic and genomic characterizations were performed on 11 Gram-negative pathogens cultured from war wounds at an intermediate evacuation hospital in Dnipro.Results:The analysis revealed both susceptible and extensively drug-resistant strains present in cultures, including high-risk global clones carrying carbapenemases.Conclusions:Globally distributed carbapenemase-producing lineages are being acquired early in the medical evacuation process.

Distinguishing hypervirulent (hvKp) from classical Klebsiella pneumoniae (cKp) strains is important for clinical care, surveillance, and research. Some combination of iucA, iroB, peg-344, rmpA, and rmpA2 are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g. siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Further, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49 K. pneumoniae strains that possessed some combination of iucA, iroB, peg-344, rmpA, and rmpA2 and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) (N=16) or cKp (N=33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid's Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression the area under the curve for biomarker count was 0.962 (P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥ 4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49) respectively. These findings can be used to inform the identification of hvKp. Importance:Hypervirulent Klebsiella pneumoniae (hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistant K. pneumoniae isolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all 5 of the biomarkers iucA, iroB, peg-344, rmpA, and rmpA2 was most accurate (94%); the presence of ≥ 4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which in turn would assist in optimizing patient care.

AbstractDistinguishing hypervirulent (hvKp) from classicalKlebsiella pneumoniae(cKp) strains is important for clinical care, surveillance, and research. Some combination ofiucA, iroB, peg-344, rmpA,andrmpA2are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g. siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Further, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49K. pneumoniaestrains that possessed some combination ofiucA, iroB, peg-344, rmpA,andrmpA2and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) (N=16) or cKp (N=33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid’s Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression the area under the curve for biomarker count was 0.962 (P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥ 4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49) respectively. These findings can be used to inform the identification of hvKp.ImportanceHypervirulentKlebsiella pneumoniae(hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistantK. pneumoniaeisolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all 5 of the biomarkersiucA, iroB, peg-344, rmpA,andrmpA2was most accurate (94%); the presence of ≥ 4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which in turn would assist in optimizing patient care.

A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts.

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip-flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis.