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Gram-negative bacilli (GNB) are currently the predominant bacterial pathogens in patients with cancer. Many GNB have become problematic due to the widespread emergence of resistance. Imipenem/relebactam (IMI/REL) is a combination of the carbapenem imipenem with relebactam, a non-β-lactam β-lactamase inhibitor. It is active against most pathogenic GNB including many that are resistant to other agents. We compared its in vitro activity to six other agents against 490 GNB recovered exclusively from patients with cancer because such data are scarce.Clinical and Laboratory Standards Institute (CLSI) microbroth dilution methods were used for susceptibility testing. Whole genome sequencing (Illumina MiSeq) was performed on 30 selected isolates.IMI/REL was active against 98% of Enterobacterales and 87% of non-Enterobacterales isolates (excluding Stenotrophomonas maltophilia). It had potent activity against extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae, and other Enterobacterales (Enterobacter cloacae, Citrobacter Spp., and Serratia Spp.) and moderate activity against carbapenem-resistant Enterobacterales. IMI/REL had potent activity against Achromobacter Spp., non-multidrug resistant Pseudomonas aeruginosa, and Sphingomonas paucimobilis and moderate activity against multidrug resistant P. aeruginosa. Overall, IMI/REL was associated with the lowest number of nonsusceptible isolates compared with six other agents (imipenem, meropenem, cefepime, piperacillin/tazobactam, amikacin, and tigecycline) commonly used in patients with cancer. Whole genome sequencing performed on 30 resistant isolates (10 each of E. coli, K. pneumonia, and P. aeruginosa) did not reveal any predominant mechanism of resistance to IMI/REL.Its in vitro activity indicates that IMI/REL might have a role to play in the treatment of Gram-negative infections in patients with cancer.

Currently, whole genome sequencing (WGS) data has not shown strong concordance withE. colisusceptibility profiles to the commonly used β-lactam/β-lactamase inhibitor (BL/BLI) combinations: ampicillin-sulbactam (SAM), amoxicillin-clavulanate (AMC), and piperacillin-tazobactam (TZP). Progressive resistance to these BL/BLIs in absence of cephalosporin resistance, also known as extended-spectrum resistance to BL/BLI (ESRI), has been suggested to primarily result from increased copy numbers ofblaTEMvariants, which is not routinely assessed in WGS data. We sought to determine whether addition of gene amplification could improve genotype-phenotype associations through WGS analysis of 147E. colibacteremia isolates with increasing categories of BL/BLI non-susceptibility ranging from ampicillin-susceptible to fully resistant to all three BL/BLIs. Consistent with a key role ofblaTEMin ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encodedblaTEM. Evidence ofblaTEMamplification (i.e.,blaTEMgene copy number estimates > 2×) was present in 40/112 (36%) strains. There were positive correlations betweenblaTEMcopy numbers with minimum inhibitory concentrations (MICs) of AMC and TZP (P-value < 0.05), but not for SAM (P-value = 0.09). The diversity of β-lactam resistance mechanisms, including non-ceftriaxone hydrolyzingblaCTX-Mvariants,blaOXA-1, as well asampCandblaTEMstrong promoter mutations, were greater in AMC and TZP non-susceptible strains but rarely observed within SAM and AMP non-susceptible isolates. Our study indicates a comprehensive analysis of WGS data, including β-lactamase encoding gene amplification, can help categorizeE. coliwith AMC or TZP non-susceptibility but that discerning the transition from SAM susceptible to non-susceptible using genetic data requires further refinement.ImportanceThe increased feasibility of whole genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptibleE. colibacteremia samples to determine how increasing dosage of narrow-spectrum β-lactamase encoding genes in conjunction with other diverse BL/BLI genetic determinants contribute to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of β-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

A major cause of mortality in hematologic malignancy patients is BSI. Previous studies have demonstrated that bacterial translocation from the GI microbiome is a major source of BSIs and is often preceded by increased levels of the infectious taxa in the GI (>30% abundance by 16S rRNA sequencing).

BackgroundBacterial infections are common in patients with cancer, and many bacteria have developed resistance to currently used antibiotics.ObjectivesWe evaluated the in vitro activity of eravacycline (a recently developed fluorocycline) and comparators against bacterial pathogens isolated from patients with cancer.MethodsAntimicrobial susceptibility testing was performed using CLSI-approved methodology and interpretive criteria for 255 Gram-positive and 310 Gram-negative bacteria. MIC and susceptibility percentage were calculated according to CLSI and FDA breakpoints when available.ResultsEravacycline had potent activity against most Gram-positive bacteria, including MRSA. Of 80 Gram-positive isolates with available breakpoints, 74 (92.5%) were susceptible to eravacycline. Eravacycline had potent activity against most Enterobacterales, including ESBL-producing organisms. Of 230 Gram-negative isolates with available breakpoints, 201 (87.4%) were susceptible to eravacycline. Eravacycline had the best activity among comparators against carbapenem-resistant Enterobacterales, with 83% susceptibility. Eravacycline was also active against many non-fermenting Gram-negative bacteria, with the lowest MIC90 value among comparators.ConclusionsEravacycline was active against many clinically significant bacteria isolated from patients with cancer, including MRSA, carbapenem-resistant Enterobacterales, and non-fermenting Gram-negative bacilli. Eravacycline might play an important role in the treatment of bacterial infections in patients with cancer, and additional clinical evaluation is warranted.

BackgroundFluoroquinolones are used for infection prevention in high-risk patients with haematological malignancies. Fluoroquinolones are active against many Gram-negative bacilli (GNB) but are less active against Gram-positive organisms. We evaluated the in vitro activity of delafloxacin and selected comparators against 560 bacterial pathogens isolated exclusively from patients with cancer.MethodsAntimicrobial susceptibility testing and time-kill studies were performed using CLSI-approved methodology and interpretive criteria for 350 Gram-positive organisms and 210 GNB that had been recently isolated from patients with cancer.ResultsDelafloxacin was more active than ciprofloxacin and levofloxacin against Staphylococcus aureus and CoNS. Overall, 63% of staphylococcal isolates were susceptible to delafloxacin, 37% to ciprofloxacin and 39% to levofloxacin. Activity of delafloxacin against most Enterobacterales was similar to that of ciprofloxacin and levofloxacin. Escherichia coli and MDR Pseudomonas aeruginosa isolates had low susceptibility rates to the three tested fluoroquinolones. In time-kill studies delafloxacin and levofloxacin decreased the bacterial load to 3.0 log10 in 8 and 13 h, respectively, using 8 × MIC.ConclusionsDelafloxacin is more active than ciprofloxacin and levofloxacin against S. aureus but has substantial gaps in coverage against GNB. Resistance to all three fluoroquinolones could be high among leading GNB such as E. coli and P. aeruginosa, particularly in cancer centres where these agents are widely used as prophylactic agents.

Extended-spectrum cephalosporin resistantEscherichia coli(ESC-R-Ec) is an urgent public health threat with sequence type clonal complex 131 (STc131), phylogroup B2 strains being particularly concerning as the dominant cause of ESC-R-Ecinfections. To address the paucity of recent ESC-R-Ecmolecular epidemiology data in the United States, we used whole genome sequencing (WGS) to fully characterize a large cohort of invasive ESC-R-Ecat a tertiary care cancer center in Houston, Texas collected from 2016-2020. During the study timeframe, there were 1154 indexE. colibloodstream infections (BSIs) of which 389 (33.7%) were ESC-R-Ec. Using time series analyses, we identified a temporal dynamic of ESC-R-Ecdistinct from ESC-susceptibleE. coli(ESC-S-Ec), with cases peaking in the last six months of the calendar year. WGS of 297 ESC-R-Ecstrains revealed that while STc131 strains accounted for ∼45% of total BSIs, the proportion of STc131 strains remained stable across the study time frame with infection peaks driven by genetically heterogeneous ESC-R-Ecclonal complexes.BlaCTX-Mvariants accounted for most β-lactamases conferring the ESC-R phenotype (89%; 220/248 index ESC-R-Ec), and amplification ofblaCTX-Mgenes was widely detected in ESC-R-Ecstrains, particularly in carbapenem non-susceptible, recurrent BSI strains.BlaCTX-M-55was significantly enriched within phylogroup A strains, and we identifiedblaCTX-M-55plasmid-to-chromosome transmission occurring across non-B2 strains. Our data provide important information regarding the current molecular epidemiology of invasive ESC-R-Ecinfections at a large tertiary care cancer center and provide novel insights into the genetic basis of observed temporal variability for these clinically important pathogens.IMPORTANCEGiven thatE. coliis the leading cause of worldwide ESC-REnterobacteralesinfections, we sought to assess the current molecular epidemiology of ESC-R-Ecusing a WGS analysis of many BSIs over a five-year period. We identified fluctuating temporal dynamics of ESC-R-Ecinfections, which has also recently been identified in other geographical regions such as Israel. Our WGS data allowed us to visualize the stable nature of STc131 over the study period and demonstrate a limited, but genetically diverse group of ESC-R-Ecclonal complexes are detected during infection peaks. Additionally, we provide a widespread assessment of β-lactamase gene copy number in ESC-R-Ecinfections and delineate mechanisms by which such amplifications are achieved in a diverse array of ESC-R-Ecstrains. These data suggest that serious ESC-R-Ecinfections are driven by a diverse array of strains in our cohort and impacted by environmental factors suggesting that community-based monitoring could inform novel preventative measures.

ObjectivesTo characterize ablaCMYvariant associated with ceftazidime-avibactam (CZA) resistance from a serially collectedEscherichia coliisolate.MethodsA patient with an intra-abdominal infection due to recurrentE. coliwas treated with CZA. On day 48 of CZA therapy,E. coliwith a CZA MIC of >256 mg/L was identified from abdominal drainage. Illumina WGS was performed on all isolates to identify potential resistance mechanisms. Site-directed mutants of CMY β-lactamase were constructed to identify amino acid residues responsible for CZA resistance.ResultsWGS revealed that all three isolates wereE. coliST410. The CZA-resistant strain uniquely acquired a novel CMY β-lactamase gene, herein calledblaCMY-185, harbored on an IncIγ-type conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2 including A114E, Q120K, V211S, and N346Y and conferred high-level CZA resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced CZA susceptibility. However, double and triple mutants containing N346Y previously associated with CZA resistance in other AmpC enzymes, conferred CZA MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to the steric hindrance between the side chain of Y346 and the sulfate group of avibactam.ConclusionWe identified CZA resistance inE. coliassociated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer CZA resistance.

In the United States, vanB-mediated resistance in enterococci is rare. We characterized three sequence type (ST) 6, vancomycin-resistant Enterococcus faecalis isolates causing bacteremia in unique patients in spatiotemporally distinct settings. Isolates were recovered between 2018 and 2020 in two cities in the United States (Houston, TX; Miami, FL). The isolates harbored the vanB operon on a chromosomally located Tn1549 transposon, and epidemiological data suggested multiple introductions of the vanB gene cluster into ST6 E. faecalis.

Background Whole-genome sequencing has gained interest for assessing antimicrobial resistance (AMR). However, multiple studies have shown a poor correlation between the β-lactam/β-lactamase inhibitor (BL/BLI) phenotype and β-lactamase gene presence/absence. In a recent publication, we found that amplification of β-lactamase encoding genes potentially contribute to BL/BLI resistance and here we explore if gene amplifications allow for better BL/BLI genotype/phenotype correlations. Methods We selected 109 E. coli bacteremia isolates and performed ETests (bioMerieux, Inc) to determine AST profiles using the following BL/BLI antibiotics: ampicillin/sulbactam (SAM), amoxicillin/clavulanic acid (AMC), and piperacillin/tazobactam (TZP). CLSI M100 guidelines (2018) were used to stratify isolates into 4 groups: SAM/AMC/TZP susceptible (Group 1), SAM resistant only (Group 2), SAM/AMC resistant (Group 3), and SAM/AMC/TZP resistant (Group 4). Short-read whole genome sequencing was performed on these isolates and analyzed by identifying AMR genes, establishing multi-locus sequence type (MLST), estimating copy number variants (CNV), and investigating blaTEM-1 promoter regions in relation to phenotype. A t-Distributed Stochastic Neighbor Embedding (t-SNE) clustering method and a core-genome, maximum-likelihood phylogeny was generated to group isolates based on AMR gene content and CNV data. Results We found 34 different MLST groups with the three most common sequence types ST131 (n=41), ST1193 (n=12), and ST648 (n=6). Group 1 and Group 2 isolates had similar copy number estimates of β-lactamase blaTEM-1 (1.8X and 1.5X respectively) while there was an increase for both Group 3 (3.7X) and Group 4 (8.2X) isolates (Table 1). Additionally, blaOXA-1 was primarily present in Group 4 isolates (63%) with elevated copy numbers (5.2X). We did not observe associations of blaTEM-1 promoter regions with each of the BL/BLI Groups. Our t-SNE analysis shows that Group 1, Group 3, and Group 4 isolates cluster independently. Core Genome Maximum-likelihood Phylogeny. Midpoint-rooted core-genome maximum-likelihood phylogeny comparing core elements (MLST) to non core elements (CNV). AMR CNV and presence/absence associate better with BL/BLI phenotypes than do core elements. Conclusion There is a clear delineation between fully susceptible and fully resistant BL/BLI E. coli isolates when gene amplification and presence/absence of narrow-spectrum β-lactamases are considered, which should be considered for BL/BLI resistance prediction models. Disclosures David E. Greenberg, MD, Shionogi: Grant/Research Support.

Background Penicillin-binding protein 4 (PBP4) is a low affinity PBP that has been associated with decreased susceptibility to penicillins in Enterococcus faecalis (Efs). In vitro data have shown that changes in the promoter region leading to increased pbp4 gene expression and amino acid changes resulting in active site remodeling contribute to this phenotype. There is limited data on the prevalence of these strains in the United States. We investigated β-lactam susceptibility trends in association with variations in PBP4 (allotypes) and the upstream promoter region. Methods Efs bloodstream isolates (n=184) were selected from the multicenter VENOUS cohort from 2016 to 2021. Whole genome sequencing (WGS) was performed on all isolates, and changes in the pbp4 gene and promoter region 200 bp upstream of the start codon were identified using Efs JH2-2 as reference. Broth microdilution (BMD) testing for ampicillin (AMP), penicillin (PCN), piperacillin (PIP), and imipenem (IMI) was performed for 81 isolates. Analysis of MICs vs. WGS results was performed. Results A total of 31 PBP4 allotypes and 10 promoter variations were identified. ST6 isolates most frequently carried the promoter mutation ΔA117 (P6), previously shown to increase expression of the pbp4 gene, with allotype 1 PBP4 (Fig 1). ST179 isolates most frequently carried the JH2-2 wild type promoter (P1) with the allotype 30 PBP4. All isolates were susceptible to AMP (MIC50 ≤1 µg/mL, MIC90 2 µg/mL) and PCN (MIC50 ≤2 µg/mL, MIC90 4 µg/mL; Table 1). PIP was the least potent β-lactam, with an MIC50 4 µg/mL and an MIC90 of 8 µg/mL. Isolates with the P6 promoter had significantly higher piperacillin MICs (p< 0.0001) as compared to P1. Figure 1.Phylogenetic Tree of E. faecalis Strains Conclusion Changes in the pbp4 gene promoter correlated with an increase in PIP MICs. Caution should be used when choosing β-lactams other than AMP for definitive treatment deep-seated Efs infections. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support William R. Miller, MD, Entasis Therapeutics: Grant/Research Support|UpToDate: Royalties - Topic Contributor.

Background Patients with leukemia are disproportionately vulnerable to infections caused by S. maltophilia and mortality approaches 100% in patients with hemorrhagic pneumonia. Trimethoprim-sulfamethoxazole (SXT) is the drug of choice for treatment of infections caused by S. maltophilia however allergies, toxicity, and resistance may preclude its use. We sought to characterize clinical outcomes of patients with leukemia and S. maltophilia bloodstream infection (BSI). Methods Patients with leukemia are disproportionately vulnerable to infections caused by S. maltophilia and mortality approaches 100% in patients with hemorrhagic pneumonia. Trimethoprim-sulfamethoxazole (SXT) is the drug of choice for treatment of infections caused by S. maltophilia however allergies, toxicity, and resistance may preclude its use. We sought to characterize clinical outcomes of patients with leukemia and S. maltophilia bloodstream infection (BSI). Results Ninety-six patients with leukemia and S. maltophilia BSI were identified. The most common diagnosis was acute myeloid leukemia (60%); 31% had prior stem cell transplant. The most common single identified source was respiratory (31%) followed by central venous catheter (CVC, 26%). A single clear source was unable to be identified in 33%.Overall 14-day mortality was 31%. After excluding eleven patients who died within 24 hours, most patients received SXT (72%), followed by ceftazidime (40%), tigecycline (TGC, 40%), and minocycline (26%). SXT was associated with reduced mortality (hazard ratio [HR] 0.15, 95% confidence interval [CI] 0.05 – 0.44, p < 0.01) while TGC was associated with increased mortality (HR 3.13, 95% CI 1.00 – 9.73, p = 0.05). All patients who died were neutropenic at baseline and none had a CVC infection. Conclusion S. maltophilia BSI is associated with high mortality in patients with leukemia, particularly those with neutropenia at baseline. CVC infection alone was not associated with mortality. SXT is associated with reduced mortality relative to other antimicrobials. Alternative agents should be used with caution in these vulnerable patients. Disclosures Samuel L. Aitken, PharmD, MPH, Entasis Therapeutics: Advisor/Consultant|GlaxoSmithKline: Advisor/Consultant|Melinta: Grant/Research Support|Shionogi: Advisor/Consultant Pablo C. Okhuysen, MD, AstraZeneca: Stocks/Bonds|Beam Therapeutics: Stocks/Bonds|Biontech: Stocks/Bonds|Deinove: Grant/Research Support|Ferring: Advisor/Consultant|Glaxo Smith Kleine: Stocks/Bonds|Johnson and Johnson: Stocks/Bonds|Melinta: Grant/Research Support|Merck Sharp & Dohme Corp: Grant/Research Support|Moderna: Stocks/Bonds|Napo Pharmaceuticals: Advisor/Consultant|Napo Pharmaceuticals: Grant/Research Support|Novavax: Stocks/Bonds|Pfizer: Stocks/Bonds|Summit: Advisor/Consultant|Summit: Grant/Research Support.

Background Non-E. faecium non-E. faecalis (NFF) enterococci are a heterogeneous group of organisms consisting of enterococci with intrinsic low-level vancomycin resistance as well as several other species known to infect humans. Patients with cancer are at increased risk for bloodstream infections (BSIs) with NFF enterococci, but their optimal treatment and outcomes have not been extensively described. Methods We conducted a retrospective review of patients (pts) with blood cultures positive for all NFF by searching the microbiology database at a major cancer center in Houston, TX from 2016 to 2021. Patients were included if they were ≥ 18 years old, had ≥ 1 blood culture with NFF enterococci, and had a repeat blood culture within 7 days (d) of the index culture, to permit outcome analysis. Outcomes were a) in-hospital mortality, b) microbiological failure (blood culture clearance >4 d after index culture), and c) recurrence of bacteremia (new positive blood culture < 14 d after eradication). Results Sixty-two unique pts had NFF enterococcal BSI. Patients with hematological malignancy made up 53.2% of the cohort (78% had leukemia). The majority (83%) of solid malignancies were pancreatic, biliary, or intestinal in origin. In the 30 d preceding NFF enterococcal BSI, 79% of pts had exposure to antibiotics. The NFF species isolated were E. gallinarum (50%), E. casseliflavus (30.6%), E. avium (11.3%), E. raffinosus (3.2%), E. hirae (3.2%), and E. durans (1.6%). Only 23.3% of NFF enterococci were susceptible to vancomycin, and 53.8% were susceptible to linezolid. Most (62.9%) pts received combination therapy; the most frequent was daptomycin plus a beta-lactam (33.9%). Bacteremia recurred in 8.1% of pts, 27.4% had in-hospital mortality, and 4.8% had microbiological failure. Conclusion This retrospective review provides preliminary insight into the treatments, and outcomes of cancer pts with NFF enterococcal BSIs. The NFF enterococci isolates in this study had a higher rate of linezolid non-susceptibility than previously reported. Molecular investigation of these NFF enterococcal isolates is needed to elucidate the mechanisms and transmission of antibiotic resistance. Comparison of BSIs caused by NFF enterococci to E. faecium/E. faecalis will provide insight to physicians caring for these pts. Disclosures Cesar A. Arias, MD, PhD, Entasis: Grant/Research Support|MeMed Diagnostics Ltd: Grant/Research Support|Merck: Grant/Research Support.

Background Blood stream infections (BSI) are a common cause of morbidity and mortality in immunocompromised patients. It was previously reported that intestinal domination (defined as occupation of ≥ 30% of the microbiota by a single bacterial taxon) by Enterococcus and Proteobacteria often preceded enterococcal and gram-negative BSI. Methods 16s rRNA sequencing was used to analyze the saliva and stool of 73 acute leukemia patients with BSIs to determine the correlation between the taxa present ( > 0% relative abundance), colonizing ( > 3%) or dominating ( > 30%) a patient’s microbiome and the BSI agent. Species level confirmation of these findings was performed via digital droplet PCR (ddPCR) and LEfSe analysis was used to determine enrichment of infectious taxa at either collection site. Whole genome sequencing and antimicrobial susceptibilities on all BSI isolates. Results We collected simultaneous patient stool and blood isolates from 11 Pseudomonas aeruginosa, 14 Escherichia coli, 4 Klebsiella sp., 8 Enterococcus sp., 18 viridans group streptococci (VGS), 13 Staphylococcus epidermidis, and 5 Staphylococcus aureus BSIs. Mann-Whitney testing confirmed that individuals with P. aeruginosa (oral P =.004, stool P < .001), E. coli (stool P < .001), and VGS infections (oral P =.001) had significantly higher relative abundance of those respective genera compared to other BSI patients, and this appeared to be site specific. LEfSe analysis confirmed enrichment of Pseudomonas in the saliva and stool, Escherichia in the stool, and Streptococcus in the saliva of respective infections. However, while 64 patients showed presence of the infectious genera in the stool and/or saliva at time of BSI, only 7 exhibited domination. ddPCR was used to confirm not only species specificity, but the detection of those antibiotic resistance determinants (CTX-14, CTX-15, Van A and CfrA) found in the BSI isolates within concurrent stool samples. Conclusion It appears that GI domination by the infecting organism was not present at the time of most BSIs. However, in scenarios where the etiological agent was detectable in the saliva or stool, so were the antibiotic resistant determinants. Furthermore, certain infections exhibited a significant difference in oral or stool presence by the same bacteria they were infected with. Disclosures All Authors: No reported disclosures.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant ( n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila , a species of mucin-degrading bacteria ( P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

Antimicrobial resistance-encoding mobile genetic elements (MGEs) may contribute to the disease potential of bacterial pathogens. We previously described the association of Group A Streptococcus (GAS) derived from invasive disease with increasingly frequent antimicrobial resistance (AMR). We hypothesized that a 65-kb AMR-encoding MGE (ICESpyM92), highly conserved among closely related emergent invasive emm92 GAS, contributes to GAS disease potential. Here, we provide evidence that a combination of ICESpyM92- and core genome-dependent differential gene expression (DGE) contributes to invasive disease phenotypes of emergent emm92 GAS. Using isogenic ICESpyM92 mutants generated in distinct emm92 genomic backgrounds, we determined the presence of ICESpyM92 enhances GAS virulence in a mouse subcutaneous infection model. Measurement of in vitro and ex vivo DGE indicates ICESpyM92 influences GAS global gene expression in a background-dependent manner. Our study links virulence and AMR on a unique MGE via MGE-related DGE and highlights the importance of investigating associations between AMR-encoding MGEs and pathogenicity.

Ceftriaxone-resistant (CRO-R)This is a prospective cohort of patients withNotable differences between patients infected with CRO-R and CRO-SPatients with CRO-R

Background Ceftolozane/tazobactam is a β-lactam/β-lactamase inhibitor combination with activity against a variety of Gram-negative bacteria, including MDR Pseudomonas aeruginosa. This agent is approved for hospital-acquired and ventilator-associated bacterial pneumonia. However, most real-world outcome data come from small observational cohorts. Thus, we sought to evaluate the utilization of ceftolozane/tazobactam at multiple tertiary hospitals in Houston, TX, USA. Methods We conducted a multicentre retrospective study of patients receiving at least 48 h of ceftolozane/tazobactam therapy from January 2016 through to September 2019 at two hospital systems in Houston. Demographic, clinical and microbiological data were collected, including the infecting bacterial isolate, when available. The primary outcome was composite clinical success at hospital discharge. Secondary outcomes included in-hospital mortality and clinical disposition at 14 and 30 days post ceftolozane/tazobactam initiation. Multivariable logistic regression analysis was used to identify predictors of the primary outcome and mortality. Recovered isolates were tested for susceptibility to ceftolozane/tazobactam and underwent WGS. Results A total of 263 patients were enrolled, and composite clinical success was achieved in 185 patients (70.3%). Severity of illness was the most consistent predictor of clinical success. Combination therapy with ceftolozane/tazobactam and another Gram-negative-active agent was associated with reduced odds of clinical success (OR 0.32, 95% CI 0.16–0.63). Resistance to ceftolozane/tazobactam was noted in 15.4% of isolates available for WGS; mutations in ampC and ftsI were common but did not cluster with a particular ST. Conclusions Clinical success rate among this patient cohort treated with ceftolozane/tazobactam was similar compared with previous experiences. Ceftolozane/tazobactam remains an alternative agent for treatment of susceptible isolates of P. aeruginosa.

Skin and soft tissue infections among the non-human immunodeficiency virus infected immunosuppressed population are a serious and growing concern. Many pathogens can cause cutaneous infections in these patients owing to the highly varied and profound immune deficits. Although patients can be infected by typical organisms, the diversity and antimicrobial-resistant nature of the organisms causing these infections result in significant morbidity and mortality. The diagnostic approach to these infections in immunocompromised hosts can differ dramatically depending on the potential causative organisms. An understanding of new immunosuppressive treatments and evolving antimicrobial resistance patterns are required to optimally manage these difficult cases.

The Practice Guidelines Committee of the American Society of Transplantation and Cellular Therapy partnered with its Transplant Infectious Disease Special Interest Group to update its 2009 compendium-style infectious diseases guidelines for hematopoietic cell transplantation (HCT). A completely fresh approach was taken, with the goal of better serving clinical providers by publishing each stand-alone topic in the infectious diseases series in a concise format of frequently asked questions (FAQs), tables, and figures [1]. Adult and pediatric infectious diseases and HCT content experts developed and then answered FAQs, and then finalized topics with harmonized recommendations that were made by assigning a strength of recommendation ranging from A to E paired with a level of supporting evidence graded I to III. The first topic in the series focuses on potentially life-threatening infections in HCT caused by Enterobacterales, relevant infection risk factors, and practical considerations regarding prevention and treatment of these infections in the setting of emerging multidrug resistance.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram‐positive bacteria. Most functional assessments of CcpA, including interaction with its key co‐factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA‐mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP‐seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL‐8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221‐CcpA‐V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP‐seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild‐type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr‐dependent and independent activities of CcpA in a key pathogenic bacterium., The carbon catabolite protein, CcpA, regulates carbohydrate utilization and virulence in concert with its phosphorylated co‐factor histine containing protein (HPr~P). In this study we engineered a CcpA isoform that does not interact with HPr~P. Using a combination of ChIPseq and RNAseq we report the in vivo binding sites of CcpA in group A Streptococcus. We also demonstrate that CcpA retains the ability to bind and regulate genes even when it cannot interact with HPr~P.

Stenotrophomonas maltophilia is a non-fermenting, Gram-negative bacillus that has emerged as an opportunistic nosocomial pathogen. Its intrinsic multidrug resistance makes treating infections caused by S. maltophilia a great clinical challenge. Clinical management is further complicated by its molecular heterogeneity that is reflected in the uneven distribution of antibiotic resistance and virulence determinants among different strains, the shortcomings of available antimicrobial susceptibility tests and the lack of standardized breakpoints for the handful of antibiotics with in vitro activity against this microorganism. Herein, we provide an update on the most recent literature concerning these issues, emphasizing the impact they have on clinical management of S. maltophilia infections.

Stenotrophomonas maltophilia is a non-fermenting, Gram-negative bacillus that has emerged as an opportunistic nosocomial pathogen. Its intrinsic multidrug resistance makes treating infections caused by S. maltophilia a great clinical challenge. Clinical management is further complicated by its molecular heterogeneity that is reflected in the uneven distribution of antibiotic resistance and virulence determinants among different strains, the shortcomings of available antimicrobial susceptibility tests and the lack of standardized breakpoints for the handful of antibiotics with in vitro activity against this microorganism. Herein, we provide an update on the most recent literature concerning these issues, emphasizing the impact they have on clinical management of S. maltophilia infections.

The prevalence of carbapenem-resistant Klebsiella pneumoniae (CR Kp ) infections in nosocomial settings remains a public health challenge. High-risk clones such as clonal group 258 (CG258) are particularly concerning due to their association with bla KPC carriage, which can severely complicate antimicrobial treatments.

Failure to maintain segregation of oral and gut microbial communities has been linked to several diseases. We sought to characterize oral-fecal microbiome community coalescence, ectopic extension of oral bacteria, clinical variables contributing to this phenomenon, and associated infectious consequences by analyzing the 16S rRNA V4 sequences of longitudinal fecal (n=551) and oral (n=737) samples from 97 patients with acute myeloid leukemia (AML) receiving induction chemotherapy (IC). Clustering observed in permutation based multivariate analysis of variance (PERMANOVA) of Bray-Curtis dissimilarity and PCoA plot of UniFrac distances between intra-patient longitudinal oral-stool sample pairs suggested potential oral-stool microbial community coalescence. Bray-Curtis dissimilarities and UniFrac distances were used to create an objective definition of microbial community coalescence. We determined that only 23 of the 92 patients exhibited oral-stool community coalescence. This was validated through a linear mixed model which determined that patients who experienced coalescence had an increased proportion of shared to unique OTUs between their oral-stool sample pairs over time compared to non-coalesced patients. Evaluation of longitudinal microbial characteristics revealed that patients who experienced coalescence had increased stool abundance of Streptococcus and Stenotrophomonas compared to non-coalesced patients. When treated as a time-varying covariate, each additional day of linezolid (HR 1.15, 95% CI 1.06 – 1.24, P

Background Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. Objectives To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. Methods This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT–PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. Results Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. Conclusions These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Background The role of enteropathogenic Escherichia coli (EPEC) as a cause of diarrhea in cancer and immunocompromised patients is controversial. Quantitation of fecal bacterial loads has been proposed as a method to differentiate colonized from truly infected patients. Methods We studied 77 adult cancer and immunosuppressed patients with diarrhea and EPEC identified in stools by FilmArray, 25 patients with pathogen-negative diarrhea, and 21 healthy adults without diarrhea. Stools were studied by quantitative polymerase chain reaction (qRT-PCR) for EPEC genes eaeA and lifA/efa-1 and strains characterized for virulence factors and adherence to human intestinal enteroids (HIEs). Results Patients with EPEC were more likely to have community-acquired diarrhea (odds ratio, 3.82 [95% confidence interval, 1.5–10.0]; P = .008) compared with pathogen-negative cases. Although EPEC was identified in 3 of 21 (14%) healthy subjects by qPCR, the bacterial burden was low compared to patients with diarrhea (≤55 vs median, 6 × 104 bacteria/mg stool; P < .001). Among EPEC patients, the bacterial burden was higher in those who were immunosuppressed (median, 6.7 × 103 vs 55 bacteria/mg; P < .001) and those with fecal lifA/ifa-1 (median, 5 × 104 vs 120 bacteria/mg; P = .015). Response to antimicrobial therapy was seen in 44 of 48 (92%) patients with EPEC as the sole pathogen. Antimicrobial resistance was common and strains exhibited distinct patterns of adherence with variable cytotoxicity when studied in HIEs. Cancer care was delayed in 13% of patients. Conclusions Immunosuppressed cancer patients with EPEC-associated diarrhea carry high burden of EPEC with strains that are resistant to antibiotics, exhibit novel patterns of adherence when studied in HIEs, and interfere with cancer care.

Background Vancomycin-resistant enterococci (VRE) are major therapeutic challenges. Prospective contemporary data characterizing the clinical and molecular epidemiology of VRE bloodstream infections (BSIs) are lacking. Methods The Vancomycin-Resistant Enterococcal BSI Outcomes Study (VENOUS I) is a prospective observational cohort of adult patients with enterococcal BSI in 11 US hospitals. We included patients with Enterococcus faecalis or Enterococcus faecium BSI with ≥1 follow-up blood culture(s) within 7 days and availability of isolate(s) for further characterization. The primary study outcome was in-hospital mortality. Secondary outcomes were mortality at days 4, 7, 10, 12, and 15 after index blood culture. A desirability of outcome ranking was constructed to assess the association of vancomycin resistance with outcomes. All index isolates were subjected to whole genome sequencing. Results Forty-two of 232 (18%) patients died in hospital and 39 (17%) exhibited microbiological failure (lack of clearance in the first 4 days). Neutropenia (hazard ratio [HR], 3.13), microbiological failure (HR, 2.4), VRE BSI (HR, 2.13), use of urinary catheter (HR, 1.85), and Pitt BSI score ≥2 (HR, 1.83) were significant predictors of in-hospital mortality. Microbiological failure was the strongest predictor of in-hospital mortality in patients with E faecium bacteremia (HR, 5.03). The impact of vancomycin resistance on mortality in our cohort changed throughout the course of hospitalization. Enterococcus faecalis sequence type 6 was a predominant multidrug-resistant lineage, whereas a heterogeneous genomic population of E faecium was identified. Conclusions Failure of early eradication of VRE from the bloodstream is a major factor associated with poor outcomes.

Although prokaryotic DNA methylation investigations have long focused on immunity against exogenous DNA, it has been recently recognized that DNA methylation impacts gene expression and phase variation in Streptococcus pneumoniae and Streptococcus suis. A comprehensive analysis of DNA methylation is lacking for beta-hemolytic streptococci, and thus we sought to examine DNA methylation in the major human pathogen group A Streptococcus (GAS). Using a database of 224 GAS genomes encompassing 80 emm types, we found that nearly all GAS strains encode a type I restriction modification (RM) system that lacks the hsdS′ alleles responsible for impacting gene expression in S. pneumoniae and S. suis. The GAS type I system is located on the core chromosome, while sporadically present type II orphan methyltransferases were identified on prophages. By combining single-molecule real-time (SMRT) analyses of 10 distinct emm types along with phylogenomics of 224 strains, we were able to assign 13 methylation patterns to the GAS population. Inactivation of the type I RM system, occurring either naturally through phage insertion or through laboratory-induced gene deletion, abrogated DNA methylation detectable via either SMRT or MinION sequencing. Contrary to a previous report, inactivation of the type I system did not impact transcript levels of the gene (mga) encoding the key multigene activator protein (Mga) or Mga-regulated genes. Inactivation of the type I system significantly increased plasmid transformation rates. These data delineate the breadth of the core chromosomal type I RM system in the GAS population and clarify its role in immunity rather than impacting Mga regulon expression. IMPORTANCE The advent of whole-genome approaches capable of detecting DNA methylation has markedly expanded appreciation of the diverse roles of epigenetic modification in prokaryotic physiology. For example, recent studies have suggested that DNA methylation impacts gene expression in some streptococci. The data described herein are from the first systematic analysis of DNA methylation in a beta-hemolytic streptococcus and one of the few analyses to comprehensively characterize DNA methylation across hundreds of strains of the same bacterial species. We clarify that DNA methylation in group A Streptococcus (GAS) is primarily due to a type I restriction modification (RM) system present in the core genome and does not impact mga-regulated virulence gene expression, but does impact immunity against exogenous DNA. The identification of the DNA motifs recognized by each type I RM system may assist with optimizing methods for GAS genetic manipulation and help us understand how bacterial pathogens acquire exogenous DNA elements.

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria, including group A Streptococcus (GAS), and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA sequencing on isogenic deletion mutants fixed in a LiaFSR “always on” (ΔliaF) or “always off” (ΔliaR) state. Transcriptome analyses of ΔliaF and ΔliaR in GAS showed near perfect inverse correlation, including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Furthermore, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, ΔliaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription with ΔliaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease—a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system cross talk shared by many Gram-positive pathogens.

Clonal replacement is a major driver for changes in bacterial disease epidemiology. Recently, it has been proposed that episodic emergence of novel, hypervirulent clones of group A Streptococcus (GAS) results from acquisition of a 36-kb DNA region leading to increased expression of the cytotoxins Nga (NADase) and SLO (streptolysin O). We previously described a gene fusion event involving the gene encoding the GAS M protein (emm) and an adjacent M-like protein (enn) in the emm4 GAS population, a GAS emm type that lacks the hyaluronic acid capsule. Using whole-genome sequencing of a temporally and geographically diverse set of 1,126 isolates, we discovered that the North American emm4 GAS population has undergone clonal replacement with emergent GAS strains completely replacing historical isolates by 2017. Emergent emm4 GAS strains contained a handful of small genetic variations, including the emm-enn gene fusion, and showed a marked in vitro growth defect compared to historical strains. In contrast to other previously described GAS clonal replacement events, emergent emm4 GAS strains were not defined by acquisition of exogenous DNA and had no significant increase in transcript levels of nga and slo toxin genes via RNA sequencing and quantitative real-time PCR analysis relative to historic strains. Despite the in vitro growth differences, emergent emm4 GAS strains were hypervirulent in mice and ex vivo growth in human blood compared to historical strains. Thus, these data detail the emergence and dissemination of a hypervirulent acapsular GAS clone defined by small, endogenous genetic variation, thereby defining a novel model for GAS strain replacement. IMPORTANCE Severe invasive infections caused by group A Streptococcus (GAS) result in substantial morbidity and mortality in children and adults worldwide. Previously, GAS clonal strain replacement has been attributed to acquisition of exogenous DNA leading to novel virulence gene acquisition or increased virulence gene expression. Our study of type emm4 GAS identified emergence of a hypervirulent GAS clade defined by variation in endogenous DNA content and lacking augmented toxin gene expression relative to replaced strains. These findings expand our understanding of the molecular mechanisms underlying bacterial clonal emergence.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKp) are urgent public health threats. Worldwide dissemination of CRKp has been largely attributed to the clonal group (CG) 258. However, recent evidence indicates the global emergence of a CRKp CG307 lineage. Houston, Texas is the first large city in the US with co-circulation of both CRKp CG307 and CG258. We sought to characterize the genomic and clinical factors contributing to the parallel endemic spread of CG258 and CG307.MethodsCRKp isolates were collected as part of the prospective, Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacterales 2 (CRACKLE-2) study. Hybrid short-read and long-read genome assemblies were generated from 119 CRKp isolates (95 originated from Houston hospitals). A comprehensive characterization of phylogenies, gene transfer, and plasmid content with pan-genome analysis were performed on all CRKp isolates. Plasmid mating experiments were performed with CG307 and CG258 isolates of interest. An inverse-probability weighted Desirability of Ordinal Outcome Ranking (DOOR) analysis was conducted to determine if patients infected/colonized with CG307 had differences in overall clinical outcomes from patients infected/colonized with CG258.ResultsDissection of the accessory genomes suggested independent evolution and limited horizontal gene transfer between CG307 and CG258 lineages. CG307 contained a diverse repertoire of mobile genetic elements harboring carbapenemases, which were shared with other non-CG258 K. pneumoniae isolates. Three unique clades of Houston CG307 isolates contained a diverse repertoire of mobile genetic elements harboring carbapenemases and clustered distinctly from other global CG307 isolates. CG307 were often isolated from the urine of hospitalized patients, likely serving as important reservoirs for genes encoding carbapenemases and extended-spectrum beta-lactamases. The DOOR probability estimate (64%; 95% CI: 48, 79) of our Houston-based cohort suggested that there was a general trend for patients infected/colonized with CG307 to have more favorable outcomes than patients infected/colonized with CG258.ConclusionsOur findings suggest parallel co-circulation of high-risk lineages with potentially divergent evolution. CG307 is widely circulating CRKp clone in the Houston region with the potential to transfer major resistance determinants to other non-CG258 CRKp lineages. Our findings provide major insights into the mechanism of epidemic spread of CRKp.

BACKGROUND: The treatment of osteomyelitis can be challenging because of poor antibiotic penetration into the infected bone and toxicities associated with prolonged antibiotic regimens to control infection. Irreversible electroporation (IRE), a percutaneous image-guided ablation technology in which the targeted delivery of high-voltage electrical pulses permanently damages the cell membrane, has been shown to effectively control bacterial growth in various settings. However, IRE for the management of bone infections has yet to be evaluated. QUESTIONS/PURPOSES: We aimed to evaluate IRE for treating osteomyelitis by assessing (1) the efficacy of IRE to suppress the in vitro growth of a clinical isolate of S. aureus, alone or combined with cefazolin; and (2) the effects of IRE on the in vivo treatment of a rabbit model of osteomyelitis. METHODS: S. aureus strain UAMS-1 expanded in vitro to the log phase was subjected to an electric field of 2700 V/cm, which was delivered in increasing numbers of pulses. Immediately after electroporation, bacteria were plated on agar plates with or without cefazolin. The number of colony-forming units (CFUs) was scored the following day. ANOVA tests were used to analyze in vitro data. In a rabbit osteomyelitis model, we inoculated the same bacterial strain into the radius of adult male New Zealand White rabbits. Three weeks after inoculation, all animals (n = 32) underwent irrigation and débridement, as well as wound culture of the infected forelimb. Then, they were randomly assigned to one of four treatment groups (n = eight per group): untreated control, cefazolin only, IRE only, or combined IRE + cefazolin. Serial radiography was performed to assess disease progression using a semiquantitative grading scale. Bone and soft-tissue specimens from the infected and contralateral forelimbs were collected at 4 weeks after treatment for bacterial isolation and histologic assessment using a semiquantitative scale. RESULTS: The in vitro growth of S. aureus UAMS-1 was impaired by IRE in a pulse-dependent fashion; the number of CFUs/mL was different among seven pulse levels, namely 0, 10, 30, 60, 90, 120, and 150 pulses. With the number of CFUs/mL observed in untreated controls set as 100%, 10 pulses rendered a median of 50.2% (range 47.1% to 58.2%), 30 pulses rendered a median of 2.7% (range 2.5% to 2.8%), 60 pulses rendered a median of 0.014% (range 0.012% to 0.015%), 90 pulses rendered a median of 0.004% (range 0.002% to 0.004%), 120 pulses rendered a median of 0.001% (range 0.001% to 0.001%), and 150 pulses rendered a median of 0.001% (range 0.000% to 0.001%) (Kruskal-Wallis test: p = 0.003). There was an interaction between the effect of the number of pulses and the concentration of cefazolin (two-way ANOVA: F [8, 30] = 17.24; p < 0.001), indicating that combining IRE with cefazolin is more effective than either treatment alone at suppressing the growth of S. aureus UAMS-1. Likewise, the clinical response in the rabbit model (the percentage of animals without detectable residual bacteria in the bone and surrounding soft tissue after treatment) was better in the combination group than in the other groups: control, 12.5% (one of eight animals); IRE only, 12.5% (one of eight animals); cefazolin only, 25% (two of eight animals); and IRE + cefazolin, 75% (six of eight animals) (two-sided Fisher’s exact test: p = 0.030). CONCLUSIONS: IRE effectively suppressed the growth of S. aureus UAMS-1 and enhanced the antibacterial effect of cefazolin in in vitro studies. When translated to a rabbit osteomyelitis model, the addition of IRE to conventional parenteral antibiotic treatment produced the strongest response, which supports the in vitro findings. CLINICAL RELEVANCE: Our results show that IRE may improve the results of standard parenteral antibiotic treatment, thus setting the stage for models with larger animals and perhaps trials in humans for validation.

SummaryCatabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in non-pathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIPseq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIPseq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.Data sharing and data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

A multicenter, retrospective study of patients infected with carbapenem-resistant Pseudomonas aeruginosa who were treated with ceftolozane/tazobactam was performed. Among 35 patients, pneumonia was the most common indication and treatment was successful in 26 (74%). Treatment failure was observed in all cases where isolates demonstrated ceftolozane-tazobactam minimum inhibitory concentrations ≥8 μg/mL.

Background Oral mucositis (OM) is a debilitating sequela for patients treated for squamous cell carcinoma of the head and neck (HNSCC). This study investigated whether oral microbial features before treatment or during treatment are associated with the time to onset of severe OM in patients with HNSCC. Methods This was a cohort study of newly diagnosed patients with locoregional HNSCC who received chemotherapy with or without radiotherapy from April 2016 to September 2017. OM was based on the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 4.0. The oral microbiome was characterized on the basis of the 16S ribosomal RNA V4 region with the Illumina platform. A mixture cure model was used to generate hazard ratios for the onset of severe OM. Results Eighty-six percent of the patients developed OM (n = 57 [33 nonsevere cases and 24 severe cases]) with a median time to onset of OM of 21 days. With adjustments for age, sex, and smoking status, genera abundance was associated with the hazard for the onset of severe OM as follows: 1) at the baseline (n = 66), Cardiobacterium (P = .03) and Granulicatella (P = .04); 2) immediately before the development of OM (n = 57), Prevotella (P = .03), Fusobacterium (P = .03), and Streptococcus (P = .01); and 3) immediately before the development of severe OM (n = 24), Megasphaera (P = .0001) and Cardiobacterium (P = .03). There were no differences in α-diversity between the baseline samples and Human Microbiome Project data. Conclusions Changes in the abundance of genera over the course of treatment were associated with the onset of severe OM. The mechanism and therapeutic implications of these findings need to be investigated in future studies.

This report highlights the importance of longitudinal, parallel characterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations., Although the term “microbiome” refers to all microorganisms, the majority of microbiome studies focus on the bacteriome. Here, we characterize the oral mycobiome, including mycobiome-bacteriome interactions, in the setting of remission-induction chemotherapy (RIC) for acute myeloid leukemia (AML). Oral samples (n = 299) were prospectively collected twice weekly from 39 AML patients during RIC until neutrophil recovery. Illumina MiSeq 16S rRNA gene (V4) and internal transcribed spacer 2 (ITS2) sequencing were used to determine bacterial and fungal diversity and community composition. Intrakingdom and interkingdom network connectivity at baseline (T1) and at midpoint (T3) and a later time point (T6) were assessed via SPIEC-EASI (sparse inverse covariance estimation for ecological association inference). In this exploratory study, mycobiome α-diversity was not significantly associated with antibiotic or antifungal receipt. However, postchemotherapy mycobiome α-diversity was lower in subjects receiving high-intensity chemotherapy. Additionally, greater decreases in Malassezia levels were seen over time among patients on high-intensity RIC compared to low-intensity RIC (P = 0.003). A significantly higher relative abundance of Candida was found among patients who had infection (P = 0.008), while a significantly higher relative abundance of Fusarium was found among patients who did not get an infection (P = 0.03). Analyses of intrakingdom and interkingdom relationships at T1, T3, and T6 indicated that interkingdom connectivity increased over the course of IC as bacterial α-diversity diminished. In (to our knowledge) the first longitudinal mycobiome study performed during AML RIC, we found that mycobiome-bacteriome interactions are highly dynamic. Our study data suggest that inclusion of mycobiome analysis in the design of microbiome studies may be necessary to optimally understand the ecological and functional role of microbial communities in clinical outcomes. IMPORTANCE This report highlights the importance of longitudinal, parallel characterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations.