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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.

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.

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.

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.

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.

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.

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.

S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections., Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates.

Patients being treated for cancer are at high risk for infectious complications, generally due to colonizing organisms that gain access to sterile sites via disrupted epithelial barriers. There is an emerging understanding that the ability of bacterial pathogens, including multidrug-resistant organisms, to colonize and subsequently infect humans is largely dependent on protective bacterial species present in the microbiome. Thus, herein we review recent studies demonstrating strong correlations between the microbiome of the oncology patient and infections occurring during chemotherapy. An increased knowledge of the interplay between potential pathogens, protective commensals, and the host immune system may facilitate the development of novel biomarkers or therapeutics that could help ameliorate the toll that infections take during the treatment of cancer.

Hypervirulent disease due to group A Streptococcus (GAS) can result from strains with mutations that enhance virulence gene expression but reduce subsequent transmission. We used whole-genome sequencing to investigate intrafamilial spread among 4 siblings of infection due to a hypervirulent GAS strain that resulted in a fatality. All invasive and pharyngeal GAS isolates had an identical mutation in a gene encoding a key regulatory protein that yielded a hyperinvasive phenotype. These data challenge the prevailing theory of reduced transmission induced by mutations that lead to hypervirulent GAS by showing that spread of hypervirulent GAS may lead to clusters of invasive disease.

Bacterial infections are common in cancer patients. Ceftaroline (CFT) is a broad-spectrum cephalosporin with activity against most Gram-positive organisms (GPOs) and many Gram-negative organisms. In this study, the in vitro activity of CFT was compared with vancomycin (VAN), daptomycin (DAP), linezolid (LZD), trimethoprim/sulphamethoxazole (SXT) and tigecycline (TIG) against bacteria (predominantly blood culture isolates) isolated from cancer patients in 2014 and 2015. CFT was active against methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-susceptible coagulase-negative staphylococci (MS-CoNS) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) with MIC90 values (minimum inhibitory concentration that inhibited 90% of the isolates) of 0.25, 2.0, 0.12 and 0.5 mg/L, respectively. MIC90 values for other GPOs were: Bacillus spp., >8.0 mg/L; Corynebacterium spp., 2.0 mg/L; Micrococcus spp.

Background E. faecalis (Efc) isolates are usually susceptible to ampicillin (AMP). AMP-based regimens are the standard of care for enterococcal infections, although other antibiotics are often used as definitive treatment. We thus compared outcomes of patients with cancer and Efc bacteremia treated with AMP-containing (ACR) and non-AMP-containing antibiotic regimens (NACR). Methods A multicenter, prospective, observational cohort study conducted at MD Anderson Cancer Center, Henry Ford Hospital, and Memorial Hermann Health System. Eligible patients were ≥ 18 years old, diagnosed with cancer, and had at least one Efc bloodstream isolate collected from 12/2015 to 12/2018. Patients with polymicrobial infections were excluded. Patients were divided into two groups: i) ACR and ii) NACR. ACR included patients who received AMP at any time during treatment; other antimicrobials were permitted. NACR patients did not receive AMP at any time. The primary outcome compared desirability of outcome ranking (DOOR) between ACR and NACR at day 14. The DOOR consisted of six hierarchical levels: 1 - death; 2 - inpatient without microbiological cure (MC) and with acute kidney injury (AKI); 3 - inpatient without MC and without AKI; 4 - inpatient admitted with MC and with AKI; 5 - inpatient with MC and without AKI; 6 - alive and discharged. Comparison of DOORs between ACR and NACR was performed using inverse probability of treatment weighted (IPTW) ordered logistic regression. Results Seventy-one patients were included (ACR, n = 35; NACR, n = 36). No difference was seen in DOORs at day 14 between ACR and NACR (odds ratio [OR] 1.14, 95% Confidence Interval [CI] 0.45 – 2.92, p=0.78). No difference was observed for all-cause mortality at day 14 (OR 0.6, 95% CI 0.09 – 3.77, p=0.58) or day 30 (OR 0.42, 95% CI 0.09 – 1.94, p=0.27). Patients treated with ACR received a lower median duration of other antibiotics at any point during treatment compared to NACR: daptomycin (2 v 4 days) vancomycin (2 v 4 days), and linezolid (1 v 2 days). Conclusion Patients with cancer and Efc bloodstream infections had similar outcomes when treated with ACR and NACR. ACR were associated with less use of broad-spectrum antimicrobials. Future research should focus on the ecologic impact of use of NACR. Disclosures Marcus Zervos, MD, Melinta Therapeutics (Grant/Research Support) Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Background Vancomycin-resistant Enterococcus faecium (VREfm) are leading causes of bloodstream infections (BSI) in patients (pts) with hematological malignancies (HM). Daptomycin (DAP) is commonly used to treat VRE BSI, but DAP non-susceptibility (DAP-NS) in pts with HM is increasing. Current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility. DAP triggers the LiaFSR cell membrane stress response pathway, resulting in the extracellular release of the protein LiaX, a novel protein that functions as a regulator of the membrane response. We postulated that detection of extracellular LiaX correlates with DAP-NS in clinical strains of VREfm. Methods We used 6 well-characterized VREfm BSI isolates (2 DAP-S, 4 DAP-NS) as reference strains to optimize a whole-cell indirect enzyme-linked immunosorbent assay (ELISA) method for LiaX detection. We assessed limit of detection and reproducibility of the ELISA LiaX method. We then assessed 54 clinical VREfm BSI isolates from pts with cancer for validation. We determined DAP MICs by broth microdilution (BMD). We collected clinical and microbiological details by chart review Results The 6 reference strains showed high reproducibility with low coefficient of variation. All DAP-NS reference strains had increased detection of LiaX (p< 0.0001) compared to DAP-S reference strains. Of the 54 isolates from pts, most pts (83.3%) had HM. The source of 62.9% of VRE BSIs was determined to be gastrointestinal. Six of the 54 isolates were DAP-NS by BMD MIC. The LiaX test and MIC had categorical agreement on 56% of isolates. Of the isolates with disagreement, 19 isolates were susceptible by MIC (median 2 μg/ml) but non-susceptible by LiaX ELISA, and 5 isolates were non-susceptible by MIC (6, 8, 8, 8, and 16 μg/ml, respectively) but susceptible by LiaX ELISA. Whole-cell indirect LiaX ELISA A405nm of Efm reference strains shows ability to differentiate DAP susceptible MICs from DAP resistant MICs. DAP susceptible (MIC=2 μg/ml) Efm strains are shown in green and DAP resistant (MIC≥8 μg/ml) strains in red. DAP-S reference strains have no LiaFSR mutations. The dotted line indicates an example cutoff for DAP-S/R in this assay. *p Conclusion Detection of extracellular LiaX has important discrepancies with DAP MIC. Interestingly, LiaX may be a surrogate marker to detect strains with heightened DAP-mediated cell membrane response and potentially identify strains predisposed to DAP therapy failure. Further characterization of the discrepant isolates by genomic analyses and time-kill assays are warranted to fully validate the performance of LiaX ELISA. Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Cefiderocol inhibited 97.5% of 478 Gram-negative isolates from cancer patients at ≤4 mg/liter. It had potent activity against extended-spectrum β-lactamase-positive Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae (CRE), and nonfermenting Gram-negative bacilli, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter species isolates. Amikacin, ceftazidime-avibactam, and meropenem had appreciable activity against non-CRE Enterobacteriaceae., Cefiderocol inhibited 97.5% of 478 Gram-negative isolates from cancer patients at ≤4 mg/liter. It had potent activity against extended-spectrum β-lactamase-positive Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae (CRE), and nonfermenting Gram-negative bacilli, including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter species isolates. Amikacin, ceftazidime-avibactam, and meropenem had appreciable activity against non-CRE Enterobacteriaceae. No comparators were active against multidrug-resistant P. aeruginosa isolates. Only trimethoprim-sulfamethoxazole had appreciable activity against S. maltophilia isolates. Overall, cefiderocol was associated with the lowest level of resistance.

BackgroundCarbapenem resistant Enterobacterales (CRE) remain urgent antimicrobial resistance threats. Approximately half of CRE clinical isolates lack carbapenem hydrolyzing enzymes and develop carbapenem resistance through alternative mechanisms. The purpose of this study was to elucidate the development of carbapenem resistance mechanisms from clonal, recurrent extended-spectrum β-lactamase positive Enterobacterales (ESBL-E) bacteremia isolates in a vulnerable patient population.MethodsThis study investigated a historical, retrospective cohort of ESBL-E bacteremia cases in the University of Texas MD Anderson Cancer Center (MDACC) from January 2015 to July 2016. Phylogenetic and comparative genomic analyses were performed to identify clonal, recurrent ESBL-E isolates developing carbapenem resistance. Oxford Nanopore Technology (ONT) long-read and Illumina short-read sequencing data were used to generate consensus assemblies and to identify signatures of mobile genetic element mediated amplification and transposition of antimicrobial resistance genes. Serial passaging experiments were performed on a set of clinical ST131 ESBL-E isolates to recapitulate in vivo observations. qPCR and qRT-PCR were used to determine respective copy number and transcript levels of β-lactamase genes.Results116 ESBL-E bacteremia cases were identified, 16 of which had documented recurrent infections. Four serial, recurrent isolates displayed a carbapenem resistant phenotype, three without the acquisition of a known carbapenemase. These three isolates had non-carbapenemase-producing CRE (non-CP-CRE) mechanisms driven by IS26- and ISEcp1-mediated amplification of respective translocatable units (TU) and transposition units (TPU) harboring both blaOXA-1 and blaCTX-M variants with concomitant outer membrane porin disruption. The TU and TPU structures inserted into the open reading frames of outer membrane porin genes in a subset of non-CP-CRE isolates. Serial passage of an index ST131 ESBL-E isolate under selective carbapenem exposure resulted in chromosomal amplification of modular, TUs harboring β-lactamase genes with concomitant porin inactivation, recapitulating the in vivo carbapenem resistance progression. Long-read sequencing of two additional MDACC bacteremia strains identified similar non-CP-CRE mechanisms observed in the serial isolates.ConclusionsNon-CP-CRE de novo mechanisms were the primary driver of CRE development in recurrent bacteremia cases within this vulnerable patient population. The incorporation of long-read ONT data into AMR surveillance platforms is critical to identify high-risk CRE isolates that are difficult to identify with low-resolution phenotypic and molecular characterization methods.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

Stenotrophomonas maltophilia is an emerging multidrug-resistant (MDR) opportunistic pathogen for which new antibiotic options are urgently needed. We report our clinical experience treating a 19-year-old renal transplant recipient who developed prolonged bacteremia due to metallo-β-lactamase-producing S. maltophilia refractory to conventional treatment. The infection recurred despite a prolonged course of colistimethate sodium (colistin) but resolved with the use of a novel drug combination with clinical efficacy against the patient's S. maltophilia isolate.

Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes . The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes .

Resistance to the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam (CAZ-AVI) among carbapenem-resistant Enterobacteriaceae (CRE) has infrequently been reported in the United States. We report unexpectedly high rates of resistance to CAZ-AVI in CRE bloodstream isolates at our institution associated with the nonoutbreak spread of New Delhi metallo-β-lactamase in diverse Enterobacteriaceae species.

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains., Author summary Group A streptococcus (GAS), also known as Streptococcus pyogenes, causes a broad array of human infections of varying severity. Tight control of production of virulence factors is critical to GAS pathogenesis, and the control of virulence two-component signaling system (CovRS) is central to this process. The activity of the bifunctional histidine kinase CovS determines the phosphorylation status and thereby the activity of its cognate response regulator CovR. Herein, we sought to determine how varying CovR phosphorylation level (CovR~P) impacts GAS pathophysiology. Using three infection models, we discovered that GAS strains lacking CovS phosphatase activity resulting in high CovR~P levels had markedly impaired infectivity. Transcriptome analysis revealed that the hypovirulent phenotype of CovS phosphatase deficient strains is due to down-regulation of numerous genes encoding GAS virulence factors. We identified repression of additional virulence genes that are typically not controlled by CovR, thus expanding the CovR regulon at high CovR~P concentrations. Our data indicate that phosphatase activity of CovS sensor kinase is crucial for spatiotemporal regulation of GAS virulence gene expression. Thus, we propose that targeting the phosphatase activity of CovS sensor kinase could be a promising novel therapeutic approach to combat GAS disease.

Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.

Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non-randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD(+) hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13-fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two-component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two-component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species.

Background Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression. IMPORTANCE Streptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator CovR plays a crucial role. As an OmpR/PhoB family member, CovR is activated by phosphorylation on a conserved aspartate residue, leading to protein dimerization and subsequent binding to operator sites. Our transcriptome analysis using the monomeric phosphorylation mimic mutant CovR-D53E broadens this general notion by revealing dimerization-independent repression of a subset of CovR-regulated genes. Combined with promoter analyses, these data suggest distinct mechanisms of CovR transcriptional control, which allow for differential expression of virulence genes in response to environmental cues.

A total of 248 Gram-positive isolates from cancer patients were tested for in-vitro susceptibility to tedizolid and 3 comparator agents using CLSI broth microdilution methodology. Tedizolid inhibited 97% of isolates at ≤0.5 μg/ml. It was active against all Gram-positive species and consistently had 8 fold lower MICs than linezolid, although based on % susceptibility using CLSI breakpoints, most isolates were also susceptible to the comparators. Tedizolid was active against MRSA isolates with vancomycin MICs of ≥1.0 μg/ml.

The taxonomy of Enterobacter species is rapidly changing. Herein we report a bloodstream infection isolate originally identified as Enterobacter cloacae by Vitek2 methodology that we found to be Kosakonia radicincitans using genetic means. Comparative whole genome sequencing of our isolate and other published Kosakonia genomes revealed these organisms lack the AmpC β-lactamase present on the chromosome of Enterobacter sp. A fimbriae operon primarily found in Escherichia coli O157:H7 isolates was present in our organism and other available K. radicincitans genomes. This is the first report of a Kosakonia species, which are typically associated with plants, causing a human infection.

Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage.

The genetically diverse viridans group streptococci (VGS) are increasingly recognized as the cause of a variety of human diseases. We used a recently developed multilocus sequence analysis scheme to define the species of 118 unique VGS strains causing bacteremia in patients with cancer; Streptococcus mitis (68 patients) and S. oralis (22 patients) were the most frequently identified strains. Compared with patients infected with non–S. mitis strains, patients infected with S. mitis strains were more likely to have moderate or severe clinical disease (e.g., VGS shock syndrome). Combined with the sequence data, whole-genome analyses showed that S. mitis strains may more precisely be considered as >2 species. Furthermore, we found that multiple S. mitis strains induced disease in neutropenic mice in a dose-dependent fashion. Our data define the prominent clinical effect of the group of organisms currently classified as S. mitis and lay the groundwork for increased understanding of this understudied pathogen.

Background Concern for serious infection due to β-lactam-resistant viridans group streptococci (VGS) is a major factor driving empiric use of an anti-gram-positive antimicrobial in patients with febrile neutropenia. We sought to develop and validate a prediction model for the presence of β-lactam resistance in VGS causing bloodstream infection (BSI) in neutropenic patients. Methods Data from 569 unique cases of VGS BSI in neutropenic patients from 2000 to 2010 at the MD Anderson Cancer Center were used to develop the clinical prediction model. Validation was done using 163 cases from 2011 to 2013. In vitro activity of β-lactam agents was determined for 2011-2013 VGS bloodstream isolates. Results In vitro resistance to β-lactam agents commonly used in the empiric treatment of febrile neutropenia was observed only for VGS isolates with a penicillin minimum inhibitory concentration (MIC) of ≥ 2 µg/mL. One hundred twenty-nine of 732 patients (17%) were infected with VGS strains with a penicillin MIC ≥ 2 µg/mL. For the derivation and validation cohorts, 98% of patients infected by VGS with a penicillin MIC of ≥ 2 µg/mL had at least 1 of the following risk factors: current use of a β-lactam as antimicrobial prophylaxis, receipt of a β-lactam antimicrobial in the previous 30 days, or nosocomial VGS BSI onset. Limiting empiric anti-gram-positive therapy to neutropenic patients having at least 1 of these 3 risk factors would have reduced such use by 42%. Conclusions Simple clinical criteria can assist with targeting of anti-gram-positive therapy to febrile neutropenic patients at risk of serious β-lactam-resistant VGS infection.

The taxonomy of Enterobacter species is rapidly changing. Herein we report a bloodstream infection isolate originally identified as Enterobacter cloacae by Vitek2 methodology that we found to be Kosakonia radicincitans using genetic means. Comparative whole genome sequencing of our isolate and other published Kosakonia genomes revealed these organisms lack the AmpC β-lactamase present on the chromosome of Enterobacter sp. A fimbriae operon primarily found in Escherichia coli O157:H7 isolates was present in our organism and other available K. radicincitans genomes. This is the first report of a Kosakonia species, which are typically associated with plants, causing a human infection.

Background Staphylococcus epidermidis is a leading cause of healthcare-associated bacteremia, but the clinical course of S. epidermidis isolated from blood cultures ranges from contamination to serious deep seated infections. We tested the hypothesis that the genetic characteristics of S. epidermidis isolated from blood cultures are significantly associated with disease severity using whole genome sequencing (WGS). Methods We performed WGS of 163 S. epidermidis isolates from a large prospective multicenter, randomized control trial that assessed the safety and efficacy of algorithm-based treatment of patients with staphylococcal bacteremia (ClinicalTrials.gov #NCT01191840). Patient’s infection types were divided into simple (including possible contamination), uncomplicated and complicated bacteremia. Failure was defined as persistent signs and symptoms of infection at 72 hours, persistent bacteremia at 7 days, relapse, complications or overall mortality within 28 days of therapy. WGS was performed using Illumina Miseq, followed by in silico multi-locus sequence type identification and phylogenomic analysis using kSNP. Results Of the 163 isolates analyzed, 39 sequence types (STs) were identified with ST2 and ST5 isolates being most frequently identified (21% and 20%, respectively). ST2 and ST5 isolates were significantly more likely associated with uncomplicated and complicated infections rather than simple bacteremia (P = 0.01 by χ2 test). By multivariate regression analysis, having an ST2 or ST5 S. epidermidis bacteremia was an independent predictor of a complicated infection (odds ratio 4.28, 95% CI 1.36–13.42). Using WGS based branching points rather than sequence typing allowed for additional strengthening of the association of S. epidermidis genetic clustering and clinical infection types (figure). Although there was no significant difference in failure rates among patients infected with different STs, ST2/ST5 strains were significantly more likely to cause relapsing infections (85.7%, P = 0.02). Conclusion WGS could offer a prognostic tool in the management of S. epidermidis bacteremia. ST2 and ST5 strains may help predict a complicated bacteremia course which would warrant appropriate antimicrobial therapy. Disclosures T. L. Holland, Basilea: Consultant, Consulting fee. Motif Bio: Consultant and Scientific Advisor, Consulting fee. Theravance: Consultant, Speaker honorarium. Genentech: Consultant, Consulting fee. V. G. Fowler Jr., Debiopharm: Consultant, Consulting fee. Durata: Consultant, Consulting fee. Merck: Consultant and Scientific Advisor, Consulting fee. Cerexa/Actavis/Allergan: Grant Investigator, Grant recipient. Pfizer: Consultant and Grant Investigator, Consulting fee and Grant recipient. Advanced Liquid Logics: Grant Investigator, Grant recipient. NIH: Grant Investigator, Grant recipient. MedImmune: Consultant and Grant Investigator, Consulting fee and Grant recipient. Basilea: Consultant and Grant Investigator, Consulting fee and Grant recipient. Karius: Grant Investigator, Grant recipient. Contrafect: Consultant and Grant Investigator, Consulting fee and Grant recipient. Regeneron: Grant Investigator, Grant recipient. Genentech: Consultant and Grant Investigator, Consulting fee and Grant recipient. Affinergy: Consultant and Grant Investigator, Consulting fee and Grant recipient. Locus: Grant Investigator, Grant recipient. Medical Surface, Inc.: Grant Investigator, Grant recipient. Theravance: Consultant, Consulting fee and Speaker honorarium. Green Cross: Consultant, Speaker honorarium. Grifols: Consultant, Consulting fee. xBiotech: Consultant, Consulting fee. Achaogen: Consultant, Consulting fee. Medicines Co: Consultant, Consulting fee. Novartis: Consultant, Consulting fee. Novadigm: Consultant, Consulting fee. Bayer: Consultant, Consulting fee. Cubist: Consultant, Consulting fee. Debiopharm: Consultant, Consulting fee. Durata: Consultant, Consulting fee. I. Raad, The University of Texas MD Anderson Cancer Center: Shareholder, Licensing agreement or royalty. The Unversity of Texas MD Anderson Cancer Center: Shareholder, Dr. Raad is a co-inventor of the Nitroglycerin-Citrate-Ethanol catheter lock solution technology which is owned by the University of Texas MD Anderson Cancer Center (UTMDACC) and has been licensed to Novel Anti-Infective Technologies LLC, in which UTMDACC and Licensing agreement or royalty.

Background Vancomycin-resistant enterococci (VRE) are a major cause of nosocomial bloodstream infections. Enterococci exhibit remarkable genomic plasticity and can recombine through the acquisition of genetic material via mobile genetic elements (MGEs), including resistance genes. The accessory genome plays a major role in the evolution of enterococci within the human host. Thus, dissecting the entire genome (pan-genome) is of paramount importance to characterize the population structure of enterococci causing disease. Methods VENOUS is an ongoing prospective, observational study of adults with enterococcal bacteremia. From September 2016 to March 2018, E. faecalis (Efs) and E. faecium (Efm) were collected in 14 hospitals of a single hospital system and a major cancer center in Houston, TX, and a general hospital in Detroit, MI. Short- and long-read genomic sequencing were performed with Illumina MiSeq and Oxford Nanopore Technologies GridION X5, respectively. A proprietary bioinformatics pipeline was utilized for genome assembly and further analyses. Results 156 Efs and 98 Efm isolates from single patients were analyzed. The average proportion of core genes in each genome was 64.6% (53.0–74.1) and 49.1% (45.2–51.0) for Efs and Efm, respectively. The vanA gene cluster was identified in 5.1% (8/157) of Efs and 57.1% (56/98) of Efm. The plasmid-encoded aac(6′)-Ie-aph(2″)-Ia gene conferring high-level resistance to aminoglycosides was found in 37.6% (59/157) Efs, seven of which also possessed vanA. Long-read sequencing of vanA-harboring plasmids from a subset of VRE revealed that the vanA cluster was carried in plasmids ranging from 31.7 to 132.3 kb. Although the vanA operon was fairly conserved, insertions of MGE were identified in the intergenic regions of vanS/vanH and vanX/vanY. Furthermore, a variety of MGE insertions mediated integration of the vanA operon, including IS1216 and IS256 (figure). Conclusion Accessory genes, including AMR genes, comprise a significant proportion of the enterococcal pan-genome, indicating major genetic plasticity within these organisms. Acquired resistance genes seem to have a high degree of recombination and play a substantial role in the expansion of the genomic repertoire in clinical isolates. Disclosures Samuel L. Aitken, PharmD, Melinta Therapeutics: Grant/Research Support, Research Grant; Merck, Sharpe, and Dohme: Advisory Board; Shionogi: Advisory Board.

Background Ceftazidime–avibactam (CZA) is a novel β-lactam / β-lactamase inhibitor with in vitro activity against multidrug-resistant Gram-negatives, including those harboring CMY-2 enzymes. Treatment-emergent resistance to CZA has been described in KPC-producing Klebsiella pneumoniae but has not been described in non-carbapenemase-producing, carbapenem-resistant Enterobacteriaceae (CRE). Methods A patient with an intra-abdominal infection due to a carbapenem-resistant E. coli (ertapenem MIC 16 µg/mL; meropenem MIC 2 µg/mL; CZA MIC 2 µg/mL; carbapenemase negative) was treated with CZA. On day 48 of therapy, a CZA resistant, carbapenem-sensitive E. coli was identified from abdominal drainage (CZA MIC ≥256 µg/mL; meropenem MIC 0.19 µg/mL). Illumina MiSeq whole-genome sequencing (WGS) was performed on both isolates to identify potential resistance mechanisms. The ResFinder database was used to identify known β-lactamase enzymes, and in silico modeling of β-lactamase structure was assessed. Results WGS revealed that both isolates were ST410 E. coli, with the sole difference in β-lactam resistance determinants between the two being a novel CMY β-lactamase harbored on an Inc1-type conjugative plasmid in the second isolate. The novel CMY has 4 amino acid substitutions relative to CMY-2: A134E, Q140K, V231S, and N366Y. The V231S substitution is found in CMY-42 and has previously been associated with increased ceftazidime hydrolysis. The remaining three substitutions have not previously been identified. Previous studies have identified that substitutions at position 366 influence the rate of ceftazidime hydrolysis rate. Preliminary protein structure analysis suggests that positions 140 and 366 are in the active site. No other differences in β-lactam resistance determinants were identified between the first and second isolates. Conclusion To our knowledge, we have identified the first case of CMY-associated CZA resistance. Given the widespread and transferrable nature of CMY enzymes, this finding raises concern for additional cases of resistance with increasing usage of CZA. Further analysis is needed to identify the mechanism by which this enzyme confers CZA resistance. Disclosures Samuel L. Aitken, PharmD, Melinta Therapeutics: Grant/Research Support, Research Grant; Merck, Sharpe, and Dohme: Advisory Board; Shionogi: Advisory Board.