Background Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression.Methods We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity.Results PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors.Conclusions We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

IMPORTANCE Oral non-beta-lactam antibiotics are commonly used for empirical therapy of Staphylococcus aureus infections, especially in outpatient settings. However, little is known about potential geographic heterogeneity and temporal trends in the prevalence of S aureus resistance to non-beta-lactams in the US. OBJECTIVE To characterize the spatiotemporal trends of resistance to non-beta-lactam antibiotics among community-onset S aureus infections, including regional variation in resistance rates and geographical heterogeneity in multidrug resistance. DESIGN, SETTING, AND PARTICIPANTS This cross-sectional study used data from Veterans Health Administration clinics collected from adult outpatients with S aureus infection in the conterminous 48 states and Washington, DC, from January 1, 2010, to December 31, 2019. Data were analyzed from January to November 2023. EXPOSURES Resistance to lincosamides (clindamycin), tetracyclines, sulfonamides (trimethoprim-sulfamethoxazole [TMP-SMX]), and macrolides. MAIN OUTCOMES AND MEASURES Spatiotemporal variation of S aureus resistance to these 4 classes of non-beta-lactam antibiotics, stratified by methicillin-resistant S aureus (MRSA) and methicillin-sensitive S aureus (MSSA), and subdivided by regions of the US (Northeast, Midwest, South, and West). Trend tests and bivariate mapping were used to determine significant changes in resistant proportions over time and identify counties where rates of resistance to multiple non-beta-lactams were high. RESULTS A total of 382 149 S aureus isolates from 268 214 unique outpatients (mean [SD] age, 63.4 [14.8] years; 252 910 males [94.29%]) were analyzed. There was a decrease in the proportion of MRSA nationwide, from 53.6% in 2010 to 38.8% in 2019. Among MRSA isolates, we observed a significant increase in tetracycline resistance (from 3.6% in 2010 to 12.8% in 2019; P for trend < .001) and TMP-SMX resistance (from 2.6% in 2010 to 9.2% in 2019; P for trend < .001), modest and not significant increases in clindamycin resistance (from 24.2% in 2010 to 30.6% in 2019; P for trend = .34), and a significant decrease in macrolide resistance (from 73.5% in 2010 to 60.2% in 2019; P for trend < .001). Among MSSA isolates, significant upward trends in clindamycin, tetracyclines, and TMP-SMX resistance were observed. For example, tetracycline resistance increased from 3.7% in 2010 to 9.1% in 2019 (P for trend < .001). Regional stratification over time showed that the Northeast had slightly higher rates of clindamycin resistance but lower rates of tetracycline resistance, while the South had notably higher rates of resistance to tetracyclines and TMP-SMX, particularly among MRSA isolates. Bivariate mapping at the county scale did not indicate clear regional patterns of shared high levels of resistance to the 4 classes of antimicrobials studied. CONCLUSIONS AND RELEVANCE In this study of outpatient S aureus isolates, MRSA became less common over the 10-year period, and MRSA isolates were increasingly resistant to tetracyclines and TMP-SMX. Geographic analysis indicated no spatial overlap in counties with high rates of resistance to both tetracyclines and TMP-SMX. Examining the regional spatial variation of antibiotic resistance can inform empirical therapy recommendations and help to understand the evolution of S aureus antibiotic resistance mechanisms.

The recently updated Society for Healthcare Epidemiology of America, the Infectious Diseases Society of America, and the Association of Professionals in Infection Control practice recommendations for methicillin-resistant Staphylococcus aureus (MRSA) prevention in acute care facilities list contact precautions (CPs) for patients known to be infected or colonized with MRSA as an “essential practice,” meaning that it should be adopted in all acute care facilities. We argue that existing evidence on benefits and harms associated with CP do not justify this recommendation. There are no controlled trials that support broad use of CP for MRSA prevention. Data from hospitals that have discontinued CP for MRSA have found no impact on MRSA acquisition or infection. The burden and harms of CP remain concerning, including the environmental impact of increased gown and glove use. We suggest that CP be included among other “additional approaches” to MRSA prevention that can be implemented under specific circumstances (eg outbreaks, evidence of ongoing transmission despite application of essential practices).

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While the use of cumulative susceptibility reports, antibiograms, is recommended for improved empiric therapy and antibiotic stewardship, the predictive ability of antibiograms has not been well-studied. While enhanced antibiograms have been shown to better capture variation in susceptibility profiles by characteristics such as infection site or patient age, the potential for seasonal or spatial variation in susceptibility has not been assessed as important in predicting likelihood of susceptibility. Utilizing Staphylococcus aureus isolates obtained in outpatient settings from a nationwide provider of care, the Veterans Health Administration, and a local provider of care, the University of Iowa Hospitals and Clinics, standard, seasonal and spatial antibiograms were created for five commonly used antibiotic classes: cephalosporins, clindamycin, macrolides, tetracycline, trimethoprim/sulfamethoxazole. A total of 338,681 S. aureus isolates obtained in VHA outpatient settings from 2010 to 2019 and 6,817 isolates obtained in UIHC outpatient settings from 2014 to 2019 were used to generate and test antibiograms. Logistic regression modeling determined the capacity of these antibiograms to predict isolate resistance to each antibiotic class. All models had low predictive capacity, with areas under the curve of < 0.7. Standard antibiograms are poor in predicting S. aureus susceptibility to antibiotics often chosen by clinicians, and seasonal and spatial antibiograms do not provide an improved tool in anticipating non-susceptibility. These findings suggest that further refinements to antibiograms may be necessary to improve their utility in informing choice of effective antibiotic therapy.

Antigen presentation is critical for adaptive immune response to cancer but is often subverted to evade immune response. While hypoxia is a pervasive feature of solid tumors, it’s role in tumor antigen processing remains understudied. To assess the effects of reduced oxygen on antigen processing machinery, we used interferon (IFN)-γ to induce the expression of the immunoproteasome (IP), a proteasome isoform critical for the tumor antigen processing, in A549 human lung cancer cells cultured under 20% O2, or 2% O2. Treatment with IFN-γ induced protein expression of IP subunits LMP2, LMP7, and MECL-1 in a dose-dependent manner under 20% O2 as assessed by western blotting. In contrast, IFN-γ failed to induce protein expression of these subunits under hypoxic conditions. We observed similar blockades of other antigen processing pathway components in response to IFN-γ under hypoxic conditions including TAP, tapasin, and calreticulin, as well as secondary mediators of IFN-γ-stimulated genes including several members of the interferon regulatory factor (IRF) family. Surprisingly, under identical conditions, induction of IP subunit mRNAs appear intact under hypoxia as assessed by RT-PCR, with similar levels of LMP2, LMP7, and MECL-1 upregulated by IFN-γ regardless of oxygen level, suggesting hypoxic IP blockade occurs post-transcriptionally or post-translationally. In line with this notion, primary mediators of IFN-γ signaling, including phosphorylated (Tyr701) STAT1 levels were unaffected by oxygen levels. To investigate the mechanism behind this phenomenon, we created hypoxia-inducible factor (HIF)-1a and/or HIF-2a knockout A549 cell lines or used cobalt chloride (CoCl2), a hydroxylase inhibitor, to directly interrogate the hypoxia signaling pathway. Blockade of IP subunit induction in response to IFN-γ was not observed after CoCl2 treatment, nor did HIF-1a and/or HIF-2a genetic deletion rescue IP subunit blockade in A549 cells cultured under hypoxia, indicating that hypoxia-induced IP blockade is independent of the HIF pathway. To test if this phenomenon is regulated by epigenic means, we treated cells with 5-Azacytidine (5-aza), a cystine analog nucleoside, and observed a re-establishment of IP subunit protein expression under hypoxia in response to IFN-γ. Given that 5-aza preferentially integrates RNA over DNA, and that IP subunit mRNA levels appear unaffected by hypoxia, suggests that hypoxia-induced IP blockade may be due to hypoxia-specific cytosine modifications within mRNA transcripts. Current studies include efforts to map mRNA methylation patterns and interrogate mRNA methylation transferases under hypoxic conditions. These studies introduce a novel link between hypoxia and reduced cancer cell antigen processing machinery and may help identify druggable epigenetic targets that can be used to enhance the immunogenicity of hypoxic tumors. Citation Format: Alexis Rebecca Ramos, Matthew Smith, Heena Panchal, Adam Mailloux. Tumor hypoxia blocks the induction of antigen processing machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2997.

Krupal B. Patel, MD, MSc; Brian D. Gonzalez, PhD; Kea Turner, PhD; Amir Alishahi Tabriz, MD; Dana E. Rollison, PhD; Edmondo Robinson, MD; Cristina Naso, MEd; Xuefeng Wang, PhD; Philippe E. Spiess, MD

Abstract Background Oral non-beta-lactam antibiotics are commonly used for empiric therapy of Staphylococcus aureus infections, especially in outpatient settings. However, little is known about geographic heterogeneity in the prevalence and temporal trends of antibiotic resistance among different regions in the US. We aimed to characterize the spatiotemporal patterns of drug-resistance prevalence of S. aureus using the nationwide surveillance data from the Veterans Health Administration (VHA) system. Methods Utilizing a dataset of 383,514 S. aureus isolates obtained in outpatient settings in the VHA from 2010-2019, we explored the spatiotemporal variation of S aureus resistance to clindamycin, tetracyclines, trimethoprim-sulfamethoxazole, and macrolides, stratified by methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and subdivided by regions of the United States (Northeast, Midwest, South, and West). Results Over the ten-year study period there was a national decrease in the proportion of S. aureus isolates which were MRSA, from 54% to 39% (Figure 1). Amongst MRSA isolates (Figure 2, panels A-D), we observed stability of clindamycin resistance (from 24.5% to 31.1%), an increase in tetracycline resistance (from 3.9% to 13.1%), an increase in trimethoprim-sulfamethoxazole resistance (from 2.7% to 9.3%), and a decrease in macrolide resistance (from 72% to 60%). For MSSA (Figure 2, panels E-H), we observed relative stability of resistance over time for all four drug classes. Regional analysis (Figure 3) demonstrated that the Northeastern US had slightly higher rates of clindamycin resistance than other regions but lower rates of tetracycline resistance, while the South had notably higher rates of trimethoprim-sulfamethoxazole resistance than other regions, particularly amongst MRSA isolates. Figure 1 Frequency of S. aureus isolates by year (A), proportion of S. aureus isolates resistant to methicillin (B), and proportion of S. aureus isolates resistant to methicillin by region (C) Figure 2 Proportion of S aureus isolates resistant to Clindamycin, Tetracyclines, Trimethoprim-Sulfamethoxazole, and Macrolides, stratified by MRSA (panels A-D) and MSSA (panels E-H) Figure 3 Proportion of S aureus isolates resistant to Clindamycin, Tetracyclines, Trimethoprim-Sulfamethoxazole, and Macrolides, stratified by MRSA (panels A-D) and MSSA (panels E-H), subdivided by region Conclusion Even though the prevalence of MRSA is decreasing nationally, there are variable levels of co-resistance to non-beta-lactam antibiotics amongst S. aureus isolates. There was some regional variability in co-resistance amongst MRSA isolates, but less so for MSSA isolates. Further studies are needed to better understand which biological mechanisms mediate these differences. Disclosures Michihiko Goto, MD MSCI, Merck & Co.: Grant/Research Support

Using data from the Veterans’ Health Administration from 2010 to 2019, we examined the distribution and prevalence of community-acquired phenotypic extended-spectrum β-lactamase (ESBL) E. coli in the United States. ESBL prevalence slowly increased during the study period, and cluster analysis showed clustering in both urban and rural locations.

Authors of this Viewpoint present actionable steps for regulatory, industry, and health care organization practices to accelerate reduction of single-use plastics and help protect planetary and human health.

Novel ST398 methicillin susceptible Staphylococcus aureus (MSSA) in the United States was first observed in New York City (2004-2007); its diffusion across the country resulted in changing treatment options. Utilizing outpatient antimicrobial susceptibility data from the Veterans Health Administration from 2010 to 2019, the spatiotemporal prevalence of potential ST398 MSSA is documented.

•Intranasal mupirocin is effective pre-operatively for orthopedic and cardiac procedures.•Mupirocin is effective for nasal decolonization in the intensive care unit setting.•Intranasal povidone-iodine is most effective for pre-operative nasal decolonization.•Other decolonization agents lack sufficient data for widespread use.•Additional research on decolonizing agents is still needed.

Introduction: The impact of the COVID-19 has resulted in a decrease in presentations in emergency departments (ED).This has been influenced by societal restrictions, lockdowns and access to services.This study aims to observe the patterns in ED presentations for Bankstown-Lidcombe Hospital over the course of the initial COVID-19 pandemic wave.Methods: A retrospective review of all presentations to Bankstown-Lidcombe Hospital ED was conducted comparing monthly patient encounters from February-August for 2017-2020.Results: The total number of ED presentations was 42,225.There was a statistically significant reduction during 2020 of 9.8% between the study periods (1546 vs 1395, p = 0.014).The period of greatest reduction was seen in March-May, the strictest lockdown period for New South Wales.The admitting disciplines that showed a reduction in this time were colorectal (76.62%, p = 0.046), emergency (25.53%, p = 0.025), endocrine (31.82%, p = 0.026), orthopaedics (33.63%, p = 0.004), and vascular (35.71%, p = 0.017).The disciplines that showed an increase in presentations over the entire studied time were gynaecology (26.54%, p = 0.008), and psychiatry (17.46%, p = 0.011).Conclusion: There were significant reductions in total number of ED presentations.Notably, there were significant reductions across multiple admitting disciplines, as well as in a decrease in presentations for those in younger age groups.

An immunocompetent man in his 40s presented with 3 months of mid-thoracic back pain which progressed to include progressive paraesthesias and lower extremity weakness. Investigations revealed thoracic spine osteomyelitis with signs of cord compression. He underwent neurosurgical intervention, including laminectomy, spinal cord decompression and partial resection of an epidural mass. Initial intraoperative biopsy and surgical pathology results were concerning for an acid-fast bacillus as the causative pathogen, and the patient was given empiric therapy for presumed Mycobacterium tuberculosis. However, microbiology speciation revealed the presence of the non-tuberculous mycobacterium (NTM) Mycobacterium kansasii, which resulted in an alteration of his antimicrobial therapy. This case highlights the importance of considering NTM as a possible aetiology of spinal osteomyelitis, even among immunocompetent individuals or in low-prevalence regions.

Fluoroquinolone resistance among Enterobacteriaceae is a notable challenge for appropriate empiric therapy in outpatient settings. We describe the spatial distribution of fluoroquinolone resistance and its chronological change between 2000 and 2017 in the nationwide Veterans' Health Administration system. We found spatially concentrated increasing prevalence in the 2000s, followed by spatial dispersion in the 2010s.