Effectiveness of empiric carbapenem versus non-carbapenem therapy for extended-spectrum ß-lactamase producing Enterobacterales infections in non-intensive care unit patients: a real-world investigation in a hospital with high-prevalence of extended-spectrum ß-lactamase producing Enterobacterales.

Objective: To investigate whether empiric carbapenem therapy, compared to empiric non-carbapenem therapy, was associated with improved clinical outcomes among hospitalized, non-intensive care unit (ICU) patients with extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales infections. Methods: We performed a retrospective cohort study of adult, non-ICU patients admitted with ESBL-producing Enterobacterales infections. Primary outcome was time to clinical stability from the first empiric antibiotic dose. Secondary outcomes were early clinical response and 30-day all-cause hospital readmission. We used multivariate regression methods to examine time to clinical stability. Results: Of the 142 patients, 59 (42%) received empiric carbapenems and 83 (58%) received empiric non-carbapenems, most commonly ceftriaxone (49/83, 59%). Median age was 59 years. The most common infection source was urinary (71%). The carbapenem group had a higher proportion of patients who received antibiotics within 6 months of admission (55% vs 28%, P < .01) and history of ESBL (57% vs 17%, P < .01). There were no significant differences in hours until clinical stability between the carbapenem and non-carbapenem groups (22 (IQR: 0, 85) vs 19 (IQR: 0, 69), P = .54). Early clinical response (88% vs 90%, P = .79) and 30-day all-cause hospital readmission (17% vs 8%, P = .13) were similar between groups. Conclusion: Among hospitalized non-ICU patients with ESBL-producing Enterobacterales infection, we found no difference in time to clinical stability after the first empiric antibiotic dose between those receiving carbapenems and those who did not. Our data suggest that empiric carbapenem use may not be an important driver of clinical response in patients with less severe ESBL-producing Enterobacterales infection.

Skin and soft tissue infection incidence before and during the COVID-19 pandemic.

Skin and Soft Tissue Infections (SSTIs) are common bacterial infections. We hypothesized that due to the COVID-19 pandemic, SSTI rates would significantly decrease due to directives to avoid unneeded care and attenuated SSTIs risk behaviours. We retrospectively examined all patients with an ICD-10 diagnosis code in the Los Angeles County Department of Health Services, the second largest U.S. safety net healthcare system between 16 March 2017 and 15 March 2022. We then compared pre-pandemic with intra-pandemic SSTI rates using an interrupted time series analysis. We found 72,118 SSTIs, 46,206 during the pre-pandemic period and 25,912 during the intra-pandemic period. Pre-pandemic SSTI rate was significantly higher than the intra-pandemic rate (3.27 vs. 2.31 cases per 1,000 empanelled patient-months, P < 0.0001). The monthly SSTI cases decreased by 1.19 SSTIs/1,000 empanelled patient-months between the pre- and intra-pandemic periods (P = 0.0003). SSTI subgroups (inpatient, observation unit, emergency department, and outpatient clinics), all had significant SSTI decreases between the two time periods (P < 0.05) except for observation unit (P = 0.50). Compared to the pre-COVID-19 pandemic period, medically attended SSTI rates in our large U.S. safety net healthcare system significantly decreased by nearly 30%. Whether findings reflect true SSTI decreases or decreased health system utilization for SSTIs requires further examination.

Longitudinal analysis of SARS-CoV-2 infection and vaccination in the LA-SPARTA cohort reveals increased risk of infection in vaccinated Hispanic participants.

Introduction: SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19). Questions remain regarding correlates of risk and immune protection against COVID-19. Methods: We prospectively enrolled 200 participants with a high risk of SARS-CoV-2 occupational exposure at a U.S. medical center between December 2020 and April 2022. Participant exposure risks, vaccination/infection status, and symptoms were followed longitudinally at 3, 6, and 12 months, with blood and saliva collection. Serological response to the SARS-CoV-2 spike holoprotein (S), receptor binding domain (RBD) and nucleocapsid proteins (NP) were quantified by ELISA assay. Results: Based on serology, 40 of 200 (20%) participants were infected. Healthcare and non-healthcare occupations had equivalent infection incidence. Only 79.5% of infected participants seroconverted for NP following infection, and 11.5% were unaware they had been infected. The antibody response to S was greater than to RBD. Hispanic ethnicity was associated with 2-fold greater incidence of infection despite vaccination in this cohort. Discussion: Overall, our findings demonstrate: 1) variability in the antibody response to SARS-CoV-2 infection despite similar exposure risk; 2) the concentration of binding antibody to the SARS-CoV-2 S or RBD proteins is not directly correlated with protection against infection in vaccinated individuals; and 3) determinants of infection risk include Hispanic ethnicity despite vaccination and similar occupational exposure.

Characterization of COVID-19 Vaccine Hesitancy Among Essential Workforce Members of a Large Safety Net Urban Medical Center.

OBJECTIVES: Vaccine hesitancy among essential workers remains a significant public health challenge. We examined psychological constructs of perceived susceptibility, threat, and self-efficacy and their associations with COVID-19 vaccine hesitancy among a racially and ethnically diverse essential workforce population. METHODS: We performed a cross-sectional survey of essential workers from September-December 2020 at a large Los Angeles safety-net medical center as part of a program offering free COVID-19 serology testing. Program participants completed a standardized survey at the time of phlebotomy. Hierarchical logistic regression was utilized to determine factors independently associated with vaccine hesitancy. RESULTS: Among 1327 persons who had serology testing, 1235 (93%) completed the survey. Of these, 958 (78%) were healthcare workers. Based on expressed intent, 22% were vaccine-hesitant 78% were vaccine acceptors. In our multivariate model, vaccine hesitancy was associated with female gender [aOR = 2.09; 95% CI (1.44-3.05)], African American race [aOR = 4.32; (2.16-8.62)], LatinX ethnicity [aOR = 2.47; 95% CI (1.51-4.05)] and history of not/sometimes receiving influenza vaccination [aOR = 4.39; 95% CI (2.98-6.48)]. Compared to nurses, vaccine hesitancy was lower among physicians [aOR = 0.09; 95% CI (0.04-0.23)], non-nursing/non-physician healthcare workers [aOR = 0.55; 95% CI (0.33-0.92)], and non-healthcare care workers [aOR = 0.53; 95% CI (0.36-0.78)]. CONCLUSIONS: Among a racially/ethnically diverse group of safety net medical center essential workers, COVID-19 vaccine hesitancy was associated with racial/ethnic minority groups, employment type, and prior influenza vaccination hesitancy. Interestingly, we found no association with the Health Belief Model construct measures of perceived susceptibility, threat, and self-efficacy. Psychological constructs not assessed may be drivers of vaccine hesitancy in our population.

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection.

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.

Context mediates antimicrobial efficacy of kinocidin congener peptide RP-1.

Structure-mechanism relationships are key determinants of host defense peptide efficacy. These relationships are influenced by anatomic, physiologic and microbiologic contexts. Structure-mechanism correlates were assessed for the synthetic peptide RP-1, modeled on microbicidal domains of platelet kinocidins. Antimicrobial efficacies and mechanisms of action against susceptible ((S)) or resistant ((R)) Salmonella typhimurium (ST), Staphylococcus aureus (SA), and Candida albicans (CA) strain pairs were studied at pH 7.5 and 5.5. Although RP-1 was active against all study organisms, it exhibited greater efficacy against bacteria at pH 7.5, but greater efficacy against CA at pH 5.5. RP-1 de-energized SA and CA, but caused hyperpolarization of ST in both pH conditions. However, RP-1 permeabilized ST(S) and CA strains at both pH, whereas permeabilization was modest for ST(R) or SA strain at either pH. Biochemical analysis, molecular modeling, and FTIR spectroscopy data revealed that RP-1 has indistinguishable net charge and backbone trajectories at pH 5.5 and 7.5. Yet, concordant with organism-specific efficacy, surface plasmon resonance, and FTIR, molecular dynamics revealed modest helical order increases but greater RP-1 avidity and penetration of bacterial than eukaryotic lipid systems, particularly at pH 7.5. The present findings suggest that pH- and target-cell lipid contexts influence selective antimicrobial efficacy and mechanisms of RP-1 action. These findings offer new insights into selective antimicrobial efficacy and context-specificity of antimicrobial peptides in host defense, and support design strategies for potent anti-infective peptides with minimal concomitant cytotoxicity.

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine.

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Na(v) channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structure-function homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of anti-infective or therapeutic peptides that selectively target microbial or abnormal host cells.

Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release.

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of > or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5'-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

Modular determinants of antimicrobial activity in platelet factor-4 family kinocidins.

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38-74) and modular domains thereof (49-63 and 60-74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60-74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.

Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins.

Chemokines are small (8-12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure-activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved gamma-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 gamma-core (IL-8gamma) and alpha-helical (IL-8alpha) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8alpha motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing gamma-core motifs and microbicidal helices associated with antimicrobial activity.

Salicylic acid attenuates virulence in endovascular infections by targeting global regulatory pathways in Staphylococcus aureus.

Aspirin has been previously shown to reduce the in vivo virulence of Staphylococcus aureus in experimental endocarditis, through antiplatelet and antimicrobial mechanisms. In the present study, salicylic acid, the major in vivo metabolite of aspirin, mitigated two important virulence phenotypes in both clinical and laboratory S. aureus strains: alpha-hemolysin secretion and fibronectin binding in vitro. In addition, salicylic acid reduced the expression of the alpha-hemolysin gene promoter, hla, and the fibronectin gene promoter, fnbA. Transcriptional analysis, fluorometry, and flow cytometry revealed evidence of salicylic acid-mediated activation of the stress-response gene sigB. Expression of the sigB-repressible global regulon sarA and the global regulon agr were also mitigated by salicylic acid, corresponding to the reduced expression of the hla and fnbA genes in vitro. Studies in experimental endocarditis confirmed the key roles of both sarA and sigB in mediating the antistaphylococcal effects of salicylic acid in vivo. Therefore, aspirin has the potential to be an adjuvant therapeutic agent against endovascular infections that result from S. aureus, by downmodulating key staphylococcal global regulons and structural genes in vivo, thus abrogating relevant virulence phenotypes.

Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1.

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.