Sampling and recovery of infectious SARS-CoV-2 from high-touch surfaces by sponge stick and macrofoam swab.

Effective sampling for severe acute respiratory syndrome 2 (SARS-CoV-2) is a common approach for monitoring disinfection efficacy and effective environmental surveillance. This study evaluated sampling efficiency and limits of detection (LODs) of macrofoam swab and sponge stick sampling methods for recovering infectious SARS-CoV-2 and viral RNA (vRNA) from surfaces. Macrofoam swab and sponge stick methods were evaluated for collection of SARS-CoV-2 suspended in a soil load from 6-in2 coupons composed of four materials: stainless steel (SS), acrylonitrile butadiene styrene (ABS) plastic, bus seat fabric, and Formica. Recovery of infectious SARS-CoV-2 was more efficient than vRNA recovery on all materials except Formica (macrofoam swab sampling) and ABS (sponge stick sampling). Macrofoam swab sampling recovered significantly more vRNA from Formica than ABS and SS, and sponge stick sampling recovered significantly more vRNA from ABS than Formica and SS, suggesting that material and sampling method choice can affect surveillance results. Time since initial contamination significantly affected infectious virus recovery from all materials, with vRNA recovery showing limited to no difference, suggesting that SARS-CoV-2 vRNA can remain detectable after viral infectivity has dissipated. This study showed that a complex relationship exists between sampling method, material, time from contamination to sampling, and recovery of SARS-CoV-2. In conclusion, data show that careful consideration be used when selecting surface types for sampling and interpreting SARS-CoV-2 vRNA recovery with respect to presence of infectious virus.

Complement Receptor 3 Mediates HIV-1 Transcytosis across an Intact Cervical Epithelial Cell Barrier: New Insight into HIV Transmission in Women.

Transmission of HIV across the mucosal surface of the female reproductive tract to engage subepithelial CD4-positive T cells is not fully understood. Cervical epithelial cells express complement receptor 3 (CR3) (integrin αMß2 or CD11b/CD18). In women, the bacterium Neisseria gonorrhoeae uses CR3 to invade the cervical epithelia to cause cervicitis. We hypothesized that HIV may also use CR3 to transcytose across the cervical epithelia. Here, we show that HIV-1 strains bound with high affinity to recombinant CR3 in biophysical assays. HIV-1 bound CR3 via the I-domain region of the CR3 alpha subunit, CD11b, and binding was dependent on HIV-1 N-linked glycans. Mannosylated glycans on the HIV surface were a high-affinity ligand for the I-domain. Man5 pentasaccharide, representative of HIV N-glycans, could compete with HIV-1 for CR3 binding. Using cellular assays, we show that HIV bound to CHO cells by a CR3-dependent mechanism. Antibodies to the CR3 I-domain or to the HIV-1 envelope glycoprotein blocked the binding of HIV-1 to primary human cervical epithelial (Pex) cells, indicating that CR3 was necessary and sufficient for HIV-1 adherence to Pex cells. Using Pex cells in a Transwell model system, we show that, following transcytosis across an intact Pex cell monolayer, HIV-1 is able to infect TZM-bl reporter cells. Targeting the HIV-CR3 interaction using antibodies, mannose-binding lectins, or CR3-binding small-molecule drugs blocked HIV transcytosis. These studies indicate that CR3/Pex may constitute an efficient pathway for HIV-1 transmission in women and also demonstrate strategies that may prevent transmission via this pathway. IMPORTANCE In women, the lower female reproductive tract is the primary site for HIV infection. How HIV traverses the epithelium to infect CD4 T cells in the submucosa is ill-defined. Cervical epithelial cells have a protein called CR3 on their surface. We show that HIV-1 binds to CR3 with high affinity and that this interaction is necessary and sufficient for HIV adherence to, and transcytosis across, polarized, human primary cervical epithelial cells. This suggests a unique role for CR3 on epithelial cells in dually facilitating HIV-1 attachment and entry. The HIV-CR3 interaction may constitute an efficient pathway for HIV delivery to subepithelial lymphocytes following virus transmission across an intact cervical epithelial barrier. Strategies with potential to prevent transmission via this pathway are presented.

Residual antimicrobial coating efficacy against SARS-CoV-2.

AIMS: This study evaluated the residual efficacy of commercially available antimicrobial coatings or films against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on non-porous surfaces. METHODS AND RESULTS: Products were applied to stainless steel or ABS plastic coupons and dried overnight. Coupons were inoculated with SARS-CoV-2 in the presence of 5% soil load. Recovered infectious SARS-CoV-2 was quantified by TCID50 assay. Tested product efficacies ranged from <1.0 to >3.0 log10 reduction at a 2-h contact time. The log10 reduction in recovered infectious SARS-CoV-2 ranged from 0.44 to 3 log10 reduction on stainless steel and 0.25 to >1.67 log10 on ABS plastic. The most effective products tested contained varying concentrations (0.5%-1.3%) of the same active ingredient: 3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride. Products formulated with other quaternary ammonium compounds were less effective against SARS-CoV-2 in this test. CONCLUSIONS: The residual antimicrobial products tested showed varied effectiveness against SARS-CoV-2 as a function of product tested. Several products were identified as efficacious against SARS-CoV-2 on both stainless steel and ABS plastic surfaces under the conditions evaluated. Differences in observed efficacy may be due to variation in active ingredient formulation; efficacy is, therefore, difficult to predict based upon listed active ingredient and its concentration. SIGNIFICANCE AND IMPACT: This study highlights the formulation-specific efficacy of several products against SARS-CoV-2 and may inform future development of residual antiviral products for use on non-porous surfaces. The identification of antimicrobial coatings or films showing promise to inactivate SARS-CoV-2 suggests that these products may be worth future testing and consideration.

Efficacy of detergent-based cleaning methods against coronavirus MHV-A59 on porous and non-porous surfaces.

This study evaluated the efficacy of detergent-based surface cleaning methods against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV (5% soil load in culture medium or simulated saliva) was inoculated onto four different high-touch materials [stainless steel (SS), Acrylonitrile Butadiene Styrene plastic (ABS), Formica, seat fabric (SF)]. Immediately and 2-hr post-inoculation, coupons were cleaned (damp wipe wiping) with and without pretreatment with detergent solution or 375 ppm hard water. Results identified that physical removal (no pretreatment) removed >2.3 log10 MHV on ABS, SS, and Formica when surfaces were cleaned immediately. Pretreatment with detergent or hard water increased effectiveness over wet wiping 2-hr post-inoculation; pretreatment with detergent significantly increased (p ≤ 0.05) removal of MHV in simulated saliva, but not in culture media, over hard water pretreatment (Formica and ABS). Detergent and hard water cleaning methods were ineffective on SF under all conditions. Overall, efficacy of cleaning methods against coronaviruses are material- and matrix-dependent; pre-wetting surfaces with detergent solutions increased efficacy against coronavirus suspended in simulated saliva. This study provides data highlighting the importance of incorporating a pre-wetting step prior to detergent cleaning and can inform cleaning strategies to reducing coronavirus surface transmission.

Efficacy of chemical disinfectants against SARS-CoV-2 on high-touch surface materials.

AIMS: This study aimed to provide operationally relevant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface disinfection efficacy information. METHODS AND RESULTS: Three EPA-registered disinfectants (Vital Oxide, Peroxide, and Clorox Total 360) and one antimicrobial formulation (CDC bleach) were evaluated against SARS-CoV-2 on material coupons and were tested using Spray (no touch with contact time) and Spray & Wipe (wipe immediately post-application) methods immediately and 2 h post-contamination. Efficacy was evaluated for infectious virus, with a subset tested for viral RNA (vRNA) recovery. Efficacy varied by method, disinfectant, and material. CDC bleach solution showed low efficacy against SARS-CoV-2 (log reduction < 1.7), unless applied via Spray & Wipe. Additionally, mechanical wiping increased the efficacy of treatments against SARS-CoV-2. The recovery of vRNA post-disinfection suggested that vRNA may overestimate infectious virus remaining. CONCLUSIONS: Efficacy depends on surface material, chemical, and disinfection procedure, and suggests that mechanical wiping alone has some efficacy at removing SARS-CoV-2 from surfaces. We observed that disinfectant treatment biased the recovery of vRNA over infectious virus. SIGNIFICANCE AND IMPACT OF STUDY: These data are useful for developing effective, real-world disinfection procedures, and inform public health experts on the utility of PCR-based surveillance approaches.

Continuous Microevolution Accelerates Disease Progression during Sequential Episodes of Infection.

Bacteria adapt to dynamic changes in the host during chronic and recurrent infections. Bacterial microevolution is one type of adaptation that imparts a selective advantage. We hypothesize that recurrent episodes of disease promote microevolution through genetic mutations that modulate disease severity. We use a pre-clinical model of otitis media (OM) to determine the potential role for microevolution of nontypeable Haemophilus influenzae (NTHI) during sequential episodes of disease. Whole genome sequencing reveals microevolution of hemoglobin binding and lipooligosaccharide (LOS) biosynthesis genes, suggesting that adaptation of these systems is critical for infection. These OM-adapted strains promote increased biofilm formation, inflammation, stromal fibrosis, and an increased propensity to form intracellular bacterial communities (IBCs). Remarkably, IBCs remain for at least one month following clinical resolution of infection, suggesting an intracellular reservoir as a nidus for recurrent OM. Additional approaches for therapeutic design tailored to combat this burdensome disease will arise from these studies.

Reprioritization of biofilm metabolism is associated with nutrient adaptation and long-term survival of Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Transient Nutrient Deprivation Promotes Macropinocytosis-Dependent Intracellular Bacterial Community Development.

Nutrient limitation restricts bacterial growth in privileged sites such as the middle ear. Transient heme-iron restriction of nontypeable Haemophilus influenzae (NTHI), the major causative agent of chronic and recurrent otitis media (OM), promotes new and diverse phenotypes that can influence planktonic, biofilm, and intracellular lifestyles of NTHI. However, the bacterial responses to nutrient restriction that impact intracellular fate and survival of NTHI are unknown. In this work, we provide evidence for the role of transient heme-iron restriction in promoting the formation of intracellular bacterial communities (IBCs) of NTHI both in vitro and in vivo in a preclinical model of OM. We show that transient heme-iron restriction of NTHI results in significantly increased invasion and intracellular populations that escape or evade the endolysosomal pathway for increased intracellular survival. In contrast, NTHI continuously exposed to heme-iron traffics through the endolysosomal pathway for degradation. The use of pharmacological inhibitors revealed that prior heme-iron status does not appear to influence NTHI internalization through endocytic pathways. However, inhibition of macropinocytosis altered the intracellular fate of transiently restricted NTHI for degradation in the endolysosomal pathway. Furthermore, prevention of macropinocytosis significantly reduced the number of IBCs in cultured middle ear epithelial cells, providing evidence for the feasibility of this approach to reduce OM persistence. These results reveal that microenvironmental cues can influence the intracellular fate of NTHI, leading to new mechanisms for survival during disease progression.IMPORTANCE Otitis media is the most common bacterial infection in childhood. Current therapies are limited in the prevention of chronic or recurrent otitis media which leads to increased antibiotic exposure and represents a significant socioeconomic burden. In this study, we delineate the effect of nutritional limitation on the intracellular trafficking pathways used by nontypeable Haemophilus influenzae (NTHI). Moreover, transient limitation of heme-iron led to the development of intracellular bacterial communities that are known to contribute to persistence and recurrence in other diseases. New approaches for therapeutic interventions that reduce the production of intracellular bacterial communities and promote trafficking through the endolysosomal pathway were revealed through the use of pharmacological inhibition of macropinocytosis. This work demonstrates the importance of an intracellular niche for NTHI and provides new approaches for intervention for acute, chronic, and recurring episodes of otitis media.

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment.

A thorough understanding of the molecular details of the interactions between bacteria and host are critical to ultimately prevent disease. Recent technological advances allow simultaneous analysis of host and bacterial protein and metabolic profiles from a single small tissue sample to provide insight into pathogenesis. We used the chinchilla model of human otitis media to determine, for the first time, the most expansive delineation of global changes in protein and metabolite profiles during an experimentally induced disease. After 48 h of infection with nontypeable Haemophilus influenzae, middle ear tissue lysates were analyzed by high-resolution quantitative two-dimensional liquid chromatography-tandem mass spectrometry. Dynamic changes in 105 chinchilla proteins and 66 metabolites define the early proteomic and metabolomic signature of otitis media. Our studies indicate that establishment of disease coincides with actin morphogenesis, suppression of inflammatory mediators, and bacterial aerobic respiration. We validated the observed increase in the actin-remodeling complex, Arp2/3, and experimentally showed a role for Arp2/3 in nontypeable Haemophilus influenzae invasion. Direct inhibition of actin branch morphology altered bacterial invasion into host epithelial cells, and is supportive of our efforts to use the information gathered to modify outcomes of disease. The twenty-eight nontypeable Haemophilus influenzae proteins identified participate in carbohydrate and amino acid metabolism, redox homeostasis, and include cell wall-associated metabolic proteins. Quantitative characterization of the molecular signatures of infection will redefine our understanding of host response driven developmental changes during pathogenesis. These data represent the first comprehensive study of host protein and metabolite profiles in vivo in response to infection and show the feasibility of extensive characterization of host protein profiles during disease. Identification of novel protein targets and metabolic biomarkers will advance development of therapeutic and diagnostic options for treatment of disease.