Baseline Sequencing Surveillance of Public Clinical Testing, Hospitals, and Community Wastewater Reveals Rapid Emergence of SARS-CoV-2 Omicron Variant of Concern in Arizona, USA.

The adaptive evolution of SARS-CoV-2 variants is driven by selection for increased viral fitness in transmissibility and immune evasion. Understanding the dynamics of how an emergent variant sweeps across populations can better inform public health response preparedness for future variants. Here, we investigated the state-level genomic epidemiology of SARS-CoV-2 through baseline genomic sequencing surveillance of 27,071 public testing specimens and 1,125 hospital inpatient specimens diagnosed between November 1, 2021, and January 31, 2022, in Arizona. We found that the Omicron variant rapidly displaced Delta variant in December 2021, leading to an "Omicron surge" of COVID-19 cases in early 2022. Wastewater sequencing surveillance of 370 samples supported the synchronous sweep of Omicron in the community. Hospital inpatient COVID-19 cases of Omicron variant presented to three major hospitals 10.51 days after its detection from public clinical testing. Nonsynonymous mutations in nsp3, nsp12, and nsp13 genes were significantly associated with Omicron hospital cases compared to community cases. To model SARS-CoV-2 transmissions across the state population, we developed a scalable sequence network methodology and showed that the Omicron variant spread through intracounty and intercounty transmissions. Finally, we demonstrated that the temporal emergence of Omicron BA.1 to become the dominant variant (17.02 days) was 2.3 times faster than the prior Delta variant (40.70 days) or subsequent Omicron sublineages BA.2 (39.65 days) and BA.5 (35.38 days). Our results demonstrate the uniquely rapid sweep of Omicron BA.1. These findings highlight how integrated public health surveillance can be used to enhance preparedness and response to future variants. IMPORTANCE SARS-CoV-2 continues to evolve new variants throughout the pandemic. However, the temporal dynamics of how SARS-CoV-2 variants emerge to become the dominant circulating variant is not precisely known. Genomic sequencing surveillance offers unique insights into how SARS-CoV-2 spreads in communities and the lead-up to hospital cases during a surge. Specifically, baseline sequencing surveillance through random selection of positive diagnostic specimens provides a representative outlook of the virus lineages circulating in a geographic region. Here, we investigated the emergence of the Omicron variant of concern in Arizona by leveraging baseline genomic sequence surveillance of public clinical testing, hospitals, and community wastewater. We tracked the spread and evolution of the Omicron variant as it first emerged in the general public, and its rapid shift in hospital admissions in the state health system. This study demonstrates the timescale of public health preparedness needed to respond to an antigenic shift in SARS-CoV-2.

Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant.IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.

The prevalence of antiseptic tolerance genes among staphylococci and enterococci in a pediatric population.

OBJECTIVE: The smr and qacA/B genes in Staphylococcus aureus confer tolerance to antiseptics and are associated with nosocomial acquisition of infection and underlying medical conditions. Such antiseptic tolerance (AT) genes have also been reported in coagulase-negative staphylococci (CoNS) and enterococci, however, few data are available regarding their prevalence. We sought to describe the frequency of AT genes among bloodstream isolates of S. aureus, CoNS and enterococci at Texas Children's Hospital (TCH). METHODS: Banked CoNS, S. aureus and enterococci isolated from blood cultures collected bewteen October 1, 2016, and October 1, 2017, were obtained from the TCH clinical microbiology laboratory. All isolates underwent polymerase chain reaction (PCR) assay for the qacA/B and smr genes. Medical records were reviewed for all cases. RESULTS: In total, 103 CoNS, 19 Enterococcus spp, and 119 S. aureus isolates were included in the study, and 80.6% of the CoNS possessed at least 1 AT gene compared to 37% of S. aureus and 43.8% of E. faecalis isolates (P < .001). Among CoNS bloodstream isolates, the presence of either AT gene was strongly associated with nosocomial infection (P < .001). The AT genes in S. aureus were associated with nosocomial infection (P = .025) as well as the diagnosis of central-line-associated bloodstream infection (CLABSI; P = .04) and recent hospitalizations (P < .001). We found no correlation with genotypic AT in E. faecalis and any clinical variable we examined. CONCLUSIONS: Antiseptic tolerance is common among bloodstream staphylococci and E. faecalis isolates at TCH. Among CoNS, the presence of AT genes is strongly correlated with nosocomial acquisition of infection, consistent with previous studies in S. aureus. These data suggest that the healthcare environment contributes to AT among staphylococci.

NLRP12 provides a critical checkpoint for osteoclast differentiation.

The alternative or noncanonical nuclear factor kappa B (NF-κB) pathway regulates the osteoclast (OC) response to receptor activator of nuclear factor kappa B ligand (RANKL) and thus bone metabolism. Although several lines of evidence support the emerging concept that nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12 (NLRP12) impedes alternative NF-κB activation in innate immune cells, a functional role for NLRP12 outside an inflammatory disease model has yet to be reported. Our study demonstrates that NLRP12 has a protective role in bone via suppression of alternative NF-κB-induced osteoclastogenesis and is down-modulated in response to osteoclastogenic stimuli. Here, we show that retroviral overexpression of NLRP12 suppressed RelB nuclear translocation and OC formation. Conversely, genetic ablation of NLRP12 promoted NIK stabilization, RelB nuclear translocation, and increased osteoclastogenesis in vitro. Using radiation chimeras, we demonstrated these in vitro observations dovetail with our in vivo findings that NLRP12 deficiency leads to enhanced OC numbers accompanied by a significant decline in bone mass under physiological conditions. Consistent with the basal bone phenotype, we also observed an enhanced osteolytic response following RANKL injection over the calvaria of NLRP12-deficient chimeric mice compared with wild-type control mice. Thus, modulation of NLRP12 levels controls alternative NF-κB signaling in OC precursors, altering bone homeostasis and osteolytic responses.

Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases.

Filifactor alocis infection and inflammatory responses in the mouse subcutaneous chamber model.

Recent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10(9) F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Pathogenic microbes and community service through manipulation of innate immunity.

The periodontal pathogen Porphyromonas gingivalis undermines major components of innate immunity, such as complement, Toll-like receptors (TLR), and their crosstalk pathways. At least in principle, these subversive activities could promote the adaptive fitness of the entire periodontal biofilm community. In this regard, the virulence factors responsible for complement and TLR exploitation (gingipain enzymes, atypical lipopolysaccharide molecules, and fimbriae) are released as components of readily diffusible membrane vesicles, which can thus become available to other biofilm organisms. This review summarizes important immune subversive tactics of P. gingivalis which might enable it to exert a supportive impact on the oral microbial community.

Neutrophils in periodontal inflammation.

Neutrophils (also called polymorphonuclear leukocytes) are the most abundant leukocytes whose primary purpose as anti-microbial professional phagocytes is to kill extracellular pathogens. Neutrophils and macrophages are phagocytic cell types that along with other cells effectively link the innate and adaptive arms of the immune response, and help promote inflammatory resolution and tissue healing. Found extensively within the gingival crevice and epithelium, neutrophils are considered the key protective cell type in the periodontal tissues. Histopathology of periodontal lesions indicates that neutrophils form a 'wall' between the junctional epithelium and the pathogen-rich dental plaque which functions as a robust anti-microbial secretory structure and as a unified phagocytic apparatus. However, neutrophil protection is not without cost and is always considered a two-edged sword in that overactivity of neutrophils can cause tissue damage and prolong the extent and severity of inflammatory periodontal diseases. This review will cover the innate and inflammatory functions of neutrophils, and describe the importance and utility of neutrophils to the host response and the integrity of the periodontium in health and disease.

The C5a receptor impairs IL-12-dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone loss.

The C5a anaphylatoxin receptor (C5aR; CD88) is activated as part of the complement cascade and exerts important inflammatory, antimicrobial, and regulatory functions, at least in part, via crosstalk with TLRs. However, the periodontal pathogen Porphyromonas gingivalis can control C5aR activation by generating C5a through its own C5 convertase-like enzymatic activity. In this paper, we show that P. gingivalis uses this mechanism to proactively and selectively inhibit TLR2-induced IL-12p70, whereas the same pathogen-instigated C5aR-TLR2 crosstalk upregulates other inflammatory and bone-resorptive cytokines (IL-1ß, IL-6, and TNF-α). In vivo, the ability of P. gingivalis to manipulate TLR2 activation via the C5a-C5aR axis allowed it to escape IL-12p70-dependent immune clearance and to cause inflammatory bone loss in a murine model of experimental periodontitis. In the latter regard, C5aR-deficient or TLR2-deficient mice were both resistant to periodontal bone loss, in stark contrast with wild-type control mice, which is consistent with the interdependent interactions of C5aR and TLR2 in P. gingivalis immune evasion and induction of bone-resorptive cytokines. In conclusion, P. gingivalis targets C5aR to promote its adaptive fitness and cause periodontal disease. Given the current availability of safe and effective C5aR antagonists, pharmacological blockade of C5aR could act therapeutically in human periodontitis and reduce associated systemic risks.

Microbial hijacking of complement-toll-like receptor crosstalk.

Crosstalk between complement and Toll-like receptors (TLRs) coordinates innate immunity. We report a previously unknown immune subversion mechanism involving microbial exploitation of communication between complement and TLRs. Porphyromonas gingivalis, a major oral and systemic pathogen with complement C5 convertase-like activity, synergizes with C5a (fragment of complement protein C5) to increase cyclic adenosine monophosphate (cAMP) concentrations, resulting in suppression of macrophage immune function and enhanced pathogen survival in vitro and in vivo. This synergy required TLR2 signaling, a pertussis toxin- and thapsigargin-sensitive C5a receptor pathway, with protein kinase A and glycogen synthase kinase-3beta as downstream effectors. Antagonistic blockade of the C5a receptor abrogated this evasive strategy and may thus have important therapeutic implications for periodontitis and atherosclerosis, diseases in which P. gingivalis is implicated. This first demonstration of complement-TLR crosstalk for immunosuppressive cAMP signaling indicates that pathogens may not simply undermine complement or TLRs (or both) as separate entities, but may also exploit their crosstalk pathways.