Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 10⁵  CFUs mL^-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.

Purpose

To report a case of orbital coccidiomycosis in an otherwise healthy 11-month-old male.

Observations

An 11-month-old male presented to his pediatrician with parental complaints of swelling, erythema, and pain of the right orbit that increased over ten days in the absence of constitutional symptoms. The child's parents reported an earlier fall onto a carpeted floor. After four weeks of conservative treatment and a course of oral cephalexin, he developed a fever, increased erythema, and palpable enlargement of a mass posterior to the lower eyelid. Ultrasound revealed an encysted mass in the inferior orbit, suggestive of an abscess. Urgent ophthalmic referral led to incision and drainage via orbitotomy. Culture, histopathology, and serological testing were positive for Coccidioides spp.. Blood studies revealed mild anemia and thrombocytosis. There was complete resolution of symptoms after surgical drainage and several weeks of oral fluconazole.

Conclusion and importance

We describe a patient with orbital coccidiomycosis without apparent systemic involvement, following what was most likely an unrelated minor trauma. Despite being rare, orbital coccidiomycosis should be considered as a primary manifestation of infection when ocular inflammation is encountered, especially in endemic regions.

The COVID pandemic has put a spotlight on laboratory medicine, showcasing how vital diagnostic testing is for society and the health care system. It has also brought to light and accelerated the critical shortage of trained and experienced laboratory personnel that has been felt for decades. The need for laboratory professionals is expected to grow by 11% between 2020 and 2030, a higher rate of growth than the overall average for all other health care occupations. Here, the background to this workforce shortage is reviewed. Some proposed actions to help address the issue are put forth, including increasing awareness of the medical laboratory science profession along with bolstering training opportunities and awareness of alternate routes to obtaining certification as a medical laboratory scientist. In addition, recent survey data specifically related to the employee shortages in microbiology are presented which demonstrate that 80% of microbiology laboratories have vacant positions and that filling these positions is challenging for a number of reasons, including a lack of qualified applicants.

Unlabelled

The Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia.

Importance

This study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.

Chlamydophila pneumoniae (CPn) is a common respiratory pathogen that causes a chronic and persistent airway infection. The elderly display an increased susceptibility and severity to this infection. However, the underlying mechanisms are not well understood. Dendritic cells (DCs) are the initiators and regulators of immune responses. Therefore, we investigated the role of DCs in the age-associated increased CPn infection in vitro in humans. Though the expression of activation markers was comparable between the two age groups, DCs from aged subjects secreted enhanced levels of proinflammatory mediators such as TNF-α and CXCL-10 in response to CPn. In contrast, the secretion of IL-10 and innate interferons, IFN-α and IFN-λ, was severely impaired in DCs from aged donors. The increased activation of DCs from aged subjects to CPn also resulted in enhanced proliferation of CD4 and CD8 T cells in a DC-T coculture. Furthermore, T cells primed with CPn-stimulated DCs from aged subjects secreted increased levels of IFN-γ and reduced levels of IL-10 compared to DCs obtained from young subjects. In summary, DCs from the elderly displayed enhanced inflammatory response to CPn which may result in airway remodeling and increase the susceptibility of the elderly to respiratory diseases such as asthma.

Introduction

Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.

Methods

Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.

Results

We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.

Conclusions

We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.

Objective

To evaluate whether an ecologic inverse association exists between methicillin-susceptible Staphylococcus aureus (MSSA) prevalence and methicillin-resistant S. aureus (MRSA) prevalence in nursing homes.

Methods

We conducted a secondary analysis of a prospective cross-sectional study of S. aureus prevalence in 26 nursing homes across Orange County, California, from 2008-2011. Admission prevalence was assessed using bilateral nares swabs collected from all new residents within 3 days of admission until 100 swabs were obtained. Point prevalence was assessed from a representative sample of 100 residents. Swab samples were plated on 5% sheep blood agar and Spectra MRSA chromogenic agar. If MRSA was detected, no further tests were performed. If MRSA was not detected, blood agar was evaluated for MSSA growth. We evaluated the association between MRSA and MSSA admission and point prevalence using correlation and linear regression testing.

Results

We collected 3,806 total swabs. MRSA and MSSA admission prevalence were not correlated (r = -0.40, P = .09). However, MRSA and MSSA point prevalence were negatively correlated regardless of whether MSSA prevalence was measured among all residents sampled (r = -0.67, P = .0002) or among those who did not harbor MRSA (r = -0.41, P = .04). This effect persisted in regression models adjusted for the percentage of residents with diabetes (β = -0.73, P = .04), skin lesions (β = -1.17, P = .002), or invasive devices (β = -1.4, P = .0006).

Conclusions

The inverse association between MRSA and MSSA point prevalence and minimal association on admission prevalence suggest MSSA carriage may protect against MRSA acquisition in nursing homes. The minimal association on admission prevalence further suggests competition may occur during nursing home stays.

Objectives

To determine whether environmental cleaning and contamination are associated with variation in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) between nursing homes.

Design

Prospective study of environmental contamination and cleaning quality.

Setting

Nursing home.

Participants

Ten California nursing homes.

Measurements

Nursing homes were categorized into two groups based upon high and low differences in MRSA point prevalence and admission prevalence (delta prevalence) from nares screenings of nursing home residents. Environmental cleaning and infection control practices were evaluated by culturing common area objects for MRSA, assessing removal of intentionally applied marks visible only under ultraviolet (UV) light, and administering surveys on infection control and cleaning.

Results

Overall, 16% (78/500) of objects were MRSA positive, and 22% (129/577) of UV-visible marks were removed. A higher proportion of MRSA-positive objects was found in the high (19%) than in low (10%) nursing home groups (P = .005). Infection control and cleaning policies varied, including the frequency of common room cleaning (median 2.5 times daily, range 1-3 times daily) and time spent cleaning per room (median 18 minutes, range 7-45 minutes). In multivariate models, MRSA-positive objects were associated with high delta prevalence nursing homes (odds ratio (OR) = 2.8, P = .005), less time spent cleaning each room (OR = 2.9, P < .001), and less-frequent cleaning of common rooms (OR = 1.5, P = .01).

Conclusion

Substantial variation was found in MRSA environmental contamination, infection control practices, and cleaning quality. MRSA environmental contamination was associated with greater differences between MRSA point and admission prevalence, less-frequent common room cleaning, and less time spent cleaning per room, which suggests that modifying cleaning practices may reduce MRSA environmental contamination and burden in nursing homes.

The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5-4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.