Biology subjects

Reed, P.

Publications and source records attributed to Reed, P..

9 recordsLinked to original sources

bioRxiv2026

Species Identification and Antimicrobial Susceptibility of Mycobacterial Isolates from Dogs and Cats

Nontuberculous Mycobacterium (NTM) species are increasingly recognized as opportunistic pathogens in companion animals, yet data on species distribution and antimicrobial resistance remain limited. The goal of this study was to evaluate species identification methods and determine antimicrobial susceptibility profiles of NTM isolates from cats and dogs submitted to the University of Tennessee veterinary diagnostic laboratory. Twenty-nine NTM isolates (25 feline, 4 canine) collected between 2016 and 2022 were cultured and identified using MALDI-TOF MS and 16S rRNA sequencing. Antimicrobial susceptibility testing (AST) was performed by broth microdilution. Most isolates (27/29) were rapidly growing mycobacteria from skin or wound samples. Combined MALDI-TOF and sequencing identified 11 isolates within the Mycobacterium fortuitum complex, 10 as M. smegmatis, 4 within the M. abscessus group, and one each as M. mucogenicum and M. farcinogenes. MALDI-TOF failed to identify four isolates, including two slow-growing strains. AST revealed consistent susceptibility to amikacin and moxifloxacin, while resistance was highest to doxycycline (44%), clarithromycin (37%), and tobramycin (33%). Rapidly growing NTM predominates in feline cutaneous infections, with species-specific resistance patterns underscoring the need for accurate identification and AST to guide therapy. Importance StatementNontuberculous mycobacteria (NTM) are emerging causes of skin infections in cats and dogs, yet little is known about which species are involved or how they respond to treatment. Knowing the specific species and their resistance patterns is critical for choosing effective treatments and improving outcomes for affected animals. This study provides critical baseline data by identifying NTM species and their antimicrobial resistance patterns in companion animals. These findings highlight the need for accurate diagnostic methods and targeted therapy to improve clinical outcomes. Understanding these trends is essential for guiding veterinary care and informing future research on antimicrobial resistance in animal health.

microbiology↗

bioRxiv2026

BAY 11-7082 potentiates select β-lactams to inhibit growth of methicillin-resistant Staphylococcus aureus.

{beta}-lactams are an important class of antibiotics that target penicillin-binding proteins (PBPs) essential to bacterial cell wall synthesis. They are used to treat serious bacterial infections, including those caused by the versatile pathogen Staphylococcus aureus. Some strains carry the resistance gene, mecA, encoding a {beta}-lactam-insensitive PBP2A that allows for peptidoglycan synthesis in the presence of {beta}-lactams like methicillin, limiting treatment options. We previously identified BAY 11-7082 as having antibacterial activity against methicillin-resistant S. aureus (MRSA) and showed that it re-sensitizes MRSA to {beta}-lactams like penicillin G, making BAY 11-7082 and its analogues promising candidates for the development of an antibiotic adjuvant. Although the direct antibacterial mechanism of BAY 11-7082 remains undefined, here we aimed to better understand how it potentiates {beta}-lactam activity. We tested BAY 11-7082 in combination with a variety of {beta}-lactams and identified a subset that were synergistic in MRSA but not methicillin-susceptible S. aureus, suggesting they may directly or indirectly impact the function of PBP2A. Electron and fluorescence microscopy studies revealed that unlike other {beta}-lactam adjuvants, such as those that target the biosynthesis of wall teichoic acids (WTAs), BAY 11-7082 failed to impact cell division, disrupt PBP2 localization, or activate a sensitive reporter of cell wall damage. However, the production of WTA was necessary for BAY 11-7082-{beta}-lactam synergy. Together these data suggest its mechanism of {beta}-lactam potentiation is distinct from known compounds, making BAY 11-7082 a useful tool to better understand the complexity of resistance in MRSA.

microbiology↗

bioRxiv2025

FtsW protein-protein interactions visualized in live Staphylococcus aureus cells by FLIM-FRET

The bacterial cell cycle relies on the coordinated and dynamic interactions between division proteins and those involved in peptidoglycan (PG) synthesis. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Forster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus using fluorescent proteins. After systematically optimizing growth conditions and the analysis pipeline, we validated the method with cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped the protein interactions of the critical glycosyltransferase FtsW within the septal PG synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, an observation corroborated by an alternative FLIM-FRET method employing Halo-Tag labelled with Janelia Fluor dyes. These findings support the hypothesis that septal PG synthesis may be carried out by a complex of multimers, capable of simultaneously synthesizing more than one glycan strand. Inhibition of PG synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem weakened the interaction between PBP1 and FtsW, whereas the FtsW self-interaction was enhanced in a dose-dependent manner. In contrast, inhibition of PG synthesis by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, had little effect on these interactions. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling the activities of FtsW and PBP1, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of PG synthesis activity.

microbiology↗

bioRxiv2024

Leptospira seroprevalence in dogs, cats and horses: the effect of cross-reactivity between serovars and vaccination status on seroprevalence estimates

In this study, we estimated the Leptospira seroprevalence in dogs (n = 376), horses (n = 88), and cats (n = 169) using Microscopic Agglutination Test (MAT) against 12 Leptospira serovars. We observed a Leptospira seroprevalence of 29.41%, 47.73%, and 12.35% in dogs, horses, and cats, respectively. The highest seroprevalence was observed for serovar Autumnalis (74.55%) in dogs, and Bratislava in horses (95.24%) and cats (42.86%). We found a significant level of potential cross-reactivity between multiple Leptospira serovars tested, with highest cross-reactivity to serovar Autumnalis in dogs. The dogs that were vaccinated against Leptospira had a significantly higher seroprevalence (45.92%) compared to unvaccinated dogs (16.28%; p < 0.001). A significant difference in seroprevalence was observed in vaccinated and unvaccinated dogs to all four serovars included in the canine vaccine (p < 0.001). Among the diagnostic samples submitted from 2021-2023, 40% (103/252) of the canine serum samples were positive by MAT, with the highest positivity rate for the serovar Autumnalis. On Leptospira PCR, 10.7 % (35/325) urine samples and 5.8% (15/257) blood samples were positive. Our study findings show evidence of Leptospira exposure and potential disease in the study area. The cross-reactivity between the Leptospira serovars used in the MAT and vaccination status may also overestimate the levels of exposure.

immunology↗

bioRxiv2023

The role of GpsB in cell morphogenesis of Staphylococcus aureus

For decades, cells of the gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a SEDS (Shape Elongation Division and Sporulation) / PBP (Penicillin Binding Protein) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria the elongation machinery, known as elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins involved in the elongation of S. aureus, we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2, the only bifunctional PBP in S. aureus, with both glycosyltransferase and transpeptidase activity, and of the PBP4 transpeptidase. Increased levels of these PBPs at the cell periphery result in higher levels of peptidoglycan insertion throughout the entire cell, overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum, which leads to cell elongation. As a consequence, in the absence of GpsB, S. aureus cells become more spherical.

microbiology↗

bioRxiv2022

A CRISPRi-based genetic resource to study essential Staphylococcus aureus genes

We have optimized a CRISPR interference system to facilitate gene knockdown in the gram-positive bacterial pathogen Staphylococcus aureus. Our approach used a CRISPRi system derived from Streptococcus pyogenes, which involves the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In our system, dcas9 is expressed from a single copy in the chromosome of methicillin resistant S. aureus (MRSA) strains COL or JE2, under the control of a tightly regulated promoter, inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables efficient, inducible, knockdown of both essential and non-essential genes. Using this approach, we constructed the Lisbon CRISPRi Mutant Library (LCML) comprising 261 strains, in the JE2 background, containing sgRNAs targeting 200 essential genes/operons. This library facilitates the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library which consists of nearly 2000 strains, each carrying a transposon insertion within a non-essential gene. The availability of these two libraries will facilitate the study of S. aureus pathogenesis and biology. Abstract ImportanceStaphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a CRISPRi system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library of 261 strains which allow the depletion of essential proteins encoded in 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library (LCML), should facilitate the study of S. aureus pathogenesis and biology.

microbiology↗

bioRxiv2022

Loss of LRP1 in adult neural stem cells impairs migration to ischemic lesions

After ischemia, cells in the brain parenchyma upregulate stromal derived factor 1 (SDF1), driving chemokine receptor CXCR4-mediated migration of adult neural stem cells from the subventricular zone (SVZ) to the ischemic injury. We discovered a novel regulator of CXCR4 in neural stem cells, low-density lipoprotein receptor related protein 1 (LRP1). We employed a tamoxifen-inducible Nestin-Cre to drive expression of a tdTomato reporter and also knockout floxed LRP1 in adult mice and then subjected mice to middle-cerebral artery occlusion. Examination 2 weeks post-stroke reveals a loss of tdTomato positive cells localizing from the SVZ to the lesion. We show that loss of LRP1 disrupts CXCR4-mediated neural stem cell migration in vitro, which is likely driven by LRP1-mediated loss of CXCR4 expression in vivo. Altogether, our results suggest that LRP1 is a novel regulator of CXCR4 in neural stem cells. Highlights- LRP1 KO in adult neural stem cells disrupts migration to ischemic lesions in vivo. - LRP1 KO in adult neural stem cells disrupts migration towards SDF1 in vitro. - LRP1 positively regulates expression of CXCR4 in adult neural stem cells. eTOC blurbAdult neural stem cells can home to ischemic brain injury and are considered an important part of the repair process after stroke. However, little is known about what molecules help drive this response. The authors discovered that LRP1 is a novel regulator of CXCR4, which is essential for neural stem cell migration to ischemic injury.

neuroscience↗

bioRxiv2020

Coronavirus surveillance in Congo basin wildlife detects RNA of multiple species circulating in bats and rodents

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 Alpha- and 6 Beta-coronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

microbiology↗

bioRxiv2019

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.

microbiology↗