Small Colony Variants Research Articles

Chimeric Ligands of Pili and Lectin A Inhibit Tolerance, Persistence, and Virulence Factors of Pseudomonas aeruginosa over a Wide Range of Phenotypes.

Bacteria readily form resilient phenotypes to counter environmental and antibiotic stresses. Here, we demonstrate a class of small molecules that inhibit a wide range of Pseudomonas aeruginosa phenotypes and enable antibiotics to kill previously tolerant bacteria, preventing the transition of tolerant bacteria into a persistent population. We identified two proteins, type IV pili and lectin LecA, as receptors for our molecules by methods including a new label-free assay based on bacterial motility sensing the chemicals in the environment, the chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure–activity relationship studies reveal a molecule that inhibits only pili appendage and a class of chimeric ligands that inhibit both LecA and pili. Important structural elements of the ligand are identified for each protein. This selective ligand binding identifies the phenotypes each protein receptor controls. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and is correlated with killing previously tolerant bacteria. Inhibiting pili appendages impedes swarming and twitching motilities and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously controls the multiple signaling mechanisms preventing bacteria to elude antibiotic treatments.

Assessing the interspecies relationship betweenPseudomonas aeruginosa and Staphylococcus aureus in mixed biofilms grown in alginate beads and collagen scaffolds

Polymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold. Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.

Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

Composite inclusion complexes containing sodium alginate composite nanogels for pH-responsive valnemulin hydrochloride release

To increase the sustained and controlled release of valnemulin hydrochloride to the infected site of Staphylococcus aureus small colony variants (SCVs), pH-responsive valnemulin hydrochloride-loaded sodium alginate (SA) composite nanogels were firstly explored. The valnemulin hydrochloride was incorporated into the β-Cyclodextrins (CD) to form valnemulin hydrochloride-β-CD-inclusion complexes (IC) and IC might be taken up into SA hydrogels to form valnemulin hydrochloride-loaded SA composite nanogels. The formulation screening, characteristics, in vitro release, antibacterial activity, therapeutic effect, and biosafety of the valnemulin hydrochloride composite nanogels were performed. The optimized formula was made up of 10 mg/mL (SA) and 0.5 mg/mL (Ca2+). The mean particle diameter, polydispersity index, zeta potential, encapsulation efficiency, and loading capacity of the optimized formulation were 229.4±1.5 nm, 0.34±0.03, -33.2±2.2 mv, 84.3%±4.1%, and 8.5%±1.2%, respectively. Light microscope and scanning electron microscope images revealed that a cross-linked polymeric network was successfully prepared. The fourier transform infrared and powder X-ray diffraction showed that valnemulin hydrochloride composite nanogels were successfully formulated by complexation and ionic crosslinking. The valnemulin hydrochloride composite nanogels exhibited a biphasic pattern with sustained release of 61.4%±1.8% after 48 h at pH 5.5. The valnemulin hydrochloride composite nanogels might be used as a biocompatible preparation because of their non-toxic effect on the renal and liver. This study provides promising valnemulin hydrochloride composite nanogels to improve the treatment of the SCVs infection.

A Relevant Wound-Like in vitro Media to Study Bacterial Cooperation and Biofilm in Chronic Wounds.

Biofilm on the skin surface of chronic wounds is an important factor in the pathology, inhibiting wound healing. The polymicrobial nature of these infected wounds and bacterial interactions inside this pathogenic biofilm are the keys for understanding chronic infection. The aim of our work was to develop an innovative in vitro medium that closely mimics the chronic wound emphasizing the microbiological, cellular, and inflammatory environment of chronic wounds but also focusing on the pH found at the wound level. This new medium, called chronic wound medium (CWM), will thus facilitate the study of pathogenic biofilm organization. Clinical Staphylococcus aureus and Pseudomonas aeruginosa strains coisolated from diabetic foot infection were collected and cultivated in this new medium for 24 h in monoculture and coculture. Bacterial growth (growth curves), presence of small colony variant (SCV), biofilm formation (BioFilm Ring Test® assay, biofilm biomass quantification), and virulence (survival curve in a Caenorhabditis elegans model) were evaluated. After 24 h in the in vitro conditions, we observed that P. aeruginosa growth was not affected, compared with a control bacterial medium, whereas for S. aureus, the stationary phase was reduced by two logs. Interestingly, S. aureus growth increased when cocultured with P. aeruginosa in CWM. In coculture with P. aeruginosa, SCV forms of S. aureus were detected. Biofilm studies showed that bacteria, alone and in combination, formed biofilm faster (as soon as 3 h) than the bacteria exposed in a control medium (as soon as 5 h). The virulence of all strains decreased in the nematode model when cultivated in our new in vitro medium. Taken together, our data confirmed the impact of the chronic wound environment on biofilm formation and bacteria virulence. They indicated that P. aeruginosa and S. aureus cooperated in coinfected wounds. Therefore, this in vitro model provides a new tool for bacterial cooperation investigation and polymicrobial biofilm formation.

Assessment of Staphylococcal Clinical Isolates from Periprosthetic Joint Infections for Potential Bacteriophage Therapy.

Bacteriophage therapy is a potential adjunctive treatment for periprosthetic joint infections (PJIs) given the capabilities of bacteriophages to degrade biofilms, self-replicate, and lyse bacteria. However, many aspects of this therapeutic are ill-defined, and the narrow spectrum of bacteriophage activity along with limited available bacteriophage strains curb potential use for specific bacteria such as Staphylococcus aureus at the present time. Therefore, the aim of this study was to determine the feasibility of using bacteriophages for PJI by (1) categorizing the causative organisms in hip and knee PJI at a tertiary academic center and (2) evaluating in vitro activity of a group of bacteriophages against clinical S. aureus PJI isolates. Patients with chronic hip or knee PJI after undergoing the first stage of a 2-stage revision protocol from 2017 to 2020 were identified retrospectively by a query of the hospital billing database. The causative pathogens in 129 cases were reviewed and categorized. From this cohort, preserved S. aureus isolates were tested against a library of 15 staphylococcal bacteriophages to evaluate for bacterial growth inhibition over 48 hours. S. aureus was the most common pathogen causing PJI (26% [33] of 129 cases). Of 29 S. aureus samples that were analyzed for bacteriophage activity, 97% showed adequate growth inhibition of the predominant planktonic colonies by at least 1 bacteriophage strain. However, 24% of the 29 samples demonstrated additional smaller, slower-growing S. aureus colonies, none of which had adequate growth inhibition by any of the initial 14 bacteriophages. Of 5 secondary colonies that underwent subsequent testing with another bacteriophage with enhanced biofilm activity, 4 showed adequate growth inhibition. Effective bacteriophage therapeutics are potentially available for S. aureus PJI isolates. The differences in bacteriophage activity against the presumed small-colony variants compared with the planktonic isolates have important clinical implications. This finding suggests that bacteriophage attachment receptors differ between the different bacterial morphologic states, and supports future in vitro testing of bacteriophage therapeutics against both planktonic and stationary states of PJI clinical isolates to ensure activity.

Biofilm and Small Colony Variants-An Update on Staphylococcus aureus Strategies toward Drug Resistance.

Recently, the drawbacks arising from the overuse of antibiotics have drawn growing public attention. Among them, drug-resistance (DR) and even multidrug-resistance (MDR) pose significant challenges in clinical practice. As a representative of a DR or MDR pathogen, Staphylococcus aureus can cause diversity of infections related to different organs, and can survive or adapt to the diverse hostile environments by switching into other phenotypes, including biofilm and small colony variants (SCVs), with altered physiologic or metabolic characteristics. In this review, we briefly describe the development of the DR/MDR as well as the classical mechanisms (accumulation of the resistant genes). Moreover, we use multidimensional scaling analysis to evaluate the MDR relevant hotspots in the recent published reports. Furthermore, we mainly focus on the possible non-classical resistance mechanisms triggered by the two important alternative phenotypes of the S. aureus, biofilm and SCVs, which are fundamentally caused by the different global regulation of the S. aureus population, such as the main quorum-sensing (QS) and agr system and its coordinated regulated factors, such as the SarA family proteins and the alternative sigma factor σB (SigB). Both the biofilm and the SCVs are able to escape from the host immune response, and resist the therapeutic effects of antibiotics through the physical or the biological barriers, and become less sensitive to some antibiotics by the dormant state with the limited metabolisms.

Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that is characterized by recurrent and chronic infections of the lung predominantly by the opportunistic pathogens, Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. While S. aureus is the main colonizing bacteria of the CF lungs during infancy and early childhood, its incidence declines thereafter and infections by P. aeruginosa become more prominent with increasing age. The competitive and cooperative interactions exhibited by these two pathogens influence their survival, antibiotic susceptibility, persistence and, consequently the disease progression. For instance, P. aeruginosa secretes small respiratory inhibitors like hydrogen cyanide, pyocyanin and quinoline N-oxides that block the electron transport pathway and suppress the growth of S. aureus. However, S. aureus survives this respiratory attack by adapting to respiration-defective small colony variant (SCV) phenotype. SCVs cause persistent and recurrent infections and are also resistant to antibiotics, especially aminoglycosides, antifolate antibiotics, and to host antimicrobial peptides such as LL-37, human β-defensin (HBD) 2 and HBD3; and lactoferricin B. The interaction between P. aeruginosa and S. aureus is multifaceted. In mucoid P. aeruginosa strains, siderophores and rhamnolipids are downregulated thus enhancing the survival of S. aureus. Conversely, protein A from S. aureus inhibits P. aeruginosa biofilm formation while protecting both P. aeruginosa and S. aureus from phagocytosis by neutrophils. This review attempts to summarize the current understanding of the molecular mechanisms that drive the competitive and cooperative interactions between S. aureus and P. aeruginosa in the CF lungs that could influence the disease outcome.

Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation

Background: Small colony variants (SCVs) of bacterial pathogens are smaller, slow-growing variants which often pose a challenge to the clinical microbiologist in their identification and characterization. SCVs are receiving much attention in recent years due to their association with several types of chronic infections. In this study, we aimed to develop a suitable culture media for high frequency generation and stable maintenance of SCV of Klebsiella pneumoniae. We also intended to compare different phenotypic characteristics such as growth, antibiotic resistance pattern, and biofilm-forming potential of SCVs with the original parental strain. Methods: We used Mueller–Hinton agar containing the extract of clove (Syzygium aromaticum) for the generation of SCV. Antibiotic sensitivity was determined using disk diffusion method and minimum inhibitory concentration determinations using microdilution method. Biofilm formation was assessed using crystal violet dye binding assay. Results: Mueller–Hinton agar (MHA) containing clove (Syzygium aromaticum) extract (10% volume/volume; MHA-C10) supported generation of SCV from K. pneumoniae at high frequency. SCVs were smaller in colony size and grew slowly in comparison to the wild-type original strain. In addition, SCVs exhibited increased resistance to aminoglycoside group of antibiotics (gentamicin and kanamycin). Crystal violet dye binding spectrophotometric method showed increased biofilm formation potential by SCVs in comparison to their parental counterparts. Conclusions: The findings of this study show that MHA-C10 can be used as a bacterial culture media for the formation of SCV by K. pneumoniae. SCVs, thus, generated on MHS-C10 exhibited typical characteristics of SCVs.

Antibacterial activity of florfenicol composite nanogels against Staphylococcus aureus small colony variants.

BackgroundFlorfenicol might be ineffective for treating Staphylococcus aureus small colony variants (SCVs) mastitis.ObjectivesIn this study, florfenicol-loaded chitosan (CS)-sodium tripolyphosphate (TPP) composite nanogels were prepared to allow targeted delivery to SCV infected sites.MethodsThe formulation screening, the characteristics, in vitro release, antibacterial activity, therapeutic efficacy, and biosafety of the florfenicol composite nanogels were studied.ResultsThe optimized formulation was obtained when the CS and TPP were 10 and 5 mg/mL, respectively. The encapsulation efficiency, loading capacity, size, polydispersity index, and zeta potential of the optimized florfenicol composite nanogels were 87.3% ± 2.7%, 5.8% ± 1.4%, 280.3 ± 1.5 nm, 0.15 ± 0.03, and 36.3 ± 1.4 mv, respectively. Optical and scanning electron microscopy showed that spherical particles with a relatively uniform distribution and drugs might be incorporated in cross-linked polymeric networks. The in vitro release study showed that the florfenicol composite nanogels exhibited a biphasic pattern with the sustained release of 72.2% ± 1.8% at 48 h in pH 5.5 phosphate-buffered saline. The minimal inhibitory concentrations of commercial florfenicol solution and florfenicol composite nanogels against SCVs were 1 and 0.25 µg/mL, respectively. The time-killing curves and live–dead bacterial staining showed that the florfenicol composite nanogels were concentration-dependent. Furthermore, the florfenicol composite nanogels displayed good therapeutic efficacy against SCVs mastitis. Biological safety studies showed that the florfenicol composite nanogels might be a biocompatible preparation because of their non-toxic effects on the renal tissue and liver.ConclusionsFlorfenicol composite nanogels might improve the treatment of SCV infections.

Enhanced antibacterial activity of tilmicosin against Staphylococcus aureus small colony variants by chitosan oligosaccharide-sodium carboxymethyl cellulose composite nanogels.

BackgroundThe poor bioadhesion capacity of tilmicosin resulting in treatment failure for Staphylococcus aureus small colony variants (SASCVs) mastitis.ObjectivesThis study aimed to increase the bioadhesion capacity of tilmicosin for the SASCVs strain and improve the antibacterial effect of tilmicosin against cow mastitis caused by the SASCVs strain.MethodsTilmicosin-loaded chitosan oligosaccharide (COS)-sodium carboxymethyl cellulose (CMC) composite nanogels were formulated by an electrostatic interaction between COS (positive charge) and CMC (negative charge) using sodium tripolyphosphate (TPP) (ionic crosslinkers). The formation mechanism, structural characteristics, bioadhesion, and antibacterial activity of tilmicosin composite nanogels were studied systematically.ResultsThe optimized formulation was comprised of 50 mg/mL (COS), 32 mg/mL (CMC), and 0.25 mg/mL (TPP). The size, encapsulation efficiency, loading capacity, polydispersity index, and zeta potential of the optimized tilmicosin composite nanogels were 357.4 ± 2.6 nm, 65.4 ± 0.4%, 21.9 ± 0.4%, 0.11 ± 0.01, and -37.1 ± 0.4 mV, respectively; the sedimentation rate was one. Scanning electron microscopy showed that tilmicosin might be incorporated in nano-sized crosslinked polymeric networks. Moreover, adhesive studies suggested that tilmicosin composite nanogels could enhance the bioadhesion capacity of tilmicosin for the SASCVs strain. The inhibition zone of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels were 2.13 ± 0.07, 3.35 ± 0.11, and 1.46 ± 0.04 cm, respectively. The minimum inhibitory concentration of native tilmicosin, tilmicosin standard, and tilmicosin composite nanogels against the SASCVs strain were 2, 1, and 1 µg/mL, respectively. The in vitro time-killing curves showed that the tilmicosin composite nanogels increased the antibacterial activity against the SASCVs strain.ConclusionsThis study provides a potential strategy for developing tilmicosin composite nanogels to treat cow mastitis caused by the SASCVs strain.

Antibacterial activity of enrofloxacin loaded gelatin-sodium alginate composite nanogels against intracellular Staphylococcus aureus small colony variants.

BackgroundThe poor intracellular concentration of enrofloxacin might lead to treatment failure of cow mastitis caused by Staphylococcus aureus small colony variants (SASCVs).ObjectivesIn this study, enrofloxacin composite nanogels were developed to increase the intracellular therapeutic drug concentrations and enhance the efficacy of enrofloxacin against cow mastitis caused by intracellular SASCVs.MethodsEnrofloxacin composite nanogels were formulated by an electrostatic interaction between gelatin (positive charge) and sodium alginate (SA; negative charge) with the help of CaCl2 (ionic crosslinkers) and optimized by a single factor test using the particle diameter, zeta potential (ZP), polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) as indexes. The formation mechanism, structural characteristics, bioadhesion ability, cellular uptake, and the antibacterial activity of the enrofloxacin composite nanogels against intracellular SASCVs strain were studied systematically.ResultsThe optimized formulation was comprised of 10 mg/mL (gelatin), 5 mg/mL (SA), and 0.25 mg/mL (CaCl2). The size, LC, EE, PDI, and ZP of the optimized enrofloxacin composite nanogels were 323.2 ± 4.3 nm, 15.4% ± 0.2%, 69.6% ± 1.3%, 0.11 ± 0.02, and −34.4 ± 0.8 mV, respectively. Transmission electron microscopy showed that the enrofloxacin composite nanogels were spherical with a smooth surface and good particle size distributions. In addition, the enrofloxacin composite nanogels could enhance the bioadhesion capacity of enrofloxacin for the SASCVs strain by adhesive studies. The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were 2, 4, 4, and 8 μg/mL, respectively. The killing rate curve had a concentration-dependent bactericidal effect as increasing drug concentrations induced swifter and more radical killing effects.ConclusionsThis study provides a good tendency for developing enrofloxacin composite nanogels for treating cow mastitis caused by intracellular SASCVs and other intracellular bacterial infections.

Enhanced antibacterial activity of tilmicosin against Staphylococcus aureus small colony variants by chitosan oligosaccharide-sodium carboxymethyl cellulose composite nanogels

Enhanced antibacterial activity of tilmicosin against Staphylococcus aureus small colony variants by chitosan oligosaccharide-sodium carboxymethyl cellulose composite nanogels

Involvement of Small Colony Variant-Related Heme Biosynthesis Genes in Staphylococcus aureus Persister Formation in vitro.

Background: Persisters are important reasons for persistent infections, and they can lead to antibiotic treatment failure in patients and consequently chronic infection. Staphylococcus aureus small colony variants (SCVs) have been shown to be related to persistent infection. Mutations in the genes of the heme biosynthesis pathway lead to the formation of SCVs. However, the relationship between heme production genes and persister has not been tested.Methods:HemA and hemB were knocked out by allelic replacement from S. aureus strain USA500 separately, and then, the heme deficiency was complemented by overexpression of related genes and the addition of hemin. The stress-related persister assay was conducted. RNA-sequencing was performed to find genes and pathways involved in heme-related persister formation, and relative genes and operons were further knocked out and overexpressed to confirm their role in each process.Results: We found that heme biosynthesis deficiency can lead to decreased persister. After complementing the corresponding genes or hemin, the persister levels could be restored. RNA-seq on knockout strains showed that various metabolic pathways were influenced, such as energy metabolism, amino acid metabolism, carbohydrate metabolism, and membrane transport. Overexpression of epiF and operon asp23 could restore USA500∆hemA persister formation under acid stress. Knocking out operon arc in USA500∆hemA could further reduce USA500∆hemA persister formation under acid and oxidative stress.Conclusion: Heme synthesis has a role in S. aureus persister formation.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

116. Characterization of small colony variants from a patient with bloodstream infection of Candida glabrata

BackgroundBacterial small colony variants (SCVs) that are tolerant to commonly used antibiotics are well recognized. Clinical SCV Candida have been rarely reported. We describe SCV C. glabrata (CG) strains recovered from within a population causing bloodstream infection (BSI) in a patient (pt), which were not recognized by the micro lab. Pt J developed CG BSI shortly after liver transplant (OLTX), which was treated with voriconazole (VOR). VOR was also used for post-OLTX mold prophylaxis. 67 d after BSI, he developed intra-abdominal infection due to VOR-resistant CG. We hypothesized that BSI might be caused by an unrecognized mixed population of azole-susceptible and –resistant strains. MethodsTen colonies from small (SCV) and large colonies (LC) from blood culture (BC) agar plates underwent Illumina NextSeq WGS and phenotype testing. ResultsBCs from pt J harbored a diverse population of genetically distinct CG strains, differing by unique SNPs and indels [Fig. 1]. Gene variants identified were enriched for biological processes involved in mitochondrial processes (2.5e-9), cell adhesion (3.3e-5), and respiration (3.5e-4). Unlike LC, SCVs were fluconazole (FLU) resistant (MIC: 128 µg/mL), and exhibited enhanced CDR1 and PDR1 expression (257 ± 11, 15 ± 4, respectively). Compared to LCs, SCVs grew slowly in YPD, did not grow on media containing glycerol as sole carbon source, and were less adherent to agar. SCVs stained poorly with rhodamine 123 by fluorescence flow cytometry and had fewer mitochrondria by transmission electron microscopy, consistent with WGS findings and respiratory deficiency. SCVs were less susceptible to macrophage (J774) phagocytosis, and they were significantly outgrown by other strains in competitive infections in vitro and during disseminated candidiasis in mice. LCs incubated with FLU in vitro yielded SCVs in concentration-dependent manner. Likewise, LCVs passed through spleens of mice following IV inoculation yielded SCVs in both presence and absence of FLU. Venn diagram for 5 representative stranis of C.glabrata ConclusionMitochondrial dysfunction and SCVs may be under-recognized determinants of azole resistance in CG, if micro labs select single colonies from BCs for antifungal susceptibility testing, or in absence of prolonged incubation.Disclosures Cornelius J. Clancy, MD, Merck (Grant/Research Support) Minh-Hong Nguyen, MD, Merck (Grant/Research Support)

MicroRNA hsa-let-7a facilitates staphylococcal small colony variants survival in the THP-1 macrophages by reshaping inflammatory responses

Recent studies have provided emerging evidence of the critical involvement of microRNAs in host immune defence against bacterial infection and that likewise the expression of the miRNAs is profoundly impacted by a variety of pathogens to subvert the immune response. Here, we report the role of hsa-let-7a miRNA in response to Staphylococcus epidermidis Small Colony Variants infection. We also assessed whether the expression levels of inflammatory cytokines associated with the hsa-let-7a are manipulated by the pathogen and the effect of the IFN-γ priming on the expression of hsa-let-7a and the fate of SCVs/WTs in infected macrophages. A striking observation was the downregulation of the let-7a miRNA upon challenge of the THP-1 activated cells with the SCV isolates while no significant changes in expression were noticed after the infection of macrophages with their WT counterparts. Staphylococcus epidermidis WT and SCV strains were found to invade and survive in macrophages. A significant reduction in bacterial load for both phenotypes was observed in macrophages treated with let-7a mimic compared to untreated ones. Survival of WTs was augmented in cells treated with the inhibitor in 4 out of 5 strains as compared to the number of bacteria recovered from non-transfected cells. At the same time, let-7a inhibitor did not influence on the survival of SCVs in macrophages as their number was comparable to number recovered from non-transfected cells. When the ratio of both let-7a cytokine targets was compared, anti-inflammatory IL-10 cytokine was induced by SCVs predominantly, while the macrophage challenge with WTs was characterized by the inflammatory cytokine profile with high IL-6 and low IL-10 production. Moreover, the balance between pro-inflammatory and anti-inflammatory cytokines has been expectedly retrieved when macrophages were transfected with let-7a mimic before infection with WT or SCV strains. The results also show that IFN-γ likely regulates the macrophage environment contributing to the inflammatory response and elimination of bacteria from intracellular milieu by augmenting the synthesis of pro-inflammatory cytokines and supressing the anti-inflammatory IL-10. Our work has shown that SCVs have the potential to regulate the let-7a miRNA to balance the pro-inflammatory IL-6 with anti-inflammatory IL-10 and this mechanism is one of the ways in a complex regulatory network adopted by SCVs to promote their survival.

Characteristics of Staphylococcus aureus small colony variants isolated from wound specimen of a tertiary care hospital in China.

BackgroundSmall colony variants (SCVs) of Staphylococcus aureus (S. aureus) frequently lead to chronic and recurrent infections, but they are always ignored and there are few researches on their clinical isolates. We intended to investigate the prevalence and characteristics of S. aureus SCVs.MethodsNone‐duplicated S. aureus strains isolated from wound samples were collected from January 2018 to December 2020. The characteristics (i.e. colony morphology, growth rate, coagulase, biofilm formation, and pathogenic characteristics), antimicrobial susceptibilities, and resistance mechanisms of SCVs were also investigated. The genetic background of SCVs was analyzed through staphylococcal protein A (SPA) typing, sequence typing, and pulse field gel electrophoresis (PFGE).ResultsThree SCVs were screened from 278 S. aureus strains (1.1%). They formed pinpoint white colonies on blood agar plates with weak hemolysis. The reproduction speed in liquid medium was very slow for SCVs strains. The coagulase weakened or disappeared, and the ability to form biofilm varied greatly. Only slight inflammation was triggered when wound infected. The SPA typing was t2592, t233, and t023, and the sequence typing was ST88, ST239, and ST965, respectively. The PFGE revealed three SCVs were singletons.ConclusionsThe rate of SCVs in wound sample is low in our hospital, and the formation is associated with the usage of antimicrobial. SCVs grow slowly, and their colony morphology and biochemical characteristics are significantly different from classic S. aureus. SCVs may cause chronic infection and weak inflammation. SCVs form in resistant or susceptible strains, and there is no clonal epidemic in this hospital.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

A Human Osteocyte Cell Line Model for Studying Staphylococcus aureus Persistence in Osteomyelitis.

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.