Search Results (23)

A unique phenotype of S. aureus called S. aureus small-colony variants (SA-SCVs) are a consequential contributor to multiple infectious processes. SA-SCVs are distinguishable from wild-type S. aureus (WT-SA) by their small size, slowed growth rate, and altered biochemical reactions; these changes make SA-SCV more difficult to detect from clinical specimens using routine diagnostics. While the clinical environment of chronic wound infections has the potential to stimulate the production of SA-SCVs, studies investigating detection of SA-SCVs in chronic wounds have not been previously conducted. Chronic wound specimens found to harbor S. aureus via qPCR screening, and screened for recent aminoglycoside treatment and/or co-infected with Pseudomonas aeruginosa, were collected from a specialty wound care clinic in April 2019. In-house enrichment methods alongside culture-dependent and independent diagnostics were utilized to recover and identify SA-SCVs from these chronic wounds. Our investigation determined difficulties in recovering and identifying SA-SCVs during routine diagnostic procedures, and the potential clinical impact of wounds harboring SA-SCVs related to antimicrobial susceptibility. Full article

Staphylococcus aureus, a common pathogen, is capable of producing a significant array of toxins and can develop biofilms or small colony variants (SCVs) to evade detection by the immune system and resist the effects of antibiotics. Its ability to persist for extended periods within host cells has led to increased research interest. This review examines the process of internalization of S. aureus, highlighting the impact of its toxins and adhesion factors on host cells. It elucidates the intricate interactions between them and the host cellular environment, thereby offering potential strategies for the treatment and prevention of S. aureus infections. Full article

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu. Full article

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the Staphylococcus aureus small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-β) and TM-03 (C3-α). These molecules are equally potent against prototypical S. aureus and E. coli strains (MIC 8 and 32 µg/mL, respectively), whereas TM-02 is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable E. coli strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited E. coli ATP synthase, with K_i values of 1.1 µM and 3.5 µM for TM-02 and TM-03, respectively, and the bactericidal effect of TM-02 was linked to ATP synthase inhibition. Furthermore, TM-02 had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, TM-03 caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α. Full article

Methicillin-sensitive Staphylococcus (S.) aureus (MSSA) bacteremia remains a global challenge, despite the availability of antibiotics. Primary treatments include β-lactam agents such as cefazolin and flucloxacillin. Ongoing discussions have focused on the potential synergistic effects of combining these agents with rifampicin or fosfomycin to combat infections associated with biofilm formation. Managing staphylococcal infections is challenging due to antibacterial resistance, biofilms, and S. aureus’s ability to invade and replicate within host cells. Intracellular invasion shields the bacteria from antibacterial agents and the immune system, often leading to incomplete bacterial clearance and chronic infections. Additionally, S. aureus can assume a dormant phenotype, known as the small colony variant (SCV), further complicating eradication and promoting persistence. This study investigated the impact of antibiotic combinations on the persistence of S. aureus 6850 and its stable small colony variant (SCV strain JB1) focusing on intracellular survival and biofilm formation. The results from the wild-type strain 6850 demonstrate that β-lactams combined with RIF effectively eliminated biofilms and intracellular bacteria but tend to select for SCVs in planktonic culture and host cells. Higher antibiotic concentrations were associated with an increase in the zeta potential of S. aureus, suggesting reduced membrane permeability to antimicrobials. When using the stable SCV mutant strain JB1, antibiotic combinations with rifampicin successfully cleared planktonic bacteria and biofilms but failed to eradicate intracellular bacteria. Given these findings, it is reasonable to report that β-lactams combined with rifampicin represent the optimal treatment for MSSA bacteremia. However, caution is warranted when employing this treatment over an extended period, as it may elevate the risk of selecting for small colony variants (SCVs) and, consequently, promoting bacterial persistence. Full article

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians. Full article

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus-positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm (p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (ΔhemB) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients. Full article

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation. Full article

Intracellular bacteria are inaccessible and highly tolerant to antibiotics, hence are a major contributor to the global challenge of antibiotic resistance and recalcitrant clinical infections. This, in tandem with stagnant antibacterial discovery, highlights an unmet need for new delivery technologies to treat intracellular infections more effectively. Here, we compare the uptake, delivery, and efficacy of rifampicin (Rif)-loaded mesoporous silica nanoparticles (MSN) and organo-modified (ethylene-bridged) MSN (MON) as an antibiotic treatment against small colony variants (SCV) Staphylococcus aureus (SA) in murine macrophages (RAW 264.7). Macrophage uptake of MON was five-fold that of equivalent sized MSN and without significant cytotoxicity on human embryonic kidney cells (HEK 293T) or RAW 264.7 cells. MON also facilitated increased Rif loading with sustained release, and seven-fold increased Rif delivery to infected macrophages. The combined effects of increased uptake and intracellular delivery of Rif by MON reduced the colony forming units of intracellular SCV-SA 28 times and 65 times compared to MSN-Rif and non-encapsulated Rif, respectively (at a dose of 5 µg/mL). Conclusively, the organic framework of MON offers significant advantages and opportunities over MSN for the treatment of intracellular infections. Full article

The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of “bet-hedging” has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections. Full article

Antibiotic resistance of bacterial pathogens results from their exposure to antibiotics and this has become a serious growing problem that limits effective use of antibiotics. Resistance can arise from mutations induced by antibiotic-mediated damage with these mutants possessing reduced target sensitivity. We have studied ciprofloxacin (CIP)-mediated killing of Staphylococcus aureus and the influence of the Reactive Oxygen Species (ROS) inactivator, thiourea and the iron chelator DIBI, on initial killing by CIP and their effects on survival and outgrowth upon prolonged exposure to CIP. CIP at 2× MIC caused a rapid initial killing which was not influenced by initial bacterial iron status and which was followed by robust recovery growth over 96 h exposure. Thiourea and DIBI did slow the initial rate of CIP killing but the overall extent of kill by 24 h exposure was like CIP alone. Thiourea permitted recovery growth whereas this was strongly suppressed by DIBI. Small Colony Variant (SCV) survivors were progressively enriched in the survivor population during CIP exposure, and these were found to have stable slow-growth phenotype and acquired resistance to CIP and moxifloxacin but not to other non-related antibiotics. DIBI totally suppressed SCV formation with all survivors remaining sensitive to CIP and to DIBI. DIBI exposure did not promote resistance to DIBI. Our evidence indicates a high potential for DIBI as an adjunct to CIP and other antibiotics to both improve antibiotic efficacy and to thwart antibiotic resistance development. Full article

Small colony variants (SCVs) are clinically significant and linked to persistent infections. In this study, synchrotron-radiation-based Fourier transform infrared (SR-FTIR) is used to investigate the microspectroscopic differences between the SCVs of Staphylococcus aureus (S. aureus) and diabetic foot Staphylococcus epidermidis (S. epidermidis) in two main IR spectral regions: (3050–2800 cm⁻¹), corresponding to the distribution of lipids, and (1855–1500 cm⁻¹), corresponding to the distribution of protein amide I and amide II and carbonyl vibrations. SR-FTIR successfully discriminated between the two staphylococcal species and between the SCV and the non-SCV strains within the two IR spectral regions. Combined S. aureus SCVs (SCVhMu) showed a higher protein content relative to the non-SCV wild type. Complemented S. aureus SCV showed distinguishable differences from the SCVhMu and the wild type, including a higher content of unsaturated fatty acids. An increase in the CH₂/CH₃ ratio was detected in S. epidermidis SCV samples compared to the standard control. Protein secondary structure in standard S. epidermidis and SCVs consisted mainly of an α-helix; however, a new shoulder at 1635 cm⁻¹, assigned to β-sheets, was evident in the SCV. In conclusion, SR-FTIR is a powerful method that can discriminate between staphylococci species and to differentiate between SCVs and their corresponding natural strains. Full article

The present study discusses a biofilm-positive P. aeruginosa isolate that survives at pH levels ranging from 4.0 to 9.0. The biofilm consortia were colonized with different phenotypes i.e., planktonic, slow-growing and metabolically inactive small colony variants (SCVs). The lower base of the consortia was occupied by SCVs. These cells were strongly attached to solid surfaces and interconnected through a network of nanotubes. Nanotubes were observed at the stationary phase of biofilm indwellers and were more prominent after applying weight to the consortia. The scanning electron micrographs indicated that the nanotubes are polar appendages with intraspecies connectivity. The micrographs indicated variations in physical dimensions (length, width, and height) and a considerable reduction in volume due to weight pressure. A total of 35 cells were randomly selected. The mean volume of cells before the application of weight was 0.288 µm³, which was reduced to 0.144 µm³ after the application of weight. It was observed that a single cell may produce as many as six nanotubes, connected simultaneously to six neighbouring cells in different directions. The in-depth analysis confirmed that these structures were the intra-species connecting tools as no free nanotubes were found. Furthermore, after the application of weight, cells incapable of producing nanotubes were wiped out and the surface was covered by nanotube producers. This suggests that the nanotubes give a selective advantage to the cells to resist harsh environmental conditions and weight pressure. After the removal of weight and proper supply of nutrients, these phenotypes reverted to normal planktonic lifestyles. It is concluded that the nanotubes are not merely the phenomenon of dying cells; rather they are a connectivity tool which helps connected cells to tolerate and resist environmental stress. Full article

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. Full article

The ATP synthase has been validated as a druggable target with the approval of the ATP synthase inhibitor, bedaquiline, for treatment of drug-resistant Mycobacterium tuberculosis, a bacterial species in which the ATP synthase is essential for viability. Gene inactivation studies have also shown that the ATP synthase is essential among Streptococci, and some studies even suggest that inhibition of the ATP synthase is a strategy for the elimination of Staphylococcus aureus small colony variants with deficiencies in the electron transport chain, as well as pathogenic fungi, such as Candida albicans. Here we investigated five structurally diverse ATP synthase inhibitors, namely N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin A, tomatidine, resveratrol and piceatannol, for their growth inhibitory activity against the bacterial strains Streptococcus pyogenes, S. aureus and two isogenic small colony variants, as well as the pathogenic fungal species, C. albicans and Aspergillus niger. DCCD showed broad-spectrum inhibitory activity against all the strains (minimum inhibitory concentration (MIC) 2–16 µg/mL), except for S. aureus, where the ATP synthase is dispensable for growth. Contrarily, oligomycin A selectively inhibited the fungal strains (MIC 1–8 µg/mL), while tomatidine showed very potent, but selective, activity against small colony variants of S. aureus with compromised electron transport chain activity (MIC 0.0625 µg/mL). Small colony variants of S. aureus were also more sensitive to resveratrol and piceatannol than the wild-type strain, and piceatannol inhibited S. pyogenes at 16–32 µg/mL. We previously showed that transposon inactivation of the ATP synthase sensitizes S. aureus towards polymyxin B and colistin, and here we demonstrate that treatment with structurally diverse ATP synthase inhibitors sensitized S. aureus towards polymyxin B. Collectively, our data show that ATP synthase inhibitors can have selective inhibitory activity against pathogenic microorganisms in which the ATP synthase is essential. The data also show that the inhibition of the ATP synthase in Streptococcus pyogenes may be a new strategy for development of a narrow-spectrum antibiotic class. In other major bacterial pathogens, such as S. aureus and potentially Escherichia coli, where the ATP synthase is dispensable, the ATP synthase inhibitors may be applied in combination with antimicrobial peptides to provide new therapeutic options. Full article