The fading efficacy of antibiotics is a growing global health concern due to its life-threatening consequences and increased healthcare costs. Non-genetic mechanisms of antimicrobial resistance, such as those employed by Chlamydia pneumoniae and Chlamydia trachomatis, complicate treatment as these bacteria can enter a non-replicative, persistent state under stress, evading antibiotics and linking to inflammatory conditions. Understanding chlamydial persistence at the molecular level is challenging, and new models for studying Chlamydia-host interactions in vivo are urgently needed. Caenorhabditis elegans offers an alternative given its immune system and numerous orthologues of human genes. This study established C. elegans as an in vivo model for chlamydial infection. Both Chlamydia species reduced the worm\'s lifespan, their DNA being detectable at three- and six-days post-infection. Azithromycin at its MIC (25 nM) failed to prevent the infection-induced lifespan reduction, indicating a persister phenotype. In contrast, the methanolic extract of Schisandra chinensis berries showed anti-chlamydial activity both in vitro (in THP-1 macrophages) and in vivo, significantly extending the lifespan of infected C. elegans and reducing the bacterial load. Moreover, S. chinensis increased the transcriptional activity of SKN-1 in the worms, but was unable to impact the bacterial load or lifespan in a sek-1 defective C. elegans strain. In summary, this study validated C. elegans as a chlamydial infection model and showcased S. chinensis berries\' in vivo anti-chlamydial potential, possibly through SEK/SKN-1 signaling modulation.

The rational design of the antibiotic treatment of bacterial infections employs these drugs to reach concentrations that exceed the minimum needed to prevent the replication of the target bacteria. However, within a treated patient, spatial and physiological heterogeneity promotes antibiotic gradients such that the concentration of antibiotics at specific sites is below the minimum needed to inhibit bacterial growth. Here, we investigate the effects of sub-inhibitory antibiotic concentrations on three parameters central to bacterial infection and the success of antibiotic treatment, using in vitro experiments with Staphylococcus aureus and mathematical-computer simulation models. Our results, using drugs of six different classes, demonstrate that exposure to sub-inhibitory antibiotic concentrations not only alters the dynamics of bacterial growth but also increases the mutation rate to antibiotic resistance and decreases the rate of production of persister cells thereby reducing the persistence level. Understanding this trade-off between mutation rates and persistence levels resulting from sub-inhibitory antibiotic exposure is crucial for optimizing, and mitigating the failure of, antibiotic therapy.

In response to growing concerns about the efficacy of antibiotic treatment, there has been a significant increase in research on bacteria that are resistant to antibiotics over the past two centuries. Such investigations might bring a spotlight on the field\'s evolution and future prospects. The study was aimed at conducting a measurable bibliometric review of the scientific literature on bacterial persistence and dormancy in the 21st Century. A scientific literature published during 21st Century was analyzed to gain insights into and identify research trends and outputs in persistent bacteria. Bibliometrix (R language package) and the VOS viewer were used to conduct a bibliometric investigation to determine the globally indexed persistent bacteria research output. WoS Core Collection databases were searched for persistent bacteria selected as the subject. A total of 1,160 published documents from 495 sources from the preceding two decades were reviewed. Maximum publications of 112 were observed in 2021 with 860 citations; however, 82 publications appeared in 2015 and were able to get the highest number of citations (4,214), only 43 (3.7%) were single-authored, whereas 1,117 (96.3%) publications are the result of collaborative works. Out of the top 10 countries ranked for publications, the USA took the top spot for the most highly productive country with 435 articles. Dormancy\' appeared 2,351 times, followed by \'Escherichia coli\" (1,744, and \'Growth\' 1,184 times) in research publications on bacterial persistence research. The findings from this study will aid in the creation of strategies and guidelines for regulating and avoiding bacterial persistence status.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

UNASSIGNED: Staphylococcus aureus, a human commensal, is also one of the most common and serious pathogens for humans. In recent years, its capacity to survive and replicate in phagocytic and non-phagocytic cells has been largely demonstrated. In these intracellular niches, bacteria are shielded from the immune response and antibiotics, turning host cells into long-term infectious reservoirs. Moreover, neutrophils carry intracellular bacteria in the bloodstream, leading to systemic spreading of the disease. Despite the serious threat posed by intracellular S. aureus to human health, the molecular mechanisms behind its intracellular survival and subsequent antibiotic treatment failure remain elusive.
UNASSIGNED: We give an overview of the killing mechanisms of phagocytes and of the impressive arsenal of virulence factors, toxins and stress responses deployed by S. aureus as a response. We then discuss the different barriers to antibiotic activity in this intracellular niche and finally describe innovative strategies to target intracellular persisting reservoirs.
UNASSIGNED: Intracellular niches represent a challenge in terms of diagnostic and treatment. Further research using ad-hoc in-vivo models and single cell approaches are needed to better understand the molecular mechanisms underlying intracellular survival and tolerance to antibiotics in order to identify strategies to eliminate these persistent bacteria.

Chronic rhinosinusitis (CRS) is an inflammatory disease of the paranasal sinuses, and microbial dysbiosis associated with CRS is thought to be a key driver of host inflammation that contributes to disease progression. Staphylococcus aureus is a common upper respiratory tract (URT) pathobiont associated with higher carriage rates in CRS populations, where S. aureus-secreted toxins can be identified in CRS tissues. Although many genera of bacteria colonize the URT, few account for the majority of sequencing reads. These include S. aureus and several species belonging to the genus Corynebacterium, including Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum, which are observed at high relative abundance in the healthy URT. Studies have examined bacterial interactions between major microbionts of the URT and S. aureus, but few have done so in the context of a healthy versus diseased URT environment. Here, we examine the role of temperature in commensal, pathogen, and epithelial dynamics using an air-liquid interface cell culture model mimicking the nasal epithelial environment. Healthy URT temperatures change from the nares to the nasopharynx and are increased during disease. Temperatures representative of the healthy URT increase persistence and aggregate formation of commensal C. propinquum and C. pseudodiphtheriticum, reduce S. aureus growth, and lower epithelial cytotoxicity compared to higher temperatures correlating with the diseased CRS sinus. Dual-species colonization revealed species-specific interactions between Corynebacterium species and S. aureus dependent on temperature. Our findings suggest URT mucosal temperature plays a significant role in mediating polymicrobial and host-bacterial interactions that may exacerbate microbial dysbiosis in chronic URT diseases.IMPORTANCEChronic rhinosinusitis is a complex inflammatory disease with a significant healthcare burden. Although presence of S. aureus and microbial dysbiosis are considered mediators of inflammation in CRS, no studies have examined the influence of temperature on S. aureus interactions with the nasal epithelium and the dominant genus of the healthy URT, Corynebacterium. Interactions between Corynebacterium species and S. aureus have been documented in several studies, but none to date have examined how environmental changes in the URT may alter their interactions with the epithelium or each other. This study utilizes a polarized epithelial cell culture model at air-liquid interface to study the colonization and spatial dynamics of S. aureus and clinical isolates of Corynebacterium from people with CRS to characterize the role temperature has in single- and dual-species dynamics on the nasal epithelium.

The alarming rise in hard-to-treat bacterial infections is of great concern to human health. Thus, the identification of molecular mechanisms that enable the survival and growth of pathogens is of utmost urgency for the development of more efficient antimicrobial therapies. In challenging environments, such as presence of antibiotics, or during host infection, metabolic adjustments are essential for microorganism survival and competitiveness. Toxin-antitoxin systems (TASs) consisting of a toxin with metabolic modulating activity and a cognate antitoxin that antagonizes that toxin are important elements in the arsenal of bacterial stress defense. However, the exact physiological function of TA systems is highly debatable and with the exception of stabilization of mobile genetic elements and phage inhibition, other proposed biological functions lack a broad consensus. This review aims at gaining new insights into the physiological effects of TASs in bacteria and exploring the experimental shortcomings that lead to discrepant results in TAS research. Distinct control mechanisms ensure that only subsets of cells within isogenic cultures transiently develop moderate levels of toxin activity. As a result, TASs cause phenotypic growth heterogeneity rather than cell stasis in the entire population. It is this feature that allows bacteria to thrive in diverse environments through the creation of subpopulations with different metabolic rates and stress tolerance programs.

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.

The recurrence of bacterial infectious diseases is closely associated with bacterial persisters. This subpopulation of bacteria can escape antibiotic treatment by entering a metabolic status of low activity through various mechanisms, for example, biofilm, toxin-antitoxin modules, the stringent response, and the SOS response. Correspondingly, multiple new treatments are being developed. However, due to their spontaneous low abundance in populations and the lack of research on in vivo interactions between persisters and the host\'s immune system, microfluidics, high-throughput sequencing, and microscopy techniques are combined innovatively to explore the mechanisms of persister formation and maintenance at the single-cell level. Here, we outline the main mechanisms of persister formation, and describe the cutting-edge technology for further research. Despite the significant progress regarding study techniques, some challenges remain to be tackled.

Amoxicillin is recommended as primary treatment for community-acquired bacterial pneumonia (CABP). 5-10% of CABP cases are caused by Chlamydia pneumoniae, an obligate intracellular bacterium which responds to beta-lactam antibiotics by converting to a persistent phenotype. To support rational pharmacotherapy of C. pneumoniae infections, we investigated how clinically relevant concentrations of azithromycin and doxycycline affect amoxicillin induced C. pneumoniae persistence. Given the known role of redox state alterations in the action of bactericidal antibiotics and widespread use of redox-active dietary supplements when experiencing respiratory symptoms, we also studied how redox active compounds affect the studied antibiotic treatments. Our data demonstrate that clinically applied amoxicillin concentrations (10 and 25 mg/l) fail to eradicate C. pneumoniae infection in respiratory epithelial cells. Transmission electron microscopy (TEM) of amoxicillin-treated C. pneumoniae infected cells reveal aberrant bacterial morphology characteristic of chlamydial stress response. Amoxicillin was also found to significantly limit the antichlamydial effect of azithromycin or doxycycline. However, based on quantitative culture and quantitative PCR data, azithromycin was superior to doxycycline in C. pneumoniae eradication either as monotherapy or in combination with amoxicillin. Amoxicillin was also found to decrease respiratory epithelial cell glutathione (GSH) levels, whereas redox-active dibenzocyclooctadiene lignans increased C. pneumoniae load in amoxicillin-treated cultures up to two-fold. These data highlight the impact of relative administration time on the efficacy of antichlamydial antibiotics and indicate unfavorable interactions between amoxicillin and redox-active small molecules.