Bacterial biofilms are a significant concern in various medical contexts due to their resilience to our immune system as well as antibiotic therapy. Biofilms often require surgical removal and frequently lead to recurrent or chronic infections. Therefore, there is an urgent need for improved strategies to treat biofilm infections. Ultrasound-mediated drug delivery is a technique that combines ultrasound application, often with the administration of acoustically-active agents, to enhance drug delivery to specific target tissues or cells within the body. This method involves using ultrasound waves to assist in the transportation or activation of medications, improving their penetration, distribution, and efficacy at the desired site. The advantages of ultrasound-mediated drug delivery include targeted and localized delivery, reduced systemic side effects, and improved efficacy of the drug at lower doses. This review scrutinizes recent advances in the application of ultrasound-mediated drug delivery for treating biofilm infections, focusing on in vivo studies. We examine the strengths and limitations of this technology in the context of wound infections, device-associated infections, lung infections and abscesses, and discuss current gaps in knowledge and clinical translation considerations.

Bacteria are involved in numerous interactions during infection and among host-associated microbial populations. Salmonella enterica serovar Typhimurium is a foodborne pathogen of great importance as well as a model organism to study interactions within a microbial community. In this study, we found that S. Typhimurium becomes tolerant to azithromycin when co-cultured with lactobacilli strains. Similarly, acidified media, from cell-free supernatant of lactobacilli cultures for instance, also induced the tolerance of S. Typhimurium to azithromycin. The addition of membrane disruptors restored the normal sensitivity to azithromycin in acidified media, but not when lactobacilli were present. These results suggested that the acidification of the media led to modification in envelope homeostasis, but that a different mechanism promoted the tolerance to azithromycin in the presence of lactobacilli strains. To further understand how lactobacilli strains modify the sensitivity of S. Typhimurium to azithromycin, a high-throughput assay was performed using the single-gene deletion collection of the S. Typhimurium (1) in co-culture with Lacticaseibacillus rhamnosus and (2) in sterile acidic conditions (pH 5.5 media only). As expected, both screens identified genes involved in envelope homeostasis and membrane permeability. Our results also suggest that changes in the metabolism of S. Typhimurium induce the tolerance observed in the presence of L. rhamnosus. Our results thus highlight two different mechanisms by which lactobacilli induce the tolerance of S. Typhimurium to azithromycin.IMPORTANCEThis study provides valuable insights into the intricate interactions between bacteria during infections and within host-associated microbial communities. Specifically, it sheds light on the significant role of lactobacilli in inducing antibiotic tolerance in Salmonella enterica serovar Typhimurium, a critical foodborne pathogen and model organism for microbial community studies. The findings not only uncover the mechanisms underlying this antibiotic tolerance but also reveal two distinct pathways through which strains of lactobacilli might influence Salmonella\'s response to antibiotics. Understanding these mechanisms has the potential to enhance our knowledge of bacterial infections and may have implications for the development of strategies to combat antibiotic resistance in pathogens, such as Salmonella. Furthermore, our results underscore the necessity to explore beyond the direct antimicrobial effects of antibiotics, emphasizing the broader microbial community context.

Antibiotics combat bacteria through their bacteriostatic (by growth inhibition) or bactericidal (by killing bacteria) action. Mechanistically, it has been proposed that bactericidal antibiotics trigger cellular damage, while bacteriostatic antibiotics suppress cellular metabolism. Here, we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic-induced bacterial protective response: the stringent response. Chloramphenicol targets the ribosome to inhibit the growth of the Gram-positive bacterium Bacillus subtilis. Intriguingly, we found that chloramphenicol becomes bactericidal in B. subtilis mutants unable to produce (p)ppGpp. We observed a similar (p)ppGpp-dependent bactericidal effect of chloramphenicol in the Gram-positive pathogen Enterococcus faecalis. In B. subtilis, chloramphenicol treatment induces (p)ppGpp accumulation through the action of the (p)ppGpp synthetase RelA. (p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action. This GTP regulation is critical for preventing chloramphenicol from killing B. subtilis, as bypassing (p)ppGpp-dependent GTP regulation potentiates chloramphenicol killing, while reducing GTP synthesis increases survival. Finally, chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin, reminiscent of the clinical phenomenon of antibiotic antagonism. Taken together, our findings suggest a role of (p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram-positive bacteria, which can be exploited to potentiate the efficacy of existing antibiotics.

UNASSIGNED: The commensal skin bacterium Cutibacterium acnes plays a role in the pathogenesis of acne vulgaris and also causes opportunistic infections of implanted medical devices due to its ability to form biofilms on biomaterial surfaces. Poly-β-(1→6)-N-acetyl-D-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation and biocide resistance in a wide range of bacterial pathogens. The objective of this study was to determine whether C. acnes produces PNAG, and whether PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro.
UNASSIGNED: PNAG was detected on the surface of C. acnes cells by fluorescence confocal microscopy using the antigen-specific human IgG1 monoclonal antibody F598. PNAG was detected in C. acnes biofilms by measuring the ability of the PNAG-specific glycosidase dispersin B to inhibit biofilm formation and sensitize biofilms to biocide killing.
UNASSIGNED: Monoclonal antibody F598 bound to the surface of C. acnes cells. Dispersin B inhibited attachment of C. acnes cells to polystyrene rods, inhibited biofilm formation by C. acnes in glass and polypropylene tubes, and sensitized C. acnes biofilms to killing by benzoyl peroxide and tetracycline.
UNASSIGNED: C. acnes produces PNAG, and PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. PNAG may play a role in C. acnes skin colonization, biocide resistance, and virulence in vivo.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb\'s ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Mycobacterium abscessus is an emerging opportunistic pathogen responsible for chronic lung diseases, especially in patients with cystic fibrosis. Treatment failure of M. abscessus infections is primarily associated with intrinsic or acquired antibiotic resistance. However, there is growing evidence that antibiotic tolerance, i.e., the ability of bacteria to transiently survive exposure to bactericidal antibiotics through physiological adaptations, contributes to the relapse of chronic infections and the emergence of acquired drug resistance. Yet, our understanding of the molecular mechanisms that underlie antibiotic tolerance in M. abscessus remains limited. In the present work, a mutant with increased cross-tolerance to the first- and second-line antibiotics cefoxitin and moxifloxacin, respectively, has been isolated by experimental evolution. This mutant harbors a mutation in serB2, a gene involved in L-serine biosynthesis. Metabolic changes caused by this mutation alter the intracellular redox balance to a more reduced state that induces overexpression of the transcriptional regulator WhiB7 during the stationary phase, promoting tolerance through activation of a WhiB7-dependant adaptive stress response. These findings suggest that alteration of amino acid metabolism and, more generally, conditions that trigger whiB7 overexpression, makes M. abscessus more tolerant to antibiotic treatment.

Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics.
OBJECTIVE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections.

Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance.

Antibiotic resistance and evasion are incompletely understood and complicated by the fact that murine interval dosing models do not fully recapitulate antibiotic pharmacokinetics in humans. To better understand how gastrointestinal bacteria respond to antibiotics, we colonized germ-free mice with a pan-susceptible genetically barcoded Escherichia coli clinical isolate and administered the antibiotic cefepime via programmable subcutaneous pumps, allowing closer emulation of human parenteral antibiotic dynamics. E. coli was only recovered from intestinal tissue, where cefepime concentrations were still inhibitory. Strikingly, \"some\" E. coli isolates were not cefepime resistant but acquired mutations in genes involved in polysaccharide capsular synthesis increasing their invasion and survival within human intestinal cells. Deleting wbaP involved in capsular polysaccharide synthesis mimicked this phenotype, allowing increased invasion of colonocytes where cefepime concentrations were reduced. Additionally, \"some\" mutant strains exhibited a persister phenotype upon further cefepime exposure. This work uncovers a mechanism allowing \"select\" gastrointestinal bacteria to evade antibiotic treatment.