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Community acquired bacterial pneumoniae (CABP) infections is the major cause of mortality and morbidity, especially in elderly patients. India accounts for 23% of global pneumonia burden with case fatality rates between 14 and 30%. There is an urgent unmet medical need for safe and effective antibiotic for CABP, due to lack of effective empirical therapy because of widespread resistance to β-lactams antibiotics. On other hand, fluoroquinolone antibiotics have poor tolerability, like hypersensitive reactions and associated disabilities. Hence, our objective was to find an antibiotic having broad coverage of multidrug resistance (MDR) pathogens including typical and atypical respiratory pathogens, with good lung penetration and safety features. Nafithromycin (MIQNAF®) is a novel "lactone-ketolide" antibiotic developed by Wockhardt Ltd. for the treatment of CABP infections. Recently it has completed phase III clinical trials in India and NDA submitted to drug controller general of India (DCGI). Distinctive features of nafithromycin are ultra-short duration of therapy, oral dosing, high concentration build up in lung i.e. target organ and safety profile. Structurally, it features novel amidoxime core with 2-pyridine-1,3,4-thiadiazole biaryl tether separated with non-flexible four atom spacer having cis double bond and chiral methyl with (S)- configuration resulted in dual target interaction. The novel conformational arrangement interacts favorably with 23S rRNA and domain V of 50S ribosome subunit to elicit outstanding potency against gram-positive bacteria. The preclinical data provided strong scientific evidences for its effectiveness against difficult-to-treat respiratory tract infections (RTIs) caused by multidrug-resistant pathogens such as macrolide-resistant strains of Streptococcus pneumoniae and Streptococcus pyogenes as well as other important pathogens including Haemophilus influenzae. Upon successful phase I clinical findings, nafithromycin was granted Qualified Infectious Disease Product (QIDP) status by the US Food and Drug Administration (USFDA). Presently besides India specific phase III clinical study completion with partial funding support from Biotechnology Industry Research Assistance Council (BIRAC), it has successfully completed global phase II clinical development, including pharmacokinetic study (NCT02770404) and study for the treatment of community-acquired bacterial pneumonia (NCT02903836). In Europe it has completed single ascending dose (SAD) and multiple ascending dose (MAD) phase I pharmacokinetic studies. This mini review covers relevant published data on nafithromycin and its potential role in management of infections caused by gram-positive pathogens along with summary of different clinical trials conducted in United States, Europe and India. [ABSTRACT FROM AUTHOR]

The rise in antibiotic-resistant bacteria highlights the need for novel antimicrobial agents. This study presents the design and synthesis of a series of rhein (RH)-derived compounds with improved antimicrobial properties. The lead compound, RH17, exhibited a potent antibacterial activity against Staphylococcus aureus (S. aureus) isolates, with minimum inhibitory concentrations (MICs) ranging from 8 to 16 μg/mL. RH17 disrupted bacterial membrane stability, hindered metabolic processes, and led to an increase in reactive oxygen species (ROS) production. These mechanisms were confirmed through bacterial growth inhibition assays, membrane function assessments, and ROS detection. Notably, RH17 outperformed the parent compound RH and demonstrated bactericidal effects in S. aureus. The findings suggest that RH17 is a promising candidate for further development as an antimicrobial agent against Gram-positive pathogens, addressing the urgent need for new therapies. [ABSTRACT FROM AUTHOR]

The rise in antibiotic-resistant bacteria highlights the need for novel antimicrobial agents. This study presents the design and synthesis of a series of rhein (RH)-derived compounds with improved antimicrobial properties. The lead compound, RH17, exhibited a potent antibacterial activity against Staphylococcus aureus (S. aureus) isolates, with minimum inhibitory concentrations (MICs) ranging from 8 to 16 μg/mL. RH17 disrupted bacterial membrane stability, hindered metabolic processes, and led to an increase in reactive oxygen species (ROS) production. These mechanisms were confirmed through bacterial growth inhibition assays, membrane function assessments, and ROS detection. Notably, RH17 outperformed the parent compound RH and demonstrated bactericidal effects in S. aureus. The findings suggest that RH17 is a promising candidate for further development as an antimicrobial agent against Gram-positive pathogens, addressing the urgent need for new therapies.

Simple Summary: Complex interactions between marine Gram-positive pathogens and fish hosts in the marine environment can result in diseases of economically important finfish, which cause economic losses in the aquaculture industry. Understanding how these pathogens interact with the fish host and generate disease will contribute to efficient prophylactic measures and treatments. To our knowledge, there are no systematic reviews on marine Gram-positive pathogens. Therefore, here we reviewed the host–pathogen interactions of marine Gram-positive pathogens from the pathogen-centric and host-centric points of view. Marine Gram-positive bacterial pathogens, including Renibacterium salmoninarum, Mycobacterium marinum, Nocardia seriolae, Lactococcus garvieae, and Streptococcus spp. cause economic losses in marine fish aquaculture worldwide. Comprehensive information on these pathogens and their dynamic interactions with their respective fish–host systems are critical to developing effective prophylactic measures and treatments. While much is known about bacterial virulence and fish immune response, it is necessary to synthesize the knowledge in terms of host–pathogen interactions as a centerpiece to establish a crucial connection between the intricate details of marine Gram-positive pathogens and their fish hosts. Therefore, this review provides a holistic view and discusses the different stages of the host–pathogen interactions of marine Gram-positive pathogens. Gram-positive pathogens can invade fish tissues, evade the fish defenses, proliferate in the host system, and modulate the fish immune response. Marine Gram-positive pathogens have a unique set of virulence factors that facilitate adhesion (e.g., adhesins, hemagglutination activity, sortase, and capsules), invasion (e.g., toxins, hemolysins/cytolysins, the type VII secretion system, and immune-suppressive proteins), evasion (e.g., free radical quenching, actin-based motility, and the inhibition of phagolysosomal fusion), and proliferation and survival (e.g., heme utilization and siderophore-mediated iron acquisition systems) in the fish host. After infection, the fish host initiates specific innate and adaptive immune responses according to the extracellular or intracellular mechanism of infection. Although efforts have continued to be made in understanding the complex interplay at the host–pathogen interface, integrated omics-based investigations targeting host–pathogen–marine environment interactions hold promise for future research. [ABSTRACT FROM AUTHOR]

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum , and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1β , il8a , il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa , mxb , mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a , cd4b , ly6g6f , cd8a , cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response. [ABSTRACT FROM AUTHOR]

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1β, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

OBJECTIVES: Gram-positive infections such as those by Staphylococcus aureus have contributed to the disease burden by increasing the morbidity and mortality rates in India. This study aims to determine the prevalence and the antibiotic susceptibility pattern of Gram-positive pathogens at a tertiary care hospital, Mumbai, Maharashtra, India. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out from January, 2015 to December, 2017, at a tertiary care hospital in Mumbai, India. The clinical isolates were cultured, and identification was done using Vitek 2 culture system. The antibiotic susceptibility testing was done as per the Clinical Laboratory Standard Institute guidelines. RESULTS: Out of 2132 (29%) Gram-positive isolates, S. aureus (49%) was the most common encountered pathogen, followed by Enterococcus spp. (24.5%) and coagulase-negative Staphylococcus (16%). Majority of the S. aureus were observed in patients with skin and soft-tissue infections (61.2%) followed by those suffering from respiratory (41%) and bloodstream infections (35%). Among the infections caused by S. aureus, the prevalence of methicillin resistance was 30%. While the MRSA isolates showed lower sensitivity toward co-trimoxazole (39%), clindamycin (30%), erythromycin (23%), and ciprofloxacin (10%), they showed higher susceptibility to linezolid (98%), vancomycin (98%), and teicoplanin (98%). All the isolates were found to be sensitive to daptomycin and tigecycline. While vancomycin-resistant enterococci (VRE) formed 7.5%, the linezolid-resistant enterococcus species was as high as 4.1%. CONCLUSION: The study showed a high prevalence of MRSA and VRE, thereby emphasizing the increasing antimicrobial resistance pattern of the Gram-positive pathogens. Therefore, there is an urgent need for novel antimicrobial stewardship to restrict the ongoing resistance rate among the isolates.

Marine Gram-positive bacterial pathogens, including Renibacterium salmoninarum, Mycobacterium marinum, Nocardia seriolae, Lactococcus garvieae, and Streptococcus spp. cause economic losses in marine fish aquaculture worldwide. Comprehensive information on these pathogens and their dynamic interactions with their respective fish–host systems are critical to developing effective prophylactic measures and treatments. While much is known about bacterial virulence and fish immune response, it is necessary to synthesize the knowledge in terms of host–pathogen interactions as a centerpiece to establish a crucial connection between the intricate details of marine Gram-positive pathogens and their fish hosts. Therefore, this review provides a holistic view and discusses the different stages of the host–pathogen interactions of marine Gram-positive pathogens. Gram-positive pathogens can invade fish tissues, evade the fish defenses, proliferate in the host system, and modulate the fish immune response. Marine Gram-positive pathogens have a unique set of virulence factors that facilitate adhesion (e.g., adhesins, hemagglutination activity, sortase, and capsules), invasion (e.g., toxins, hemolysins/cytolysins, the type VII secretion system, and immune-suppressive proteins), evasion (e.g., free radical quenching, actin-based motility, and the inhibition of phagolysosomal fusion), and proliferation and survival (e.g., heme utilization and siderophore-mediated iron acquisition systems) in the fish host. After infection, the fish host initiates specific innate and adaptive immune responses according to the extracellular or intracellular mechanism of infection. Although efforts have continued to be made in understanding the complex interplay at the host–pathogen interface, integrated omics-based investigations targeting host–pathogen–marine environment interactions hold promise for future research.

Objective To investigate the clinical and laboratory characteristics of sepsis caused by different types of pathogens in preterm infants. Methods Totally 99 premature infants with sepsis treated at our hospital during May 2008 and April 2018 were analyzed. According to the results of blood culture, they were divided into gram negative bacteria group and gram-positive bacterial infection group. The clinical and laboratory results of the two groups were compared. Results The incidence of nosocomial bacterial sepsis in premature infants was 1.16% (99/8 512). Gram-positive pathogens accounted for 70.3%, with Staphylococcus epidermidis as the main pathogen (36.6%). Gram-negative pathogens accounted for 29.7%, with klebsiella pneumoniae as the main pathogen (40.0%). Compared with those in gram-positive pathogens group, the incidences of severe complications, leukocyte decline, CRP elevation, abdominal distention and thrombocytopenia in gram-negative bacteria group increased significantly (P <0.05). Compared with gram-positive pathogens group, the abandonment or mortality rate of gram-negative bacteria group was also increased significantly (P <0.05). Conclusion Gram-positive pathogens are the main pathogens causing sepsis in premature infants. Gram-negative pathogen infection is more prone to complications such as shock, central infection and abdominal distention. Laboratory tests showed leukocyte reduction and thrombocytopenia and elevated CRP. Early selection of antibiotics based on clinical and laboratory results may improve the rationality of clinical medication. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Gram-positive infections such as those by Staphylococcus aureus have contributed to the disease burden by increasing the morbidity and mortality rates in India. This study aims to determine the prevalence and the antibiotic susceptibility pattern of Gram-positive pathogens at a tertiary care hospital, Mumbai, Maharashtra, India. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out from January, 2015 to December, 2017, at a tertiary care hospital in Mumbai, India. The clinical isolates were cultured, and identification was done using Vitek 2 culture system. The antibiotic susceptibility testing was done as per the Clinical Laboratory Standard Institute guidelines. RESULTS: Out of 2132 (29%) Gram-positive isolates, S. aureus (49%) was the most common encountered pathogen, followed by Enterococcus spp. (24.5%) and coagulase-negative Staphylococcus (16%). Majority of the S. aureus were observed in patients with skin and soft-tissue infections (61.2%) followed by those suffering from respiratory (41%) and bloodstream infections (35%). Among the infections caused by S. aureus, the prevalence of methicillin resistance was 30%. While the MRSA isolates showed lower sensitivity toward co-trimoxazole (39%), clindamycin (30%), erythromycin (23%), and ciprofloxacin (10%), they showed higher susceptibility to linezolid (98%), vancomycin (98%), and teicoplanin (98%). All the isolates were found to be sensitive to daptomycin and tigecycline. While vancomycin-resistant enterococci (VRE) formed 7.5%, the linezolid-resistant enterococcus species was as high as 4.1%. CONCLUSION: The study showed a high prevalence of MRSA and VRE, thereby emphasizing the increasing antimicrobial resistance pattern of the Gram-positive pathogens. Therefore, there is an urgent need for novel antimicrobial stewardship to restrict the ongoing resistance rate among the isolates. [ABSTRACT FROM AUTHOR]

Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase–substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB–peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB–peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.

The Gram-positive anaerobic human pathogenic bacterium Atopobium vaginae causes most diagnosed cases of bacterial vaginosis as well as opportunistic infections in immunocompromised patients. In addition to its well-established role in carbohydrate metabolism, D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Streptococcus pyogenes and S. pneumoniae have been reported to act as extracellular virulence factors during streptococcal infections. Here, we report the crystal structure of GAPDH from A. vaginae (AvGAPDH) at 2.19 Å resolution. The refined model has a crystallographic Rfree of 22.6%. AvGAPDH is a homotetramer wherein each subunit is bound to a nicotinamide adenine dinucleotide (NAD+)molecule. The AvGAPDH enzyme fulfills essential glycolytic as well as moonlight (non-glycolytic) functions, both of which might be targets of chemotherapeutic intervention. We report that AvGAPDH interacts in vitro with the human C5a anaphylatoxin and inhibits C5a-specific granulocyte chemotaxis, thereby suggesting the participation of AvGAPDH in complement-targeted immunoevasion in a context of infection. The availability of high-quality structures of AvGAPDH and other homologous virulence factors from Gram-positive pathogens is critical for drug discovery programs. In this study, sequence and structural differences between AvGAPDH and related bacterial and eukaryotic GAPDH enzymes are reported in an effort to understand how to subvert the immunoevasive properties of GAPDH and evaluate the potential of AvGAPDH as a druggable target. [ABSTRACT FROM AUTHOR]

Corynebacterium pseudotuberculosis strain VD57 (Cp_VD57), a highly virulent, nonmotile, non-sporulating, and a mesophilic bacterium, was isolated from a goat's granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. Here, we describe a set of features of the strain, together with the details of its complete genome sequence and annotation. The genome comprises of a 2.5 Mbp long, single circular genome with 2,101 protein-coding genes, 12 rRNA, 49 tRNA and 47 pseudogenes and a G + C content of 52.85%. Genetic variation was detected in Cp_VD57 using C. pseudotuberculosis strain 1002 as reference, wherein small genomic insertions and deletions were identified. The comparative analysis of the genome sequence provides means to better understand the host pathogen interactions of this strain and can also help us to understand the molecular and genetic basis of virulence of this bacterium. [ABSTRACT FROM AUTHOR]

The Actinobacteria, Corynebacterium pseudotuberculosis strain P54B96, a nonmotile, non-sporulating and a mesophile bacterium, was isolated from liver, lung and mediastinal lymph node lesions in an antelope from South Africa. This strain is interesting in the sense that it has been found together with non-tuberculous mycobacteria (NTMs) which could nevertheless play a role in the lesion formation. In this work, we describe a set of features of C. pseudotuberculosis P54B96, together with the details of the complete genome sequence and annotation. The genome comprises of 2.34 Mbp long, single circular genome with 2,084 protein-coding genes, 12 rRNA, 49 tRNA and 62 pseudogenes and a G+C content of 52.19%. The analysis of the genome sequence provides means to better understanding the molecular and genetic basis of virulence of this bacterium, enabling a detailed investigation of its pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides. [Copyright &y& Elsevier]

For the majority of bacterial pathogens, acquisition of iron from host proteins is a prerequisite for growth during infection. The mechanisms by which Gram-negative bacteria obtain iron from host proteins have been well described, but only recently has substantial progress been made in identifying these mechanisms for Gram-positive bacterial pathogens. This review provides an overview of the existing knowledge on the genetic basis of iron transport for important Gram-positive pathogens. [Copyright &y& Elsevier]

Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen. Monitoring sortase activity and screening its inhibitors are of great significance to fundamental understanding of the infection mechanism and pharmaceutical development. Herein, we developed a digital single-particle imaging method to quantify sortase A (SrtA) activity based on transpeptidation-mediated assembly and enumeration of gold nanoparticles (GNPs). The assay utilizes two peptide stands, in which one has the SrtA recognition sequence LPXTG motif while the other carries an oligoglycine nucleophile at the one end and a biotin group at the other. The presence of SrtA enables the ligation of two peptides and allows for the immobilization of streptavidin-functionalized GNPs. Thus, SrtA activity can be quantified by imaging and enumeration of the surface-assembled GNPs at the single-particle level via dark-field microscopy. The single-particle method was highly sensitive to SrtA activity with a low detection limit of 7.9 pM and a wide linear dynamic range from 0.05 to 50 nM. Besides detection of SrtA in complex biological samples such as Gram-positive pathogen lysates, the proposed method was also successfully applied to estimate the half-maximal inhibitory concentration (IC 50) values of SrtA inhibitors (curcumin, berberine hydrochloride and quercetin). The present method, combining single-GNP counting and dark-field imaging, provides a facile and novel analytical tool for SrtA activity and its inhibitor screening. Image 1 • A digital single-particle sensing platform for ultra-sensitive and specific detection of sortase A is developed. • The sensing platform was applied to monitor sortase A in complex biological samples such as Gram-positive pathogen lysates. • The proposed assay can be used to evaluate the inhibition efficiency of sortase A inhibitors. • This single-particle method can be reliably used for detection of SrtA activity in serum and urine samples. [ABSTRACT FROM AUTHOR]

The capacity of bacterial pathogens to infect their hosts depends on the tight spatiotemporal regulation of virulence genes. The Listeria monocytogenes ( Lm ) metal efflux pump repressor CadC is highly expressed during late infection stages, modulating lipoprotein processing and host immune response. Here we investigate the potential of CadC as broad repressor of virulence genes. We show that CadC represses the expression of the bile salt hydrolase impairing Lm resistance to bile. During late infection, in absence of CadC-dependent repression, the constitutive bile salt hydrolase expression induces the overexpression of the cholic acid efflux pump MdrT that is unfavorable to Lm virulence. We establish the CadC regulon and show that CadC represses additional virulence factors activated by σ B during colonization of the intestinal lumen. CadC is thus a general repressor that promotes Lm virulence by down-regulating, at late infection stages, genes required for survival in the gastrointestinal tract. This demonstrates for the first time how bacterial pathogens can repurpose regulators to spatiotemporally repress virulence genes and optimize their infectious capacity.

Attachment to host tissue is a prerequisite for successful host colonization and invasion of pathogens. Many pathogenic bacteria use surface appendices, called pili, to bind and firmly attach to host tissue surfaces. Although it has been speculated that the laterally positioned D3 domain of the pilus-1 backbone protein RrgB of Streptococcus pneumoniae may promote bacterial-host interaction, via adhesion to extracellular matrix molecules, such as collagen, earlier studies showed no affinity of RrgB to collagen I. Using atomic force microscopy-based single molecule force spectroscopy combined with lateral force microscopy, we show that under mechanical load, RrgB in fact binds to human collagen I in a force-dependent manner. We observe exceptionally strong interactions, with interaction forces reaching as much as 1500 pN, and we show that high force loading and shearing rates enhance and further strengthen the interaction. In addition, the affinity of RrgB to collagen I under mechanical load not only depends on the orientation of the D3 domain but also on the orientation of the collagen fibrils, relative to the pulling direction. Both exceptionally high binding forces and force-induced bond strengthening resemble the behavior of so-called catch bonds, which have recently been observed in bacterial adhesins, but have not been reported for multimeric backbone subunits of virulence related pili.

In this protocol, we describe the application of using a high affinity monoclonal antibody generated against the major pilin protein component of the pilin structure of Enterococcus faecalis as a PET imaging agent for enterococcal endocarditis detection. The anti-pilin -mAb 64Cu conjugate was able to specifically label enterococcal endocarditis vegetation in vivo in a rodent endocarditis model. By targeting pili, a covalently linked surface antigen extending from the bacterial surface, we provided evidence that gram-positive pilin represent a logical surface antigen to define or target an infectious agent for molecularly guided imaging. Our goal in providing a detailed protocol of our efforts is to enable others to build upon this methodology to answer pertinent translational and basic research questions in the pursuit of diagnosis and treatment of infective endocarditis.

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase-substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB-peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB-peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival.

This study investigated the antimicrobial activity of Enterococcus faecium FAIR-E 198 against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Using the critical-dilution method, the bacteriocin produced by E. faecium FAIR-E 198 inhibited all L. monocytogenes strains evaluated (1,600 to 19,200 AU mL(-1)). However, none of the B. cereus and S. aureus strains investigated were inhibited. The maximum activity of this bacteriocin (800 AU mL(-1)) was observed in MRS broth, while the activity in milk was 100 AU mL(-1). In the co-cultivation test in milk, B. cereus K1-B041 was reduced to below the detection limit (1.00 log CFU mL(-1)) after 48 h. E. faecium reduced the initial L. monocytogenes Scott A population by 1 log CFU mL(-1) after 3 h at 35°C, However, the pathogen regained growth, reaching 3.68 log CFU mL(-1) after 48 h. E. faecium did not influence the growth of S. aureus ATCC 27154 during the 48 h of co-cultivation, Therefore, it can be concluded that the effectiveness of the antimicrobial activity of E. faecium FAIR-E 198 is strictly related to the species and strain of the target microorganism and to the culture medium.