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Staphylococcus haemolyticus (S. haemolyticus) is the second most commonly isolated coagulase-negative staphylococcus (CoNS) in patients with hospital-acquired infections. It can produce phenol-soluble modulin (PSM) toxins and form biofilms. Compared with the wealth of information on Staphylococcus aureus and Staphylococcus epidermidis , very little is known about S. haemolyticus. There is an urgent need to find an effective preparation to combat the harm caused by S. haemolyticus infection. Chinese herbs have been utilized to cure inflammation and infectious diseases and have a long history of anticancer function in China. Here, we modified fusaric acid characterized from the metabolites of Gibberella intermedia , an endophyte previously isolated from Polygonum capitatum. This study shows that fusaric acid analogs (qy17 and qy20) have strong antibacterial activity against S. haemolyticus. In addition, crystal violet analyses and scanning electron microscopy observations demonstrated that qy17 inhibited biofilm formation and disrupted mature biofilms of S. haemolyticus in a dose-dependent manner. Additionally, it reduced the number of live bacteria inside the biofilm. Furthermore, the antibiofilm function of qy17 was achieved by downregulating transcription factors (sigB), transpeptidase genes (srtA), and bacterial surface proteins (ebp , fbp) and upregulating biofilm-related genes and the density-sensing system (agrB). To further elucidate the bacteriostatic mechanism, transcriptomic analysis was carried out. The following antibacterial mechanisms were uncovered: (i) the inhibition of heat shock (clpB , groES , groL , grpE , dnaK , dnaJ)-, oxidative stress (aphC)- and biotin response (bioB)-related gene expression, which resulted in S. haemolyticus being unable to compensate for various stress conditions, thereby affecting bacterial growth; and (ii) a reduction in the expression of PSM-beta (PSMβ1, PSMβ2, PSMβ3) toxin- and Clp protease (clpP , clpX)-related genes. These findings could have major implications for the treatment of diseases caused by S. haemolyticus infections. Our research reveals for the first time that fusaric acid derivatives inhibit the expression of biofilm formation-related effector and virulence genes of S. haemolyticus. These findings provide new potential drug candidates for hospital-acquired infections caused by S. haemolyticus. [ABSTRACT FROM AUTHOR]

Staphylococcus haemolyticus (S. haemolyticus) is the second most commonly isolated coagulase-negative staphylococcus (CoNS) in patients with hospital-acquired infections. It can produce phenol-soluble modulin (PSM) toxins and form biofilms. Compared with the wealth of information on Staphylococcus aureus and Staphylococcus epidermidis, very little is known about S. haemolyticus. There is an urgent need to find an effective preparation to combat the harm caused by S. haemolyticus infection. Chinese herbs have been utilized to cure inflammation and infectious diseases and have a long history of anticancer function in China. Here, we modified fusaric acid characterized from the metabolites of Gibberella intermedia, an endophyte previously isolated from Polygonum capitatum. This study shows that fusaric acid analogs (qy17 and qy20) have strong antibacterial activity against S. haemolyticus. In addition, crystal violet analyses and scanning electron microscopy observations demonstrated that qy17 inhibited biofilm formation and disrupted mature biofilms of S. haemolyticus in a dose-dependent manner. Additionally, it reduced the number of live bacteria inside the biofilm. Furthermore, the antibiofilm function of qy17 was achieved by downregulating transcription factors (sigB), transpeptidase genes (srtA), and bacterial surface proteins (ebp, fbp) and upregulating biofilm-related genes and the density-sensing system (agrB). To further elucidate the bacteriostatic mechanism, transcriptomic analysis was carried out. The following antibacterial mechanisms were uncovered: (i) the inhibition of heat shock (clpB, groES, groL, grpE, dnaK, dnaJ)-, oxidative stress (aphC)- and biotin response (bioB)-related gene expression, which resulted in S. haemolyticus being unable to compensate for various stress conditions, thereby affecting bacterial growth; and (ii) a reduction in the expression of PSM-beta (PSMβ1, PSMβ2, PSMβ3) toxin- and Clp protease (clpP, clpX)-related genes. These findings could have major implications for the treatment of diseases caused by S. haemolyticus infections. Our research reveals for the first time that fusaric acid derivatives inhibit the expression of biofilm formation-related effector and virulence genes of S. haemolyticus. These findings provide new potential drug candidates for hospital-acquired infections caused by S. haemolyticus.

RationaleAlthough frequently retrieved in tracheal secretions of critically ill patients on mechanical ventilation, the existence of pneumonia caused by coagulase-negative staphylococci (CoNS) remains controversial.ObjectiveTo assess whether Staphylococcus haemolyticus (S. haemolyticus) inoculated in mice’s trachea can infect normal lung parenchyma, increasing concentrations of S. haemolyticus were intratracheally administered in 221 immunocompetent mice.MethodsEach animal received intratracheally phosphate-buffered saline (PBS) (n = 43) or live (n = 141) or inactivated (n = 37) S. haemolyticus at increasing load: 1.0 × 106, 1.0 × 107, and 1.0 × 108 colony forming units (CFU). Forty-three animals were sacrificed at 12 h and 178 were sacrificed at 36 h; 64 served for post-mortem lung histology, 157 served for pre-mortem bronchoalveolar lavage (BAL) analysis, and 42 served for post-mortem quantitative bacteriology of lung tissue. The distribution of biofilm-associated genes was investigated in the S. haemolyticus strain used in our in vivo experiment as well as among 19 other clinical S. haemolyticus strains collected from hospitals or nursing houses.Measurements and Main ResultsIntratracheal inoculation of 1.0 × 108 CFU live S. haemolyticus caused macroscopic and histological confluent pneumonia with significant increase in BAL white cell count, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP)-2. At 12 h, high concentrations of S. haemolyticus were identified in BAL. At 36 h, lung injury and BAL inflammation were less severe than at 12 h and moderate concentrations of species belonging to the oropharyngeal flora were identified in lung tissue. The inoculation of 1.0 × 106 and 1.0 × 107 CFU live S. haemolyticus caused histologic interstitial pneumonia and moderate BAL inflammation. Similar results were observed after inoculation of inactivated S. haemolyticus. Moreover, biofilm formation was a common phenotype in S. haemolyticus isolates. The low prevalence of the ica operon in our clinical S. haemolyticus strain collection indicated icaA and icaD independent-biofilm formation.ConclusionIn immunocompetent spontaneously breathing mice, inoculation of S. haemolyticus causes concentration-dependent lung infection that spontaneously recovers over time. icaA and icaD independent biofilm formation is a common phenotype in S. haemolyticus isolates.

Staphylococcus haemolyticus is a skin commensal gaining increased attention as an emerging pathogen of nosocomial infections. However, knowledge about the transition from a commensal to an invasive lifestyle remains sparse and there is a paucity of studies comparing pathogenicity traits between commensal and clinical isolates. In this study, we used a pan-genomic approach to identify factors important for infection and hospital adaptation by exploring the genomic variability of 123 clinical isolates and 46 commensal S. haemolyticus isolates. Phylogenetic reconstruction grouped the 169 isolates into six clades with a distinct distribution of clinical and commensal isolates in the different clades. Phenotypically, multi-drug antibiotic resistance was detected in 108/123 (88%) of the clinical isolates and 5/46 (11%) of the commensal isolates (p < 0.05). In the clinical isolates, we commonly identified a homolog of the serine-rich repeat glycoproteins sraP. Additionally, three novel capsular polysaccharide operons were detected, with a potential role in S. haemolyticus virulence. Clinical S. haemolyticus isolates showed specific signatures associated with successful hospital adaption. Biofilm forming S. haemolyticus isolates that are resistant to oxacillin (mecA) and aminoglycosides (aacA-aphD) are most likely invasive isolates whereas absence of these traits strongly indicates a commensal isolate. We conclude that our data show a clear segregation of isolates of commensal origin, and specific genetic signatures distinguishing the clinical isolates from the commensal isolates. The widespread use of antimicrobial agents has probably promoted the development of successful hospital adapted clones of S. haemolyticus clones through acquisition of mobile genetic elements or beneficial point mutations and rearrangements in surface associated genes.

Staphylococcus haemolyticus is one of the most significant coagulase-negative staphylococci, and it often causes severe infections. Rapid strain typing of pathogenic S. haemolyticus is indispensable in modern public health infectious disease control, facilitating the identification of the origin of infections to prevent further infectious outbreak. Rapid identification enables the effective control of pathogenic infections, which is tremendously beneficial to critically ill patients. However, the existing strain typing methods, such as multi-locus sequencing, are of relatively high cost and comparatively time-consuming. A practical method for the rapid strain typing of pathogens, suitable for routine use in clinics and hospitals, is still not available. Matrix-assisted laser desorption ionization-time of flight mass spectrometry combined with machine learning approaches is a promising method to carry out rapid strain typing. In this study, we developed a statistical test-based method to determine the reference spectrum when dealing with alignment of mass spectra datasets, and constructed machine learning-based classifiers for categorizing different strains of S. haemolyticus. The area under the receiver operating characteristic curve and accuracy of multi-class predictions were 0.848 and 0.866, respectively. Additionally, we employed a variety of statistical tests and feature-selection strategies to identify the discriminative peaks that can substantially contribute to strain typing. This study not only incorporates statistical test-based methods to manage the alignment of mass spectra datasets but also provides a practical means to accomplish rapid strain typing of S. haemolyticus.

Rationale: Although frequently retrieved in tracheal secretions of critically ill patients on mechanical ventilation, the existence of pneumonia caused by coagulase-negative staphylococci (CoNS) remains controversial. Objective: To assess whether Staphylococcus haemolyticus (S. haemolyticus) inoculated in mice's trachea can infect normal lung parenchyma, increasing concentrations of S. haemolyticus were intratracheally administered in 221 immunocompetent mice. Methods: Each animal received intratracheally phosphate-buffered saline (PBS) (n = 43) or live (n = 141) or inactivated (n = 37) S. haemolyticus at increasing load: 1.0 × 106, 1.0 × 107, and 1.0 × 108 colony forming units (CFU). Forty-three animals were sacrificed at 12 h and 178 were sacrificed at 36 h; 64 served for post-mortem lung histology, 157 served for pre-mortem bronchoalveolar lavage (BAL) analysis, and 42 served for post-mortem quantitative bacteriology of lung tissue. The distribution of biofilm-associated genes was investigated in the S. haemolyticus strain used in our in vivo experiment as well as among 19 other clinical S. haemolyticus strains collected from hospitals or nursing houses. Measurements and Main Results: Intratracheal inoculation of 1.0 × 108 CFU live S. haemolyticus caused macroscopic and histological confluent pneumonia with significant increase in BAL white cell count, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP)-2. At 12 h, high concentrations of S. haemolyticus were identified in BAL. At 36 h, lung injury and BAL inflammation were less severe than at 12 h and moderate concentrations of species belonging to the oropharyngeal flora were identified in lung tissue. The inoculation of 1.0 × 106 and 1.0 × 107 CFU live S. haemolyticus caused histologic interstitial pneumonia and moderate BAL inflammation. Similar results were observed after inoculation of inactivated S. haemolyticus. Moreover, biofilm formation was a common phenotype in S. haemolyticus isolates. The low prevalence of the ica operon in our clinical S. haemolyticus strain collection indicated icaA and icaD independent-biofilm formation. Conclusion: In immunocompetent spontaneously breathing mice, inoculation of S. haemolyticus causes concentration-dependent lung infection that spontaneously recovers over time. icaA and icaD independent biofilm formation is a common phenotype in S. haemolyticus isolates. [ABSTRACT FROM AUTHOR]

Staphylococcus haemolyticus is one of the most significant coagulase-negative staphylococci, and it often causes severe infections. Rapid strain typing of pathogenic S. haemolyticus is indispensable in modern public health infectious disease control, facilitating the identification of the origin of infections to prevent further infectious outbreak. Rapid identification enables the effective control of pathogenic infections, which is tremendously beneficial to critically ill patients. However, the existing strain typing methods, such as multi-locus sequencing, are of relatively high cost and comparatively time-consuming. A practical method for the rapid strain typing of pathogens, suitable for routine use in clinics and hospitals, is still not available. Matrix-assisted laser desorption ionization-time of flight mass spectrometry combined with machine learning approaches is a promising method to carry out rapid strain typing. In this study, we developed a statistical test-based method to determine the reference spectrum when dealing with alignment of mass spectra datasets, and constructed machine learning-based classifiers for categorizing different strains of S. haemolyticus. The area under the receiver operating characteristic curve and accuracy of multi-class predictions were 0.848 and 0.866, respectively. Additionally, we employed a variety of statistical tests and feature-selection strategies to identify the discriminative peaks that can substantially contribute to strain typing. This study not only incorporates statistical test-based methods to manage the alignment of mass spectra datasets but also provides a practical means to accomplish rapid strain typing of S. haemolyticus. [ABSTRACT FROM AUTHOR]

Staphylococcus haemolyticus is a skin commensal gaining increased attention as an emerging pathogen of nosocomial infections. However, knowledge about the transition from a commensal to an invasive lifestyle remains sparse and there is a paucity of studies comparing pathogenicity traits between commensal and clinical isolates. In this study, we used a pan-genomic approach to identify factors important for infection and hospital adaptation by exploring the genomic variability of 123 clinical isolates and 46 commensal S. haemolyticus isolates. Phylogenetic reconstruction grouped the 169 isolates into six clades with a distinct distribution of clinical and commensal isolates in the different clades. Phenotypically, multi-drug antibiotic resistance was detected in 108/123 (88%) of the clinical isolates and 5/46 (11%) of the commensal isolates (p < 0.05). In the clinical isolates, we commonly identified a homolog of the serine-rich repeat glycoproteins sraP. Additionally, three novel capsular polysaccharide operons were detected, with a potential role in S. haemolyticus virulence. Clinical S. haemolyticus isolates showed specific signatures associated with successful hospital adaption. Biofilm forming S. haemolyticus isolates that are resistant to oxacillin (mecA) and aminoglycosides (aacA-aphD) are most likely invasive isolates whereas absence of these traits strongly indicates a commensal isolate. We conclude that our data show a clear segregation of isolates of commensal origin, and specific genetic signatures distinguishing the clinical isolates from the commensal isolates. The widespread use of antimicrobial agents has probably promoted the development of successful hospital adapted clones of S. haemolyticus clones through acquisition of mobile genetic elements or beneficial point mutations and rearrangements in surface associated genes. [ABSTRACT FROM AUTHOR]

Staphylococcus haemolyticus is the second most frequently isolated CoNS from ocular infections and human blood cultures. In this study, we examined 18 ocular S. haemolyticus isolates for their capacity to form biofilm and conducted detachment assay to determine the composition of the biofilm matrix and involvement of various elements in cell lysis. PCR identified the presence of biofilm-associated genes, and ica operon and CLSM visualized the components of the biofilm matrix. We found that PIA-independent biofilm formation is the characteristic feature of S. haemolyticus isolates, irrespective of the sources of isolation, and protein or DNA or both are the major components of the biofilm matrix. Cell lysis enabling DNA release was an essential step for biofilm attachment during the initial stages of biofilm development. The srtA transcript expression study indicates its role in the early stages of biofilm development. We found the presence of antibiotic resistance genes in the eDNA and gDNA thus suggesting the possible role of biofilm in horizontal gene transfer of antibiotic resistance determinants. The overall study indicates that S. haemolyticus formed the biofilm comprising of protein or DNA or both and srtA play a role in the initial development of biofilm.

Staphylococcus haemolyticus is the second most frequently isolated CoNS from ocular infections and human blood cultures. In this study, we examined 18 ocular S. haemolyticus isolates for their capacity to form biofilm and conducted detachment assay to determine the composition of the biofilm matrix and involvement of various elements in cell lysis. PCR identified the presence of biofilm-associated genes, and ica operon and CLSM visualized the components of the biofilm matrix. We found that PIA-independent biofilm formation is the characteristic feature of S. haemolyticus isolates, irrespective of the sources of isolation, and protein or DNA or both are the major components of the biofilm matrix. Cell lysis enabling DNA release was an essential step for biofilm attachment during the initial stages of biofilm development. The srtA transcript expression study indicates its role in the early stages of biofilm development. We found the presence of antibiotic resistance genes in the eDNA and gDNA thus suggesting the possible role of biofilm in horizontal gene transfer of antibiotic resistance determinants. The overall study indicates that S. haemolyticus formed the biofilm comprising of protein or DNA or both and srtA play a role in the initial development of biofilm. [ABSTRACT FROM AUTHOR]

Coagulase-negative staphylococci (CoNS) are important nosocomial pathogens and the leading cause of sepsis. The second most frequently implicated species, after Staphylococcus epidermidis, is Staphylococcus haemolyticus. However, we have a significant lack of knowledge about what causes virulence of S. haemolyticus, as virulence factors of this pathogen have remained virtually unexplored. In contrast to the aggressive pathogen Staphylococcus aureus, toxin production has traditionally not been associated with CoNS. Recent findings have suggested that phenol-soluble modulins (PSMs), amphipathic peptide toxins with broad cytolytic activity, are widespread in staphylococci, but there has been no systematic assessment of PSM production in CoNS other than S. epidermidis. Here, we identified, purified, and characterized PSMs of S. haemolyticus. We found three PSMs of the β-type, which correspond to peptides that before were described to have anti-gonococcal activity. We also detected an α-type PSM that has not previously been described. Furthermore, we confirmed that S. haemolyticus does not produce a δ-toxin, as results from genome sequencing had indicated. All four S. haemolyticus PSMs had strong pro-inflammatory activity, promoting neutrophil chemotaxis. Notably, we identified in particular the novel α-type PSM, S. haemolyticus PSMα, as a potent hemolysin and leukocidin. For the first time, our study describes toxins of this important staphylococcal pathogen with the potential to have a significant impact on virulence during blood infection and sepsis.