Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity.

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that residues 163-168 located on the ß6/ß7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Clostridioides difficile spores stimulate inflammatory cytokine responses and induce cytotoxicity in macrophages.

Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium, and the leading cause of antibiotic-associated diarrhea worldwide. During C. difficile infection, spores germinate in the presence of bile acids into vegetative cells that subsequently colonize the large intestine and produce toxins. In this study, we demonstrated that C. difficile spores can universally adhere to, and be phagocytosed by, murine macrophages. Only spores from toxigenic strains were able to significantly stimulate the production of inflammatory cytokines by macrophages and subsequently induce significant cytotoxicity. Spores from the isogenic TcdA and TcdB double mutant induced significantly lower inflammatory cytokines and cytotoxicity in macrophages, and these activities were restored by pre-exposure of the spores to either toxins. These findings suggest that during sporulation, spores might be coated with C. difficile toxins from the environment, which could affect C. difficile pathogenesis in vivo.

Swimming Pool-Associated Vittaforma-Like Microsporidia Linked to Microsporidial Keratoconjunctivitis Outbreak, Taiwan.

We analyzed 2 batches of environmental samples after a microsporidial keratoconjunctivitis outbreak in Taiwan. Results indicated a transmission route from a parking lot to a foot washing pool to a swimming pool and suggested that accumulation of mud in the foot washing pool during the rainy season might be a risk factor.

Genetic Relationships among Multidrug-Resistant Salmonella enterica Serovar Typhimurium Strains from Humans and Animals.

We identified 20 to 22 resistance genes, carried in four incompatibility groups of plasmids, in each of five genetically closely related Salmonella enterica serovar Typhimurium strains recovered from humans, pigs, and chickens. The genes conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, fluoroquinolones, extended-spectrum cephalosporins and cefoxitin, and azithromycin. This study demonstrates the transmission of multidrug-resistant Salmonella strains among humans and food animals and may be the first identification of mphA in azithromycin-resistant Salmonella strains in Taiwan.

Veillonella Catalase Protects the Growth of Fusobacterium nucleatum in Microaerophilic and Streptococcus gordonii-Resident Environments.

The oral biofilm is a multispecies community in which antagonism and mutualism coexist among friends and foes to keep an ecological balance of community members. The pioneer colonizers, such as Streptococcus gordonii, produce H2O2 to inhibit the growth of competitors, like the mutans streptococci, as well as strict anaerobic middle and later colonizers of the dental biofilm. Interestingly, Veillonella species, as early colonizers, physically interact (coaggregate) with S. gordonii A putative catalase gene (catA) is found in most sequenced Veillonella species; however, the function of this gene is unknown. In this study, we characterized the ecological function of catA from Veillonella parvula PK1910 by integrating it into the only transformable strain, Veillonella atypica OK5, which is catA negative. The strain (OK5-catA) became more resistant to H2O2 Further studies demonstrated that the catA gene expression is induced by the addition of H2O2 or coculture with S. gordonii Mixed-culture experiments further revealed that the transgenic OK5-catA strain not only enhanced the growth of Fusobacterium nucleatum, a strict anaerobic periodontopathogen, under microaerophilic conditions, but it also rescued F. nucleatum from killing by S. gordonii A potential role of catalase in veillonellae in biofilm ecology and pathogenesis is discussed here.IMPORTANCEVeillonella species, as early colonizers, can coaggregate with many bacteria, including the initial colonizer Streptococcus gordonii and periodontal pathogen Fusobacterium nucleatum, during various stages of oral biofilm formation. In addition to providing binding sites for many microbes, our previous study also showed that Veillonella produces nutrients for the survival and growth of periodontal pathogens. These findings indicate that Veillonella plays an important "bridging" role in the development of oral biofilms and the ecology of the human oral cavity. In this study, we demonstrated that the reducing activity of Veillonella can rescue the growth of Fusobacterium nucleatum not only under microaerophilic conditions, but also in an environment in which Streptococcus gordonii is present. Thus, this study will provide a new insight for future studies on the mechanisms of human oral biofilm formation and the control of periodontal diseases.

Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein.

Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens.

Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence in Clostridioides difficile strain R20291.

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio.

Pilus gene pool variation and the virulence of Corynebacterium diphtheriae clinical isolates during infection of a nematode.

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

Structural determinants of Actinomyces sortase SrtC2 required for membrane localization and assembly of type 2 fimbriae for interbacterial coaggregation and oral biofilm formation.

As a pioneer colonizer of the oral cavity, Actinomyces oris expresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci and Actinomyces biofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a "lid" domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminated Actinomyces species biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia.

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

Clostridium butyricum therapy for mild-moderate Clostridioides difficile infection and the impact of diabetes mellitus.

The therapeutic effect of Clostridium butyricum for adults with Clostridioides difficile infection (CDI) was investigated. A retrospective study was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, between January 2013 and April 2020. The disease severity of CDI was scored based on the Clinical Practice Guidelines of the IDSA/SHEA. Treatment success was defined as the resolution of diarrhea within six days of a therapeutic intervention without the need to modify the therapeutic regimen. In total, 241 patients developed CDI during hospitalization in the study period. The treatment success rates for the 99 patients with mild-moderate CDI among them were as follows: metronidazole, 69.4%; C. butyricum, 68.2%; metronidazole plus C. butyricum, 66.7%; and oral vancomycin, 66.7% (p=1.00). Patients with treatment success were less likely to have diabetes mellitus than those with treatment failure (38.2% vs. 61.3%, p=0.05). Patients treated with C. butyricum alone or in combination with metronidazole had shorter durations of diarrhea than those treated with metronidazole alone (3.1 ± 2.0 days or 3.5 ± 2.4 days vs. 4.2 ± 3.5 days; p=0.43 or 0.71), although the differences were not statistically significant. In conclusion, the treatment success rate of C. butyricum alone or in combination with metronidazole for patients with CDI was non inferior to that of metronidazole alone. The presence of diabetes mellitus in affected individuals is a risk factor for treatment failure.

Hypervirulent Clostridioides difficile RT078 lineage isolates from the river: A potential reservoir for environmental transmission.

This is the first report to discover Clostridiodes difficile (C. difficile) ribotype RT126 and RT598 (both ribotypes belong to RT078-lineage) in a river water system in southern Taiwan. Fluoroquinolone resistance was also found. The connection between clinical isolates and those from the environment needs further investigation.

Uremic Toxin-Producing Bacteroides Species Prevail in the Gut Microbiota of Taiwanese CKD Patients: An Analysis Using the New Taiwan Microbiome Baseline.

Rationale and Objective: Gut microbiota have been targeted by alternative therapies for non-communicable diseases. We examined the gut microbiota of a healthy Taiwanese population, identified various bacterial drivers in different demographics, and compared them with dialysis patients to associate kidney disease progression with changes in gut microbiota. Study Design: This was a cross-sectional cohort study. Settings and Participants: Fecal samples were obtained from 119 healthy Taiwanese volunteers, and 16S rRNA sequencing was done on the V3-V4 regions to identify the bacterial enterotypes. Twenty-six samples from the above cohort were compared with fecal samples from 22 peritoneal dialysis and 16 hemodialysis patients to identify species-level bacterial biomarkers in the dysbiotic gut of chronic kidney disease (CKD) patients. Results: Specific bacterial species were identified pertaining to different demographics such as gender, age, BMI, physical activity, and sleeping habits. Dialysis patients had a significant difference in gut microbiome composition compared to healthy controls. The most abundant genus identified in CKD patients was Bacteroides, and at the species level hemodialysis patients showed significant abundance in B. ovatus, B. caccae, B. uniformis, and peritoneal dialysis patients showed higher abundance in Blautia producta (p ≤ 0.05) than the control group. Pathways pertaining to the production of uremic toxins were enriched in CKD patients. The abundance of the bacterial species depended on the type of dialysis treatment. Conclusion: This study characterizes the healthy gut microbiome of a Taiwanese population in terms of various demographics. In a case-control examination, the results showed the alteration in gut microbiota in CKD patients corresponding to different dialysis treatments. Also, this study identified the bacterial species abundant in CKD patients and their possible role in complicating the patients' condition.

Differential response of Streptococcus mutans towards friend and foe in mixed-species cultures.

In the oral biofilm, the 'mitis' streptococci are among the first group of organisms to colonize the tooth surface. Their proliferation is thought to be an important factor required for antagonizing the growth of cariogenic species such as Streptococcus mutans. In this study, we used a three-species mixed culture to demonstrate that another ubiquitous early colonizing species, Veillonella parvula, can greatly affect the outcome of the competition between a pair of antagonists such as S. mutans and Streptococcus gordonii. Transcriptome analysis further revealed that S. mutans responds differentially to its friend (V. parvula) and foe (S. gordonii). In the mixed culture with S. gordonii, all but one of the S. mutans sugar uptake and metabolic genes were downregulated, while genes for alternative energy source utilization and H2O2 tolerance were upregulated, resulting in a slower but persistent growth. In contrast, when cultured with V. parvula, S. mutans grew equally well or better than in monoculture and exhibited relatively few changes within its transcriptome. When V. parvula was introduced into the mixed culture of S. mutans and S. gordonii, it rescued the growth inhibition of S. mutans. In this three-species environment, S. mutans increased the expression of genes required for the uptake and metabolism of minor sugars, while genes required for oxidative stress tolerance were downregulated. We conclude that the major factors that affect the competition between S. mutans and S. gordonii are carbohydrate utilization and H2O2 resistance. The presence of V. parvula in the tri-species culture mitigates these two major factors and allows S. mutans to proliferate, despite the presence of S. gordonii.

Advances in the Application of Nanomaterials as Treatments for Bacterial Infectious Diseases.

Bacteria-targeting nanomaterials have been widely used in the diagnosis and treatment of bacterial infectious diseases. These nanomaterials show great potential as antimicrobial agents due to their broad-spectrum antibacterial capacity and relatively low toxicity. Recently, nanomaterials have improved the accurate detection of pathogens, provided therapeutic strategies against nosocomial infections and facilitated the delivery of antigenic protein vaccines that induce humoral and cellular immunity. Biomaterial implants, which have traditionally been hindered by bacterial colonization, benefit from their ability to prevent bacteria from forming biofilms and spreading into adjacent tissues. Wound repair is improving in terms of both the function and prevention of bacterial infection, as we tailor nanomaterials to their needs, select encapsulation methods and materials, incorporate activation systems and add immune-activating adjuvants. Recent years have produced numerous advances in their antibacterial applications, but even further expansion in the diagnosis and treatment of infectious diseases is expected in the future.

Preliminary crystallographic study of the Streptococcus agalactiae sortases, sortase A and sortase C1.

Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. In Streptococcus agalactiae (GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) from S. agalactiae strain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form.

The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile.

Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis.

Micro-colonization of arsenic-resistant Staphylococcus sp. As-3 on arsenopyrite (FeAsS) drives arsenic mobilization under anoxic sub-surface mimicking conditions.

We investigated the subsurface biomatrix of the most abundant As-mineral, arsenopyrite (FeAsS), and meticulously studied a potential biogenic arsenic mobilization phenomenon. An arsenic-resistant [up to 7.5 mM As(III) and 200 mM As(V)] and arsenate-reducing bacterial strain (Staphylococcus sp. As-3) was isolated from a sediment core sample taken from the Budai borehole, on the southwestern coast of Taiwan. Isolate As-3 could reduce 5 mM As(V) to 3.04 mM in 96 h, generating 1.6 mM As(III) under anoxic conditions. Isolate As-3, which adsorbed As(V) up to 19.02 mg g-1 (cdw) and As(III) up to 0.46 mg g-1 (cdw), demonstrated effective As-bioaccumulating ability, as corroborated by a TEM-EDS analysis. Under anaerobic batch conditions, isolate As-3 micro-colonies could grow on as well as interact with arsenopyrite (FeAsS), mobilizing arsenic into soluble phase as As(III) and As(V). Using synchrotron radiation-based FTIR micro-spectroscopy, various functional group signatures and critical chemical bonds enabling a direct interaction with arsenopyrite were underpinned, such as a potential P-OFe bond involved in facilitating bacteria-mineral interaction. Using atomic force microscopy, we analyzed the scattered bacterial cell arrangement and structure and measured various biomechanical properties of micro-colonized Staphylococcus sp. As-3 cells on arsenopyrite. We suggest that the release of organic acids from As-3 drives soluble arsenic release in the aqueous phase under anoxic conditions through oxidative dissolution. Furthermore, arsC-encoding putative cytoplasmic arsenic reductase sequencing and transcript characterization indicated that arsC plays a possible role in the reduction of moderately soluble As(V) to highly soluble toxic As(III) under anoxic conditions. Thus, we suggest that firmicutes such as Staphylococcus sp. As-3 may play an important role in microbially-mediated arsenic mobilization, leading to arsenic release in the sub-surface niche.

Indocyanine Green-Mediated Photodynamic Therapy Reduces Methicillin-Resistant Staphylococcus aureus Drug Resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) skin-wound infections are associated with considerable morbidity and mortality. Indocyanine green (ICG), a safe and inexpensive dye used in clinical imaging, can be activated by near-infrared in photodynamic therapy (PDT) and photothermal therapy (PTT) to effectively kill MRSA. However, how this treatment affects MRSA drug sensitivity remains unknown. The drug-sensitivity phenotypes, bacterial growth rate, and cell-wall thickness of three MRSA strains were analyzed after ICG-PDT. Drug-resistant gene expressions were determined by polymerase chain reaction (PCR) and quantitative reverse transcription (qRT)-PCR. Related protein expressions were examined with immunoblotting. Drug sensitivity was further evaluated in animal models. MRSA that survived the treatment grew faster, and the cell wall became thinner compared to parental cells. These cells became more sensitive to oxacillin, which was partly related to mecA complex gene deletion. Skin necrosis caused by ICG-PDT-treated MRSA infection was smaller and healed faster than that infected with parental cells. With oxacillin therapy, no bacteria could be isolated from mouse lung tissue infected with ICG-PDT-treated MRSA. ICG-PDT drives MRSA toward an oxacillin-sensitive phenotype. It has the potential to develop into an alternative or adjuvant clinical treatment against MRSA wound infections.