Microbiota and metabolic adaptation shape Staphylococcus aureus virulence and antimicrobial resistance during intestinal colonization.

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC , which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment. Importance: The fitness of MRSA depends on its ability to colonize. A key, underappreciated observation is that gut colonization frequently serves as the site for MRSA infections, especially among vulnerable groups such as children and hospitalized adults. By evolving MRSA strains in germ-free mice, we identify molecular mechanisms underlying how MRSA exploits a depletion in host microbiota to enhance gut colonization fitness. This work points to bacterial colonization factors that may be targetable. Our findings indicate that adaptive changes in MRSA often reduce its antimicrobial resistance and virulence, and are suppressed by the presence of native commensal bacteria. This work helps explain the ecology of pathoadaptive variants that thrive in hospital settings but falter under colonization conditions in healthy hosts. Additionally, it illustrates the potential adverse effects of prolonged, broad-spectrum empirical antimicrobial therapy and adds a new type of weight to calls for microbiota transplantation to reduce colonization by antimicrobial-resistant pathogens.

Eubacterium callanderi bacteremia: A case report.

Eubacterium species are a group of obligated anaerobic gram-positive bacilli that are recognized as commensals of the gastrointestinal tract flora. Cases of bacteremia mediated by Eubacterium are rare. This report describes a case of bacteremia caused by Eubacterium callanderi in an 82-year-old female with a history of a cecal perforation secondary to an obstructing sigmoid stricture. The results showed the utility of using whole genome sequencing to identify the causative agent and underlined the significance to identify anaerobic organisms in diagnostic microbiology practice and to perform antimicrobial susceptibility testing to guide therapy and enhance patient outcomes.

Staphylococcus aureus skin colonization is mediated by SasG lectin variation.

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. The initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus: SasG-I and SasG-II. Structural analyses of SasG-II identify a nonaromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicate that SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment results in different binding profiles between SasG-I and SasG-II on skin cells. In addition, SasG-mediated adhesion is recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

Glutamate-dependent arginine biosynthesis requires the inactivation of spoVG, sarA, and ahrC in Staphylococcus aureus.

Genome sequencing has demonstrated that Staphylococcus aureus encodes arginine biosynthetic genes argDCJBFGH synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, S. aureus does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by S. aureus that facilitate growth in CDM-R. However, these selected mutants synthesize arginine utilizing proline as a substrate rather than glutamate. In this study, we demonstrate that the ectopic expression of the argDCJB operon supports the growth of S. aureus in CDM-R, thus documenting the functionality of this pathway. Furthermore, suppressor mutants of S. aureus JE2 putA::Tn, which is defective in synthesizing arginine from proline, were selected on CDM-R agar. Genome sequencing revealed that these mutants had compensatory mutations within both spoVG, encoding an ortholog of the Bacillus subtilis stage V sporulation protein, and sarA, encoding the staphylococcal accessory regulator. Transcriptional studies document that argD expression is significantly increased when JE2 spoVG sarA was grown in CDM-R. Lastly, we found that a mutation in ahrC was required to induce argD expression in JE2 spoVG sarA when grown in an arginine-replete medium (CDM), suggesting that AhrC also functions to repress argDCJB in an arginine-dependent manner. In conclusion, these data indicate that the argDCJB operon is functional when transcribed in vitro and that SNPs within potential putative regulatory proteins are required to alleviate the repression.IMPORTANCEAlthough Staphylococcus aureus has the capability to synthesize all 20 amino acids, it is phenotypically auxotrophic for several amino acids including arginine. This work identifies putative regulatory proteins, including SpoVG, SarA, and AhrC, that function to inhibit the arginine biosynthetic pathways using glutamate as a substrate. Understanding the ultimate mechanisms of why S. aureus is selected to repress arginine biosynthetic pathways even in the absence of arginine will add to the growing body of work assessing the interactions between metabolism and S. aureus pathogenesis.

Disinfection of vascular catheter connectors that are protected by antiseptic caps is unnecessary.

OBJECTIVE: Determination of whether vascular catheter disinfecting antiseptic-containing caps alone are effective at decreasing microbial colonization of connectors compared to antiseptic-containing caps plus a 5-second alcohol manual disinfection. SETTING: The study was conducted in a 718-bed, tertiary-care, academic hospital. PATIENTS: A convenience sample of adult patients across intensive care units and acute care wards with peripheral and central venous catheters covered with antiseptic-containing caps. METHODS: Quality improvement study completed over 5 days. The standard-of-care group consisted of catheter connectors with antiseptic-containing caps cleaned with a 5-second alcohol wipe scrub prior to culture. The comparison group consisted of catheter connectors with antiseptic-containing caps without a 5-second alcohol wipe scrub prior to culture. The connectors were pressed directly onto blood agar plates and incubated. Plates were assessed for growth after 48-72 hours. RESULTS: In total, 356 catheter connectors were cultured: 165 in the standard-of-care group, 165 in the comparison group, and 26 catheters connectors without an antiseptic-containing cap, which were designated as controls. Overall, 18 catheter connectors (5.06%) yielded microbial growth. Of the 18 connectors with microbial growth, 2 (1.21%) were from the comparison group, 1 (0.61%) was from the standard-of-care group, and 15 were controls without an antiseptic-containing cap. CONCLUSIONS: Bacterial colonization rates were similar between the catheter connectors cultured with antiseptic-containing caps alone and catheter connectors with antiseptic-containing caps cultured after a 5-second scrub with alcohol. This finding suggests that the use of antiseptic-containing caps precludes the need for additional disinfection.

Staphylococcus aureus skin colonization is mediated by SasG lectin variation.

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. Initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus, SasG-I and SasG-II. Structural analyses of SasG-II identified a unique non-aromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicated SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment resulted in different binding profiles between SasG-I and SasG-II on skin cells. Additionally, SasG-mediated adhesion was recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

A critical role for staphylococcal nitric oxide synthase in controlling flavohemoglobin toxicity.

Most coagulase-negative staphylococcal species, including the opportunistic pathogen Staphylococcus epidermidis, struggle to maintain redox homeostasis and grow under nitrosative stress. Under these conditions, growth can only resume once nitric oxide (NO) is detoxified by the flavohemoglobin Hmp. Paradoxically, S. epidermidis produces endogenous NO through its genetically encoded nitric oxide synthase (seNOS) and heavily relies on its activity for growth. In this study, we investigate the basis of the growth advantage attributed to seNOS activity. Our findings reveal that seNOS supports growth by countering Hmp toxicity. S. epidermidis relies on Hmp activity for its survival in the host under NO stress. However, in the absence of nitrosative stress, Hmp generates significant amounts of the harmful superoxide radical (O2•-) from its heme prosthetic group which impedes growth. To limit Hmp toxicity, nitrite (NO2-) derived from seNOS promotes CymR-CysK regulatory complex activity, which typically regulates cysteine metabolism, but we now demonstrate to also repress hmp transcription. These findings reveal a critical mechanism through which the bacterial NOS-Hmp axis drives staphylococcal fitness.

Metabolic reprogramming and altered cell envelope characteristics in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.

The Challenge of Emerging Resistant Gram-Positive Pathogens in Hip and Knee Periprosthetic Joint Infections.

➤: An increase in resistant bacterial pathogens has occurred over the last 4 decades. ➤: Careful patient selection and improving or correcting risk factors for periprosthetic joint infection (PJI) before elective surgical treatment are strongly recommended. ➤: Appropriate microbiological methods, including those used to detect and grow Cutibacterium acnes, are recommended. ➤: Antimicrobial agents used in the prevention or management of infection should be selected appropriately and the duration of therapy should be carefully considered in order to mitigate the risk of developing bacterial resistance. ➤: Molecular methods including rapid polymerase chain reaction (PCR) diagnostics, 16S sequencing, and/or shotgun and/or targeted whole-genome sequencing are recommended in culture-negative cases of PJI. ➤: Expert consultation with an infectious diseases specialist (if available) is recommended to assist with the appropriate antimicrobial management and monitoring of patients with PJI.

A Transducing Bacteriophage Infecting Staphylococcus epidermidis Contributes to the Expansion of a Novel Siphovirus Genus and Implies the Genus Is Inappropriate for Phage Therapy.

The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis-infecting phage, Lacachita, and compare its genome with those of five other phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but Lacachita is capable of transducing antibiotic resistance and conferring phage resistance to transduced cells. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that Lacachita may be temperate and members of this novel genus are not suitable for phage therapy. IMPORTANCE This project describes the discovery of a culturable bacteriophage infecting Staphylococcus epidermidis that is a member of a rapidly growing novel siphovirus genus. A member of this genus was recently characterized and proposed for phage therapy, as there are few phages currently available to treat S. epidermidis infections. Our data contradict this, as we show Lacachita is capable of moving DNA from one bacterium to another, and it may be capable of maintaining itself in a plasmid-like state in infected cells. These phages' putative plasmid-like extrachromosomal state appears to be due to a simplified maintenance mechanism found in true plasmids of Staphylococcus and related hosts. We suggest Lacachita and other identified members of this novel genus are not suitable for phage therapy.

60-year-old male with rapidly progressive pneumocephalus caused by Clostridium septicum in the setting of an occult colonic adenocarcinoma.

BACKGROUND: Disseminated Clostridium septicum infection is an uncommon complication associated with malignancies, particular colonic adenocarcinoma. The organism appears to preferentially colonize large masses in rare individuals and subsequently seed the blood via mucosal ulceration. This has rarely been reported to lead to central nervous system infection and, in several cases, rapidly progressive pneumocephalus. In the few cases reported, this was a universally fatal condition. The current case adds to the reports of this extremely rare complication and provides a unique and complete clinicopathologic characterization with autopsy examination, microscopy, and molecular testing. CASE PRESENTATION: A 60-year-old man with no known past medical history was discovered having seizure-like activity and stroke-like symptoms. Blood cultures turned positive after six hours. Imaging revealed a large, irregular cecal mass as well as 1.4 cm collection of air in the left parietal lobe that progressed to over 7 cm within 8 h. By the following morning, the patient had lost all neurologic reflexes and died. Post-mortem examination revealed brain tissue with multiple grossly evident cystic spaces and intraparenchymal hemorrhage, while microscopic exam showed diffuse hypoxic-ischemic injury and gram-positive rods. Clostridium septicum was identified on blood cultures and was confirmed in paraffin embedded tissue from the brain by 16 S ribosomal sequencing and from the colon by C. septicum specific PCR. CONCLUSIONS: C. septicum is an anaerobic, gram-positive rod that can become invasive and is strongly associated with gastrointestinal pathology including colonic adenocarcinomas. Central nervous system infection with rapidly progressive pneumocephalus is a rarely reported and universally fatal complication of disseminated C. septicum infection.

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to ß-lactam antibiotics.

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in ß-lactam resistance, which will support efforts to identify new drug targets and reintroduce ß-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other ß-lactam resistant pathogens. Author summary: High-level resistance to penicillin-type (ß-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of ß-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

Mechanistic basis of staphylococcal interspecies competition for skin colonization.

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required for Staphylococcus epidermidis skin colonization and compare its characteristics to the lectin domain from the orthologous Staphylococcus aureus adhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap-and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition of S. aureus colonization.

Proline transporters ProT and PutP are required for Staphylococcus aureus infection.

Proline acquired via specific transporters can serve as a proteinogenic substrate, carbon and nitrogen source, or osmolyte. Previous reports have documented that Staphylococcus aureus, a major community and nosocomial pathogen, encodes at least four proline transporters, PutP, OpuC, OpuD, and ProP. A combination of genetic approaches and 3H-proline transport assays reveal that a previously unrecognized transporter, ProT, in addition to PutP, are the major proline transporters in S. aureus. Complementation experiments using constitutively expressed non-cognate promoters found that proline transport via OpuD, OpuC, and ProP is minimal. Both proline biosynthesis from arginine and proline transport via ProT are critical for growth when S. aureus is grown under conditions of high salinity. Further, proline transport mediated by ProT or PutP are required for growth in media with and without glucose, indicating both transporters function to acquire proline for proteinogenic purposes in addition to acquisition of proline as a carbon/nitrogen source. Lastly, inactivation of proT and putP resulted in a significant reduction (5 log10) of bacterial burden in murine skin-and-soft tissue infection and bacteremia models, suggesting that proline transport is required to establish a S. aureus infection.

Staphylococcal Corneocyte Adhesion: Assay Optimization and Roles of Aap and SasG Adhesins in the Establishment of Healthy Skin Colonization.

Staphylococcus aureus is an opportunistic pathogen that causes the majority of wound and soft tissue infections. The accumulation-associated protein (Aap) from S. epidermidis and surface protein G (SasG) from S. aureus are cell wall-anchored (CWA) proteins known to be important in adhesion to healthy corneocytes from human skin. We investigated the mechanisms by which S. aureus colonizes healthy human skin by developing an optimized corneocyte adhesion assay. Trypan blue was used for enhanced red autofluorescent visualization of corneocytes with an overlay of green-fluorescent bacteria. The percent area of bacterial adhesion for images acquired by a fluorescence microscope was quantified using Fiji ImageJ. Using this optimized imaging procedure, differences in adhesion between various species and strains of staphylococci were measured. The ability of purified SasG to reduce Staphylococcus epidermidis adhesion was investigated in order to determine if these CWA proteins can compete for binding sites. To further test CWA-mediated adhesion, we engineered a nonadhering S. carnosus strain to express full-length SasG from two methicillin-resistant S. aureus (MRSA) strains. Finally, we demonstrated that the SasG A domain was a critical region of this surface protein for adherence to healthy human corneocytes. The developed imaging and expression methods are useful for studying staphylococcal adhesion to healthy human skin and have the potential to be used with a wide variety of fluorescently labeled organisms on both healthy and disease-state (such as atopic dermatitis) corneocytes. IMPORTANCE The skin is the largest organ of the human body and acts as a shield against hazards such as harmful bacteria like Staphylococcus aureus. A diverse skin microbiota and immune cross talk control S. aureus numbers. S. aureus can bind to healthy skin and subsequently proliferate when the skin barrier is compromised, such as in a wound or in patients with atopic dermatitis (AD). It is important to understand these mechanisms in an effort to prevent pathogenic bacteria from causing infection. We describe an augmented corneocyte adhesion assay using fluorescence microscopy to study binding of various staphylococcal species to healthy human skin cells. In addition, we tested the ability of homologous proteins from different staphylococcal species to reduce binding, and developed a new S. carnosus expression system to test individual protein binding properties. Our newly developed methods and findings will enhance the understanding of how staphylococci bind to healthy human skin.

Neuroinvasive West Nile virus infections after solid organ transplantation: Single center experience and systematic review.

West Nile virus (WNv) is a major cause of viral encephalitis in the United States. WNv infection is usually asymptomatic or a limited febrile illness in the immunocompetent hosts, although a small percentage can develop neuroinvasive disease. Neuroinvasive disease due to WNv in solid organ transplant recipients occurs at higher rates than observed in the general population and can have long term neurological sequalae. METHODS: We retrospectively reviewed medical records of all solid organ transplant recipients at our institution who tested positive for WNv from 2010 to 2018. Two reviewers performed electronic searches of Medline, Embase, Cochrane Library of literature of WNv infections in SOT. Descriptive statistics were performed on key variables. RESULTS: Eight recipients (mean age 54, five males) were diagnosed with neuroinvasive WNv infection at our institution. Distribution of infection was as follows: five kidney transplants, one in each kidney-pancreas, liver, and lung. Diagnoses included meningitis (3), encephalitis (1), meningo-encephalitis (4). Median time from transplant to infection was 49.8 months (2.7-175.4). No infections were considered donor-derived. Five patients received treatment with IVIG. Six patients were alive at median follow-up of 49.5 months (21.7-116.8). We identified 29 studies published from 2002 to 2019. Median time from transplant to infection was 14.2 months, with similar allograft distribution; 53% were donor-derived infections. CONCLUSION: WNv infections in solid organ transplant recipients can be a consequence of organ donation or can be acquired via the community. Infections can be more severe in SOT recipients and lead to neuroinvasive disease.

Catabolic Ornithine Carbamoyltransferase Activity Facilitates Growth of Staphylococcus aureus in Defined Medium Lacking Glucose and Arginine.

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.

The staphylococcal biofilm protein Aap mediates cell-cell adhesion through mechanically distinct homophilic and lectin interactions.

The accumulation phase of staphylococcal biofilms relies on both the production of an extracellular polysaccharide matrix and the expression of bacterial surface proteins. A prototypical example of such adhesive proteins is the long multidomain protein Aap (accumulation-associated protein) from Staphylococcus epidermidis, which mediates zinc-dependent homophilic interactions between Aap B-repeat regions through molecular forces that have not been investigated yet. Here, we unravel the remarkable mechanical strength of single Aap-Aap homophilic bonds between living bacteria and we demonstrate that intercellular adhesion also involves sugar binding through the lectin domain of the Aap A region. We find that the mechanical force needed to unfold individual ß-sheet-rich G5-E domains from the Aap B-repeat regions is very high, ranging from 300 up to 1,000 pN at high loading rates, indicating these are extremely stable. This high mechanostability provides a means to the cells to form highly adhesive and cohesive biofilms capable of sustaining high physiological shear stress. Importantly, we identify a previously undescribed role of Aap in bacterial-bacterial adhesion, that is, heterophilic sugar binding by a specific lectin domain located in the N-terminal A region, which might be important to establish initial contacts between cells before strong homophilic bonds come into play. This study emphasizes the remarkable mechanical and binding properties of Aap as well as its wide diversity of adhesive functions.

Whole-Genome Sequences of Staphylococcus aureus Isolates from Positive Blood Cultures.

Staphylococcus aureus is a major cause of skin and soft tissue infections as well as bloodstream infections worldwide. Here, we report the draft genome sequences of 18 deidentified S. aureus clinical strains collected from positive blood cultures.