Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells.

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.

Microbial communities in Cerrado soils under native vegetation subjected to prescribed fire and under pasture

The objective of this work was to evaluate the effects of fire regimes and vegetation cover on the structure and dynamics of soil microbial communities, through phospholipid fatty acid (PLFA) analysis. Comparisons were made between native areas with different woody covers ("cerrado stricto sensu" and "campo sujo"), under different fire regimes, and a 20-year-old active palisadegrass pasture in the Central Plateau of Brazil. Microbial biomass was higher in the native plots than in the pasture, and the highest monthly values were observed during the rainy season in the native plots. No significant differences were observed between fire regimes or between communities from the two native vegetation types. However, the principal component (PC) analysis separated the microbial communities by vegetation cover (native x pasture) and season (wet x dry), accounting for 45.8% (PC1 and PC3) and 25.6% (PC2 and PC3), respectively, of the total PLFA variability. Changes in land cover and seasonal rainfall in Cerrado ecosystems have significant effects on the total density of soil microorganisms and on the abundance of microbial groups, especially Gram-negative and Gram-positive bacteria.

The role of laparoscopic biopsies in lumbar spondylodiscitis.

Infection of an intervertebral disk is a serious condition. Diagnosis often is elusive and difficult. It is imperative to obtain appropriate microbiological specimens before initiation of treatment. The authors describe a 51-year-old woman with lumbar spondylodiscitis that was because of infection after the placement of an epidural catheter for postoperative analgesia. A spinal magnetic resonance imaging confirmed the diagnosis, but computed tomography-guided fine needle biopsy did not provide adequate material for a microbiologic diagnosis. Laparoscopic biopsies of the involved disk provided good specimens and a diagnosis of Propionibacterium acnes infection. The authors believe that this minimally invasive procedure should be performed when computed tomography-guided fine needle biopsy does not provide a microbiologic diagnosis in spondylodiscitis.

In vitro selection of bacteria with potential for use as probiotics in marine shrimp culture

The objective of this work was to isolate strains of lactic acid bacteria with probiotic potential from the digestive tract of marine shrimp (Litopenaeus vannamei), and to carry out in vitro selection based on multiple characters. The ideotype (ideal proposed strain) was defined by the highest averages for the traits maximum growth velocity, final count of viable cells, and inhibition halo against nine freshwater and marine pathogens, and by the lowest averages for the traits duplication time and resistance of strains to NaCl (1.5 and 3%), pH (6, 8, and 9), and biliary salts (5%). Mahalanobis distance (D²) was estimated among the evaluated strains, and the best ones were those with the shortest distances to the ideotype. Ten bacterial strains were isolated and biochemically identified as Lactobacillus plantarum (3), L. brevis (3), Weissella confusa (2), Lactococcus lactis (1), and L. delbrueckii (1). Lactobacillus plantarum strains showed a wide spectrum of action and the largest inhibition halos against pathogens, both Gram-positive and negative, high growth rate, and tolerance to all evaluated parameters. In relation to ideotype, L. plantarum showed the lowest Mahalanobis (D²) distance, followed by the strains of W. confusa, L. brevis, L. lactis, and L. delbrueckii. Among the analyzed bacterial strains, those of Lactobacillus plantarum have the greatest potential for use as a probiotic for marine shrimp.

Actinobaculum schaalii: clinical observation of 20 cases.

Actinobaculum schaalii is a new species that has so far been isolated from human blood, urine and pus. Its importance has probably been underestimated and other Actinobaculum spp. may also have been underdiagnosed. This retrospective study comprises all known cases of A. schaalii infections identified since 2004 in the canton of Neuchâtel (170,000 inhabitants), Switzerland. Strains were cultivated and isolated in the bacteriology laboratory using its routine procedure. Identification included a Rapid ID 32 A strip (bioMérieux) and 16S rRNA gene sequencing. Twenty-one positive samples were found in 19 patients (11 male, 8 female) of all ages (range 16-91 years): 10 from urine (50%), six from blood (30%), one from both blood and urine (5%), and three from pus (15%). Thirteen out of 17 (76%) cases with either blood or urine specimens had underlying genitourinary tract pathologies. When urine cultures were positive for A. schaalii, leucocytes were found in all samples (10/10, 100%) but all nitrite tests were negative (10/10, 100%). The onset of appropriate treatment was delayed due to the diminished sensitivity of A. schaalii to the antibiotics commonly used for UTIs (i.e. ciprofloxacin and trimethoprim/sulfamethoxazole) and to the delay in microbiological diagnosis. A. schaalii should specifically be searched in all cases of leukocyturia with a negative nitrite test but with Gram-positive rods in the Gram stain, in patients with underlying genitourinary tract pathology, instead of dismissing these findings as clinically irrelevant colonization by coryneform bacteria. This infection may be much more common than previously thought.

Targeted therapy against multi-resistant bacteria in leukemic and hematopoietic stem cell transplant recipients: guidelines of the 4th European Conference on Infections in Leukemia (ECIL-4, 2011).

The detection of multi-resistant bacterial pathogens, particularly those to carbapenemases, in leukemic and stem cell transplant patients forces the use of old or non-conventional agents as the only remaining treatment options. These include colistin/polymyxin B, tigecycline, fosfomycin and various anti-gram-positive agents. Data on the use of these agents in leukemic patients are scanty, with only linezolid subjected to formal trials. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for their use in these patient populations. Targeted therapy should be based on (i) in vitro susceptibility data, (ii) knowledge of the best treatment option against the particular species or phenotype of bacteria, (iii) pharmacokinetic/pharmacodynamic data, and (iv) careful assessment of the risk-benefit balance. For infections due to resistant Gram-negative bacteria, these agents should be preferably used in combination with other agents that remain active in vitro, because of suboptimal efficacy (e.g., tigecycline) and the risk of emergent resistance (e.g., fosfomycin). The paucity of new antibacterial drugs in the near future should lead us to limit the use of these drugs to situations where no alternative exists.

Propionibacterium acnes Promotes Th17 and Th17/Th1 Responses in Acne Patients.

Propionibacterium acnes is a Gram-positive commensal bacterium thought to be involved in the pathogenesis of acne vulgaris. Although the ability of P. acnes in the initiation of pro-inflammatory responses is well documented, little is known about adaptive immune responses to this bacterium. The observation that infiltrating immune cells consist mainly of CD4(+) T cells in the perifollicular space of early acne lesions suggests that helper T cells may be involved in immune responses caused by the intra-follicular colonization of P. acnes. A recent report showing that P. acnes can induce IL-17 production by T cells suggests that acne might be a T helper type 17 (Th17)-mediated disease. In line with this, we show in this work that, in addition to IL-17A, both Th1 and Th17 effector cytokines, transcription factors, and chemokine receptors are strongly upregulated in acne lesions. Furthermore, we found that, in addition to Th17, P. acnes can promote mixed Th17/Th1 responses by inducing the concomitant secretion of IL-17A and IFN-γ from specific CD4(+) T cells in vitro. Finally, we show that both P. acnes-specific Th17 and Th17/Th1 cells can be found in the peripheral blood of patients suffering from acne and, at lower frequencies, in healthy individuals. We therefore identified P. acnes-responding Th17/Th1 cells as, to our knowledge, a previously unreported CD4(+) subpopulation involved in inflammatory acne.

Quinupristin-dalfopristin combined with beta-lactams for treatment of experimental endocarditis due to Staphylococcus aureus constitutively resistant to macrolide-lincosamide-streptogramin B antibiotics.

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS(B)) than against MLS(B)-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. beta-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D-beta-lactam interaction was independent of MLS(B) or beta-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D-beta-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS(B)-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (P < 0.05). Importantly, Q-D prevented the growth of highly beta-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D-beta-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.

Targeting Enterococcus faecalis Biofilms with Phage Therapy.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

The UlaG protein familly defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events.

Vancomycin-associated nephrotoxicity, a case control study

Background: Vancomycin is a cornerstone antibiotic for the management of severe Gram positive infections. However, high doses of vancomycin are associated with a risk of nephrotoxicity. This study aimed to evaluate the relationship between the evolution of vancomycin trough concentration and the occurrence of nephrotoxicity, and to identify risk factors for both vancomycin-associated nephrotoxicity and vancomycin overexposure. Methods: A total of 1240 patients records from our hospital therapeutic drug monitoring database between 2007 and 2011 were screened and grouped according to predefined criteria defining vancomycin overexposure (one or more occurrence of a trough level ≥ 20 mg/L) and treatment-related nephrotoxicity (rise of serum creatinine by ≥ 50% over baseline). A representative sample of 150 cases was selected for in depth analysis. Weighted logistic regression analyses were used to test associations between vancomycin overexposure, nephrotoxicity and other predictors of interest. Results: Patients with high trough concentrations were found to be more likely to develop nephrotoxicity (odds ratio: 4.12; p <0.001). Specific risk factors, notably concomitant nephrotoxic treatments and comorbid conditions (heart failure), were found to independently increase the risk of either nephrotoxicity or vancomycin exposure. Finally, the exploration of temporal relationships between variations of vancomycin trough concentrations and creatinine levels were in line with circular causality with some antecedence of vancomycin on creatinine changes. Conclusion: Our results confirm the important nephrotoxic potential of vancomycin and indicate that the utilisation of this drug deserves thorough individualization for conditions susceptible to increase its concentration exposure and reactive adjustment based on therapeutic drug monitoring.

Occurrence and new mutations involved in rifampicin-resistant Propionibacterium acnes strains isolated from biofilm or device-related infections.

We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.

Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria.

Vaccination aims at generating memory immune responses able to protect individuals against pathogenic challenges over long periods of time. Subunit vaccine formulations based on safe, but poorly immunogenic, antigenic entities must be combined with adjuvant molecules to make them efficient against infections. We have previously shown that gas-filled microbubbles (MB) are potent antigen-delivery systems. This study compares the ability of various ovalbumin-associated MB (OVA-MB) formulations to induce antigen-specific memory immune responses and evaluates long-term protection toward bacterial infections. When initially testing dendritic cells reactivity to MB constituents, palmitic acid exhibited the highest degree of activation. Subcutaneous immunization of naïve wild-type mice with the OVA-MB formulation comprising the highest palmitic acid content and devoid of PEG2000 was found to trigger the more pronounced Th1-type response, as reflected by robust IFN-γ and IL-2 production. Both T cell and antibody responses persisted for at least 6 months after immunization. At that time, systemic infection with OVA-expressing Listeria monocytgenes was performed. Partial protection of vaccinated mice was demonstrated by reduction of the bacterial load in both the spleen and liver. We conclude that antigen-bound MB exhibit promising properties as a vaccine candidate ensuring prolonged maintenance of protective immunity.

A retrospective study of deep sternal wound infections: clinical and microbiological characteristics, treatment, and risk factors for complications.

Deep sternal wound infection (DSWI) is a feared complication following cardiac surgery. This study describes clinical, microbiological, and treatment outcomes of DSWI and determines risk factors for complications. Of 55 patients with DSWI, 66% were male and mean age was 68.2years. Initial sternotomy was for coronary artery bypass graft in 49% of patients. Sternal debridement at mean 25.4±18.3days showed monomicrobial (94%), mainly Gram-positive infection. Secondary sternal wound infection (SSWI) occurred in 31% of patients, was mostly polymicrobial (71%), and was predominantly due to Gram-negative bacilli. Risk factors for SSWI were at least 1 revision surgery (odds ratio [OR] 4.8 [95% confidence interval {CI} 1.0-22.4], P=0.047), sternal closure by muscle flap (OR 4.6 [1.3-16.8], P=0.02), delayed sternal closure (mean 27 versus 14days, P=0.03), and use of vacuum-assisted closure device (100% versus 58%, P=0.008). Hospital stay was significantly longer in patients with SSWI (69days versus 48days, P=0.04).

Vancomycin-associated nephrotoxicity, a case control study

Vancomycin is a cornerstone antibiotic for the management of severe Gram positive infections. However, high doses of vancomycin are associated with a risk of nephrotoxicity. This study aimed to evaluate the relationship between the evolution of vancomycin trough concentration and the occurrence of nephrotoxicity, and to identify risk factors for both vancomycin-associated nephrotoxicity and vancomycin overexposure.