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1.  Genome Sequence of Bacterial Interference Strain Staphylococcus aureus 502A 
Genome Announcements  2014;2(2):e00284-14.
Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s and early 1970s. Infants were deliberately colonized with 502A with the goal of preventing colonization with more invasive strains. We present the completed genome sequence of this organism.
doi:10.1128/genomeA.00284-14
PMCID: PMC3983310  PMID: 24723721
2.  Bordetella holmesii: initial genomic analysis of an emerging opportunist 
Pathogens and disease  2013;67(2):132-135.
Bordetella holmesii is an emerging opportunistic pathogen that causes respiratory disease in healthy individuals and invasive infections among patients lacking splenic function. We used 16S rRNA analysis to confirm B. holmesii as the cause of bacteremia in a child with sickle cell disease. Semiconductor-based draft genome sequencing provided insight into B. holmesii phylogeny and potential virulence mechanisms and also identified a toluene-4-monoxygenase locus unique among bordetellae.
doi:10.1111/2049-632X.12028
PMCID: PMC3653170  PMID: 23620158
Bordetella holmesii; genome; asplenia; opportunistic
3.  Klebsiella pneumoniae K1 Liver Abscess and Septic Endophthalmitis in a U.S. Resident 
Journal of Clinical Microbiology  2013;51(3):1049-1051.
Klebsiella pneumoniae K1 is a major agent of hepatic abscess with metastatic disease in East Asia, with sporadic reports originating elsewhere. We report a case of abscess complicated by septic endophthalmitis caused by a wzyAKpK1-positive Klebsiella strain in a U.S. resident, raising concern for global emergence.
doi:10.1128/JCM.02853-12
PMCID: PMC3592058  PMID: 23303492
4.  Cigarette Smoke Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress 
Infection and Immunity  2012;80(11):3804-3811.
The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biofilm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biofilm dispersal, and increased transcription of biofilm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biofilms. Exposure of bacteria to hydrogen peroxide alone increased biofilm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biofilm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding fibronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized fibronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans.
doi:10.1128/IAI.00689-12
PMCID: PMC3486061  PMID: 22890993
5.  Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity 
Journal of Antimicrobial Chemotherapy  2012;67(12):2870-2872.
Background
Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown.
Methods
We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro.
Results
RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation.
Conclusions
These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.
doi:10.1093/jac/dks305
PMCID: PMC3494843  PMID: 22855857
defensin; vaginolysin; bacterial vaginosis; biofilm
6.  Crystal structures of respiratory pathogen neuraminidases 
Biochemical and biophysical research communications  2009;380(3):10.1016/j.bbrc.2009.01.108.
Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.
doi:10.1016/j.bbrc.2009.01.108
PMCID: PMC3836282  PMID: 19284989
Streptococcus pneumoniae; Pseudomonas aeruginosa; neuraminidase; crystal structure; pneumonia
7.  Differential Binding of IgG and IgA to Mucus of the Female Reproductive Tract 
PLoS ONE  2013;8(10):e76176.
Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.
doi:10.1371/journal.pone.0076176
PMCID: PMC3788792  PMID: 24098437
8.  Trends and Significance of VRE Colonization in the ICU: A Meta-Analysis of Published Studies 
PLoS ONE  2013;8(9):e75658.
Background
The burden and significance of vancomycin-resistant enterococci (VRE) colonization in the ICU is not clearly understood.
Methods
We searched PubMed and EMBASE up to May 2013 for studies reporting the prevalence of VRE upon admission to the ICU and performed a meta-analysis to assess rates and trends of VRE colonization. We calculated the prevalence of VRE on admission and the acquisition (colonization and/or infection) rates to estimate time trends and the impact of colonization on ensuing VRE infections.
Findings
Across 37 studies (62,959 patients at risk), the estimated prevalence of VRE on admission to the ICU was 8.8% (7.1-10.6). Estimates were more consistent when cultures were obtained within 24 hours from admission. The VRE acquisition rate was 8.8% (95% CI 6.9-11.0) across 26 evaluable studies (35,364 patients at risk). Across US studies, VRE acquisition rate was 10.2% (95% CI 7.7-13.0) and demonstrated significant decline in annual trends. We used the US estimate of colonization on admission [12.3% (10.5-14.3)] to evaluate the impact of VRE colonization on admission in overall VRE prevalence. We demonstrated that VRE colonization on admission is a major determinant of the overall VRE burden in the ICU. Importantly, among colonized patients (including admitted and/or acquired cases) the VRE infection rates vary widely from 0-45% (with the risk of VRE bacteremia being reported from 0-16%) and <2% among those without a proven colonization.
Conclusion
In summary, up to 10.6% of patients admitted in the ICU are colonized with VRE on admission and a similar percentage will acquire VRE during their ICU stay. Importantly, colonization on admission is a major determinant of VRE dynamics in the ICU and the risk of VRE-related infections is close related to colonization.
doi:10.1371/journal.pone.0075658
PMCID: PMC3785502  PMID: 24086603
9.  Predictors of Staphylococcus aureus Rectovaginal Colonization in Pregnant Women and Risk for Maternal and Neonatal Infections 
Background.
Staphylococcus aureus infections are increasing among pregnant and postpartum women and neonates, but risk factors for S. aureus colonization in pregnancy and the association between maternal colonization and infant infections are not well defined. We sought to identify risk factors for maternal S. aureus rectovaginal colonization and assess colonization as a risk factor for infections among mothers and infants.
Methods.
We conducted a retrospective cohort study of pregnant women and their infants. Demographic and clinical data, including S. aureus infections that occurred in mothers from 3 months before to 3 months after delivery and in infants during the first 3 months of life, were extracted from electronic medical records. Predictors for maternal S. aureus rectovaginal colonization were assessed through multivariable logistic regression analysis.
Results.
The cohort included 2702 women and 2789 infants. The prevalence of maternal rectovaginal colonization with methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) was 13% and 0.7%. Independent predictors of colonization included multigravidity, human immunodeficiency virus seropositivity, and group B Streptococcus colonization. S. aureus colonization was associated with an increased risk of infection in mothers (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.4–8.8) but not in their infants (OR, 1.9; 95% CI, .6–5.6). The frequency of S. aureus infections was 0.8% in mothers and 0.7% in infants.
Conclusions.
S. aureus rectovaginal colonization was associated with an increased risk of infections in women but not in their infants. The frequency of MRSA infections was low. These data suggest that routine MRSA screening of pregnant women may not be indicated.
doi:10.1093/jpids/pis001
PMCID: PMC3656550  PMID: 23687569
10.  Microbiota of the upper and lower genital tract 
Summary
Our understanding of the bacterial species inhabiting the female genital tract has been limited primarily by our ability to detect them. Early investigations using microscopy and culture-based techniques identified lactobacilli as the predominant members of the vaginal microbiota and suggested that these organisms might serve a protective function at the mucosal surface. Improvements in cultivation techniques and the development of molecular-based detection strategies validated these early findings and enabled us to recognize that the microbiota of the female genital tract is much more complex than previously suspected. Disruption of the vaginal microbial community due to invasion of exogenous organisms or by overgrowth of one or more endogenous species has important health implications for both the mother and newborn.
doi:10.1016/j.siny.2011.08.006
PMCID: PMC3242913  PMID: 21920833
Bacterial vaginosis; Lactobacilli; Vaginal microbiota; Vaginitis
11.  Genome Sequence of the Human Abscess Isolate Streptococcus intermedius BA1 
Genome Announcements  2013;1(1):e00117-12.
Streptococcus intermedius is a human pathogen with a propensity for abscess formation. We report a high-quality draft genome sequence of S. intermedius strain BA1, an isolate from a human epidural abscess. This sequence provides insight into the biology of S. intermedius and will aid investigations of pathogenicity.
doi:10.1128/genomeA.00117-12
PMCID: PMC3569275  PMID: 23405291
12.  Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria 
Cellular microbiology  2007;9(5):1343-1351.
During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defense. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signaling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabeled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus α-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.
doi:10.1111/j.1462-5822.2006.00878.x
PMCID: PMC1867455  PMID: 17474907
13.  EPITHELIAL CELLS ARE SENSITIVE DETECTORS OF BACTERIAL PORE-FORMING TOXINS 
The Journal of biological chemistry  2006;281(18):12994-12998.
Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins – including pneumolysin from Streptococcus pneumoniae, α-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis – at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial PFT, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.
doi:10.1074/jbc.M511431200
PMCID: PMC1586115  PMID: 16520379
14.  Synonymous Codon Ordering: A Subtle but Prevalent Strategy of Bacteria to Improve Translational Efficiency 
PLoS ONE  2012;7(3):e33547.
Background
In yeast coding sequences, once a particular codon has been used, subsequent occurrence of the same amino acid tends to use codons sharing the same tRNA. Such a phenomenon of co-tRNA codons pairing bias (CTCPB) is also found in some other eukaryotes but it is not known whether it occurs in prokaryotes.
Methodology/Principal Findings
In this study, we focused on a total of 773 bacterial genomes to investigate their synonymous codon pairing preferences. After calculating the actual frequencies of synonymous codon pairs and comparing them with their expected values, we detected an obvious pairing bias towards identical codon pairs. This seems consistent with the previously reported CTCPB phenomenon, since identical codons are certainly read by the same tRNA. However, among co-tRNA but non-identical codon pairs, only 22 were often found overrepresented, suggesting that many co-tRNA codons actually do not preferentially pair together in prokaryotes. Therefore, the previously reported co-tRNA codons pairing rule needs to be more rigorously defined. The affinity differences between a tRNA anticodon and its readable codons should be taken into account. Moreover, both within-gene-shuffling tests and phylogenetic analyses support the idea that translational selection played an important role in shaping the observed synonymous codon pairing pattern in prokaryotes.
Conclusions
Overall, a high level of synonymous codon pairing bias was detected in 73% investigated bacterial species, suggesting the synonymous codon ordering strategy has been prevalently adopted by prokaryotes to improve their translational efficiencies. The findings in this study also provide important clues to better understand the complex dynamics of translational process.
doi:10.1371/journal.pone.0033547
PMCID: PMC3303843  PMID: 22432034
15.  Shiga Toxin Binding to Glycolipids and Glycans 
PLoS ONE  2012;7(2):e30368.
Background
Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable.
Methodology
We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells.
Results
By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells.
Conclusions
Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED50) of protein synthesis was about 10−11 M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.
doi:10.1371/journal.pone.0030368
PMCID: PMC3278406  PMID: 22348006
16.  Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum 
BMC Microbiology  2011;11:239.
Background
Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis, wound infections, and a variety of occasional invasive diseases. Since its initial discovery in 1946, this Gram positive organism has been known to have hemolytic activity, yet no hemolysin has been previously reported. A. haemolyticum also displays variable hemolytic activity on laboratory blood agar that is dependent upon which species the blood is derived.
Results
Here we describe a cholesterol-dependent cytolysin (CDC) secreted by A. haemolyticum, designated arcanolysin (aln), which is present in all strains (n = 52) tested by DNA dot hybridization. Among the known CDCs, ALN is most closely related to pyolysin (PLO) from Trueperella (formerly Arcanobacterium) pyogenes. The aln probe, however, did not hybridize to DNA from T. pyogenes. The aln open reading frame has a lower mol %G+C (46.7%) than the rest of the A. haemolyticum genome (53.1%) and is flanked by two tRNA genes, consistent with probable acquisition by horizontal transfer. The ALN protein (~ 64 kDa) contains a predicted signal sequence, a putative PEST sequence, and a variant undecapeptide within domain 4, which is typically important for function of the toxins. The gene encoding ALN was cloned and expressed in Escherichia coli as a functional recombinant toxin. Recombinant ALN had hemolytic activity on erythrocytes and cytolytic activity on cultured cells from human, rabbit, pig and horse origins but was poorly active on ovine, bovine, murine, and canine cells. ALN was less sensitive to inhibition by free cholesterol than perfringolysin O, consistent with the presence of the variant undecapeptide.
Conclusions
ALN is a newly identified CDC with hemolytic activity and unique properties in the CDC family and may be a virulence determinant for A. haemolyticum.
doi:10.1186/1471-2180-11-239
PMCID: PMC3215231  PMID: 22029628
17.  Inerolysin, a Cholesterol-Dependent Cytolysin Produced by Lactobacillus iners▿  
Journal of Bacteriology  2010;193(5):1034-1041.
Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.
doi:10.1128/JB.00694-10
PMCID: PMC3067590  PMID: 21169489
18.  The Ngal Reporter Mouse Detects the Response of the Kidney to Injury in Real Time 
Nature medicine  2011;17(2):216-222.
Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential characteristics which link the protein to the injured organ have not yet been described. We generated an NGAL-reporter mouse by inserting a di-fusion reporter gene, Luciferase2(Luc2)/mCherry(mC) into the Ngal locus. The Ngal-Luc2/mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real-time. In the kidney, Ngal-Luc2/mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ and cellular specific relationship with tubular stress, which quantitatively paralleled urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2/mC mice could also track both the onset and the resolution of the injury, and monitor the actions of NF-κB inhibitors and antibiotics in the case of infection. Accordingly, the imaging of Ngal-Luc2/mC mice and cells identified injurious and reparative agents which effect kidney damage.
doi:10.1038/nm.2290
PMCID: PMC3059503  PMID: 21240264
19.  Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract 
mBio  2011;2(3):e00016-11.
ABSTRACT
The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4−/−, Myd88−/−, Trif−/−, and Nod2−/− mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.
IMPORTANCE
The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.
doi:10.1128/mBio.00016-11
PMCID: PMC3101776  PMID: 21586648
20.  Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages 
PLoS ONE  2014;9(2):e87980.
Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.
doi:10.1371/journal.pone.0087980
PMCID: PMC3912197  PMID: 24498419
21.  Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci 
PLoS ONE  2014;9(1):e88136.
Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.
doi:10.1371/journal.pone.0088136
PMCID: PMC3909332  PMID: 24498253
22.  Trends in Methicillin-Resistant Staphylococcus aureus Anovaginal Colonization in Pregnant Women in 2005 versus 2009▿  
Journal of Clinical Microbiology  2010;48(10):3675-3680.
In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.
doi:10.1128/JCM.01129-10
PMCID: PMC2953117  PMID: 20686089
23.  Decline in Varicella-Related Ambulatory Visits and Hospitalizations in the United States Since Routine Immunization Against Varicella 
Background
Widespread varicella vaccination has led to substantial decreases in varicella-related mortality and hospitalizations. The impact of the vaccine on ambulatory care utilization is poorly defined.
Objective
To determine trends in varicella-related ambulatory care and hospital discharges before and after vaccine licensure.
Design, Setting, and Participants
Estimates of varicella-related ambulatory and hospital discharges were calculated for the pre- (1993–1995) and post- (1996–2004) vaccine licensure periods using the National Ambulatory Medical Care Survey, National Hospital Ambulatory Medical Care Survey and National Hospital Discharge Survey.
Main Outcome Measure
Ambulatory and hospital discharge rates for varicella.
Results
The rate of varicella-related ambulatory discharges decreased by 66% from 106.6/100,000 (95 % CI: 80.5–132.6) in the pre-licensure period to 36.4/100,000 population (95% CI: 29.3–43.5) in the post-licensure period (P<0.001). The decrease was significant across all age groups <45 years, with the greatest reduction (98%) occurring among patients 0–4 years of age. The incidence of varicella-related hospital discharges decreased by 53% from 30.9/100,000 (95% CI: 24.4–37.3) to 14.5/100,000 population (95% CI: 12.1–16.8) (P<0.001). This difference was significant among patients <14 years of age. Rates of varicella-related ambulatory discharges decreased significantly for both whites and non-whites in the post-licensure period, but post-licensure ambulatory discharge rates remained higher for non-whites than for whites. Decreases in varicella-related hospital discharges were statistically significant for whites and non-whites. Racial differences in the incidence of varicella-related hospital discharges also persisted following vaccine licensure.
Conclusions
Varicella-related ambulatory visits and hospitalizations have decreased significantly in the period after licensure of the varicella vaccine.
doi:10.1097/INF.0b013e3181bbf2a0
PMCID: PMC2924155  PMID: 19949362
varicella; chickenpox; vaccine; varicella; epidemiology; United States
24.  Iron Traffics in Circulation Bound to a Siderocalin (Ngal)-Catechol Complex 
Nature chemical biology  2010;6(8):602-609.
The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal [known as Neutrophil Gelatinase Associated Lipocalin, Siderocalin, Lipocalin 2] sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography, and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH sensitive mechanism. Since catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal:catechol:iron complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal:siderophore interactions, but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.
doi:10.1038/nchembio.402
PMCID: PMC2907470  PMID: 20581821
25.  Cigarette Smoke Inhibits Airway Epithelial Cell Innate Immune Responses to Bacteria ▿  
Infection and Immunity  2010;78(5):2146-2152.
The human upper respiratory tract, including the nasopharynx, is colonized by a diverse array of microorganisms. While the host generally exists in harmony with the commensal microflora, under certain conditions, these organisms may cause local or systemic disease. Respiratory epithelial cells act as local sentinels of the innate immune system, responding to conserved microbial patterns through activation of signal transduction pathways and cytokine production. In addition to colonizing microbes, these cells may also be influenced by environmental agents, including cigarette smoke (CS). Because of the strong relationship among secondhand smoke exposure, bacterial infection, and sinusitis, we hypothesized that components in CS might alter epithelial cell innate immune responses to pathogenic bacteria. We examined the effect of CS condensate (CSC) or extract (CSE) on signal transduction and cytokine production in primary and immortalized epithelial cells of human or murine origin in response to nontypeable Haemophilus influenzae and Staphylococcus aureus. We observed that epithelial production of interleukin-8 (IL-8) and IL-6 in response to bacterial stimulation was significantly inhibited in the presence of CS (P < 0.001 for inhibition by either CSC or CSE). In contrast, epithelial production of beta interferon (IFN-β) was not inhibited. CSC decreased NF-κB activation (P < 0.05) and altered the kinetics of mitogen-activated protein kinase phosphorylation in cells exposed to bacteria. Treatment of CSC with antioxidants abrogated CSC-mediated reduction of epithelial IL-8 responses to bacteria (P > 0.05 compared to cells without CSC treatment). These results identify a novel oxidant-mediated immunosuppressive role for CS in epithelial cells.
doi:10.1128/IAI.01410-09
PMCID: PMC2863539  PMID: 20194598

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