bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonconcordance with Surgical Site Infection Prevention Guidelines and Rates of Surgical Site Infections for General Surgical, Neurological, and Orthopedic Procedures
Authors: Young Barnaby , Ng Tat Ming , Teng Christine , Ang Brenda , Tai Hwei Yee , Lye David C.
Published: August 2011

Surgical site infection (SSI) is a common and preventable complication of surgery, but the relative importance of individual measures recommended by guidelines has not been determined. Elective general surgical, neurological, and orthopedic procedures requiring antibiotic prophylaxis from a 3-month period were retrospectively studied to determine concordance with SSI prevention guidelines and to identify factors which predicted the development of SSIs. A total of 216 surgeries were reviewed, with 18 SSIs (8.3%). A mean of 1.4 antibiotic prophylaxis errors per surgery were identified, with correct antibiotic type identified for 64%, antibiotic timing for 83%, supplemental antibiotic dosing for 34%, and antibiotic duration of less than 24 h for 44%. Normothermia was present in 79% of surgeries, and normoglycemia was present in 17%. Univariate analysis of the SSI rate identified four significant factors. Antibiotic prophylaxis for less than 24 h postoperatively (odds ratio [OR], 0.213; 95% confidence interval [95% CI]0.060 to 0.757) and neurosurgery (OR, 0.118; 95% CI, 0.15 to 0.903) correlated with a reduced risk of SSI. The mean number of prophylaxis errors (OR, 1.6; 95% CI, 1.02 to 2.4) and a duration of surgical drainage for more than 3 days (OR, 2.679; 95% CI, 1.009 to 7.113) predicted SSI. By multivariate analysis, errors in individual antibiotic prophylaxis measures were not significantly associated with SSI; however, the presence of more than two errors was significant (OR, 4.030; 95% CI, 1.018 to 15.96). A strong correlation was identified between the degree of concordance to SSI prevention guidelines and the SSI rate (
P
= 0.001, Mantel-Haenszel linear-by-linear association chi-square test).
In Vitro
and
In Vivo
Activities of Novel, Semisynthetic Thiopeptide Inhibitors of Bacterial Elongation Factor Tu
Authors: Leeds J. A. , LaMarche M. J. , Brewer J. T. , Bushell S. M. , Deng G. , Dewhurst J. M. , Dzink-Fox J. , Gangl E. , Jain A. , Lee L. , Lilly M. , Manni K. , Mullin S. , Neckermann G. , Osborne C. , Palestrant D. , Patane M. A. , Raimondi A. , Ranjitkar S. , Rann E. M. , Sachdeva M. , Shao J. , Tiamfook S. , Whitehead L. , Yu D.
Published: August 2011

Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the
in vitro
and
in vivo
activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant
Staphylococcus aureus
and vancomycin-resistant enterococci (MIC
90
≤ 0.25 μg/ml) but weaker against the streptococci (MIC
90
≥ 4 μg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the
tuf
gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal
S. aureus
infections with 50% effective doses (ED
50
) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic
E. faecalis
infections with ED
50
of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active
in vitro
and
in vivo
activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.
Characterization of the Retrocyclin Analogue RC-101 as a Preventative of Staphylococcus aureus Nasal Colonization
Authors: Lamers Ryan P. , Eade Colleen R. , Waring Alan J. , Cole Amy L. , Cole Alexander M.
Published: August 2011

Nasal colonization ofStaphylococcus aureusis a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization ofS. aureushas become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic θ-defensin analogue RC-101 as a promising anti-S. aureusagent for nasal decolonization. RC-101 exhibits bactericidal effects againstS. aureuswith the use ofin vitroepithelium-free systems, while also preventing the pathogen's proliferation and attachment in anex vivohuman nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 μM elicited significant reductions inS. aureusgrowth in epithelium-free systems, with 10 μM concentrations being completely bactericidal for all strains tested, including USA300. Inex vivonasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation ofS. aureuson human nasal epithelia. Reductions inS. aureusviability were evident in these assays, with as little as 1 μg of peptide per tissue, while 10 μg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 μM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative ofS. aureusnasal colonization.
Dissemination of an NDM-2-Producing Acinetobacter baumannii Clone in an Israeli Rehabilitation Center
Authors: Espinal P. , Fugazza G. , López Y. , Kasma M. , Lerman Y. , Malhotra-Kumar S. , Goossens H. , Carmeli Y. , Vila J.
Published: August 2011

New Delhi metallo-β-lactamase (NDM-1) was initially identified in various
Enterobacteriaceae
and recently in
Acinetobacter baumannii
. This study described the clonal dissemination of an NDM-2-producing
A. baumannii
isolate in an Israeli rehabilitation ward and the genetic surroundings of the gene. The
bla
NDM-2
gene was surrounded by the
ble
and
trpF
genes downstream and two copies of the IS
Aba125
on both sides. These are the first NDM-producing
A. baumannii
strains in Israel from patients with no previous travel or hospitalization on the Indian subcontinent.
The MUT056399 Inhibitor of FabI Is a New Antistaphylococcal Compound
Authors: Escaich S. , Prouvensier L. , Saccomani M. , Durant L. , Oxoby M. , Gerusz V. , Moreau F. , Vongsouthi V. , Maher Kirsty , Morrissey Ian , Soulama-Mouze C.
Published: August 2011

MUT056399 is a highly potent new inhibitor of the FabI enzyme of both
Staphylococcus aureus
and
Escherichia coli. In vitro
, MUT056399 was very active against
S. aureus
strains, including methicillin-susceptible
S. aureus
(MSSA), methicillin-resistant
S. aureus
(MRSA), linezolid-resistant, and multidrug-resistant strains, with MIC
90
s between 0.03 and 0.12 μg/ml. MUT056399 was also active against coagulase-negative staphylococci, with MIC
90
s between 0.12 and 4 μg/ml. The antibacterial spectrum is consistent with specific FabI inhibition with no activity against bacteria using FabK but activity against FabI-containing Gram-negative bacilli.
In vitro
, resistant clones of
S. aureus
were obtained at a low frequency. All of the resistant clones analyzed were found to contain mutations in the
fabI
gene.
In vivo
, MUT056399, administered subcutaneously, protected mice from a lethal systemic infection induced by MSSA, MRSA, and vancomycin-intermediate
S. aureus
strains (50% effective doses ranging from 19.3 mg/kg/day to 49.6 mg/kg/day). In the nonneutropenic murine thigh infection model, the same treatment with MUT056399 reduced the bacterial multiplication of MSSA and MRSA in the thighs of immunocompetent mice. These properties support MUT056399 as a very promising candidate for a novel drug to treat severe staphylococcal infections.
Influence of Pore Structure on the Effectiveness of a Biogenic Carbonate Surface Treatment for Limestone Conservation
Authors: De Muynck Willem , Leuridan Stijn , Van Loo Denis , Verbeken Kim , Cnudde Veerle , De Belie Nele , Verstraete Willy
Published: August 2011

A ureolytic biodeposition treatment was applied to five types of limestone in order to investigate the effect of pore structure on the protective performance of a biogenic carbonate surface treatment. Protective performance was assessed by means of transport and degradation processes, and the penetration depth of the treatment was visualized by microtomography. Pore size governs bacterial adsorption and hence the location and amount of carbonate precipitated. This study indicated that in macroporous stone, biogenic carbonate formation occurred to a larger extent and at greater depths than in microporous stone. As a consequence, the biodeposition treatment exhibited the greatest protective performance on macroporous stone. While precipitation was limited to the outer surface of microporous stone, biogenic carbonate formation occurred at depths of greater than 2 mm for Savonnières and Euville. For Savonnières, the presence of biogenic carbonate resulted in a 20-fold decreased rate of water absorption, which resulted in increased resistance to sodium sulfate attack and to freezing and thawing. While untreated samples were completely degraded after 15 cycles of salt attack, no damage was observed in biodeposition-treated Savonnières. From this study, it is clear that biodeposition is very effective and more feasible for macroporous stones than for microporous stones.
Abundance and Distribution of Synechococcus spp. and Cyanophages in the Chesapeake Bay
Authors: Wang Kui , Wommack K. Eric , Chen Feng
Published: August 2011

Despite the increasing knowledge of
Synechococcus
spp. and their co-occurring cyanophages in oceanic and coastal water, little is known about their abundance, distribution, and interactions in the Chesapeake Bay estuarine ecosystem. A 5-year interannual survey shows that
Synechococcus
spp. and their phages are persistent and abundant members of Chesapeake Bay microbial communities.
Synechococcus
blooms (10
6
cells ml
−1
) were often observed in summer throughout the Bay, contributing 20 to 40% of total phytoplankton chlorophyll
a
. The distribution of phycoerythrin-containing (PE-rich)
Synechococcus
cells appeared to mostly correlate with the salinity gradient, with higher abundances at higher salinities. Cyanophages infectious to
Synechococcus
were also abundant (up to 6 × 10
5
viruses ml
−1
by the most probable number assay) during summer months in the Bay. The covariation in abundance of
Synechococcus
spp. and cyanophages was evident, although the latitude of observed positive correlation varied in different years, mirroring the changing environmental conditions and therefore the host-virus interactions. The impacts of cyanophages on host
Synechococcus
populations also varied spatially and temporally. Higher phage-related
Synechococcus
mortality was observed in drought years. Virus-mediated host mortality and subsequent liberation of dissolved organic matter (DOM) may substantially influence oceanic biogeochemical processing through the microbial loop as well as the microbial carbon pump. These observations emphasize the influence of environmental gradients on natural
Synechococcus
spp. and their phage population dynamics in the estuarine ecosystem.
Relevance of Bifidobacterium animalis subsp. lactis Plasminogen Binding Activity in the Human Gastrointestinal Microenvironment
Authors: Candela Marco , Turroni Silvia , Centanni Manuela , Fiori Jessica , Bergmann Simone , Hammerschmidt Sven , Brigidi Patrizia
Published: August 2011

Human plasmin(ogen) is regarded as a component of the molecular cross talk between the probiotic species
Bifidobacterium animalis
subsp.
lactis
and the human host. However, up to now, only
in vitro
studies have been reported. Here, we demonstrate that the probiotic strain
B. animalis
subsp.
lactis
BI07 is capable of recruiting plasmin(ogen) present at physiological concentrations in crude extracts from human feces. Our results provide evidence that supports the significance of the
B. lactis
-plasmin(ogen) interaction in the human gastrointestinal tract.
Association of Antibiotic Resistance in Agricultural Escherichia coli Isolates with Attachment to Quartz
Authors: Liu Ping , Soupir Michelle L. , Zwonitzer Martha , Huss Bridgette , Jarboe Laura R.
Published: August 2011

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203
Escherichia coli
isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (
P
< 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (
P
< 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms.
Increased Thermal and Osmotic Stress Resistance in Listeria monocytogenes 568 Grown in the Presence of Trehalose Due to Inactivation of the Phosphotrehalase-Encoding Gene
treA
Authors: Ells Timothy C. , Truelstrup Hansen Lisbeth
Published: August 2011

The food-borne pathogen
Listeria monocytogenes
is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including
Escherichia coli
, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in
L. monocytogenes
, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the
treA
gene. Here we report on an isogenic
treA
mutant of
L. monocytogenes
568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in
L. monocytogenes
, and we demonstrate that
lmo1254
(
tre
A
) in
L. monocytogenes
568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the
treA
gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the
treA
mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.
Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation
Authors: Sanchez-Salas Jose-Luis , Setlow Barbara , Zhang Pengfei , Li Yong-qing , Setlow Peter
Published: August 2011

The first ∼10% of spores released from sporangia (early spores) during
Bacillus subtilis
sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca
2+
but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.
Acquired Antibiotic Resistance: Are We Born with It?
Authors: Zhang Lu , Kinkelaar Daniel , Huang Ying , Li Yingli , Li Xiaojing , Wang Hua H.
Published: August 2011

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including
tet
(M),
ermB
,
sul2
, and
bla
TEM
were detected in infant subjects.
Enterococcus
spp.,
Staphylococcus
spp.,
Klebsiella
spp.,
Streptococcus
spp., and
Escherichia coli
/
Shigella
spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.
Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2
Authors: Lee Chia-Ni , Tseng Tsai-Tien , Lin Juey-Wen , Fu Yung-Chieh , Weng Shu-Fen , Tseng Yi-Hsiung
Published: August 2011

Acinetobacter baumannii
is an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistant
A. baumannii
isolates has increased in recent years. Directed toward phage therapy, a lytic phage of
A. baumannii
, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging to
Myoviridae
), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of the
A. baumannii
isolates tested, which were all multiple drug resistant, but not other bacteria. Mg
2+
enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded by
A. baumannii
strain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 of
Klebsiella
phage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein of
A. baumannii
ACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.
Cell Surface Attachment Structures Contribute to Biofilm Formation and Xylem Colonization by Erwinia amylovora
Authors: Koczan Jessica M. , Lenneman Bryan R. , McGrath Molly J. , Sundin George W.
Published: August 2011

Biofilm formation plays a critical role in the pathogenesis of
Erwinia amylovora
and the systemic invasion of plant hosts. The functional role of the exopolysaccharides amylovoran and levan in pathogenesis and biofilm formation has been evaluated. However, the role of biofilm formation, independent of exopolysaccharide production, in pathogenesis and movement within plants has not been studied previously. Evaluation of the role of attachment in
E. amylovora
biofilm formation and virulence was examined through the analysis of deletion mutants lacking genes encoding structures postulated to function in attachment to surfaces or in cellular aggregation. The genes and gene clusters studied were selected based on
in silico
analyses. Microscopic analyses and quantitative assays demonstrated that attachment structures such as fimbriae and pili are involved in the attachment of
E. amylovora
to surfaces and are necessary for the production of mature biofilms. A time course assay indicated that type I fimbriae function earlier in attachment, while type IV pilus structures appear to function later in attachment. Our results indicate that multiple attachment structures are needed for mature biofilm formation and full virulence and that biofilm formation facilitates entry and is necessary for the buildup of large populations of
E. amylovora
cells in xylem tissue.
Modification of the Technical Properties of Lactobacillus johnsonii NCC 533 by Supplementing the Growth Medium with Unsaturated Fatty Acids
Authors: Muller J. A. , Ross R. P. , Sybesma W. F. H. , Fitzgerald G. F. , Stanton C.
Published: August 2011

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain
Lactobacillus johnsonii
NCC 533, such as heat and acid tolerance, and inhibition of
Salmonella enterica
serovar Typhimurium infection. Our results showed that the membrane composition and morphology of
L. johnsonii
NCC 533 were significantly changed by supplementing a minimal
Lactobacillus
medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 μg/ml). The subsequent acid and heat tolerance of
L. johnsonii
decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 μg/ml). Following acid exposure, significantly higher (
P
< 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min),
L. johnsonii
was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore,
L. johnsonii
grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in
S. enterica
serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.
Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli
Authors: Yan Dalai , Lenz Peter , Hwa Terence
Published: August 2011

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in
Escherichia coli
. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.
Whole-Genome Sequencing and Phenotypic Analysis of Bacillus subtilis Mutants following Evolution under Conditions of Relaxed Selection for Sporulation
Authors: Brown Christopher T. , Fishwick Laura K. , Chokshi Binna M. , Cuff Marissa A. , Jackson Jay M. , Oglesby Travis , Rioux Alison T. , Rodriguez Enrique , Stupp Gregory S. , Trupp Austin H. , Woollcombe-Clarke James S. , Wright Tracy N. , Zaragoza William J. , Drew Jennifer C. , Triplett Eric W. , Nicholson Wayne L.
Published: August 2011

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of
Bacillus subtilis
evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol.
77:
4105–4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of
sigW
, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by
B. subtilis
subsp.
spizizenii
and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of
sigW
. The results suggest that propagation of
B. subtilis
for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.
Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil
Authors: Uhlik Ondrej , Strejcek Michal , Junkova Petra , Sanda Miloslav , Hroudova Miluse , Vlcek Cestmir , Mackova Martina , Macek Tomas
Published: August 2011

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with
Arthrobacter
,
Serratia
,
Rhodococcus
, and
Rhizobium
being predominant.
Arthrobacter
isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.
Identification of a Transcriptional Repressor Involved in Benzoate Metabolism in Geobacter bemidjiensis
Authors: Ueki Toshiyuki
Published: August 2011

Subsurface environments contaminated with aromatic compounds can be remediated
in situ
by
Geobacter
species. A transcription factor that represses expression of
bamA
, a benzoate-inducible gene, in
Geobacter bemidjiensis
during growth with acetate was identified. It is likely that this repressor also regulates other genes involved in aromatic compound metabolism.
Bacterial Display and Screening of Posttranslationally Thioether-Stabilized Peptides
Authors: Bosma Tjibbe , Kuipers Anneke , Bulten Erna , de Vries Louwe , Rink Rick , Moll Gert N.
Published: August 2011

A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in
Lactococcus lactis
, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.
High-Throughput Typing Method To Identify a Non-Outbreak-Involved Legionella pneumophila Strain Colonizing the Entire Water Supply System in the Town of Rennes, France
Authors: Sobral D. , Le Cann P. , Gerard A. , Jarraud S. , Lebeau B. , Loisy-Hamon F. , Vergnaud G. , Pourcel C.
Published: August 2011

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of
Legionella pneumophila
in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.
Chemoenzymatic Synthesis of
N
-Acetyl-
d
-Neuraminic Acid from
N
-Acetyl-
d
-Glucosamine by Using the Spore Surface-Displayed
N
-Acetyl-
d
-Neuraminic Acid Aldolase
Authors: Gao Chao , Xu Xiaoman , Zhang Xifeng , Che Bin , Ma Cuiqing , Qiu Jianhua , Tao Fei , Xu Ping
Published: August 2011

Chemoenzymatic synthesis of
N
-acetyl-
d
-neuraminic acid from
N
-acetyl-
d
-glucosamine using the spore surface-displayed
N
-acetyl-
d
-neuraminic acid aldolase at a high concentration (53.9 g liter
−1
) was achieved in this study. Thus, displaying a target enzyme on the surface of spores might be an alternative for integration of biocatalytic conversion into chemical synthesis.
Initiation of Polyene Macrolide Biosynthesis: Interplay between Polyketide Synthase Domains and Modules as Revealed via Domain Swapping, Mutagenesis, and Heterologous Complementation
Authors: Heia Sondre , Borgos Sven E. F. , Sletta Håvard , Escudero Leticia , Seco Elena M. , Malpartida Francisco , Ellingsen Trond E. , Zotchev Sergey B.
Published: August 2011

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT
0
in the nystatin PKS loading module NysA with the propionate-specific AT
1
from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT
0
domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT
0
and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT
0
domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a
rimA
-deficient mutant of the rimocidin/CE-108 producer
Streptomyces diastaticus
. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT
0
domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of
Streptomyces noursei
.
Helicobacter pylori Activates Calpain via Toll-Like Receptor 2 To Disrupt Adherens Junctions in Human Gastric Epithelial Cells
Authors: O'Connor Pamela M. , Lapointe Tamia K. , Jackson Shannon , Beck Paul L. , Jones Nicola L. , Buret Andre G.
Published: August 2011

Helicobacter pylori
is a risk factor for the development of gastritis, gastroduodenal ulcers, and gastric adenocarcinoma.
H. pylori
-induced disruption of epithelial adherens junctions (AJs) is thought to promote the development of severe disease; however, the mechanisms whereby
H. pylori
alters AJ structure remain incompletely understood. The present study demonstrates that
H. pylori
infection in human patients is associated with elevated serum levels of an 80-kDa E-cadherin ectodomain, whose presence is independent of the presence of serum antibodies against CagA.
In vitro
, a heat-labile
H. pylori
surface component activates the host protease calpain in human gastric MKN45 cells independently of the virulence factors CagA and VacA.
H. pylori
-induced calpain activation results in cleavage of E-cadherin to produce a 100-kDa truncated form and induce relocalization of E-cadherin and β-catenin. Stimulation of MKN45 cells with the toll-like receptor 2 (TLR2) ligand P3C activated calpain and disrupted E-cadherin and β-catenin in a pattern similar to that induced by
H. pylori
. Inhibition of TLR2 prevented
H. pylori
-induced calpain activation and AJ disassembly. Together, these findings identify a novel pathway whereby
H. pylori
activates calpain via TLR2 to disrupt gastric epithelial AJ structure.
The LuxS-Dependent Quorum-Sensing System Regulates Early Biofilm Formation by Streptococcus pneumoniae Strain D39
Authors: Vidal Jorge E. , Ludewick Herbert P. , Kunkel Rebekah M. , Zähner Dorothea , Klugman Keith P.
Published: August 2011

Streptococcus pneumoniae
is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The
luxS
-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of
S. pneumoniae
biofilm formation. Our results demonstrate that the
luxS
gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The
luxS
gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating
S. pneumoniae
biofilms, at 24 h postinoculation, two different D39Δ
luxS
mutants produced ∼80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with
luxS
, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39Δ
luxS
. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of
lytA
, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of
ply
, which encodes the pneumococcal pneumolysin. In conclusion, the
luxS
-controlled QS system is a key regulator of early biofilm formation by
S. pneumoniae
strain D39.
Identification of an ATPase, MsmK, Which Energizes Multiple Carbohydrate ABC Transporters in Streptococcus pneumoniae
Authors: Marion Carolyn , Aten Andrew E. , Woodiga Shireen A. , King Samantha J.
Published: August 2011

Streptococcus pneumoniae
is the leading cause of community-acquired pneumonia and results in over 1 million deaths each year worldwide. Asymptomatic colonization of the airway precedes disease, and acquisition of carbohydrates from the host environment is necessary for bacterial survival. We previously demonstrated that
S. pneumoniae
cleaves sialic acid from human glycoconjugates to be used as a carbohydrate source. The
satABC
genes are required for growth and import of sialic acid. The
satABC
genes are predicted to encode components of an ABC transporter but not the ATPases essential to energize transport. As this subunit is essential, an ATPase must be encoded elsewhere in the genome. We identified
msmK
as a candidate based on similarity to other known carbohydrate ATPases. Recombinant MsmK hydrolyzed ATP, revealing that MsmK is an ATPase. An
msmK
mutant was reduced in growth on and transport of sialic acid, demonstrating that MsmK is the ATPase energizing the sialic acid transporter. In addition to s
atABC
,
S. pneumoniae
contains five other loci that are predicted to encode CUT1 family carbohydrate ABC transporter components; each of these lacks a predicted ATPase. Data indicate that
msmK
is also required for growth on raffinose and maltotetraose, which are the substrates of two other characterized carbohydrate ABC transporters. Furthermore, an
msmK
mutant was reduced in airway colonization. Together, these data imply that
in vivo
, MsmK energizes multiple carbohydrate transporters in
S. pneumoniae
. This is the first demonstration of a shared ATPase in a pathogenic bacterium.
Host Cytosolic Phospholipase A
2
α Contributes to Group B Streptococcus Penetration of the Blood-Brain Barrier
Authors: Maruvada Ravi , Zhu Longkun , Pearce Donna , Sapirstein Adam , Kim Kwang Sik
Published: August 2011

Group B
Streptococcus
(GBS) is the most common bacterium causing neonatal meningitis, and neonatal GBS meningitis continues to be an important cause of mortality and morbidity. Here we provide the first direct evidence that host cytosolic phospholipase A
2
α (cPLA
2
α) contributes to type III GBS invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier and penetration into the brain, the key step required for the development of GBS meningitis. This was shown by our demonstration that pharmacological inhibition and gene deletion of cPLA
2
α significantly decreased GBS invasion of the HBMEC monolayer and penetration into the brain. cPLA
2
α releases arachidonic acid from membrane phospholipids, and we showed that the contribution of cPLA
2
α to GBS invasion of HBMEC involved lipoxygenated metabolites of arachidonic acid, cysteinyl leukotrienes (LTs). In addition, type III GBS invasion of the HBMEC monolayer involves protein kinase Cα (PKCα), as shown by time-dependent PKCα activation in response to GBS as well as decreased GBS invasion in HBMEC expressing dominant-negative PKCα. PKCα activation in response to GBS, however, was abolished by inhibition of cPLA
2
α and cysteinyl LTs, suggesting that cPLA
2
α and cysteinyl LTs contribute to type III GBS invasion of the HBMEC monolayer via PKCα. These findings demonstrate that specific host factors involving cPLA
2
α and cysteinyl LTs contribute to type III GBS penetration of the blood-brain barrier and their contribution involves PKCα.
Course of Infection with the Emergent Pathogen Brucella microti in Immunocompromised Mice
Authors: Jiménez de Bagüés María P. , de Martino Alba , Quintana Juan F. , Alcaraz Ana , Pardo Julián
Published: August 2011

A new
Brucella
species,
Brucella microti
, has been isolated from wild rodents and found to be pathogenic in mice. The biological relevance of this new mouse pathogen is clear, as it allows us to study
Brucella
infection in a species-specific model. The course of infection in wild-type (wt) and immunodeficient mice that lack B (Jh), T and B (SCID), or T, B, and NK (SCID.Beige) cells was analyzed over 3 weeks. wt mice completely cleared bacteria from the liver and spleen after that time. However, SCID mice showed a much higher bacterial load in the spleen and liver than wt and Jh mice after 1 week and maintained the same level during the next 2 weeks. All mice tested survived for the 3 weeks. In contrast, the bacterial levels in mice that lacked NK cell activity progressively increased and these mice succumbed to infection after 16 to 18 days. Histopathology analysis of infected mice showed extensive areas of necrotic tissue and thrombosis in liver after 1 week in all infected SCID.Beige mice but were not seen in either SCID or wt animals. These processes were dramatically increased after 21 days, corresponding with the death of SCID.Beige animals. Our results indicate that T and/or B cells are required for the control of infection with the mouse pathogen
Brucella microti
in liver and spleen but that NK cells are crucial for survival in the absence of B and T cells. In addition, they suggest that controlled granuloma formation is critical to clear this type of infection in wt mice.
Filifactor alocis Has Virulence Attributes That Can Enhance Its Persistence under Oxidative Stress Conditions and Mediate Invasion of Epithelial Cells by Porphyromonas gingivalis
Authors: Aruni A. Wilson , Roy Francis , Fletcher H. M.
Published: August 2011

Filifactor alocis
, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of
F. alocis
and its ability to interact with
Porphyromonas gingivalis
W83, several clinical isolates of
F. alocis
were characterized.
F. alocis
showed nongingipain protease and sialidase activities.
In silico
analysis revealed the molecular relatedness of several virulence factors from
F. alocis
and
P. gingivalis
. In contrast to
P. gingivalis
,
F. alocis
was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with
P. gingivalis
or
F. alocis
monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The
F. alocis
clinical isolate had an increased invasive capacity in coculture with
P. gingivalis
compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that
F. alocis
has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.