Health Education and Behavioral

Applied and Environmental Microbiology, January 2010, p. 434-440, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:46:00 +0000

Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China,¹ Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China,² Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany,³ Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521⁴

Received 7 September 2009/ Accepted 10 November 2009

A genetically engineered Escherichia coli strain coexpressingorganophosphorus hydrolase (OPH) and methyl parathion hydrolase(MPH) was constructed for the first time by cotransforming twocompatible plasmids. Since these two enzymes have differentsubstrate specificities, the coexpression strain showed a broadersubstrate range than strains expressing either one of the hydrolases.To reduce the mass transport limitation of organophosphates(OPs) across the cell membrane, MPH and OPH were simultaneouslytranslocated to the periplasm and cell surface of E. coli, respectively,by employing the twin-arginine translocation (Tat) pathway andice nucleation protein (INP) display system. The resulting recombinantstrain showed sixfold-higher whole-cell activity than the controlstrain expressing cytosolic OP hydrolases.The correct localizationof MPH and OPH was demonstrated by cell fractionation, immunoblotting,and enzyme activity assays. No growth inhibition was observedfor the recombinant E. coli strain, and suspended cultures retainedalmost 100% of the activity over a period of 2 weeks. Owingto its high level of activity and superior stability, the recombinantE. coli strain could be employed as a whole-cell biocatalystfor detoxification of OPs. This strategy of utilizing dual translocationpathways should open up new avenues for cotranslocating multiplefunctional moieties to different extracytosolic compartmentsof a bacterial cell.

Applied and Environmental Microbiology, January 2010, p. 394-399, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:40:00 +0000

Effectiveness of Liquid Soap and Hand Sanitizer against Norwalk Virus on Contaminated Hands

Center for Global Safe Water, Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, Georgia 30322,¹ Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695²

Received 21 July 2009/ Accepted 12 November 2009

Disinfection is an essential measure for interrupting humannorovirus (HuNoV) transmission, but it is difficult to evaluatethe efficacy of disinfectants due to the absence of a practicablecell culture system for these viruses. The purpose of this studywas to screen sodium hypochlorite and ethanol for efficacy againstNorwalk virus (NV) and expand the studies to evaluate the efficacyof antibacterial liquid soap and alcohol-based hand sanitizerfor the inactivation of NV on human finger pads. Samples weretested by real-time reverse transcription-quantitative PCR (RT-qPCR)both with and without a prior RNase treatment. In suspensionassay, sodium hypochlorite concentrations of 160 ppm effectivelyeliminated RT-qPCR detection signal, while ethanol, regardlessof concentration, was relatively ineffective, giving at mosta 0.5 log₁₀ reduction in genomic copies of NV cDNA. Using theAmerican Society for Testing and Materials (ASTM) standard fingerpad method and a modification thereof (with rubbing), we observedthe greatest reduction in genomic copies of NV cDNA with theantibacterial liquid soap treatment (0.67 to 1.20 log₁₀ reduction)and water rinse only (0.58 to 1.58 log₁₀ reduction). The alcohol-basedhand sanitizer was relatively ineffective, reducing the genomiccopies of NV cDNA by only 0.14 to 0.34 log₁₀ compared to baseline.Although the concentrations of genomic copies of NV cDNA wereconsistently lower on finger pad eluates pretreated with RNasecompared to those without prior RNase treatment, these differenceswere not statistically significant. Despite the promise of alcohol-basedsanitizers for the control of pathogen transmission, they maybe relatively ineffective against the HuNoV, reinforcing theneed to develop and evaluate new products against this importantgroup of viruses.

Applied and Environmental Microbiology, January 2010, p. 627-630, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:39:00 +0000

Diverse Cadmium Resistance Determinants in Listeria monocytogenes Isolates from the Turkey Processing Plant Environment

Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina 27695

Received 23 July 2009/ Accepted 15 November 2009

Two different cadA cadmium resistance determinants (cadA1, firstidentified in Tn5422, andcadA2, associated with pLM80) weredetected among cadmium-resistant Listeria monocytogenesstrainsfrom turkey processing plants. Prevalence of cadA1 versus cadA2was serotype associated. Cadmium-resistant isolates that werealso resistant to benzalkonium chloride (BC) were more likelyto harbor cadA2 alone or together with cadA1 than isolates thatwere cadmium resistant but BC susceptible.

pplied and Environmental Microbiology, January 2010, p. 584-588, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:33:00 +0000

Detection of Infective Poliovirus by a Simple, Rapid, and Sensitive Flow Cytometry Method Based on Fluorescence Resonance Energy Transfer Technology

Department of Environmental Sciences, University of California, Riverside, California 92521,¹Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521²

Received 31 July 2009/ Accepted 12 November 2009

The rapid and effective detection of virus infection is criticalfor clinical management and prevention of disease spread duringan outbreak. Several methods have been developed for this purpose,of which classical serological and viral nucleic acid detectionare the most common. We describe an alternative approach thatutilizes engineered cells expressing fluorescent proteins undergoingfluorescence resonance energy transfer (FRET) upon cleavageby the viral 2A protease (2A^pro) as an indication of infection.Quantification of the infectious-virus titers was resolved byusing flow cytometry, and utility was demonstrated for the detectionof poliovirus 1 (PV1) infection. Engineered buffalo green monkeykidney (BGMK) cells expressing the cyan fluorescent protein(CFP)-yellow fluorescent protein (YFP) substrate linked by acleavage recognition site for PV1 2A^pro were infected with differenttiters of PV1. After incubation at various time points, cellswere harvested, washed, and subjected to flow cytometry analysis.The number of infected cells was determined by counting thenumber of cells with an increased CFP-to-YFP ratio. As earlyas 5 h postinfection, a significant number of infected cells(3%) was detected by flow cytometry, and cells infected withonly 1 PFU were detected after 12 h postinfection. When appliedto an environmental water sample spiked with PV1, the flow cytometry-basedassay provided a level of sensitivity similar to that of theplaque assay for detecting and quantifying infectious virusparticles. This approach, therefore, is more rapid than plaqueassays and can be used to detect other viruses that frequentlydo not form clear plaques on cell cultures.

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:29:00 +0000

Gene Clusters Involved in Isethionate Degradation by Terrestrial and Marine Bacteria

Department of Biology, The University, D-78457 Konstanz, Germany,¹ Department of Microbiology, Oregon State University, Corvallis, Oregon 97331,² Agroscope Changins-Wädenswil ACW, Swiss Federal Research Station, Schloss, Postfach, CH-8820 Wädenswil, Switzerland³

Received 29 July 2009/ Accepted 10 November 2009

Ubiquitous isethionate (2-hydroxyethanesulfonate) is dissimilatedby diverse bacteria. Growth ofCupriavidus necator H16 withisethionate was observed, as was inducible membrane-bound isethionatedehydrogenase (IseJ) and inducible transcription of the genespredicted to encode IseJ and a transporter (IseU). Biodiversityin isethionate transport genes was observed and investigatedby transcription experiments.

Applied and Environmental Microbiology, January 2010, p. 609-617, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:28:00 +0000

Deep Sequencing of a Dimethylsulfoniopropionate-Degrading Gene (dmdA) by Using PCR Primer Pairs Designed on the Basis of Marine Metagenomic Data

Departments of Microbiology,¹ Marine Sciences, University of Georgia, Athens, Georgia 30602²

Received 1 June 2009/ Accepted 16 November 2009

In silico design and testing of environmental primer pairs withmetagenomic data are beneficial for capturing a greater proportionof the natural sequence heterogeneity in microbial functionalgenes, as well as for understanding limitations of existingprimer sets that were designed from more restricted sequencedata. PCR primer pairs targeting 10 environmental clades andsubclades of the dimethylsulfoniopropionate (DMSP) demethylaseprotein, DmdA, were designed using an iterative bioinformaticapproach that took advantage of thousands of dmdA sequencescaptured in marine metagenomic data sets. Using the bioinformaticallyoptimized primers, dmdA genes were amplified from compositefree-living coastal bacterioplankton DNA (from 38 samples over5 years and two locations) and sequenced using 454 technology.An average of 6,400 amplicons per primer pair represented morethan 700 clusters of environmental dmdA sequences across allprimers, with clusters defined conservatively at >90% nucleotidesequence identity (95% amino acid identity). Degenerate andinosine-based primers did not perform better than specific primerpairs in determining dmdA richness and sometimes captured alower degree of richness of sequences from the same DNA sample.A comparison of dmdA sequences in free-living versus particle-associatedbacteria in southeastern U.S. coastal waters showed that sequencerichness in some dmdA subgroups differed significantly betweensize fractions, though most gene clusters were shared (52 to91%) and most sequences were affiliated with the shared clusters(90%). The availability of metagenomic sequence data has significantlyenhanced the design of quantitative PCR primer pairs for thiskey functional gene, providing robust access to the capabilitiesand activities of DMSP demethylating bacteria in situ.

Applied and Environmental Microbiology, January 2010, p. 400-405, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:25:00 +0000

Biosynthesis and Degradation of H₂O₂ by Vaginal Lactobacilli

Area de Microbiología, Instituto Universitario de Biotecnología, Universidad de Oviedo, Oviedo,¹Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, Spain²

Received 10 July 2009/ Accepted 15 November 2009

Hydrogen peroxide production by vaginal lactobacilli representsone of the most important defense mechanisms against vaginalcolonization by undesirable microorganisms. To quantify theability of a collection of 45 vaginal Lactobacillus strainsto generate H₂O₂, we first compared three published colorimetricmethods. It was found that the use of DA-64 as a substrate renderedthe highest sensitivity, while tetramethyl-benzidine (TMB) maintainedits linearity from nanomolar to millimolar H₂O₂ concentrations.Generation of H₂O₂ was found to be especially common and strongfor L. jensenii strains, while it was variable among L. crispatusand L. gasseristrains. Biosynthesis of H₂O₂ only occurred uponagitation of the cultures, but the H₂O₂-producing machinerywas already present in them before aeration started. Calcium,magnesium, manganese, and zinc ions did not affect H₂O₂ production,while Cu²⁺ inhibited the growth ofLactobacillus jensenii CECT4306, which was chosen as a model strain. Cultures with Fe³⁺,hemin, and hemoglobin did not accumulate H₂O₂. Fe³⁺ activatedan extracellular peroxidase that destroyed the H₂O₂ being producedby the cultures. This protected the lactobacilli against itsantimicrobial effect. The production of the enzyme appears tobe constitutive, the Fe³⁺ ions being a necessary cofactor ofthe reaction.

Applied and Environmental Microbiology, January 2010, p. 387-393, Vol. 76, No. 2

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:21:00 +0000

Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies

Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin—Madison, Madison, Wisconsin 53706,¹ School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom²

Received 20 August 2009/ Accepted 9 November 2009

Clostridium botulinum produces the most poisonous natural toxinknown and is a perennial concern to the food industry and toregulatory agencies due to the potential threat of food-bornebotulism. To ensure the botulinal safety of foods, rigorousfood challenge testing to validate food-processing conditionsand food formulations has been routinely performed. Detectionof the botulinum neurotoxin is performed by using a mouse bioassayand/or in vitro assays. There has been considerable interestby the food industry and regulatory agencies in minimizing oreven replacing the use of animals in these challenge studies.In addition, due to stringent select-agent regulations, thetesting of various foods using toxigenic C. botulinum strainsrequires facilities and personnel that are certified for workwith this organism. For this purpose we propose to generatesets of nontoxigenic C. botulinum strains from proteolytic andnonproteolytic groups that differ from the wild-type strainsonly by their inability to produce botulinum neurotoxin. Inthis initial study we describe the generation of a nontoxigenicmutant of C. botulinum strain 62A using the ClosTron mutagenesissystem by inserting a group II intron into the botulinum neurotoxintype A gene (bont/A). The mutant clones were nontoxigenic asdetermined by Western blots and mouse bioassays but showed physiologicalcharacteristics, including growth properties and sporulation,that were similar to those of the parent strain in laboratorymedia. Additional studies will be required to evaluate comparablecharacteristics in various food matrices. The availability ofsuitable nontoxigenic C. botulinum strains for food challengestudies will be beneficial for enhancing the botulinal safetyof foods as well as increasing the biosafety of workers andmay eliminate the use of laboratory animals.

Journal of Virology, February 2010, p. 1221-1236, Vol. 84, No. 3

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 16:10:00 +0000

Immune Evasion Proteins of Murine Cytomegalovirus Preferentially Affect Cell Surface Display of Recently Generated Peptide Presentation Complexes

Institute for Virology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany

Received 2 October 2009/ Accepted 6 November 2009

For recognition of infected cells by CD8 T cells, antigenicpeptides are presented at the cell surface, bound to major histocompatibilitycomplex class I (MHC-I) molecules. Downmodulation of cell surfaceMHC-I molecules is regarded as a hallmark function of cytomegalovirus-encodedimmunoevasins. The molecular mechanisms by which immunoevasinsinterfere with the MHC-I pathway suggest, however, that thisdownmodulation may be secondary to an interruption of turnoverreplenishment and that hindrance of the vesicular transportof recently generated peptide-MHC (pMHC) complexes to the cellsurface is the actual function of immunoevasins. Here we haveused the model of murine cytomegalovirus (mCMV) infection toprovide experimental evidence for this hypothesis. To quantitatepMHC complexes at the cell surface after infection in the presenceand absence of immunoevasins, we generated the recombinant virusesmCMV-SIINFEKL and mCMV-m06m152-SIINFEKL, respectively, expressingthe K^b-presented peptide SIINFEKL with early-phase kineticsin place of an immunodominant peptide of the viral carrier proteingp36.5/m164. The data revealed 10,000 K^b molecules presentingSIINFEKL in the absence of immunoevasins, which is an occupancyof10% of all cell surface K^b molecules, whereas immunoevasinsreduced this number to almost the detection limit. To selectivelyevaluate their effect on preexisting pMHC complexes, cells wereexogenously loaded with SIINFEKL peptide shortly after infectionwith mCMV-SIINFEKA, in which endogenous presentation is preventedby an L174A mutation of the C-terminal MHC-I anchor residue.The data suggest that pMHC complexes present at the cell surfacein advance of immunoevasin gene expression are downmodulateddue to constitutive turnover in the absence of resupply.

Clinical Microbiology Reviews, January 2010, p. 14-34, Vol. 23, No. 1

IkrarChandraBachtiar@gmail.com (IKRAR CHANDRA BACHTIAR, S.KM) — Tue, 19 Jan 2010 15:53:00 +0000

Rifampin Combination Therapy for Nonmycobacterial Infections

Summary: The increasing emergence of antimicrobial-resistantorganisms, especially methicillin-resistant Staphylococcus aureus(MRSA), has resulted in the increased use of rifampin combinationtherapy. The data supporting rifampin combination therapy innonmycobacterial infections are limited by a lack of significantlycontrolled clinical studies. Therefore, its current use is basedupon in vitro or in vivo data or retrospective case series,all with major limitations. A prominent observation from thisreview is that rifampin combination therapy appears to haveimproved treatment outcomes in cases in which there is a loworganism burden, such as biofilm infections, but is less effectivewhen effective surgery to obtain source control is not performed.The clinical data support rifampin combination therapy for thetreatment of prosthetic joint infections due to methicillin-sensitiveS. aureus (MSSA) after extensive debridement and for the treatmentof prosthetic heart valve infections due to coagulase-negativestaphylococci. Importantly, rifampin-vancomycin combinationtherapy has not shown any benefit over vancomycin monotherapyagainst MRSA infections either clinically or experimentally.Rifampin combination therapy with daptomycin, fusidic acid,and linezolid needs further exploration for these severe MRSAinfections. Lastly, an assessment of the risk-benefits is neededbefore the addition of rifampin to other antimicrobials is consideredto avoid drug interactions or other drug toxicities.