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1.  The conserved upstream region of lscB/C determines expression of different levansucrase genes in plant pathogen Pseudomonas syringae 
BMC Microbiology  2014;14:79.
Pseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight of soybean plants. It produces the exopolysaccharide levan by the enzyme levansucrase. Levansucrase has three gene copies in PG4180, two of which, lscB and lscC, are expressed while the third, lscA, is cryptic. Previously, nucleotide sequence alignments of lscB/C variants in various P. syringae showed that a ~450-bp phage-associated promoter element (PAPE) including the first 48 nucleotides of the ORF is absent in lscA.
Herein, we tested whether this upstream region is responsible for the expression of lscB/C and lscA. Initially, the transcriptional start site for lscB/C was determined. A fusion of the PAPE with the ORF of lscA (lscB UpN A) was generated and introduced to a levan-negative mutant of PG4180. Additionally, fusions comprising of the non-coding part of the upstream region of lscB with lscA (lscB Up A) or the upstream region of lscA with lscB (lscA Up B) were generated. Transformants harboring the lscB UpN A or the lscB Up A fusion, respectively, showed levan formation while the transformant carrying lscA Up B did not. qRT-PCR and Western blot analyses showed that lscB UpN A had an expression similar to lscB while lscB Up A had a lower expression. Accuracy of protein fusions was confirmed by MALDI-TOF peptide fingerprinting.
Our data suggested that the upstream sequence of lscB is essential for expression of levansucrase while the N-terminus of LscB mediates an enhanced expression. In contrast, the upstream region of lscA does not lead to expression of lscB. We propose that lscA might be an ancestral levansucrase variant upstream of which the PAPE got inserted by potentially phage-mediated transposition events leading to expression of levansucrase in P. syringae.
PMCID: PMC3973379  PMID: 24670199
Pseudomonas syringae; Levansucrase; Expression; Exopolysaccharides; Levan; Evolution
2.  A functional peptidoglycan hydrolase characterized from T4SS in 89K pathogenicity island of epidemic Streptococcus suis serotype 2 
BMC Microbiology  2014;14:73.
Streptococcus suis serotype 2 (S. suis 2) has evolved efficient mechanisms to cause streptococcal toxic shock syndrome (STSS), which is a new emerging infectious disease linked to S. suis. We have previously reported that a type IV secretion system (T4SS) harbored by the specific 89K pathogenicity island (PAI) of S. suis 2 contributes to the development of STSS and mediates horizontal transfer of 89K. However, the 89K T4SS machinery assembly in vivo and in vitro is poorly understood, and the component acting directly to digest the bacterial cell wall needs to be identified.
The virB1-89K gene product encoded in the 89K PAI is the only one that shows similarity to the Agrobacterium VirB1 component and contains a conserved CHAP domain that may function in peptidoglycan hydrolysis, which makes it a plausible candidate acting as a hydrolase against the peptidoglycan cell wall to allow the assembly of the T4SS apparatus. In the current study, the CHAP domain of VirB1-89K from S. suis 89K PAI was cloned and over-expressed in Escherichia coli, and its peptidoglycan-degrading activity in vitro was determined. The results indicated that the VirB1-89K CHAP domain can degrade the peptidoglycan layer of bacteria. Deletion of virB1-89K reduces significantly, but does not abolish, the virulence of S. suis in a mouse model.
The experimental results presented here suggested that VirB1-89K facilitates the assembly of 89K T4SS apparatus by catalyzing the degradation of the peptidoglycan cell wall, thus contributing to the pathogenesis of S. suis 2 infection.
PMCID: PMC3974602  PMID: 24655418
Streptococcus suis serotype 2; Streptococcal toxic shock syndrome; Type IV secretion system; Pathogenicity island; Assembly; Peptidoglycan hydrolase
3.  Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry 
BMC Microbiology  2014;14:74.
Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram.
Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates.
Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.
PMCID: PMC3977698  PMID: 24655432
Ochrobactrum anthropi; rep-PCR fingerprinting; MALDI-TOF MS; Strain typing
4.  The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E. coli is improved by combination with miconazole and polymyxin B nonapeptide 
BMC Microbiology  2014;14:65.
Nitric oxide (NO) is produced as part of the host immune response to bacterial infections, including urinary tract infections. The enzyme flavohemoglobin, coded by the hmp gene, is involved in protecting bacterial cells from the toxic effects of NO and represents a potentially interesting target for development of novel treatment concepts against resistant uropathogenic bacteria. The aim of the present study was to investigate if the in vitro antibacterial effects of NO can be enhanced by pharmacological modulation of the enzyme flavohemoglobin.
Four clinical isolates of multidrug-resistant extended-spectrum β-lactamase (ESBL)-producing uropathogenic E. coli were included in the study. It was shown that the NO-donor substance DETA/NO, but not inactivated DETA/NO, caused an initial growth inhibition with regrowth noted after 8 h of exposure. An hmp-deficient strain showed a prolonged growth inhibition in response to DETA/NO compared to the wild type. The imidazole antibiotic miconazole, that has been shown to inhibit bacterial flavohemoglobin activity, prolonged the DETA/NO-evoked growth inhibition. When miconazole was combined with polymyxin B nonapeptide (PMBN), in order to increase the bacterial wall permeability, DETA/NO caused a prolonged bacteriostatic response that lasted for up to 24 h.
An NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.
PMCID: PMC3984681  PMID: 24629000
Uropathogenic E. coli; Extended-spectrum β-lactamase; Nitric oxide; Polymyxin B nonapeptide
5.  Starvation can diversify the population structure and virulence strategies of an environmentally transmitting fish pathogen 
BMC Microbiology  2014;14:67.
Generalist bacterial pathogens, with the ability for environmental survival and growth, often face variable conditions during their outside-host period. Abiotic factors (such as nutrient deprivation) act as selection pressures for bacterial characteristics, but their effect on virulence is not entirely understood. “Sit and wait” hypothesis expects that long outside-host survival selects for increased virulence, but maintaining virulence in the absence of hosts is generally expected to be costly if active investments are needed. We analysed how long term starvation influences bacterial population structure and virulence of an environmentally transmitting fish pathogen Flavobacterium columnare.
F. columnare populations in distilled water and in lake water were monitored for 5 months. During the experiment, the population structure of F. columnare diversified by rough and soft colony morphotypes appearing among the ancestral rhizoid ones. After 5 months starvation in lake water, the virulence of the starved and ancestral bacterial isolates was tested. The starved rhizoid isolates had significantly higher virulence than the ancestral rhizoid, whereas the virulence of the rough isolates was low.
We suggest that F. columnare population diversification is an adaptation to tolerate unpredictable environment, but may also have other biological significance. Maintaining and increasing virulence ensures efficient invasion into the host especially under circumstances when the host density is low or the outside-host period is long. Changing from rhizoid into a rough morphotype has trade-offs in making bacteria less virulent and unable to exploit the host, but may ensure bacterial survival under unpredictable conditions. Our study gives an example how abiotic selection can diversify virulence of environmentally transmitting bacterial pathogen.
PMCID: PMC3984704  PMID: 24629049
Flavobacterium columnare; Starvation; Trade-off; Transmission; Virulence
6.  Inhibition of HSP70 reduces porcine reproductive and respiratory syndrome virus replication in vitro 
BMC Microbiology  2014;14:64.
Successful viral infection requires the involvement of host cellular factors in their life cycle. Heat shock protein 70 (HSP70) can be recruited by numerous viruses to promote the folding, maturation, or assembly of viral proteins. We have previously shown that HSP70 is significantly elevated in porcine reproductive and respiratory syndrome virus (PRRSV)-infected lungs, suggesting HSP70 may play a potential role during PRRSV infection. In this study, we tried to investigate the role of HSP70 during PRRSV infection.
In this study, we observed that PRRSV infection induced the expression of HSP70. The down-regulation of HSP70 using quercetin, a HSPs synthesis inhibitor, or small interfering RNAs (siRNA) reduced the viral protein level and viral production. Notably, these inhibitory effects on PRRSV infection could be attenuated by heat shock treatment. In addition, HSP70 was found to colocalize with the viral double-stranded RNA (dsRNA) and knockdown of HSP70 decreased the dsRNA levels, suggesting HSP70 is involved in the formation of viral replication and transcription complex (RTC) and thus affects the viral replication.
Our study revealed that HSP70 is an essential host factor required for the replication of PRRSV. The inhibition of HSP70 significantly reduced PRRSV replication, which may be applied as an effective antiviral strategy.
PMCID: PMC3984673  PMID: 24625230
PRRSV; HSP70; DsRNA; Replication; Antiviral
7.  Application of a target enrichment-based next-generation sequencing protocol for identification and sequence-based prediction of pneumococcal serotypes 
BMC Microbiology  2014;14:60.
The use of whole-genome sequencing in microbiology at a diagnostic level, although feasible, is still limited by the expenses associated and by the complex bioinformatics pipelines in data analyses. We describe the use of target enrichment-based next-generation sequencing for pneumococcal identification and serotyping as applied to the polysaccharide 23 valent vaccine serotypes as an affordable alternative to whole genome sequencing.
Correct identification of Streptococcus pneumoniae and prediction of common vaccine serotypes: 12 to serotype level and the rest to serogroup levels were achieved for all serotypes with >500 reads mapped against serotypes sequences. A proportion-based criterion also enabled the identification of two serotypes present in the same sample, thus indicating the possibility of using this method in detecting co-colonizing serotypes. The results obtained were comparable to or an improvement on the currently existing molecular serotyping methods for S. pneumoniae in relation to the polysaccharide vaccine serotypes.
We propose that this method has the potential to become an affordable and adaptable alternative to whole-genome sequencing for pneumococcal identification and serotyping.
PMCID: PMC3984734  PMID: 24612771
Next generation sequencing (NGS); Streptococcus pneumoniae; Serotyping
8.  Atypical Listeria innocua strains possess an intact LIPI-3 
BMC Microbiology  2014;14:58.
Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes.
In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin.
Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.
PMCID: PMC3974016  PMID: 24606727
9.  Potent bactericidal efficacy of copper oxide impregnated non-porous solid surfaces 
BMC Microbiology  2014;14:57.
The role of fomites and the environment in nosocomial infections is becoming widely recognized. In this paper we discuss the use of Cupron copper oxide impregnated non-porous solid surface in the hospital setting and present in vitro testing data via USA Environmental Protection Agency (EPA) approved testing protocols that demonstrate the efficacy of these products to assist in reduction in environmental contamination and potentially nosocomial infections.
The two countertops tested passed all the acceptance criteria by the EPA (>99.9% kill within 2 hours of exposure) killing a range of bacterial pathogens on the surface of the countertops even after repeated exposure of the countertops to the pathogen, and multiple wet and dry abrasion cycles.
Cupron enhanced EOS countertops thus may be an important adjunct to be used in hospital settings to reduce environmental bioburden and potentially nosocomial infections.
PMCID: PMC3973859  PMID: 24606672
Copper oxide; EPA registered; Antimicrobial surface; Environmental bioburden
10.  Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa 
BMC Microbiology  2014;14:53.
Mixed microbial infections of the respiratory tracts with P. aeruginosa and A. fumigatus capable of producing biofilms are commonly found in cystic fibrosis patients. The primary objective of this study was to develop an in vitro model for P. aeruginosa and A. fumigatus polymicrobial biofilm to study the efficacy of various antimicrobial drugs alone and in combinations against biofilm-embedded cells. Simultaneous static cocultures of P. aeruginosa and sporelings were used for the development of in vitro P. aeruginosa-A. fumigatus polymicrobial biofilm in SD broth in 24-well cell culture plates at 35°C, and the biofilm formation was monitored microscopically and spectrophotometrically. Using P. aeruginosa-A. fumigatus sporelings cocultures we examined the effects of various antimicrobial drugs alone and in combination against polymicrobial biofilm by CFU and tetrazolium reduction assays.
In simultaneous static cocultures P. aeruginosa cells killed A. fumigatus conidia, whereas the bacterial cells showed no substantial fungicidal effect on sporelings grown for 12 h or longer at 35°C. Monospecies cultures of P. aeruginosa produced loosely adhered monomicrobial biofilm and addition of 10% bovine serum to the growth medium inhibited the formation of monomicrobial biofilm by P. aeruginosa whereas it produced tightly adhered polymicrobial biofilm in the presence of A. fumigatus mycelial growth. A. fumigatus produced firmly adherent monomicrobial and polymicrobial biofilms. A comparison of CFU and MTT assays showed that the latter is unsuitable for studying the effectiveness of antimicrobial treatment against polymicrobial biofilm. Tobramycin alone and in combination with posaconazole was highly effective against monomicrobial and polymicrobial biofilms of P. aeruginosa whereas cefepime alone and in combination with posaconazole showed excellent activity against monomicrobial biofilm of P. aeruginosa but was less effective against polymicrobial biofilm. Monomicrobial and polymicrobial biofilms of A. fumigatus showed similar susceptibility to posaconazole with and without the antibacterial drug.
Simultaneous static coculture of A. fumigatus sporelings grown for 12 h or longer was superior to ungerminated conidia with P. aeruginosa for the development of A. fumigatus-P. aeruginosa biofilm. P. aeruginosa-A. fumigatus polymicrobial biofilm shows differential susceptibility to antimicrobial drugs whereas the susceptibility of A. fumigatus to antimicrobial drugs was unchanged.
PMCID: PMC3973989  PMID: 24588809
11.  Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting 
BMC Microbiology  2014;14:52.
Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes.
We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping.
We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.
PMCID: PMC3946169  PMID: 24575831
Meyerozyma guilliermondii; Emerging infectious yeasts; ITS-RFLP; mtDNA-RFLP; PFGE karyotyping; Soibum
12.  Diffusible signal factor (DSF) quorum sensing signal and structurally related molecules enhance the antimicrobial efficacy of antibiotics against some bacterial pathogens 
BMC Microbiology  2014;14:51.
Extensive use of antibiotics has fostered the emergence of superbugs that are resistant to multidrugs, which becomes a great healthcare and public concern. Previous studies showed that quorum sensing signal DSF (diffusible signal factor) not only modulates bacterial antibiotic resistance through intraspecies signaling, but also affects bacterial antibiotic tolerance through interspecies communication. These findings motivate us to exploit the possibility of using DSF and its structurally related molecules as adjuvants to influence antibiotic susceptibility of bacterial pathogens.
In this study, we have demonstrated that DSF signal and its structurally related molecules could be used to induce bacterial antibiotic susceptibility. Exogenous addition of DSF signal (cis-11-methyl-2-dodecenoic acid) and its structural analogues could significantly increase the antibiotic susceptibility of Bacillus cereus, possibly through reducing drug-resistant activity, biofilm formation and bacterial fitness. The synergistic effect of DSF and its structurally related molecules with antibiotics on B. cereus is dosage-dependent. Combination of DSF with gentamicin showed an obviously synergistic effect on B. cereus pathogenicity in an in vitro model. We also found that DSF could increase the antibiotic susceptibility of other bacterial species, including Bacillus thuringiensis, Staphylococcus aureus, Mycobacterium smegmatis, Neisseria subflava and Pseudomonas aeruginosa.
The results indicate a promising potential of using DSF and its structurally related molecules as novel adjuvants to conventional antibiotics for treatment of infectious diseases caused by bacterial pathogens.
PMCID: PMC3941561  PMID: 24575808
13.  High-throughput screening and whole genome sequencing identifies an antimicrobially active inhibitor of Vibrio cholerae 
BMC Microbiology  2014;14:49.
Pathogenic serotypes of Vibrio cholerae cause the life-threatening diarrheal disease cholera. The increasing development of bacterial resistances against the known antibiotics necessitates the search for new antimicrobial compounds and targets for this pathogen.
A high-throughput screening assay with a Vibrio cholerae reporter strain constitutively expressing green fluorescent protein (GFP) was developed and applied in the investigation of the growth inhibitory effect of approximately 28,300 structurally diverse natural compounds and synthetic small molecules. Several compounds with activities in the low micromolar concentration range were identified. The most active structure, designated vz0825, displayed a minimal inhibitory concentration (MIC) of 1.6 μM and a minimal bactericidal concentration (MBC) of 3.2 μM against several strains of V. cholerae and was specific for this pathogen. Mutants with reduced sensitivity against vz0825 were generated and whole genome sequencing of 15 pooled mutants was carried out. Comparison with the genome of the wild type strain identified the gene VC_A0531 (GenBank: AE003853.1) as the major site of single nucleotide polymorphisms in the resistant mutants. VC_A0531 is located on the small chromosome of V. cholerae and encodes the osmosensitive K+-channel sensor histidine kinase (KdpD). Nucleotide exchange of the major mutation site in the wild type strain confirmed the sensitive phenotype.
The reporter strain MO10 pG13 was successfully used for the identification of new antibacterial compounds against V. cholerae. Generation of resistant mutants and whole genome sequencing was carried out to identify the histidine kinase KdpD as a novel antimicrobial target.
PMCID: PMC3937525  PMID: 24568688
Vibrio cholerae; Small molecules; Histidine kinase inhibitor; KdpD; whole genome sequencing
14.  The Escherichia coli O157:H7 bovine rumen fluid proteome reflects adaptive bacterial responses 
BMC Microbiology  2014;14:48.
To obtain insights into Escherichia coli O157:H7 (O157) survival mechanisms in the bovine rumen, we defined the growth characteristics and proteome of O157 cultured in rumen fluid (RF; pH 6.0-7.2 and low volatile fatty acid content) obtained from rumen-fistulated cattle fed low protein content “maintenance diet” under diverse in vitro conditions.
Bottom-up proteomics (LC-MS/MS) of whole cell-lysates of O157 cultured under anaerobic conditions in filter-sterilized RF (fRF; devoid of normal ruminal microbiota) and nutrient-depleted and filtered RF (dRF) resulted in an anaerobic O157 fRF-and dRF-proteome comprising 35 proteins functionally associated with cell structure, motility, transport, metabolism and regulation, but interestingly, not with O157 virulence. Shotgun proteomics-based analysis using isobaric tags for relative and absolute quantitation used to further study differential protein expression in unfiltered RF (uRF; RF containing normal rumen microbial flora) complemented these results.
Our results indicate that in the rumen, the first anatomical compartment encountered by this human pathogen within the cattle gastrointestinal tract (GIT), O157 initiates a program of specific gene expression that enables it to adapt to the in vivo environment, and successfully transit to its colonization sites in the bovine GIT. Further experiments in vitro using uRF from animals fed different diets and with additional O157 strains, and in vivo using rumen-fistulated cattle will provide a comprehensive understanding of the adaptive mechanisms involved, and help direct evolution of novel modalities for blocking O157 infection of cattle.
PMCID: PMC3936929  PMID: 24559513
O157; Rumen; LC-MS/MS; iTRAQ; Proteome; Growth-patterns
15.  There is a specific response to pH by isolates of Haemophilus influenzae and this has a direct influence on biofilm formation 
BMC Microbiology  2014;14:47.
Haemophilus influenzae colonizes the nasopharynx as a commensal. Strain-specific factors allow some strains to migrate to particular anatomical niches, such as the middle ear, bronchi or blood, and induce disease by surviving within the conditions present at these sites in the body. It is established that H. influenzae colonization and in some cases survival is highly dependent on their ability to form a biofilm. Biofilm formation is a key trait in the development of chronic infection by certain isolates. This is exemplified by the contrast between the biofilm-forming strains found in middle ear infections and those isolates that survive within the blood and are rarely associated with biofilm development.
Screening a group of H. influenzae strains revealed only slight variations in their growth across a range of pH conditions. However, some isolates responded to a pH of 8.0 by the formation of a biofilm. While the type b capsular blood isolate Eagan did not form a biofilm and grew at the same rate regardless of pH 6.8-8.0, transcriptomic analyses demonstrated that at pH 8.0 it uniquely induced a gluconate-uptake and metabolism pathway, which concurrently imports H+. A non-typeable H. influenzae, isolated from the middle ear, induced biofilm formation at pH 8.0, and at this pH it induced a series of iron acquisition genes, consistent with previous studies linking iron homeostasis to biofilm lifestyle.
Different strains of H. influenzae cope with changes in environmental factors using strain-specific mechanisms. These pathways define the scope and mode of niche-survival for an isolate. The pH is a property that is different from the middle ear (at least pH 8.0) compared to other sites that H. influenzae can colonize and infect. The transcriptional response to increasing pH by H. influenzae varies between strains, and pH is linked to pathways that allow strains to either continue free-living growth or induction of a biofilm. We showed that a biofilm-forming isolate induced iron metabolism pathways, whereas a strain that does not form biofilm at increasing pH induced mechanisms for growth and pH homeostasis based on sugar acid transport.
PMCID: PMC3938079  PMID: 24555828
Biofilm; H. influenzae; pH stress; Transcriptomics
16.  Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA 
BMC Microbiology  2014;14:46.
The antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD.
The current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter.
From the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.
PMCID: PMC3945005  PMID: 24555804
Antimetabolite toxin; mgo operon; GacS/GacA; Plant-microbe interaction
17.  A novel reporter system for neutralizing and enhancing antibody assay against dengue virus 
BMC Microbiology  2014;14:44.
Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies.
In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals.
This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies.
PMCID: PMC3930823  PMID: 24548533
Dengue virus; Neutralizing antibody; Enhancing antibody; Luciferase assay
18.  Phylogenetic analysis of faecal microbiota from captive cheetahs reveals underrepresentation of Bacteroidetes and Bifidobacteriaceae 
BMC Microbiology  2014;14:43.
Imbalanced feeding regimes may initiate gastrointestinal and metabolic diseases in endangered felids kept in captivity such as cheetahs. Given the crucial role of the host’s intestinal microbiota in feed fermentation and health maintenance, a better understanding of the cheetah’s intestinal ecosystem is essential for improvement of current feeding strategies. We determined the phylogenetic diversity of the faecal microbiota of the only two cheetahs housed in an EAZA associated zoo in Flanders, Belgium, to gain first insights in the relative distribution, identity and potential role of the major community members.
Taxonomic analysis of 16S rRNA gene clone libraries (702 clones) revealed a microbiota dominated by Firmicutes (94.7%), followed by a minority of Actinobacteria (4.3%), Proteobacteria (0.4%) and Fusobacteria (0.6%). In the Firmicutes, the majority of the phylotypes within the Clostridiales were assigned to Clostridium clusters XIVa (43%), XI (38%) and I (13%). Members of the Bacteroidetes phylum and Bifidobacteriaceae, two groups that can positively contribute in maintaining intestinal homeostasis, were absent in the clone libraries and detected in only marginal to low levels in real-time PCR analyses.
This marked underrepresentation is in contrast to data previously reported in domestic cats where Bacteroidetes and Bifidobacteriaceae are common residents of the faecal microbiota. Next to methodological differences, these findings may also reflect the apparent differences in dietary habits of both felid species. Thus, our results question the role of the domestic cat as the best available model for nutritional intervention studies in endangered exotic felids.
PMCID: PMC3936777  PMID: 24548488
Bacterial community sequencing; Exotic felids; Wildlife conservation; Zoo nutrition
19.  Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous system 
BMC Microbiology  2014;14:40.
Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates.
We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells.
Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes.
PMCID: PMC3931295  PMID: 24533538
Bacterial pathogenesis; Chlamydia; Yersinia; Type III secretion; Effectors
20.  Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication 
BMC Microbiology  2014;14:42.
Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler’s diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model.
HLHK9∆ureA, HLHK9∆ureC, HLHK9∆ureD and HLHK9∆ureE all exhibited no urease activity. HLHK9∆arcA1 and HLHK9∆arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9∆arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9∆ureA/arcA1/arcA2 and HLHK9∆arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9∆ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly reduced (p < 0.01).
In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.
PMCID: PMC3936950  PMID: 24533585
Laribacter hongkongensis; Acid resistance; Arginine deiminase pathway; Microbe-host interaction
21.  High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk 
BMC Microbiology  2014;14:41.
Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years.
We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8).
The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes.
PMCID: PMC3936997  PMID: 24533573
Coxiella burnetii; Q fever; Environmental detection; Genotyping; Phylogeography; Multispacer typing;  SNP typing; Canonical SNP; CanSNP
22.  Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye 
BMC Microbiology  2014;14:38.
The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus.
The detection limit of LAMP using in vitro RNA transcripts was 3.6×10 copies·μL-1, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples.
The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.
PMCID: PMC3930758  PMID: 24524254
Astrovirus; Loop-mediated isothermal amplification; Hydroxynaphthol blue
23.  Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3 
BMC Microbiology  2014;14:37.
Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI.
Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells.
The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our understanding of the molecular basis of Hsp16.3-facilitated Mtb survival in macrophages.
PMCID: PMC3925440  PMID: 24521422
microRNAs; Macrophages; Mycobacterium; Tuberculosis; Small heat shock protein; Latent tuberculosis infection
24.  Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis 
BMC Microbiology  2014;14:36.
The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains.
The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum.
Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action.
PMCID: PMC3930553  PMID: 24521354

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