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1.	Tomenius, Henrik, et al. (author) The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract 2006 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 6, s. 27- Journal article (peer-reviewed)abstract Background: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. Results: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1: 1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. Conclusion: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model.
2.	Rodin, S., et al. (author) Performance of a 70-mer oligonucleotide microarray for genotyping of Campylobacter jejuni 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8 Journal article (peer-reviewed)abstract BACKGROUND: Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection. RESULTS: With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of C. jejuni. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance. CONCLUSION: In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of Campylobacter jejuni isolates, with resolution outperforming MLST.
3.	Allhorn, Maria, et al. (author) EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8:1 Journal article (peer-reviewed)abstract ABSTRACT: BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSIONS: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.
4.	Butler, Éile, et al. (author) Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress 2013 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 13 Journal article (peer-reviewed)abstract Background: Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity. Results: The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further. Conclusions: The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee's food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of these secreted proteins may have a putative antimicrobial function. In the future, understanding these processes in this complicated environment may lead to novel applications of honey crop LAB proteins.
5.	Drasbek, M, et al. (author) Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA 2004 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 4 Journal article (peer-reviewed)abstract Background: Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples. Results: A recombinant protein derived from the P116 protein and one from the P1 protein were used in two ELISA tests, rP116-ELISA and rP1-ELISA. Human serum samples from patients with atypical pneumonia were tested and compared to the results of the complement fixation test. There was a good agreement between the two tests but the rP1-ELISA showed the best discrimination between positive and negative samples. Conclusion: Two ELISA tests based on recombinant proteins have been analysed and compared to the complement fixation test results. The two ELISA tests were found suitable for use in serodiagnostics of M. pneumoniae infections. The use of specific antigens eliminates the risk of cross reaction to an immune response against other bacteria.
6.	Lood, Rolf, et al. (author) Inducible Siphoviruses in superficial and deep tissue isolates of Propionibacterium acnes 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8 Journal article (peer-reviewed)abstract Background: Propionibacterium acnes is a commensal of human skin but is also known to be involved in certain diseases, such as acne vulgaris and infections of orthopaedic implants. Treatment of these conditions is complicated by increased resistance to antibiotics and/or biofilm formation of P. acnes bacteria. P. acnes can be infected by bacteriophages, but until recently little has been known about these viruses. The aim of this study was to identify and characterize inducible phages from P. acnes on a genetic and morphological basis. Results: More than 70% (65/92) of P. acnes isolates investigated have inducible phages, classified morphologically as Siphoviruses. The phages have a head of 55 nm in diameter and a tail of 145 155 nm in length and 9-10 nm in width. There was no difference in carriage rate of phages between P. acnes isolates from deep infections and isolates from skin. However, there was a significant lower carriage rate of phages in P. acnes biotype IB, mostly attributed to the low carriage rate of inducible phages in biotype IB isolated from deep tissue. Most phages have a strong lytic activity against all P. acnes isolates with inducible phages, but have less lytic activity against isolates that have no prophages. Phages only infected and lysed P. acnes and not other closely related propionibacteria. All phages could infect and lyse their non-induced parental host, indicating that these prophages do not confer superinfection immunity. The phages have identical protein pattern as observed on SDS-PAGE. Finally, sequencing of two phage genes encoding a putative major head protein and an amidase and showed that the phages could be divided into different groups on a genetic basis. Conclusion: Our findings indicate that temperate phages are common in P. acnes, and that they are a genetically and functionally homogeneous group of Siphoviruses. The phages are specific for P. acnes and do not seem to confer superinfection immunity.
7.	Nilsson, Anna, et al. (author) Polymerase chain reaction is superior to serology for the diagnosis of acute Mycoplasma pneumoniae infection and reveals a high rate of persistent infection 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8 Journal article (peer-reviewed)abstract Background: Diagnosis of Mycoplasma pneumoniae (MP) infection is traditionally based on serology, which may require more than two weeks for diagnostic antibodies to develop. PCR-based methods offer earlier diagnosis. During a community outbreak of MP infection, we compared seminested and real-time PCR of oropharyngeal swabs with serology for diagnosis of MP infection at different time points after disease onset. PCR-positive individuals were followed longitudinally to assess the persistence of MP DNA in throat secretions. We also studied carriage of MP among household contacts and school children. Results: MP infection was diagnosed in 48 of 164 patients with respiratory tract infection. Fortyfive (29%) had detectable MP DNA in oropharynx. A significant increase in MP IgG IgG titre or MP IgM antibodies was detected in 44/154 (27%) subjects. Two MP PCR-positive patients lacked antibody responses. Sera were missing from another two patients. The agreement between serology and PCR was good,. = 0.90. During the first three weeks after disease onset the performance of PCR was excellent and all patients but one were detected. In contrast, only 21% of the patients with confirmed MP infection were positive by serum 1 during the first symptomatic week (56% during the second and 100% during the third week). Only 1/237 (0.4%) school children was positive by PCR. This child had respiratory symptoms. Eighteen of 22 (75%) symptomatic household contacts were MP PCR positive. Persistence of MP DNA in the throat was common. Median time for carriage of MP DNA was 7 weeks after disease onset (range 2 days-7 months). Adequate antibiotic treatment did not shorten the period of persistence. Bacterial load, measured by quantitative real-time PCR declined gradually, and all followed patients eventually became PCR-negative. Conclusion: PCR is superior to serology for diagnosis of MP infection during the early phases of infection. Persistent, sometimes long-term, carriage of MP DNA in the throat is common following acute infection, and is not affected by antibiotic therapy. Asymptomatic carriage of MP even during an outbreak is uncommon.
8.	Salerno, Paola, et al. (author) Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation 2013 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 13 Journal article (peer-reviewed)abstract Background: The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. Results: We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. Conclusion: Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.
9.	Sjögren, Jonathan, et al. (author) Study of the IgG endoglycosidase EndoS in group A streptococcal phagocyte resistance and virulence 2011 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 11 Journal article (peer-reviewed)abstract Background: The secreted enzyme EndoS, an endoglycosidase from Streptococcus pyogenes, hydrolyzes the N-linked glycan of the constant region of immunoglobulin G (IgG) heavy chain and renders the antibody unable to interact with Fc receptors and elicit effector functions. In this study we couple targeted allelic replacement mutagenesis and heterologous expression to elucidate the contribution of EndoS to group A Streptococcus (GAS) phagocyte resistance and pathogenicity in vitro and in vivo. Results: Knocking out the EndoS gene in GAS M1T1 background revealed no significant differences in bacterial survival in immune cell killing assays or in a systemic mouse model of infection. However, exogenous addition and heterologous expression of EndoS was found to increase GAS resistance to killing by neutrophils and monocytes in vitro. Additionally, heterologous expression of EndoS in M49 GAS increased mouse virulence in vivo. Conclusions: We conclude that in a highly virulent M1T1 background, EndoS has no significant impact on GAS phagocyte resistance and pathogenicity. However, local accumulation or high levels of expression of EndoS in certain GAS strains may contribute to virulence.
10.	Wallin-Carlquist, Nina, et al. (author) Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus 2010 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10 Journal article (peer-reviewed)abstract Background: The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results: The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions: Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.
11.	Sullivan, Patrick F, et al. (author) An unbiased metagenomic search for infectious agents using monozygotic twins discordantfor chronic fatigue 2011 In: BMC Microbiology. - : BMC. - 1471-2180. ; 11:2 Journal article (peer-reviewed)abstract Background: Chronic fatigue syndrome is an idiopathic syndrome widely suspected of having an infectious orimmune etiology. We applied an unbiased metagenomic approach to try to identify known or novel infectiousagents in the serum of 45 cases with chronic fatigue syndrome or idiopathic chronic fatigue. Controls were theunaffected monozygotic co-twins of cases, and serum samples were obtained at the same place and time.Results: No novel DNA or RNA viral signatures were confidently identified. Four affected twins and no unaffectedtwins evidenced viremia with GB virus C (8.9% vs. 0%, p = 0.019), and one affected twin had previously undetectedhepatitis C viremia. An excess of GB virus C viremia in cases with chronic fatigue requires confirmation.Conclusions: Current, impairing chronic fatigue was not robustly associated with viremia detectable in serum.
12.	Ryberg, Anna, et al. (author) Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8, s. 175- Journal article (peer-reviewed)abstract Background: Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent Helicobacter pylori and human host cellular DNA genotyping analysis using PCR-based methods. Results: Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using H. pylori-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (IL1B) and Interferon-gamma receptor (IFNGR1) SNP analysis, and Interleukin-1 receptor antagonist (IL1RN) variable tandem repeats (VNTR) in intron 2. Finally, regions of the vacA-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. H. pylori specific multiplex PCR assays revealed the presence of H. pylori cagA and vacA genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of H. pylori were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, IL1B and IFNGR1-SNP and IL1RN-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged vacA-PCR amplicons revealed the presence of highly diverse H. pylori vacA-s/i/m regions. Conclusion: The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.
13.	Abdeldaim, Guma, 1969-, et al. (author) Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis 2010 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10, s. 310- Journal article (peer-reviewed)abstract Background. Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. Results. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. Conclusions. The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.
14.	Agervald, Åsa, et al. (author) Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8, s. 69- Journal article (peer-reviewed)abstract Background: The maturation of hydrogenases into active enzymes is a complex process and e. g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hypgenes in Nostoc sp. strain PCC 7120. Results: RTPCRs demonstrated that the six hypgenes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hypoperon, and two downstream ORFs from the hypgenes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifUlike protein. Orthologues of the four ORFs asr0689alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2fixing cyanobacteria, but are absent in non N2fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hypoperon, appearing between 11 and 79 times in the genome. Conclusion: This study demonstrated that five ORFs upstream of the hypgene cluster are cotranscribed with the hypgenes, and identified three TSPs in the extended hypgene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.
15.	Ahlinder, Jon, et al. (author) Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays 2012 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12, s. 220- Journal article (peer-reviewed)abstract Background: Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results: In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions: Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.
16.	Ankarklev, Johan, 1980-, et al. (author) Allelic sequence heterozygosity in single Giardia parasites 2012 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12 Journal article (peer-reviewed)abstract Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.
17.	Bang, Charlotte Sahlberg, 1967-, et al. (author) The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E-coli is improved by combination with miconazole and polymyxin B nonapeptide 2014 In: BMC Microbiology. - London : BioMed Central. - 1471-2180. ; 14 Journal article (peer-reviewed)abstract Background: 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.Results: Four clinical isolates of multidrug-resistant extended-spectrum beta-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.Conclusion: An NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.
18.	Bergh Drott, Johanna, et al. (author) Propionibacterium acnes infection induces upregulation of inflammatory genes and cytokine secretion in prostate epithelial cells 2010 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 10, s. 126- Journal article (peer-reviewed)abstract Background: The immune stimulating bacterium Propionibacterium acnes is a frequent colonizer of benign and malignant prostate tissue. To understand the pathogenesis of the earliest phase of this infection, we examined the P. acnes triggered immune response in cultivated prostate epithelial cells.Results: Prostate epithelial cells are triggered to secrete IL-6, IL-8 and GM-CSF when infected with P. acnes. The secretion of cytokines is accompanied by NFκB related upregulation of the secreted cytokines as well as several components of the TLR2-NFκB signaling pathway.Conclusions: P. acnes has potential to trigger a strong immune reaction in the prostate glandular epithelium. Upon infection of prostate via the retrograde urethral route, the induced inflammatory reaction might facilitate bacterial colonization deeper in the prostate tissue where persistent inflammation may impact the development of prostate diseases as hyperplasia and/or malignancy.
19.	Björndal, Asa Szekely, et al. (author) Ebola virus infection inversely correlates with the overall expression levels of promyelocytic leukaemia (PML) protein in cultured cells 2003 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 3, s. 6- Journal article (peer-reviewed)abstract We have established a simple fixation and immunofluorescence staining procedure that allows specific co-detection and precise sub-cellular localization of the PML nuclear bodies and the Ebola virus encoded proteins NP, VP35 and VP40 in formaldehyde treated cells. Interferon-alpha treatment delays virus production in vitro. Intact PML bodies may play an anti-viral role in Ebola infected cells.
20.	Bröms, Jeanette E., et al. (author) A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552 2013 In: BMC Microbiology. - London, England : BioMed Central. - 1471-2180. ; 13, s. 96- Journal article (peer-reviewed)abstract Background: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.
21.	Chooneea, Darren, et al. (author) Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique 2010 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10 Journal article (peer-reviewed)abstract Abstract: Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI(TM) FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches. Results: In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B(12), LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. Conclusion: Using a multi-step digest approach the LPI(TM) technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI(TM) technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.
22.	Demirel, Isak, 1987-, et al. (author) Comparison of host response mechanisms evoked by extended spectrum beta lactamase (ESBL)- and non-ESBL-producing uropathogenic E. coli 2013 In: BMC Microbiology. - London, United Kingdom : BioMed Central. - 1471-2180. ; 13 Journal article (peer-reviewed)abstract Background: Infections caused by extended spectrum beta-lactamases (ESBL)-producing bacteria have been emerging worldwide and the majority of ESBL-producing E. coli strains are isolated from patients with urinary tracts infections. The purpose of this study was to compare the host-response mechanisms in human polymorphonucleated leukocytes (PMN) and renal epithelial cells when stimulated by ESBL-or non-ESBL-producing uropathogenic E. coli (UPEC) isolates. The host-pathogen interaction of these ESBL-producing strains in the urinary tract is not well studied.Results: The ability of ESBL strains to evoke ROS-production from PMN cells was significantly higher than that of the non-ESBL strains. The growth of ESBL strains was slightly suppressed in the presence of PMN compared to non-ESBL strains after 30 min and 2 h, but the opposite was observed after 5 and 6 h. The number of migrating PMN was significantly higher in response to ESBL strains compared to non-ESBL strains. Stimulation of A498 cells with ESBL strains elicited lower production of IL-6 and IL-8 compared to non-ESBL strains.Conclusion: Significant differences in host-response mechanisms were identified when host cells were stimulated by ESBL-or non-ESBL producing strains. The obtained results on the early interactions of ESBL-producing strains with the host immune system may provide valuable information for management of these infections.
23.	Devine, Ellenor, et al. (author) Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 2009 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 9, s. 53- Journal article (peer-reviewed)abstract Background: The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results: In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for[NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion: Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the[NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.
24.	Drobni, Mirva, et al. (author) Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities. 2006 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 43:6 Journal article (peer-reviewed)abstract BACKGROUND: Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galbeta binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. RESULTS: Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4) and genospecies 2 (n = 4), both of which harboured variant Galbeta-dependent hemagglutination (HA) types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8-66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (> 97.2%) within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. CONCLUSION: The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent oligomerization of FimA subunit proteins in solution was indicated.
25.	Dryselius, Rikard, et al. (author) Variable coordination of cotranscribed genes in Escherichia coli following antisense repression 2006 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 6, s. 97- Journal article (peer-reviewed)abstract Background: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.
26.	Dubey, Mukesh, et al. (author) Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea 2014 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 14 Journal article (peer-reviewed)abstract Background: Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.Results: Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the Delta Hyd3 strain, but not for the Delta Hyd1 strain. Furthermore, enhanced root colonization ability for the Delta Hyd1 Delta Hyd3 strain was found in comparison to WT.Conclusions: These results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.
27.	Feliziani, Constanza, et al. (author) Immunodominant proteins α-1 giardin and β-giardin are expressed in both assemblages A and B of Giardia lamblia 2011 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 11, s. 233- Journal article (peer-reviewed)abstract BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
28.	Forslund, Anna-Lena, et al. (author) The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis 2010 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10, s. 227- Journal article (peer-reviewed)abstract This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.
29.	Fredriksson, Johan, 1979, et al. (author) Diversity and dynamics of Archaea in an activated sludge wastewater treatment plant 2012 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12 Journal article (peer-reviewed)abstract BACKGROUND: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. RESULTS: The Archaea community was specialized and dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6-% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. CONCLUSIONS: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.
30.	Garcia-Aljaro, Cristina, et al. (author) Quorum-sensing regulates biofilm formation in Vibrio scophthalmi 2012 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 12, s. 287- Journal article (peer-reviewed)abstract Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits.Results: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study.Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.
31.	Holmqvist, Marie, et al. (author) Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 29133 2009 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 9, s. 54- Journal article (peer-reviewed)abstract Background: In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H-2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H-2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H-2. Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the hupSL promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the hupSL promoter, truncated versions of the hupSL promoter were fused to either gfp or luxAB, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively. Results: Interestingly, all hupsSL promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity. Conclusion: Obtained data suggest that the hupSL promoter in N. punctiforme is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (- 57 to tsp) is enough for a high and heterocyst specific expression of hupSL. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.
32.	Honn, Marie, 1982-, et al. (author) The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS 2012 In: BMC Microbiology. - London : BioMed Central. - 1471-2180. ; 12, s. 14- Journal article (peer-reviewed)abstract Background: The Francisella tularensis protein MglA performs complex regulatory functions since it influences the expression of more than 100 genes and proteins in F. tularensis. Besides regulating the igl operon, it has been suggested that it also regulates several factors such as SspA, Hfq, CspC, and UspA, all important to stress adaptation. Therefore, it can be hypothesized that MglA plays an important role for Francisella stress responses in general and for the oxidative stress response specifically.Results: We investigated the oxidative stress response of the Delta mglA mutant of the live vaccine strain (LVS) of F. tularensis and found that it showed markedly diminished growth and contained more oxidized proteins than the parental LVS strain when grown in an aerobic milieu but not when grown microaerobically. Moreover, the Delta mglA mutant exhibited an increased catalase activity and reduced expression of the fsl operon and feoB in the aerobic milieu. The mutant was also found to be less susceptible to H2O2. The aberrant catalase activity and gene expression was partially normalized when the Delta mglA mutant was grown in a microaerobic milieu.Conclusions: Altogether the results show that the Delta mglA mutant exhibits all the hallmarks of a bacterium subjected to oxidative stress under aerobic conditions, indicating that MglA is required for normal adaptation of F. tularensis to oxidative stress and oxygen-rich environments.
33.	Jayaprakash, Kartheyaene, 1983-, et al. (author) Gingipains from Porphyromonas gingivalis play a significant role in induction and regulation of CXCL8 in THP-1 cells 2014 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 14 Journal article (peer-reviewed)abstract Background: Porphyromonas gingivalis is an important bacterial etiological agent involved in periodontitis. The bacterium expresses two kinds of cysteine proteases called gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). This study evaluated the interaction between P. gingivalis and THP-1 cells, a widely used monocytic cell line, in vitro with a focus on CXCL8 at the gene and protein levels and its fate thereafter in cell culture supernatants. THP-1 cells were stimulated with viable and heat-killed wild-type strains ATCC 33277 or W50 or viable isogenic gingipain mutants of W50, E8 (Rgp mutant) or K1A (Kgp mutant), for 24 hours.Results: ELISA and qPCR results show an elevated CXCL8 expression and secretion in THP-1 cells in response to P. gingivalis, where the heat-killed ATCC33277 and W50 induced higher levels of CXCL8 in comparison to their viable counterparts. Furthermore, the Kgp-deficient mutant K1A caused a higher CXCL8 response compared to the Rgp-deficient E8. Chromogenic quantification of lipopolysaccharide (LPS) in supernatant showed no significant differences between viable and heat killed bacteria except that W50 shed highest levels of LPS. The wild-type strains secreted relatively more Rgp during the co-culture with THP-1 cells. The CXCL8 degradation assay of filter-sterilized supernatant from heat-killed W50 treated cells showed that Rgp was most efficient at CXCL8 hydrolysis. Of all tested P. gingivalis strains, adhesion and internalization in THP-1 cells was least conspicuous by Rgp-deficient P. gingivalis (E8), as demonstrated by confocal imaging.Conclusions: W50 and its Kgp mutant K1A exhibit a higher immunogenic and proteolytic function in comparison to the Rgp mutant E8. Since K1A differs from E8 in the expression of Rgp, it is rational to conclude that Rgp contributes to immunomodulation in a more dynamic manner in comparison to Kgp. Also, W50 is a more virulent strain when compared to the laboratory strain ATCC33277.
34.	Johansson, Åsa, et al. (author) The detection and verification of carbapenemases using ertapenem and Matrix Assisted Laser Desorption Ionization-Time of Flight 2014 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 14 Journal article (peer-reviewed)abstract Background: The increase in carbapenemase producing Enterobacteriaceae and Pseudomonas aeruginosa is a significant threat to modern medicine. A rapid detection of carbapenemase production in Klebsiella pneumoniae and Pseudomonas aeruginosa is of importance for the institution of correct antibiotic treatment and infection control measures.Results: Standardised inoculums of K. pneumoniae or P. aeruginosa were incubated at 37 degrees C with ertapenem in 15 and 120 min followed by centrifugation. The supernatant was applied on a steel target plate, covered with HCCA matrix and analysed using a Microflex(TM) (Bruker Daltonics) in the mass range of 4-600 Da. The assay detected and separated KPC from other carbapenemases in K. pneumoniae after only 15 min incubation. In P. aeruginosa, however, only 8/14 isolates of VIM-producing P. aeruginosa were detected. None of the tested carbapenemase negative isolates displayed a pattern of hydrolysis of ertapenem.Conclusions: This assay allows for a very rapid detection and verification of KPC (45 min including the preparation steps) and MBL production (150 min) in K. pneumoniae and can be performed using standard matrix. However, the study revealed the need for optimization of the substrate/species combination in assays for the detection of carbapenemases in P. aeruginosa using MALDI-TOF.
35.	Jonas, Kristina, et al. (author) Roles of curli, cellulose and BapA in Salmonella biofilm morphology studied by atomic force microscopy 2007 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 7 Journal article (peer-reviewed)abstract Background: Curli, cellulose and the cell surface protein BapA are matrix components in Salmonella biofilms. In this study we have investigated the roles of these components for the morphology of bacteria grown as colonies on agar plates and within a biofilm on submerged mica surfaces by applying atomic force microscopy (AFM) and light microscopy. Results: AFM imaging was performed on colonies of Salmonella Typhimurium grown on agar plates for 24 h and on biofilms grown for 4, 8, 16 or 24 h on mica slides submerged in standing cultures. Our data show that in the wild type curli were visible as extracellular material on and between the cells and as fimbrial structures at the edges of biofilms grown for 16 h and 24 h. In contrast to the wild type, which formed a three-dimensional biofilm within 24 h, a curli mutant and a strain mutated in the global regulator CsgD were severely impaired in biofilm formation. A mutant in cellulose production retained some capability to form cell aggregates, but not a confluent biofilm. Extracellular matrix was observed in this mutant to almost the same extent as in the wild type. Overexpression of CsgD led to a much thicker and a more rapidly growing biofilm. Disruption of BapA altered neither colony and biofilm morphology nor the ability to form a biofilm within 24 h on the submerged surfaces. Besides curli, the expression of flagella and pili as well as changes in cell shape and cell size could be monitored in the growing biofilms. Conclusion: Our work demonstrates that atomic force microscopy can efficiently be used as a tool to monitor the morphology of bacteria grown as colonies on agar plates or within biofilms formed in a liquid at high resolution.
36.	Karched, Maribasappa, et al. (author) Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form 2008 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 28:8:18 Journal article (peer-reviewed)abstract Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 um), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 um) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSIONS: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.
37.	Karlsson, Anneli, et al. (author) Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden 2012 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 12:129 Journal article (peer-reviewed)abstract UNLABELLED: ABSTRACT:BACKGROUND: Chronic gastritis, peptic ulcer disease, and gastric cancer have been shown to be related to infection with Helicobacter pylori (H. pylori). Two major virulence factors of H. pylori, CagA and VacA, have been associated with these sequelae of the infection. In this study, total DNA was isolated from gastric biopsy specimens to assess the cagA and vacA genotypes.RESULTS: Variations in H. pylori cagA EPIYA motifs and the mosaic structure of vacA s/m/i/d regions were analysed in 155 H. pylori-positive gastric biopsies from 71 individuals using PCR and sequencing. Analysis of a possible association between cagA and vacA genotypes and gastroduodenal pathogenesis was made by logistic regression analysis. We found that H. pylori strains with variation in the number of cagA EPIYA motif variants present in the same biopsy correlated with peptic ulcer, while occurrence of two or more EPIYA-C motifs was associated with atrophy in the gastric mucosa. No statistically significant relation between vacA genotypes and gastroduodenal pathogenesis was observed.CONCLUSIONS: The results of this study indicate that cagA genotypes may be important determinants in the development of gastroduodenal sequelae of H. pylori infection. In contrast to other studies, vacA genotypes were not related to disease progression or outcome. In order to fully understand the relations between cagA, vacA and gastroduodenal pathogenesis, the mechanisms by which CagA and VacA act and interact need to be further investigated.
38.	Karlsson, Mattias, et al. (author) Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4 2012 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 12, s. 15- Journal article (peer-reviewed)abstract Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation.Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR 1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR 1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB.Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR 1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance.
39.	Kivi, Marten, et al. (author) Helicobacter pylori genome variability in a framework of familial transmission 2007 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 7, s. 54- Journal article (peer-reviewed)abstract Background: Helicobacter pylori infection is exceptionally prevalent and is considered to be acquired primarily early in life through person-to-person transmission within the family. H. pylori is a genetically diverse bacterial species, which may facilitate adaptation to new hosts and persistence for decades. The present study aimed to explore the genetic diversity of clonal isolates from a mother and her three children in order to shed light on H. pylori transmission and host adaptation. Results: Two different H. pylori strains and strain variants were identified in the family members by PCR-based molecular typing and sequencing of five loci. Genome diversity was further assessed for 15 isolates by comparative microarray hybridizations. The microarray consisted of 1,745 oligonucleotides representing the genes of two previously sequenced H. pylori strains. The microarray analysis detected a limited mean number (+/- standard error) of divergent genes between clonal isolates from the same and different individuals (1 +/- 0.4, 0.1%, and 3 +/- 0.3, 0.2%, respectively). There was considerable variability between the two different strains in the family members (147 +/- 4, 8%) and for all isolates relative to the two sequenced reference strains (314 +/- 16, 18%). The diversity between different strains was associated with gene functional classes related to DNA metabolism and the cell envelope. Conclusion: The present data from clonal H. pylori isolates of family members do not support that transmission and host adaptation are associated with substantial sequence diversity in the bacterial genome. However, important phenotypic modifications may be determined by additional genetic mechanisms, such as phase-variation. Our findings can aid further exploration of H. pylori genetic diversity and adaptation.
40.	Klint, Markus, et al. (author) Mosaic structure of intragenic repetitive elements in histone H1-like protein Hc2 varies within serovars of Chlamydia trachomatis 2010 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 10, s. 81- Journal article (peer-reviewed)abstract BACKGROUND: The histone-like protein Hc2 binds DNA in Chlamydia trachomatis and is known to vary in size between 165 and 237 amino acids, which is caused by different numbers of lysine-rich pentamers. A more complex structure was seen in this study when sequences from 378 specimens covering the hctB gene, which encodes Hc2, were compared. RESULTS: This study shows that the size variation is due to different numbers of 36-amino acid long repetitive elements built up of five pentamers and one hexamer. Deletions and amino acid substitutions result in 14 variants of repetitive elements and these elements are combined into 22 configurations. A protein with similar structure has been described in Bordetella but was now also found in other genera, including Burkholderia, Herminiimonas, Minibacterium and Ralstonia.Sequence determination resulted in 41 hctB variants that formed four clades in phylogenetic analysis. Strains causing the eye disease trachoma and strains causing invasive lymphogranuloma venereum infections formed separate clades, while strains from urogenital infections were more heterogeneous. Three cases of recombination were identified. The size variation of Hc2 has previously been attributed to deletions of pentamers but we show that the structure is more complex with both duplication and deletions of 36-amino acid long elements. CONCLUSIONS: The polymorphisms in Hc2 need to be further investigated in experimental studies since DNA binding is essential for the unique biphasic life cycle of the Chlamydiacae. The high sequence variation in the corresponding hctB gene enables phylogenetic analysis and provides a suitable target for the genotyping of C. trachomatis.
41.	Lavigne, Rob, et al. (author) Classification of Myoviridae bacteriophages using protein sequence similarity. 2009 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 9, s. 224- Journal article (peer-reviewed)abstract BACKGROUND: We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages. RESULTS: CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses." CONCLUSION: The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae.
42.	Lindgren, Marie, et al. (author) The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection 2013 In: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 13 Journal article (peer-reviewed)abstract Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.
43.	Lindmark, Barbro, 1963-, et al. (author) Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni 2009 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 16:9, s. 220- Journal article (peer-reviewed)abstract BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.
44.	Löfström, Charlotta, et al. (author) Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella 2009 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 9 Journal article (peer-reviewed)abstract Background. One of the major sources of human Salmonella infections is meat. Therefore, efficient and rapid monitoring of Salmonella in the meat production chain is necessary. Validation of alternative methods is needed to prove that the performance is equal to established methods. Very few of the published PCR methods for Salmonella have been validated in collaborative studies. This study describes a validation including comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal) of a same-day, non-commercial real-time PCR method for detection of Salmonella in meat and carcass swabs. Results. The comparative trial was performed against a reference method (NMKL-71:5, 1999) using artificially and naturally contaminated samples (60 minced veal and pork meat samples, 60 poultry neck-skins, and 120 pig carcass swabs). The relative accuracy was 99%, relative detection level 100%, relative sensitivity 103% and relative specificity 100%. The collaborative trial included six laboratories testing minced meat, poultry neck-skins, and carcass swabs as un-inoculated samples and samples artificially contaminated with 110 CFU/25 g, and 10100 CFU/25 g. Valid results were obtained from five of the laboratories and used for the statistical analysis. Apart from one of the non-inoculated samples being false positive with PCR for one of the laboratories, no false positive or false negative results were reported. Partly based on results obtained in this study, the method has obtained NordVal approval for analysis of Salmonella in meat and carcass swabs. The PCR method was transferred to a production laboratory and the performance was compared with the BAX Salmonella test on 39 pork samples artificially contaminated with Salmonella. There was no significant difference in the results obtained by the two methods. Conclusion. The real-time PCR method for detection of Salmonella in meat and carcass swabs was validated in comparative and collaborative trials according to NordVal recommendations. The PCR method was found to perform well. The test is currently being implemented for screening of several hundred thousand samples per year at a number of major Danish slaughterhouses to shorten the post-slaughter storage time and facilitate the swift export of fresh meat. © 2009 Löfström et al; licensee BioMed Central Ltd.
45.	Oscarsson, Jan, et al. (author) Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells 2008 In: BMC Microbiology. - London : Springer Science and Business Media LLC. - 1471-2180. ; 8, s. 206- Journal article (peer-reviewed)abstract Background: Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells. Results: By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1 beta, TNF-alpha, IL-6, IL-8, MIP-1 beta) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity. Conclusion: A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.
46.	Palm, Eleonor, 1980-, et al. (author) Porphyromonas gingivalis downregulates the immune response of fibroblasts 2013 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 13, s. 155- Journal article (peer-reviewed)abstract Background: Porphyromonas gingivalis is a key pathogen in periodontitis, an inflammatory disease leading to destruction of bone and tooth-supporting tissue. P. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. Accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. The aim of this study was to determine if P. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. Human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-alpha (TNF-alpha) and cocultured with P. gingivalis. Gingipain inhibitors were used to explore the effect of gingipains. CXCL8 levels were determined with ELISA and the relative levels of various inflammatory mediators were determined by a cytokine assay.Results: TNF-alpha-triggered CXCL8 levels were completely abolished by viable P. gingivalis, whereas heat-killed P. gingivalis did not suppress CXCL8. Accumulation of CXCL8 was partially restored by an arginine-gingipain inhibitor. Furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable P. gingivalis.Conclusion: These findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. In addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells.
47.	Pant, N., et al. (author) Effective prophylaxis against rotavirus diarrhea using a combination of Lactobacillus rhamnosus GG and antibodies 2007 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 7 Journal article (peer-reviewed)abstract Background. Rotavirus is a worldwide cause of infectious infantile diarrhea that claims over 600,000 lives annually. Recently, two new vaccine candidates have been developed but their efficacy in developing countries, still remains to be proven. Oral delivery of specific immunoglobulins provides passive immunity and is a fast acting treatment for rotavirus diarrhea. Probiotic bacteria have also gained considerable attention lately as treatment for rotavirus diarrhea. Here we report an evaluation of the therapeutic potential of different probiotics and their combination with anti - rotavirus antibodies in a mouse model of rotavirus diarrhea. Results. Of the six probiotic bacteria tested, Lactobacillus rhamnosus strain GG had the strongest influence in reducing prevalence, duration and severity of diarrhea and was therefore chosen for combination treatment with immunoglobulins. The combination treatment reduced the diarrhea outcome measures significantly, prevented histopathological changes and reduced the virus load in the intestines. Conclusion. The advantages associated with immunoglobulins and probiotics based therapy is that the treatment provides a rapid therapeutic effect and is cost efficient. These components do not require special storage conditions and could potentially complement the rehydration therapy that is currently used. © 2007 Pant et al, licensee BioMed Central Ltd.
48.	Romano, Sara, et al. (author) Multilocus sequence typing supports the hypothesis that Ochrobactrum anthropi displays a human-associated subpopulation. 2009 In: BMC microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 9:267 Journal article (peer-reviewed)abstract BACKGROUND: Ochrobactrum anthropi is a versatile bacterial species with strains living in very diverse habitats. It is increasingly recognized as opportunistic pathogen in hospitalized patients. The population biology of the species particularly with regard to the characteristics of the human isolates is being investigated. To address this issue, we proposed a polyphasic approach consisting in Multi-Locus Sequence Typing (MLST), multi-locus phylogeny, genomic-based fingerprinting by pulsed-field gel electrophoresis (PFGE) and antibiotyping. RESULTS: We tested a population of 70 O. anthropi clinical (n=43) and environmental (n=24) isolates as well as the type strain O. anthropi ATCC49188T and 2 strains of Ochrobactrum lupini and Ochrobactrum cytisi isolated from plant nodules. A Multi-Locus Sequence Typing (MLST) scheme for O. anthropi is proposed here for the first time. It was based on 7 genes (3490 nucleotides) evolving mostly by neutral mutations. The MLST approach suggested an epidemic population structure. A major clonal complex corresponded to a human-associated lineage since it exclusively contained clinical isolates. Genomic fingerprinting separated isolates displaying the same sequence type but it did not detect a population structure that could be related to the origin of the strains. None of the molecular method allowed the definition of particular lineages associated to the host-bacteria relationship (carriage, colonisation or infection). Antibiotyping was the least discriminative method. CONCLUSION: The results reveal a human-associated subpopulation in our collection of strains. The emergence of this clonal complex was probably not driven by the antibiotic selective pressure. Therefore, we hypothesise that the versatile species O. anthropi could be considered as a human-specialized opportunistic pathogen.
49.	Rosales, S, et al. (author) Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras 2010 In: BMC microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 10, s. 208- Journal article (peer-reviewed)
50.	Sabala, I., et al. (author) Anti-staphylococcal activities of lysostaphin and LytM catalytic domain 2012 In: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12:1 Journal article (peer-reviewed)abstract Background: Lysostaphin and the catalytic domain of LytM cleave pentaglycine crossbridges of Staphylococcus aureus peptidoglycan. The bacteriocin lysostaphin is secreted by Staphylococcus simulans biovar staphylolyticus and directed against the cell walls of competing S. aureus. LytM is produced by S. aureus as a latent autolysin and can be activated in vitro by the removal of an N-terminal domain and occluding region. Results: We compared the efficacies of the lysostaphin and LytM catalytic domains using a newly developed model of chronic S. aureus infected eczema. Lysostaphin was effective, like in other models. In contrast, LytM was not significantly better than control. The different treatment outcomes could be correlated with in vitro properties of the proteins, including proteolytic stability, affinity to cell wall components other than peptidoglycan, and sensitivity to the ionic milieu. Conclusions: Although lysostaphin and LytM cleave the same peptide bond in the peptidoglycan, the two enzymes have very different environmental requirements what is reflected in their contrasting performance in mouse eczema model.

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