MBL2 gene affect the concentration and functionality of the protein and are associated with enhanced susceptibility to severe malaria in African children. Most MBL2 typing strategies were designed to the analysis of selected variants, the significance of whole haplotypes is poorly known. In this work, a new typing strategy was developed and validated in an association analysis of MBL2 with adult asymptomatic infection.Mannose binding lectin (MBL) has an important role in the activation of the complement system and opsonization of pathogenic microorganisms. Frequent polymorphisms found in the MBL2 allele-specific fragments of 144 healthy Gabonese adults were amplified by using haplotype-specific sequencing (HSS), a new strategy that combines sequence-specific PCR and sequence-based typing. The Gabonese were investigated for the presence of Plasmodium falciparum parasitaemia by the amplification of parasite genes, immunochromatographic antigen detection and microscopic analysis. HSS results were also compared with a previously used real-time PCR (RT-PCR) method in 72 Euro-Brazilians.H, L; X, Y; P, Q) and exon 1  variants. The MBL2*LYPA/LYPA genotype was associated with the absence of asymptomatic infection (P = 0.017), whereas the MBL2*LYQC haplotype and YA/YO + YO/YO genotypes were associated with positive parasite counts in asymptomatic adults . The associations were specific to LYPA  and LYQC (Y16578) and would not have been revealed by standard genotyping, as there was no association with LYPA and LYQC haplotypes carrying new polymorphisms defined by sequence-based typing. In contrast, HSS and RT-PCR produced very similar results in the less diverse European-derived population.Fourteen polymorphisms were identified beside the commonly investigated promoter (P. falciparum-infected adults. In populations with high nucleotide diversity, it allowed for the identification of associations with fine-scaled haplotypes that would not have been found using common typing techniques. In this preliminary study, MBL2 haplotypes or SNPs linked to them were found associated with susceptibility to infection and parasitaemia control of asymptomatic adults.In this work, a new typing strategy for a highly polymorphic gene was developed and validated focusing on the asymptomatic status of  Plasmodium falciparum infection causes between one and two million deaths annually, mostly in African children less than 5 years of age. This means that every 30 seconds a child dies of malaria. Those who survive became increasingly immune to severe malaria with each disease episode . This association was specific to the LYQC haplotype .LYPA/LYPA homozygotes in the adult non-parasitized group but none in the asymptomatic group . This association was specific to LYPA : frequencies of all other genotypes with LYPA-similar haplotypes presented no significant differences. To look for the effect of X, Y and A, O variants, known to modulate the concentration of high-order MBL oligomers, the genotypes were grouped as: YA/YA, YA/XA, XA/XA, XA/YO, YA/YO and YO/YO ].Genotype frequencies from both Gabonese groups were at Hardy and Weinberg equilibrium table . There wMBL2 genotyping techniques, based on restriction fragment length polymorphisms (RFLP) of PCR products [H/L, X/Y, P/Q and A/B/C/D variants and thus of the HYPA (reference sequence: Y16581), HYPD (Y16582), LXPA (Y16580), LYPA (Y16577), LYPB (Y16579), LYQA (Y16576) and LYQC (Y16578) haplotypes. Newer methods that allow the identification of other SNPs rely on maximum likelihood methods for phasing. It was established in previous work [LYPA haplotypes in the Gabonese population are not identical with the reference sequence Y16577. These haplotypes carry additional variants, the most frequent being g.388G>A. Around 17% of the LYQA haplotypes are not identical with Y16576 and around 13% of the LYQC haplotypes are not identical with Y16578 in the same population. In contrast, only 1.4% of the Euro-Brazilian group presented uncommon SNPs. This is mainly due to the higher heterozygosity and nucleotide diversity of the MBL2 promoter region in African, compared to non-African populations [MBL2 disease association studies with Afro-derived populations.There are numerous published products , sequencproducts ,31, denaproducts , real-tiproducts  and sequproducts . Most ofLYPA Y1657, LYPB . Both offer the possibility of isolating large fragments, even the whole homologue chromosomes of heterozygote individuals . Clonings before ,36.MBL2 physical haplotyping with SSP primers began in 2002 and led to the discovery of LYPD, a previously unrecognized MBL2 haplotype [D variant with the HYP promoter was not absolute, as formerly assumed. In contrast, other haplotypes whose existence was predicted by statistical algorithms have physically not been confirmed, including HXPA and LYQB [aplotype , and to and LYQB .MBL2 association study including only truly non-parasitized and asymptomatic P. falciparum infected adults. In contrast, other authors evaluated the association of O alleles with asymptomatic infection in children. The long-lasting asymptomatic status of infected African children depends on the number of previous malaria attacks [O alleles with asymptomatic infection in a follow-up study with 158 Gabonese schoolchildren [MBL2 genotyping method [This work is the first  attacks . This cochildren , but in children . A reasog method .P. falciparum infection is highly prevalent in the Gabonese adult population [sine qua non condition for the maintenance of semi-immunity, which is lost within some weeks outside an endemic area [LYPA/LYPA genotype with protection against asymptomatic parasitaemia was found. None of the LYPA-similar haplotypes were associated with the infection, and so the association would not have been revealed by conventional MBL2 typing techniques. LYPA is probably an ancient haplotype, with a wide geographical distribution. It occurred in nine different African groups, being less common in East Asian and rare in Amerindian(-similar) populations [LYPA (Y16577) haplotypes carry a linked SNP (-1165 G>T), which could actually be responsible for the observed association. This SNP was only found in African populations [Asymptomatic pulation . It seemmic area . It coululations ,34. In tulations .MBL2*LYQC variant, YO/YO and YA/YO genotypes with microscopically detectable parasites. These preliminary findings in adults correlate well with some studies in children. In Ghanaian infected children, the YO/YO genotype was found to be associated with higher parasitaemia [LYQC haplotypesas well as A/O genotypes, with higher susceptibility to P. falciparum infection [P. falciparum may also change with the acquisition of semi immunity as individuals grow older, depending on the frequency of disease episodes. The replication of the results in a larger adult setting, as well as functional in vitro and in vivo studies, would help to clarify this suggestion.There was also an association of the sitaemia  and grounfection . DespiteMBL2 haplotypes or SNPs linked to them were associated with the susceptibility to infection and with parasitaemia control of asymptomatic adults.HSS was validated as a fast and highly informative method for analyzing small-sized haplotypes of polymorphic genes in populations with high nucleotide diversity, as are the vast majority of Afro-derived ethnic groups. It is more affordable than HSE, which relies on magnetic beads, and as cloning, which requires living cells. In this preliminary study, The authors declare that they have no competing interests.MBL2 genotyping and statistical analysis, and drafted the manuscript. IJM-R helped in interpretation of the data and to draft the manuscript. MLAP, SI and BL collected the Euro-Brazilian and Gabonese samples, respectively. PGK participated in the design of the study and revised the manuscript for important intellectual content. JFJK coordinated the study, participated in the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript.ABWB developed the HSS method, performed the MBL2 nucleotide changes and haplotype frequencies in the Gabonese samples according to parasitaemia status. The data show the haplotype frequency in Gabonese individuals with and without detected parasites. The positions corresponding to the SNPs and to the deletion (position of the first deleted nucleotide) are shown in the first row (Reference sequence: Y16577). In bold: significant different frequencies. LYPAs1, LYPAs2 and LYPAs3:LYPA-similar haplotypes; LYQAs1 and LYQAs2: LYQA-similar haplotypes; LYQCs1, LYQCs2 and LYQCs3:LYQC-similar haplotypes. PCR polymerase chain reaction, TBS thick blood smear, – negative result, + positive result positive result, N number of chromosomes. All PCR-individuals were also negative in a Rapid Diagnosis Test. In SNP database: g.273G>C as rs11003125, g.388G>A as rs7100749, g.396A>C as rs11003124, g.456G>T as rs35615810, g.474A>G as rs7084554, g.478G>A as ss107796301, g.482A>G as ss107796302, g.487A>G as rs36014597, g.495delAAAGAG as rs10556764, g.578G>A as rs35236971, g.602G>C as rs7096206, g.659C>T as ss107796304, g.712A>T as ss107796305, g.753C>T as rs11003123, g.797C>A as rs45602536, g.826C>T as rs7095891, g.925C>G as ss107796306, g.926T>G as ss107796307, g.1052G>A as rs1800450, g.1061G>A as rs1800451. All ss numbers were submitted by the authors to the SNP database and will be changed to rs numbers in the near future.Click here for file


Note: This section lists opinions by different doctors on specific problems. These opinions are their personal opinions and not necessarily supported by any data. The section only serves to offer options/opinions in different difficult to treat conditions.Question asked: Can Becker’s nevus be effectively treated? Is it worth treating at all?Responses:Beckers nevus has hairs that should be treated first with a long pulsed Nd-YAG laser followed by (a) 1 pass of 532 nm 1-2 J/s 4 mm spot size, which reduces the epidermal pigment that is pronounced in Beckers nevus followed by (b) 1064 nm 4 mm spot size 10-13 J/s till bleeding points for good results. We have tried this in our few cases.Dr. SG Parasramani, Lilavati Hospital, MumbaiThe best approach to a Becker’s nevus is to use a hair reduction laser along with either a Q-switched laser to lighten the lesion or a fractionated ablative laser. I have tried the Q-switched laser alone but the response was less than satisfactory. In my view, its not worth attempting. If a patient seeks treatment, we proceed only after a test patch.sanjeev.aurangabadkar@gmail.comDr. Sanjeev Aurangabadkar, Skin and Laser Clinic, Hyderabad, India. E-mail: Non-hairy Beckers nevi pigmentation can be improved by an ER:YAG laser but hairy Beckers nevi is a challenge. Here, the hairs are very resistant to therapy by either diode or Nd:YAG laser. Somehow, the response of Beckers nevi in males is less effective compared with females.drhmomprakash@yahoo.comDr. Omprakash, National Skin & Hair Centre, Bangalore. E-mail: We have treated more than 15 cases of Becker’s nevus on Harmony from Alma lasers. We have tried different options for pigment reduction, including Q-switched 1064–532 pixel, Er–YAG, LEO 540, 570, 650. Most consistent results obtained were with LEO 540 Harmony.niteendhepe@yahoo.comDr. Dhepe, Consultant Dermatologist, Skin City, Pune, India. E-mail: Question asked: Can keratosis pilaris be treated by laser?Responses:To the best of my best knowledge, nothing can be done on a long-term basis for keratosis pilaris. I tried many things  but improvement did not last longer than 2 months with any modality.madatto@skinpulse.chDr. Maurice Adatto, Switzerland. E-mail: Laser hair removal cannot work in keratosis pilaris as the hairs are too small and contain little melanin. Other forms of laser also do not work consistently or produce lasting results.alapidot@netvision.net.ilDr. Moshe Lapidoth, Israel. E-mail:


Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species. Legionella longbeachae, found in potting soil, and L. pneumophila, present in aquatic environments, are opportunistic human pathogens that cause Legionnaires' disease, a severe and often fatal pneumonia. The analysis and comparison of the genome sequences of four L. longbeachae genomes together with the study of its gene expression program and virulence pattern in different infection models provides important new insight on the organism's lifestyle and virulence strategies. L. longbeachae harbors a unique repertoire of secreted substrates, many of which encode eukaryotic like domains that may help the pathogen to subvert host functions and cause disease. Curiously, L. longbeachae may also be able to interact with plants. Several proteins present mainly in plants and phytopathogenic bacteria and several enzymes that might confer the ability to degrade plant material were identified in its genome. Interestingly, L. longbeachae encodes a chemotaxis system but no flagella, in contrast L. pneumophila encodes flagella but no chemotaxis system. It will be an interesting aspect of future research to understand these peculiarities. Finally, the genome sequence and analysis reported here will aid in understanding how L. longbeachae causes disease and will open new possibilities to develop tools for rapid identification and risk prediction of L. longbeachae infection. Legionella longbeachae is one species of the family Legionellaceae that causes legionellosis, an atypical pneumonia that can be fatal if not promptly treated. While Legionella pneumophila is the leading cause of legionellosis in the USA and Europe, and is associated with around 91% of the cases worldwide, L. longbeachae is responsible for approximately 30% of legionellosis cases in Australia and New Zealand and nearly 50% in South Australia L. longbeachae but most of the human cases of legionellosis are due to Sg1 strains L. pneumophila, which inhabits aquatic environments, L. longbeachae is found predominantly in potting soil and is transmitted by inhalation of dust from contaminated soils L. longbeachae but a few studies suggest considerable differences with respect to L. pneumophila. In contrast, the intracellular life cycle of L. pneumophila is well characterized  L. longbeachae possesses all genes necessary to code a Dot/Icm T4SS L. pneumophila and L. longbeachae share common effectors.Efficient formation of the L. pneumophila, A/J, C57BL/6 and BALB/c mice are all permissive for replication of L. longbeachaeL. pneumophila has been attributed to polymorphisms in Nod-like receptor apoptosis inhibitory protein 5 (naip5) allele L. pneumophila replication is restricted due to flagellin dependent caspase-1 activation through Naip5-Ipaf and early macrophage cell death by pyroptosis. Why L. longbeachae, in contrast to L. pneumophila, is able to replicate in macrophages of all three different mouse strains is still not understood.Another interesting difference between these two species is their ability to colonize the lungs of mice. While only A/J mice are permissive for replication of L. longbeachae Sg1 strain isolated in Australia and compare this genome to three L. longbeachae draft genome sequences (one Sg1 and two Sg2 strains) and the published genome sequences of four L. pneumophila strains L. longbeachae. This has allowed us to propose answers for the questions raised above and brings exciting new insight into the varying adaptation to different ecological niches and different intracellular life cycles of Legionella species.In this study we report the complete genome sequencing and analysis of a clinical L. longbeachae NSW150 genome consists of a 4,077,332-bp chromosome and a 71,826-bp plasmid with an average GC content of 37.11% and 38.19%, respectively  are L. longbeachae specific with respect to L. pneumophila Paris, Lens, Philadelphia and Corby , encoded ORFs showing high amino acid sequence similarity , strain 98072 (Sg2) and strain C-4E7 (Sg2) were deep sequenced using the Illumina technology and then compared to the genome of strain NSW150. We obtained a coverage of 93–96% for each genome with respect to the NSW150 genome  and three putative type I secretion systems (T1SS) are present. However, the Lss T1SS might not be functional in L. longbeachae as only LssXYZA are conserved (55 to 82% identity with strain Paris) and the two essential components LssB (ABC transporter-ATP binding) and LssD (HlyD family secretion protein) are missing. In contrast, the two additional putative T1SS, encoded by the genes llo2283-llo2288 and llo0441-llo0444 appeared to be functional. Furthermore, two HlyD-like proteins (Llo2901 and Llo0979) localized next to ABC transporters (Llo2900 and Llo0980-Llo0981) were present, but no contiguous outer membrane protein was found. However, these proteins could also be part of T1SS and function together with a genetically unlinked outer membrane component, similar to what is seen for the Hly T1SS of Escherichia coli and may thus constitute two additional T1SS. Finally, L. longbeachae encodes four type IV secretion systems (T4SS). The Lvh T4ASS of L. pneumophila is absent from L. longbeachae but we identified three other type-IVA secretion systems. One T4ASS is present on the plasmid and the other two are embedded on putative mobile genomic islands (GI) in the chromosome. llo1819-llo1929 (GI-1) of around 120 kb is bordered by Ser and Arg tRNAs and carries a gene coding for a phage integrase (llo1819). The second cluster (GI-2) of 106 kb spans from the integrase coding gene llo2859 to llo2960ab and is also bordered by a Met tRNA. Most of the proteins encoded on GI-2 are of unknown function. However both islands code for several proteins, which may be dedicated to stress response. On GI-1, Llo1862 and Llo1863llo1863 are homologous to DNA polymerase IV subunit C and D respectively, involved in the SOS repair pathway. On GI-2 are the OsmC-like protein Llo2923, the putative universal stress proteins Llo2926, Llo2927, Llo2929 and the predicted trancriptional regulator Llo2913 with S24 peptidase domain. Indeed, the S24 peptidase family includes LexA, a transcriptional repressor of SOS response genes to DNA damage. Several transporters were also identified on GI-2: Llo2918 of the MFS superfamily, the Na/H exchange protein Llo2930 and the putative T1SS proteins Llo2900 and Llo2901 discussed above. It possesses in addition a putative restriction/modification system encoded by llo2865, llo2866 and llo2867.L. pneumophila virulence is the Dot/Icm type IV secretion system. This T4BSS is also present in L. longbeachae and the general organization of the genomic region encoding it is conserved with protein identities of 47 to 92% with respect to that of L. pneumophila. This is similar to what has been reported previously for other Legionella species L. longbeachae the icmR gene is replaced by the ligB gene, however, the encoded proteins have been shown to perform similar functions L. longbeachae is 477 amino acids larger than that of L. pneumophila. DotG is part of the core transmembrane complex of the secretion system and it is composed of three domains: a transmembrane N-terminal domain, a central region composed of 42 repeats of 10 amino acid and a C-terminal region homologous to VirB10. The central region of DotG from L. longbeachae comprises approximately 90 repeats. It will be challenging to understand the possible impact of this modification on the function of the type-IV secretion system.Central to the establishment of the intracellular replicative niche and to L. longbeachae we constructed a deletion mutant in the L. longbeachae NSW150 gene llo0364, homologous to dotA of L. pneumophila and tested its ability to replicate compared to the wild type strain in A. castellanii and the lungs of A/J mice. We found that L. longbeachae NSW150 infects A. castellanii in a comparable manner to L. pneumophila and that the dotA mutant was strongly attenuated for intracellular growth in A. castellanii, similar to what is seen for a L. pneumophila dotA mutant   to anchor the effector proteins SidC, SidM/DrrA, LpnE and LidA to the membrane of the replication vacuole L. longbeachae may employ an additional strategy to interfere with the host PI as Llo0793 is homologous to a mammalian PI metabolizing enzyme phosphatidylinositol-4-phosphate 5-kinase and it is tempting to speculate that this protein allows direct modulation of the host cell PI levels.Although Llo2249) , which mL. pneumophila is able to target microtubule-dependent vesicular transport. AnkX/AnkN, for example, prevents microtubule-dependent vesicular transport interfering with the fusion of the L. pneumophila-containing vacuole with late endosomes L. longbeachae, however L. longbeachae did encode a putative tubulin-tyrosine ligase (TTL) Llo2200, which adds to the 19 bacterial TTL identified to date. TTL catalyzes the ATP-dependent post-translational addition of a tyrosine to the carboxy terminal end of detyrosinated alpha-tubulin. Although the exact physiological function of alpha-tubulin has so far not been established, it has been linked to altered microtubule structure and function L. pneumophila Dot/Icm substrates. Interestingly, 23 of the 29 ankyrin proteins identified in the L. pneumophila strains are absent from the L. longbeachae genome, however L. longbeachae encodes 23 specific ankyrin repeat proteins  domain. SH2 domains, in eukaryotes, have regulatory functions in various intracellular signaling cascades. Furthermore, L. longbeachae encodes two proteins (Llo1404 and Llo2643) with pentatricopeptide repeat (PPR) domains. This family seems to be greatly expanded in plants, where they appear to play essential roles in organellar RNA metabolism Ralstonia solanacearum and the facultative photosynthetic bacterium Rhodobacter sphaeroides.L. pneumophila (Lgt) and related to the group of clostridial glucosylating cytotoxins has been described L. longbeachae encodes two putative specific cytotoxic glucosyltransferases Llo1721 and Llo1578. They share only low homology with the L. pneumophila Lgt proteins with 23% protein identity over 62% of the protein length and 36% protein identity over 32% of the length, respectively. However, the DXD motif that is critical for enzymatic activity of clostridial enzymes is conserved suggesting that these enzymes might also be active in L. longbeachae. We also identified Llo3231 as another putative specific glucosyltransferase with a DXD motif, distantly related to the L. pneumophila SetA protein (23% protein identity over 67% of the protein length). SetA is known to cause delay in vacuolar trafficking L. longbeachae does not encode a homologue of the L. pneumophila structural toxin protein RtxA, however we identified a homolog of the TcaZ toxin (Llo1558) present in the insect pathogen Photorhabdus luminescensRecently, a family of cytotoxic glucosyltransferases produced by L. longbeachae encodes a variety of proteins probably devoted to the metabolism of compounds present in plant cell walls, going in hand with the fact that that bacterium can be isolated from composted plant material. The main components of the plant cell wall are cellulose, hemicellulose and pectin. Cellulose utilization by microorganisms involves endo-1,4-beta-glucanases, cellobiohydrolases and β-glucosidases, that act synergically to convert cellulose to glucose. Examination of the L. longbeachae genome sequence revealed the presence of twelve such cellulolytic enzymes. Five glucanases, four cellobiohydrolases and three β-glucosidases are present. Interestingly, L. pneumophila also encodes two putative endo-1,4-beta-glucanases and one putative β-glucosidase but does not encode any cellobiohydrolase.L. longbeachae encodes three pectin lyases . The last two proteins possess a signal peptide and may therefore be secreted. Pectin lyases are virulence factors usually found in phytopathogenic microorganisms that degrade the pectic component of the plant cell wall. In addition to these specific enzymes and similar to L. pneumophila, L. longbeachae encodes a protein homologous to endo-1,4-beta-xylanase. Endo-1,4-beta-xylanase hydrolyses xylan the most common hemicellulose polymer in the plant kingdom and the second most abundant polysaccharide on earth. So, unlike L. pneumophila, which does not possess cellobiohydrolase and pectin lyase, L. longbeachae seems to be fully equipped to utilize cellulose as a carbon source  that are predicted to be secreted proteins. However, the homologue of ChiA from L. pneumophila that was shown to be involved in infection of lungs of A/J mice L. longbeachae.Within the plant cell wall, the cellulose microfibrils are linked via hemicellulosic tethers to form the cellulose-hemicellulose network, which is embedded in the pectin matrix. To gain access to cellulose in plant material, pectin and hemicellulose hydrolysis is necessary. Interestingly, n source . Soil baL. longbeachae encodes a putative cyanophycin synthase (Llo2537) and therefore may be able to synthesize cyanophycin. Cyanophycin is an amino acid polymer composed of an aspartic acid backbone and arginine side groups. It serves as a storage compound for nitrogen, carbon and energy in many cyanobacteria. Acinetobacter baylyi strain ADP1 was the first non-cyanobacterial strain shown to synthesize cyanophycin, a metabolic capacity that is still restricted to only few prokaryotes L. longbeachae also harbors a putative cyanophycinase (Llo2536) enabling the degradation of cyanophycin to dipeptides and a dipeptidase (Llo2535) necessary to hydrolyze beta-Asp-Arg dipeptides. L. longbeachae may thus be able to completely utilize cyanophycin, providing a mechanism for energy supply under substrate-limited conditions.Interestingly, L. longbeachae NSW150 we identified two gene clusters encoding proteins that are predicted to be involved in production of lipopolysaccharide (LPS) and/or capsule  that associate with CheA or cytoplasmic chemosensory receptors are present. Ligand-binding to receptors regulates the autophosphorylation activity of CheA in these complexes. The CheA phosphoryl group is subsequently transferred to CheY, which then diffuses away to the flagellum where it modulates motor rotation. Adaptation to continuous stimulation is mediated by a methyltransferase CheR encoded by llo3299 in L. longbeachae. Together, these proteins represent an evolutionarily conserved core of the chemotaxis pathway, common to many bacteria and archea L. drancourtii LLAP12 L. pneumophila. The flanking genomic regions are highly conserved among L. longbeachae and all L. pneumophila strains sequenced, suggesting that L. pneumophila, although it encodes flagella has lost the chemotaxis system encoding genes.Although L. longbeachae possesses a long pilus-like structure. Indeed, all genes necessary to code for type IV pili are present in the genome of L. longbeachae and are, with 63–88% amino acid similarity, highly conserved between L. longbeachae and L. pneumophila. Taken together genome analysis revealed interesting features of the Legionella genomes: both encode pilus-like structures, in contrast L. longbeachae encodes a chemotaxis system but no flagella, and L. pneumophila encodes flagella but no chemotaxis system. It will be an interesting aspect of future research to understand these particular features of the two Legionella species.We also observed using electron microscopy  that L. L. pneumophila genomes and consistent with its intracellular lifestyle, the regulatory repertoire of L. longbeachae is rather small. Genome analysis identified 121 transcriptional regulators (113–116 in the four sequenced L. pneumophila genomes), which represent only 3.0% of the predicted genes . This is significantly different in L. longbeachae, as we identified only 14 GGDEF/EAL domain-containing regulators, despite the larger size of the L. longbeachae genome. Furthermore, this group of regulators may fulfill specific functions in L. longbeachae, since most of the regulators possess no orthologs in the L. pneumophila genomes  of which only one (Llo0181) is also conserved in L. pneumophila. LadC, an adenylate cyclase of L. pneumophila that was shown to have a significant role in the initiation of infection in vitro and in vivoL. longbeachae. As shown for Pseudomonas aeruginosa, these class III adenylate cyclases may sense environmental signals ranging from nutritional content of the surrounding media to the presence of host cells and control virulence gene expression accordingly L. pneumophila, again indicating specific regulatory circuits for L. longbeachae. This high number of proteins that may sense cAMP indicates the potential importance of this signaling molecule in L. longbeachae.In L. longbeachae. Likewise, the two-component systems PmrAB (Llo1159/Llo1158) and CpxRA (Llo1781/Llo1782) that regulate the Dot/Icm T4SS system and some of its substrates are both conserved in L. longbeachaeIn contrast, the regulators shown to be important for growth phase and life cycle dependent gene expression, such as the two component system LetA/LetS (Lllo2653/llo1235), the RNA-binding protein CsrA (Llo2071), the two small RNAs RsmY and RsmZ regulating CsrA L. pneumophila exhibits at least two developmental stages, a replicative/avirulent and a transmissive/virulent phase that are each characterized by the expression of specific traits L. pneumophila between the two phases of its life cycle L. longbeachae had a similar biphasic life cycle we studied its gene expression program in exponential and post exponential growth phase in vitro. A multiple-genome microarray was constructed containing 10 692 gene-specific oligonucleotides representing 3567 genes predicted in the genome and on the plasmid and 3010 oligonucleotides specific for intergenic regions. RNA of in vitro grown bacteria was sampled at OD 2.5  and at OD 3.7  and the global gene expression program was compared.It has been shown in several studies that L. longbeachae were upregulated in the exponential (E) phase , the NADH deshydrogenase (nuo genes), most of the genes involved in translocation systems (sec genes) and several enzymatic activities , the regulatory protein RecX and several proteins related to starvation and stress are not upregulated in E phase L. longbeachae. There are only 11 genes specific for L. longbeachae and induced in E phase, all of which code proteins for which no function could be predicted.Overall, 187 genes in tivities . HoweverL. longbeachae, of which only 53 are also among the 441 PE phase genes of L. pneumophila. Interestingly, 208 of the genes upregulated in PE in L. longbeachae have no orthologs in L. pneumophila, and for 70% of these no function could be predicted. Thus the response of L. longbeachae to PE phase growth is distinct from that of L. pneumophila. In particular we observed differences in the expression profiles of many factors known to be involved in L. pneumophila virulence. For example, of the genes coding putative substrates of the Dot/Icm secretion system only few, sidC (llo3098), sdhB (llo2439), sidE homologue (llo2210), sdeC/laiC (llo3092) and sdeB/laiB (llo3095) are upregulated in post-exponential phase. However, several of the newly identified putative substrates are induced in L. longbeachae in PE phase. These comprise seven proteins homologous to Sid proteins of L. pneumophila , three genes coding homologues of EnhA , three ankyrin proteins  and a putative serine threonine kinase (Llo1139). However, clear differences in gene expression between L. pneumophila and L. longbeachae exist and the switch from replicative to transmissive phase seems to be less pronounced in L. longbeachae than in L. pneumophila. Interestingly, the genes coding the stationary phase sigma factor RpoS and the sigma factor 28 (FliA) and CsrA, all involved in the regulation of the biphasic life cycle of L. pneumophila are not differentially regulated in L. longbeachae. In contrast, seven GGDEF/EAL domain-containing regulators  and four cAMP binding proteins  are induced in PE phase. Thus cyclic di-GMP and cAMP may be important signaling molecules for regulating PE phase traits of L. longbeachae. According to our transcriptome analysis, the switch in the lifecycle of L. longbeachae appears less pronounced as compared to L. pneumophila, and regulation may be achieved mainly by secondary messenger molecules.In PE phase 313 genes are upregulated in L. longbeachae is the second leading cause of Legionnaires' disease in the world and a major cause of pneumonia in Australia and New Zealand. Yet, still very little is known about its virulence strategies and the genetic basis of virulence and niche adaptation. Analysis of the genome sequences of four L. longbeachae strains and its comparison with the published L. pneumophila genomes has uncovered important differences in the genetic repertoire of the two species and suggests different strategies for intracellular replication and niche adaptation.L. pneumophila, L. longbeachae encodes a type IVB secretion system homologous to the Dot/Icm system. Inactivation of the type IV secretion system, through deletion of the dotA gene, showed that it is essential for virulence, as the dotA mutant had a severe growth defect in A. castellanii infection and could not establish an infection in the lungs of A/J mice. Despite this resemblance to L. pneumophila, the secreted effectors are very different as only 44% of the known L. pneumophila substrates were conserved in L. longbeachae. However, like L. pneumophila, many of them have eukaryotic domains or resemble eukaryotic proteins. Thus a large cohort of eukaryotic-like proteins was also a specific feature of the L. longbeachae genomes. An emerging theme in bacterial virulence is the evolution of virulence factors that can mimic the activities of Ras small GTPases  using Velvet (version 0.7.55) Definition of coding sequences and annotation were done as described dotA mutant strain, the chromosomal region containing the dotA gene was PCR-amplified with the primers dotA-for CTCGCGCATTGGAACTTTAT and dotA-rev TTCGCTCATAAACCGCTCTT. The product was cloned into the pGEM-T Easy vector (Promega) yielding pGEM-dotA. We performed inverse PCR on pGEM-dotA with primers dotAinv-for CGCGGATCCCCGCATTTAATACGCCAAAC and dotAinv-rev CGCGGATCCAAGGTTTTGCGTTGGATAGG containing BamHI overhangs allowing internal deletion of 2582 bp in dotA. PCR product was digested with BamHI and ligated to the kanamycin resistance cassette, which was amplified via PCR from the plasmid pGEM-KanR, using primers containing BamHI restriction sites at the ends (Kan-BamHI-for TGCAGGTCGACTCAGAGGAT Kan-BamHI-rev CGCGGATCCGAGCTCGGTACC). Linearized vector was electroporated in L. longbeachae to obtain dotA::Km mutant.To construct the in vivo growth of L. longbeachae and its dotA deletion mutant in A. castellanii we followed a protocol previously described A. castellanii were washed in infection buffer  and adjusted to 105–106 cells per ml. Stationary phase Legionella grown on BCYE agar, diluted in water were mixed with A. castellanii at a MOI of 0.1. After allowing invasion for 1 hour at 37°C the A. castellanii layer was washed twice with infection buffer (start point of time-course experiment). Intracellular multiplication was monitored using a 300 µl sample, which was centrifuged (14 000 rpm) and vortexed to break up amoeba. The number of colony forming units (CFU) of Legionellae was determined by plating on BCYE agar. Each infection was carried out in duplicates.For L. longbeachae NSW150 and the dotA::Km derivative within A/J mice was examined via competition assays and in single infections, as described previously 5 CFU of each L. longbeachae strain under investigation. At 24 and 72 h following inoculation, mice were sacrificed and their lung tissue isolated. Tissue was homogenized, and complete host cell lysis was achieved by incubation in 0.1% saponin for 15 min at 37°C. Serial dilutions of the homogenate were plated onto both plain and antibiotic-selective BCYE agar to determine the number of viable bacteria and the ratio of wild-type to mutant bacteria colonizing the lung in mixed infections.The comparative virulence of Bacteria were transferred to Formvar-carbon-coated copper grids after glow discharged for 3′, stained with 1% uranyl acetate for 35sec, air dried and observed under a Jeol 1200EXII transmission electron microscope  operated at 80kV. Digital acquisition was performed with a Mega View camera and the Analysis pro software version 3,1 .L. pneumophila Paris and L. longbeachae NSW150 were amplified with genomic DNA of strain Paris and NSW150 respectively. Primers were designed using the Primer 3 software to amplify a specific fragment of about 1 -3kb respectively for each region (melting temperatures 58°C). Amplification reactions were performed in a 50-µl reaction volume containing 6 ng of chromosomal DNA. The size of each PCR product was verified on agarose gels. Primers used are listed in PCR for the regions containing the flagella biosynthesis coding genes in strain L. longbeachae strain NSW150 was cultured in N-(2-acetamido)-2-aminoethanesulphonic acid (ACES)-buffered yeast extract broth or on ACES-buffered charcoal –yeast (BCYE) extract agar at 37C. Total RNA was extracted as previously described L. longbeachae was harvested for RNA isolation at the exponential (OD 2.5) and post-exponential phase (OD 3.7). RNA was prepared from three independent cultures and each RNA sample was hybridized twice to the microarrays (dye swap). RNA was reverse-transcribed with Superscript indirect cDNA kit (Invitrogen) and labeled with Cy5 or Cy3 (Amersham Biosciences) according to the supplier's instructions. The microarray containing 13 710 60mer oligonucleotides specific for 3567 predicted genes of the genome, the plasmid and all intergenic regions longer than 200nts has been designed using the program OligoArray (http://berry.engin.umich.edu/oligoarray/). Based on these sequences a custom oligonucleotide array was manufactured (Agilent Technologies) with a final density of 15K. For hybridization, Cy3 and Cy5 target quantities were normalized at 150 pmol. Arrays were scanned using an Axon 4000B scanner with fixed PMT (PMT  =  550 for Cy3 and 650 for Cy5). Data were acquired and analyzed by Genepix Pro 5.0 (Axon Instrument). Spots were excluded from analysis in case of high local background fluorescence slide abnormalities or weak intensity. Data normalization and differential analysis were conducted using the R software (http://www.r-project.org). For each gene 3 probes were present on the microarray. Data for which at least 2 of the 3 probes gave a significant and non-contradictory result were taken into account. A loess normalization http://www.bioconductor.org/packages/bioc/html/marray.html). Differential analysis was carried out separately for each comparison between two time points, using the VM method (VarMixt package L. longbeachae NSW150 genome is accessible at the LegioList Web Server (http://genolist.pasteur.fr/LegioList and http://genolist.pasteur.fr/) and under the EMBL/Genbank Accession number: FN650140 the L. longbeachae NSW150 plasmid under the EMBL/Genbank Accession number: FN650141. Due to new regulations for genome sequence submissions to EMBL/Genbank the gene names (locus_tag), which are e.g. llo0001 in the article and in the Institut Pasteur databases had to be changed to LLO_0001 in the sequence submission. According to the standards for genome sequences published by Chain and colleagues L. pneumophila NSW150 genome sequence can be defined as “Finished” and the three Solexa genome sequence drafts can be defined as “High-Quality Draft”. The complete dataset for the transcriptome analysis is available at http://genoscript.pasteur.fr in a MIAME compliance public database maintained at the Institut Pasteur and was submitted to the ArrayExpress database maintained at http://www.ebi.ac.uk/microarray-as/ae/ under the Accession number: A-MEXP-1779.The sequence and the annotation of the Figure S1L. longbeachae CDS in the different COG groups. 2,506 CDS are classified in at least one COG group. Since several genes are assigned to multiple categories, the total number of assignments is greater than the number of ORFs in the genome.Classification of the (11.39 MB TIF)Click here for additional data file.Figure S2L. pneumophila strain Paris and L. longbeachae NSW150. The plot was created using the mummer software package (http://mummer.sourceforge.net/).Synteny plot of the chromosomes of (6.88 MB TIF)Click here for additional data file.Figure S3L. longbeachae and L. pneumophila. (A) Synteny LinePlot between the L. longbeachae plasmid and the plasmids of L. pneumophila strain Lens and Paris, respectively. Orthologous genes are defined by bi-directional blastP best hits (BDBH) or a blastP alignment threshold of 35% sequence identity over 80% of the length of the smaller protein. The gap parameter, representing the maximum number of consecutive genes that are not involved in a synteny group was 3. (B) Percentage of aminoacid identity among Tra proteins of the L. longbeachae and the L. pneumophila strain Lens as compared to the Tra region of strain Paris. (C) Venn diagram showing the common and specific gene content of the plasmids of L. pneumophila strains Paris, Lens and L. longbeachae NSW150.Comparison of the plasmids identified in (17.22 MB TIF)Click here for additional data file.Figure S4L. longbeachae ATCC39462 (Sg1) and C-4E7 (Sg2) with respect to the completely sequence genome of L. longbeachae NSW 150 (Sg1). Outer circle, Mapping of SNPs between L. longbeachae Sg1 (NSW150) and Sg2 (C-4E7), central circle in green, sequence couverage of mapped reads of strain ATCC39462 on the NSW150 genome, inner circle; SNP distributon among the two Sg1 strains sequenced. 1426 SNPs are located in 7 genomic regions; region 1: llo0557-llo0587 containing 112 SNPs; region 2: llo0643-llo0653, carries an integrase gene and contains 152 SNPs; region 3: llo0814-llo0841 containing 38 SNPs; region 4: llo0943-llo0952, carries an integrase gene and contains 152 SNPs; region 5: llo1813-llo1886, carries many tra- like genes and contains 651 SNPs; region 6: llo2119-llo2142, contains 89 SNPs, region 7: llo3148-llo3180, carries genes encoding the putative capsule and contains 166 SNPs.Distribution of SNPs along the chromosome of (10.54 MB TIF)Click here for additional data file.Figure S5L. longbeachae proteins identified in the genome of strain NSW150. PFAM was used to align the different sequences (http://pfam.sanger.ac.uk/).Aminoacid alignment of the RAS-domains of different (14.63 MB TIF)Click here for additional data file.Figure S6L. longbeachae Sg1 and Sg2 using the ARTEMIS comparison tool. Note the nearly perfect alignment of the four segments with only two regions differing between Sg1 and Sg2. Furthermore, the putative LPS-coding region of the two strains of the same Sg line perfectly up with a over 90% nucleotide identity. Specific regions and the predicted proteins encoded are depicted below.Alignment of the putative LPS-encoding region of (8.74 MB TIF)Click here for additional data file.Table S1L. longbeachae NSW150 protein coding genes and their distribution within functional categories.(0.03 MB DOC)Click here for additional data file.Table S2L. longbeachae without orthologues in any of the four sequenced L. pneumophila genomes.Specific genes of (0.50 MB DOC)Click here for additional data file.Table S3L. pneumophila in L. longbeachae.Distribution of known and predicted Dot/Icm substrates of (0.31 MB DOC)Click here for additional data file.Table S4L. longbeachae and its comparison to L. pneumophila Paris.Putative capsule and LPS encoding genes in (0.14 MB DOC)Click here for additional data file.Table S5L. longbeachae.Analysis of the FlgD, FleR/S, and FliA/FleN encoding regions in (0.05 MB DOC)Click here for additional data file.Table S6L. longbeachae and their orthologs in L. pneumophila.Transcriptional regulators identified in (0.30 MB DOC)Click here for additional data file.Table S7L. longbeachae in exponential growth phase.Genes upregulated in (0.23 MB DOC)Click here for additional data file.Table S8L. longbeachae in post-exponetial growth phase.Genes upregulated in (0.35 MB DOC)Click here for additional data file.Table S9Sequence of primers used to amplify putative flagella gene encoding regions.(0.04 MB DOC)Click here for additional data file.


Klebsiella pneumoniae and he improved clinically with a seven-day course of intravenous co-amoxiclav and metronidazole. Two weeks later he was readmitted to hospital with impaired consciousness and septic shock, and died three days later in intensive care. Post mortem examination revealed bacterial meningitis and an infected pancreatic pseudocyst. Klebsiella pneumoniae was isolated from the pancreas and meninges.We report a case of a 49-year-old male with a history of chronic alcoholism and evidence of a pancreatic pseudocyst on CT scanning. He presented with a 3-days history of fever, loss of appetite and upper abdominal pain. Blood cultures grew  His CRP was 38.5 mg/L, albumin 32 g/L, bilirubin 16 umol/L, alanine-aminotransferase (ALT) 24IU/L and amylase 48 IU/L. Urinalysis revealed no nitrites or leucocytes. Blood cultures grew Klebsiella pneumoniae, resistant to ampicillin but susceptible to amoxicillin/clavulanic acid by BSAC disc diffusion methods. Abdominal CT scan with contrast revealed evidence of a pseudocyst in the pancreas but no evidence of infection. Radiologically, there was no evidence of cholelithiasis, obstruction of the common bile duct or cholecystitis. Intravenous (iv) antibiotics, amoxicillin/clavulanic acid 1.2 g, thrice daily, (tds) and metronidazole 500 mg tds, were commenced with one dose of gentamicin 400 mg (iv). Antibiotics were continued for seven days. The patient improved clinically and was discharged home. The source of his bacteraemia remained undefined.A 49-year-old man was admitted to hospital with a three day history of fever, loss of appetite and upper abdominal pain. He was of Indian origin, lived in the UK and denied any recent travel. Fifteen years previously, he was diagnosed with alcohol-related chronic pancreatitis. On admission his temperature was 38.5°C, pulse rate 142/minute, respiratory rate 24/minute and blood pressure 116/80 mmHg. He was conscious and orientated and had no meningism. Abdominal examination revealed tenderness in the right hypochondrium. White blood count was 8.5 × 102 saturation 98%, pulse 142/min and BP 140/90. White cell count was 10.6 × 109/l, haemoglobin 10.6 g/dl and platelet count 199 × 109/l. His CRP was elevated at 236.1 mg/L. Liver function tests were normal. Blood culture again grew Klebsiella pneumoniae, resistant to ampicillin. Prior to blood culture results, empirical treatment with piperacillin/tazobactam (iv) 4.5 g tds was commenced. Four hours later, the patient became unresponsive and his Glascow Coma Score (GCS) fell to 6/15. An arterial blood gas revealed metabolic acidosis with respiratory compensation. CT scan of the head was normal. The patient was transferred to ITU, intubated, ventilated and maintained on the same antibiotic regime. Two days later he died. A post mortem revealed an infected pancreatic pseudocyst and bacterial meningitis. Tissue and pus from both sites grew Klebsiella pneumoniae. Pancreatic, blood and meningeal K pneumoniae isolates were sent for typing to the UK Health Protection Agency (HPA) Enteric Pathogen laboratory. All isolates had indistinguishable serotypes (K2) and a pulse field gel electrophoresis pattern designated 'King 15'.Two weeks later the patient was readmitted with a four day history of rigors. On admission, his temperature was 40°C, OK pneumoniae causes infection of bile and urine, sterile fluids in close proximity with the gastrointestinal tract. Complications of primary sites of infection are uncommon but liver and prostatic abscesses do occur. Dissemination of K. pneumoniae to secondary sites is rare but septic arthritis, vertebral osteomyelitis and endophthalmitis have been described.Like other members of the family enterobacteriaceae, S. pneumoniae and N. meningitides are the commonest causes of bacterial meningitis. Bacterial meningitis caused by K. pneumoniae is uncommon but some cases have been reported, especially in Taiwan. Over a four-year period, 27 Taiwanese patients were diagnosed with K. pneumoniae meningitis[K. pneumoniae as a cause of bacterial meningitis rose from 8% to 18%[K. pneumoniae meningitis have occurred, predominantly in other parts of Asia [In adults, eningitis and in a8% to 18%. Outside of Asia -7 but al of Asia  and Nort of Asia -14. The  of Asia .K. pneumoniae isolates with hypermucoviscosity are, 'in-vivo', anti-opsonic, anti-phagocytic and associated with severe disease[magA and rmpA that regulate extracapsular polysaccharide synthesis, are probably the most important virulence determinants for K. pneumoniae[Phenotypically, e disease. Isolateneumoniae.K pneumoniae meningitis despite adequate and prolonged antibiotic treatment. Different choices of empirical intravenous cephalosporins do not alter clinical outcomes[Clinical outcomes are poor in cases of  outcomes. In the Pancreatic pseudocysts are an uncommon complication of pancreatitis. They arise from disruptions of the pancreatic duct due to pancreatitis and extravasation of enzymatic material. Over 75% of cases are caused by alcohol and most pseudocysts resolve spontaneously. The optimal management of pancreatic pseudocysts remains debatable although drainage should be performed when symptoms persist or complications arise. Infection occurs in approximately 10% of cases and thisK. pneumoniae bacteraemia. Remarkably, it caused bacterial meningitis following initial clinical improvement with a seven-day course of appropriate antibiotics. Had we suspected the pseudocyst was infected we would have advised drainage and a longer course of treatment. Rarely, outside Asia, and in alcoholic patients with underlying co-morbidities, virulent strains of K. pneumoniae may disseminate from primary sites of infection and cause bacterial meningitis.To our knowledge this is the first case of an infected pancreatic pseudocyst to cause The author(s) declare that they have no competing interests.AO wrote the case history and researched the discussion. MM and SL were involved in the patient's management and gave antimicrobial advice. GS performed the post mortem. All authors have read and approved the final manuscript.Written informed consent was obtained from the patient's relative for publication of this case report.


Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks.Leptospira serogroups  in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates.In this study, the O-antigen gene clusters of four epidemic Leptospira.The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of  Leptospira, a slender and flexuous spirochaete with tight coils, contribute to Leptospirosis [Leptospira genus has been divided into 20 species based on DNA-DNA hybridization studies. Pathogenic species include L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. weilii, L. santarosai, L. alexanderi and L. alstonii [Leptospira can also be classified into about 250 serovars based on the lipopolysaccharide (LPS) structure. Antigenically related serovars have been grouped into at least 24 serogroups [spirosis . The Lepalstonii . Leptosprogroups .Leptospira serotyping is performed by microscopic agglutination test (MAT) using antisera raised in rabbits against the corresponding standard references strains. This typing method is laborious and time consuming [Leptospirosis exists widely in both temperate and tropical climates and has become a serious public health threat in both developed and developing countries. Human infection results from exposure to the urine of infected animals, either directly or via contaminated soil or water,8. The consuming .E. coli [S. enterica [S. boydii [Y. pseudotuberculosis [Chemical, immunochemical and ultrastructural data on LPS show that the epitope for serovar specificity is the O-antigen ,12. ReceE. coli , S. enteenterica , S. boyd. boydii , and Y. rculosis . Target Leptospira. Compared with O-genotyping method, the results of these methods are not easy to analyze. Lacking of sequences of O-antigen gene clusters from various serogroups, this kind of O-genotyping has not been developed in Leptospira, however.DNA-based typing methods, including variable-number tandem-repeat (VNTR) typing -20, inseLeptospira were not conservative, especially in the 5'-proximal end [It has been confirmed that genetic variation in the O-antigen gene cluster underlies the structural variation in the O-antigen ,29. It himal end . In thisimal end . Analyziimal end  that tarAll strains, including 75 reference strains and 40 isolated strains, were tested by MAT with standard rabbit serum. The results are shown in additional file We assigned functions of all ORFs by comparing homology genes. Most of predicted proteins are shown to be related to O-antigen biosynthesis except for some hypothetical proteins  and wzy (encode O-antigen polymerase), are O-genotyping targets, usually. Previous analysis of the O-antigen of Leptospira showed that the biosynthesis of LPS in Leptospira is a Wzy-dependent pathway [For typing bacteria, several different approaches have been used in bacteria -17. Thes pathway ,33. In c pathway , our comIn silico analysis of sequence of O-antigen clusters in 8 representative strains  showed that several glycosyltransferase genes and sugar synthesis genes may be serogroup-specific. Primer pairs were designed to target these genes and PCR were performed.To identify highly specific genes for PCR typing, we analyzed all predicted ORFs by the BLAST program. First, we selected genes that exhibit less than 70% amino acid similarity with their counterpart genes. Second, we compared these selected genes with draft data generated by 454 sequencing and discarded genes with more than 70% nucleotide similarity to any sequence in the draft data. Leptospira are available, only six serogroups of strains have been discriminated so far. There are also six strains cannot be discriminated by both MAT and O-genotyping in clinical isolates. We proposed that they are from other serogroups which beyond the field we can characterize.Analyzing the PCR products, we excluded primer pairs that could generate false-positive results in strains belonging to other serogroups and selected primer pairs that could discriminate as many strains belonging to the serogroups to be tested as possible. The primer pairs listed in Table Leptospira O-antigen-encoding genes, as a credible and convenient method for epidemiological research. To our knowledge, this work is the first to discriminate serogroups of leptospira based on the presence or absence of a PCR product.We found that six O-antigen-specific genes can be used to discriminate certain serogroups. We verified this DNA-based typing approach, which based on detecting The reference strains and clinical strains are listed in additional file Leptospira cell suspensions (representing 18 serogroups) were added to serially diluted standard hyperimmune rabbit serum  in 6-well flat-bottom microtiter plates and incubated at 37°C for 1 h. Agglutination was examined by dark-field microscopy at 100× magnification. The reported titer was calculated as the reciprocal of the highest dilution of serum that agglutinated at least 50% of the cells for each serovar used. Serogroups (serovars in parentheses) included in the antigen panel were as follows: Australis , Autumnalis , Ballum , Bataviae (Bataviae), Canicola (Canicola), Celledoni (Anhoa), Grippotyphosa (Grippotyphosa), Hebdomadis (Hebdomadis), Icterohaemorrhagiae (Lai), Javanica (Javanica), Manhao (Qingshui), Mini (Mini), Pomona (Pomona), Pyrogenes (Pyrogenes), Sejroe (Wolffi), and Tarassovi (Tarassovi).The MAT was performed according to the standard procedure  with minhttp://opal.biology.gatech.edu/GeneMark/. ORFs were assigned functions based on a comparison with the most significant homologues in the NCBI databases and are summarized in the supplemental material.Genomic DNA was prepared with a bacterial DNA minikit  as previously described . The gen2, 100 mM deoxynucleoside triphosphates, 0.1 μM of each primer, 2.5 U Taq DNA polymerase (Takara), 50 ng template DNA and PCR-grade water. Thermal PCR conditions were as follow: initial denaturation, 95°C for 2 min; 30 cycles of 30 s at 95°C (denaturation), 30 s  at temperatures varying according to the Tm of the primer pair (annealing temperatures are listed in Table The primers for the serogroup-specific PCR are listed in Table leptospira strains of S. enteritidis H9812 and S. aureus N315.The specificity of each PCR was assessed using 75 reference strains, 40 isolates and the non-Nucleotide sequences are available under the following accession numbers: O-antigen gene clusters of strains Gui44, Lin4, Lin6 and C401 are FJ976886, FJ976887, FJ976888 and FJ976889, respectively.CSC and XKG designed the research project and prepared the manuscript. CSC, YZZ and ZY carried out sequencing and data analysis. XFX and XGJ performed the strains culture and MAT. XLL, PH and JHQ performed PCR assays. GPZ and SYW participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Table S1: Results of reference strains discriminated with O-genotyping. Details about 75 reference strains and O-genotyping results are included in this table.Click here for fileTable S2: Results of clinical strains discriminated with O-genotyping. Details about 40 clinical strains and O-genotyping results are included in this table.Click here for fileTables S3-S6. Table S3: Putative genes in the L. interrogans serogroup Canicola serovar Canicola str.gui44 O-antigne gene clusterDetails about putative genes in the L. interrogans serogroup Canicola serovar Canicola str.gui44 O-antigne gene cluster are included in this table. Table S4: Putative genes in the L. interrogans serogroup Autumnalis serovar Autumnalis str.lin4 O-antigne gene clusterDetails about putative genes in the L. interrogans serogroup Autumnalis serovar Autumnalis str.lin4 O-antigne gene cluster are included in this table. Table S5: Putative genes in the L. interrogans serogroup Grippotyphosa serovar Linhai str.lin6 O-antigne gene clusterDetails about putative genes in the L. interrogans serogroup Grippotyphosa serovar Linhai str.lin6 O-antigne gene cluster are included in this table. Table S6: Putative genes in the L. interrogans serogroup Hebdomadis serovar Hebdomadis str.C401 O-antigne gene cluster. Details about putative genes in the L. interrogans serogroup Hebdomadis serovar Hebdomadis str.C401 O-antigne gene cluster are included in this table.Click here for file


Mycobacterium avium subsp. hominissuis (MAH), an agent of Mycobacterium avium complex (MAC) lung disease, is often mosaic and is composed of chromosomal segments originating from different lineages. This makes it difficult to infer the MAH lineage in a simple experimental set-up. To overcome this difficulty, we sought to identify chromosomal marker genes containing lineage-specific alleles by genome data mining.The rapid identification of lineage remains a challenge in the genotyping of clinical isolates of recombinogenic pathogens. The chromosome of M. avium subspecies were distinguishable. A recombination-cold region of 116 kb contains an insertion hotspot and is flanked by a mammalian cell-entry protein operon where allelic variants have previously been reported to occur. Hence, we speculate that the acquisition of lineage- or strain-specific insertions has introduced homology breaks in the chromosome, thereby reducing the chance of interlineage recombination.We conducted genetic population structure analysis, phylogenetic analysis, and a survey of historical recombination using data from 125 global MAH isolates. Six MAH lineages  were identified in the current dataset. One P-450 gene (locus_tag MAH_0788/MAV_0940) in the recombination-cold region was found to have multiple alleles that could discriminate five lineages. By combining the information about allele type from one additional gene, the six MAH lineages as well as other M. avium. The single nucleotide polymorphism typing approach targeting multiallelic loci in recombination-cold regions will facilitate the epidemiological study of MAC, and may also be useful for equivalent studies of other nontuberculous mycobacteria potentially carrying mosaic genomes.The allele types of the newly identified marker genes can be used to predict major lineages of  Mycobacterium avium complex (MAC), consisting of M. avium, M. intracellualare, and several rarely reported taxa, is the most common non-tuberculous mycobacterial group causing pulmonary disease in Asia and Europe . C. C33]. Cference) , and , and MAH_0809 , but not in MAH_2714   has been used as a subspecies identification marker in the genotyping of MAC isolates [hsp65 gene tree, the isolates were not clustered per lineage  tended to cluster at one node in the trees of MAH_0766 , MAH_0771 , MAH_0788   and the sugA gene encoding the trehalose ABC transporter , could additively inform the core genome lineage (or association of BAPS subpopulations) of MAH isolates as well as M. avium subspecies.Because the members of SC2 and SC4 showed identical sequences in the MAH_0788 ortholog sequence, the MAH_0788 ortholog nucleotide sequence alone cannot distinguish between SC2 and SC4. Furthermore, SC4 members do not carry unique SNPs in software , which pHi score < − 2.0) includes a previously reported insertion hotspot containing clustered tRNA genes and an East Asian lineage-unique segment , but the equivalent segments are missing in M. ulcerans, M. abscessus, M. tuberculosis, M. canettii, and M. smegmatis , each from a different lineage. The 116 kb recombination-cold region  and EA2 , were predominantly found in clinical isolates in Japan, and two others, SC2  and SC4 (with a large number of recombinations), were commonly found in clinical and environmental isolates in Germany. SC3 and SC1 have also frequently been found in the USA  typing. This enables the identification of MAC taxa at the subspecies level, but not at the lineage level , located in the recombination-cold region between the SC2 and SC4 populations, enabled the distinction between SC2, SC4, and MAA. As lineage indicative SNPs are distributed in close proximity within MAH_0788, PCR amplification and Sanger sequencing of an approximately 600 bp-long segment covering positions 705 to 1269 will discriminate the five major lineages. Similarly, sequencing a short segment in MAV_1375, from position 78 to 168, will distinguish SC2 from SC4. We have also listed the secondary marker genes, the MAH_0771 locus for five-lineage distinction, and the MAV_2820 (MAH_2466) locus for SC2 and SC4 distinction  and its neighboring genes, subB (ferredoxin) and phbB (acetoacetyl-CoA reductase), as the region containing these genes is conserved in at least five NTM species for which the complete chromosome sequences are available. It remains to be determined whether a recombination-free or cold region also exists in the genomes of other mycobacterial species, which are reported to possess mosaic genomes [The recombination cold-region identified in the present study contains previously reported clustered tRNA genes that potentially serve as an insertion hotspot . This reeumoniae . We newleumoniae . It is rs aureus . Moreovee genome . However genomes –49.Six MAH lineages were identified in the currently available data of global MAH isolates. We found that the P-450 gene in the recombination-cold region contained allelic variations that can distinguish between five major lineages, despite the genome-wide occurrence of interlineage recombination. Furthermore, allele information of one additional gene allowed the distinction between six lineages. We propose the minimum-SNP typing approach focusing on a multiallelic locus in a recombination-cold region as a novel epidemiological genotyping method that can easily be applied to all bacteria without obvious virulence markers.M. avium subsp. avium (MAA), subsp. silvaticum (MAS), and subsp. paratuberculosis (MAP), genome sequences of a type or well-known strain ATCC 25291, ATCC 49884, and K-10 were obtained from RefSeq . We used the sequences from strains AH16 (GenBank accession no. CP012885.1), ATCC12478 (CP006835.1), ATCC 13950 (RefSeq accession no. NC_016946.1), M (NC_010612.1), Agy99 (NC_008611.1), ATCC 19977 (NC_010397.1), H37Rv (NC_000962.2), CIPT 140010059 (NC_015848.1), and MC2 155 (NC_008596.1) as the reference sequences for M. chimaera, M. kansasii, M. intracellulare, M. marinum, M. ulcerans, M. abscessus, M. tuberculosis, M. canettii, and M. smegmatis.Genomic and associated metadata of 125 MAH isolates were retrieved from the PATRIC database in February 2018 . InformaM. avium lineage was defined using a non-phylogenetic method, namely BAPS and its related program, fastGEAR, which clusters individuals based on genetic linkage patterns after detecting recombinations between BAPS subpopulation groups called sequence clusters (SCs) [The rs (SCs) , 32. BAPrs (SCs) . It was rs (SCs) , 50, allrs (SCs) . To geners (SCs) . Polymorrs (SCs) . The outrs (SCs)  to inferFiltered polymorphic sites were combined with intervening reference genome sequences in a multi-fasta format, and used as input for fastGEAR (running on MATLAB compiler runtime v 9.0.1). In the lineage estimation by fastGEAR, the distance between two isolates is defined as 1 – the proportion of chromosomal fragments sharing the ancestry (PSA), and the lineage is determined as a result of hierarchical clustering . fastGEAHi statistic known as the realized recombination rate (intensity of recombination relative to genome average) inferred by OrderedPainting [Hi < − 2.0 (sites ranked in the bottom 2.55%) were regarded as recombination-cold regions, and were used to screen for single copy core orthologous genes (see below) embedded within these regions.To evaluate recombination frequency throughout the chromosome, we used the Painting . In the A total of 31,598 filtered polymorphic sites were obtained for the core genome of 54 isolates consisting of only SC2 and SC4 members, using the complete genome sequence of strain Mah104 as a reference. Filtered polymorphic sites of this SC2/SC4 dataset, obtained as described above, were used for phylogenetic analysis and recombination detection using Gubbins software . This soTo identify core genes in the MAH population, homologous gene clustering was performed using the CD-HIT algorithm implemented in Roary v 3.7.0 software . The 125H [Codon alignments were analyzed to calculate the average pairwise nucleotide diversity, haplotype diversity, and number of alleles in the alignment using the PopGenome package of R . Genes fH ) > 0.6 ahttp://tree.bio.ed.ac.uk/software/figtree/). The gene trees where most of the isolates of one lineage clustered on a single node were selected as the trees of the lineage marker gene.The codon alignments of marker gene candidates were further converted to Phylip format using a Perl script, and used to infer the maximum likelihood phylogenetic tree in PhyML based on the GTR + G model . The allThe structures of completely sequenced chromosomes were compared and visualized in GenomeMatcher software  using thAdditional file 1. Information of data sources used in this study.Additional file 2. Difference in the number and total fragment lengths of recent recombination events between MAH lineages.Additional file 3. Phylogenetic trees based on codon alignments of marker gene candidates and hsp65. (i) MAH_0788/MAV_0940 ortholog, (ii) MAH_0771/MAV_0930 ortholog, (iii) MAH_0766/MAV_0925 ortholog, (iv) MAA_0809/MAV_0960 ortholog, (v) MAH_2714/ MAV_2410 ortholog (inadequate marker gene), (vi) hsp65 ortholog, (vii) MAV_1375 ortholog, (viii) MAV_2820 ortholog Trees were constructed using the GTR + G model in PhylML with 100 times bootstrapping run. Values by the branch indicate the number of bootstrap supports. Allele of strain A5 was used as an out group for the analysis in panels i–vi. The out group was not used for tree construction in panels vii and viii. Alignment information is shown by the tree as follows: n.allele, number of alleles in the alignment (population); nuc.diversity, average pairwise nucleotide diversity (number of site differences) in the alignment; hap.diversity, probability of haplotype  differences in the alignment; alignment length, length of alignment without gaps.Additional file 4. Alignment of lineage-specific alleles. (A) MAH_0788/MAV_0940 locus (cinA/P-450 gene). (B) MAH_1236/MAV_1375 locus (sugA gene). Polymorphic sites were indicated by distinct color. Sites used to distinguish among M. avium lineages were indicated by asterisks under the alignment.Additional file 5. Phylogeny inference of SC2 and SC4 members by Gubbins. (Left) Phylogenetic tree based on recombination-tract free alignments. Scale bar indicate the number of SNPs. SC2 members were shown in green, while SC4 members were shown in magenta. (Right) Location of recombination tracts. Recombination tracts introduced in internal braches were shown in red. Recombination tracts unique to terminal branch is shown in blue. (PDF 822 kb)Additional file 6. Insertions near the recombination-cold region. (A) Similarity between two chromosomes. Locations of the recombination-cold region and the MCE operon locus 3 are indicated by white horizontal lines. The similarity between two genomes was determined by the blastn algorithm implemented in GenomeMatcher software [int) or excisionase (xis). The insert in strain H87 contain 31 copies of recombinase genes. (C) Conservation of marker genes in the MAH recombination-cold region in M. chimaera, M. intracellulare, M. marinum, and M. kansasii. (D) Absence of marker genes in M. abscessus, M. ulcerans, M. tuberculosis, M. canettii, and M. smegmatis. (PDF 7032 kb)software . Genomic


Meloidogyne incognita was published in 2008 and additional genome and transcriptome data became available later on. However, lack of a publically available annotation for M. incognita genome and transcriptome(s) limits the use of this data for functional and comparative genomics by the interested researchers. Here we present a comprehensive annotation for the M. incognita proteome data available at INRA Meloidogyne Genomic Resources page (https://meloidogyne.inra.fr/Downloads/Meloidogyne-incognita-V2-2017) and European Nucleotide Archive (ENA) (accession number: ERP009887) using a multi-pronged approach.Root-knot nematodes are devastating pathogens of crop plants. The draft genome of southern root-knot nematode  As compared to the 67.7% of the total proteins annotated by the standard approach using RefSeq database, the multi-pronged approach resulted in annotation of 73% of the proteome.A comprehensive annotation for the •M. incognita proteome data can be helpful for a large number of researchers who are using RNA-seq data for understanding the biology of M. incognita for applied purposes. The availability and access of this annotation would help the researchers globally in a manner that they need not assemble their RNA-Seq data to construct transcriptome for various experiments and then annotate it. Instead, the researchers can simply map their RNA-Seq data to the available cDNA using recent tools such as Kallisto, Salmon, Sailfish etc., and use the provided annotation to interpret their experimental findings quickly.The annotation of •Present annotation would save significant time and computing resources required for the assembly and annotation, and allow the researchers to focus on answering the biological questions faster. This would be highly beneficial for development of novel strategies to combat this global pest menace.1M. incognita was published in 2008 M. incognitaper se is not available in the public databases, thereby limiting the direct use of sequence information by the interested researchers for making sense of their own experiment-specific transcriptome data. The latest genome analysis of M. incognita in 2017 C. elegans and Nematoda proteins followed by NCBI-FLINK based annotation for the Gene Ontology (GO), 29,621 proteins could be annotated  was the most enriched protein domain in M. incognita, followed by protein kinase-like (IPR011009) and protein kinase domains (IPRO00719)  . The anaRO00719) . The annM. incognita proteome dataset annotated 30,400 proteins. These proteins were then assembled into respective KOG functional classes. The highest number of proteins (3984) grouped into signal transduction mechanisms, followed by 3937 proteins enriched in cell motility category (M. incognita proteome (clade IV) to protein sequence data of Trichinella spiralis (clade I), Ascaris lumbricoides (clade III), and Caenorhabditis elegans (clade V), and with plant parasitic nematodes Globodera pallida and M. hapla (both clade IV). M. incognita shared 3650 ortholog protein families with T. spiralis, 6054 with C. elegans and 6149 with A. lumbricoides, whereas 3326 ortholog protein families were common to all the compared nematodes (M. incognita genome/transcriptome. We could find 45 RNAi effector proteins whereas 27 have been reported earlier M. incognita effector proteins were identified (Using EuKaryotic Orthologous Groups (KOG) to find ortholog and paralog in category . A searcematodes A. Howeveematodes B. Lastlyentified .Fig. 5ThM. incognita proteome dataset to 73% as compared to 67.7% by the standard RefSeq based method  C. elegans and Nematoda proteins with a cut-off set to e-value = 10−3 and query coverage of > 60% https://www.ncbi.nlm.nih.gov/Structure/flink/flink.cgi] through which the genes were assigned GO IDs for each of three ontology terms  and retrieved the KEGG pathways. To enrich and refine the obtained annotation further, domain level analysis was done using InterProScan −3. To better understand functions and interactions, all annotated genes were also mapped against the KEGG database for a pathway-based analysis using the online KEGG Automatic Annotation Server (KAAS) (http://www.genome.jp/kegg/kaas/). KEGG Orthology (KO) assignment was obtained using the GHOSTX which is a homology search tool, detects remote homologues like BLAST but 100 times more efficient than BLAST and bi-directional best hit (BBH) method A. lumbricoides, C. elegans, G. pallida, M. hapla, and T. spiralis were downloaded from Wormbase ParaSite (http://parasite.wormbase.org/index.html). OrthoMCL was run with default parameters. The proteins belonging to RNAi pathway were analysed by comparing with C. elegans RNAi pathway homologues. The carbohydrate active enzymes (CAZymes) were identified by using Carbohydrate Active Enzymes database (http://www.cazy.org/) M. incognita proteome at e-value = 10−3, query coverage > 60%, and percent identity > 90%.The protein sequence file used for annotation was obtained from the INRA