To control the double burden of communicable and non-communicable diseases (NCDs), in the developing world, understanding the patterns of morbidity and healthcare-seeking is critical. The objective of this cross-sectional study was to determine the distribution, predictors and inter-relationship of perceived morbidity and related healthcare-seeking behavior in a poor-resource setting.Between October 2013 and July 2014, 43999 consenting subjects were recruited from 10107 households in Malda district of West Bengal state in India, through multistage random sampling, using probability proportional-to-size. Information on socio-demographics, behaviors, recent ailments, perceived severity and healthcare-seeking were analyzed in SAS-9.3.2.Pri)=0.76, 95% confidence interval=0.71-0.83) and for Govt. healthcare provider(AORGovt)=0.80(0.68-0.95)], females [AORGovt=0.80(0.73-0.88)], Muslims [AORPri=0.85(0.69-0.76) and AORGovt=0.92(0.87-0.96)], backward castes [AORGovt=0.93(0.91-0.96)] and rural residents [AORPri=0.82(0.75-0.89) and AORGovt=0.72(0.64-0.81)] had lower odds of visiting qualified practitioners. Apparently less severe NCDs , gastrointestinal [AORPri=0.28(0.24-0.33) & AORGovt=0.69(0.58-0.81)], respiratory [AORPri=0.35(0.32-0.39) & AORGovt=0.46(0.41-0.52)] and skin infections [AORPri=0.65(0.55-0.77)] were also less often treated by qualified practitioners. Better education [AORPri=1.91(1.65-2.22) for ≥graduation], sanitation [AORPri=1.58(1.42-1.75)] and access to safe water [AORPri=1.33(1.05-1.67)] were associated with healthcare-seeking from qualified private practitioners. Longstanding NCDs  and serious infections [typhoid: AORPri=2.86(2.04-4.03)] were also more commonly treated by qualified private practitioners. Potential limitations included temporal ambiguity, reverse causation, generalizability issues and misclassification.Recent illnesses were reported by 55.91% (n=24600) participants. Among diagnosed ailments (n=23626), 50.92% (n=12031) were NCDs. Respiratory ), gastrointestinal  and musculoskeletal  problems were predominant. Non-qualified practitioners treated 53.16% (n=13074) episodes. Older children/adolescents [adjusted odds ratio for private healthcare providers(AORIn this poor-resource setting with high morbidity, ailments and their perceived severity were important predictors for healthcare-seeking. Interventions to improve awareness and healthcare-seeking among under-privileged and vulnerable population with efforts to improve the knowledge and practice of non-qualified practitioners probably required urgently. Demographic ageing, unplanned urbanization and unhealthy lifestyles are the major contributors for the changing pattern of disease in recent years, from communicable to non-communicable diseases (NCDs), globally.–3 This eDespite remarkable progress in socio-economic development and having an overarching aim of addressing the health needs through several comprehensive programs, health outcomes in India remained poor. During 2012, approximately 60% deaths were attributed to NCDs  and 28% to communicable, maternal, perinatal and nutritional conditions in this country.,11 EvideIndividual healthcare-seeking pattern in a community is determined by complex interrelationships between socio-economic and physical environment along with individual characteristics and behaviors. Thus heaRelevant researches on morbidity and healthcare-seeking ever conducted in India were mostly limited to urban areas of southern and western part while eastern region remained largely understudied. Malda isHence, a community-based cross-sectional study was designed involving a representative population of Malda to understand the distribution of the perceived morbidity and healthcare-seeking behavior, their predictors and inter-relationship.The study protocol was reviewed and approved by the Ethics Committee of the National Institute of Cholera and Enteric Diseases, Kolkata. Written informed consent left thumb impression  was obtained from residents older than 18 years and from the guardians of residents aged 1 to 17 years. Written assent was additionally obtained from residents aged 12 to 17 years.Based on the 2011 census data, the urban area of the Malda district was divided into two broad urban administrative divisions termed as Municipalities . Each Municipality was further subdivided into smaller administrative units called Wards . Using probability proportional to size (PPS) determined by the total number of households in the Wards, 4 Wards in Old Malda and 12 Wards in English Bazar were selected randomly. The rural area of the district consisted of 3701 villages and 27 rural towns from which similarly using PPS, 25 villages/census towns were selected randomly. Using an exhaustive house-list of the urban and rural areas, each selected municipal ward and village/rural town was categorized into several segments (considered as Primary Sampling Unit: PSU), each having 125 households (defined as those who shared the cooking-pot in each dwelling). Next, 4012 urban and 6095 rural households (maintaining the population ratio) were selected from the whole district, through multistage random sampling, using PPS. Thus, 16 municipal wards in urban and 24 villages/towns in rural area were selected. In each selected ward/village from the list of segments two were selected randomly and all households were surveyed there after collecting written informed consent from the residents.All the individuals residing in the selected households were interviewed at home by trained interviewers, using a structured, pre-tested, bi-lingual  questionnaire. Information was collected on socio-demographic and related variables such as age, gender, religion, caste, education level and occupation of the household members, maximum education level among adults in the house, house ownership, residential area, type and location of water source, water treatment at home, material used for cooking and domestic light source. Housing type was classified as Kachha , Pacca  and Semi-pacca . Sanitation level of toilet use practices were categorized as poor (if the household had no toilet and the members used open space/field/jungle for defecation), good (for households having toilets with flush to piped sewer system/flush to septic tank) and all others  as average.Based on the information regarding household assets (enquired using an appropriate list of assets), number of cattle, goats/sheep, poultry, place for keeping them and the aforementioned household information, wealth index was calculated by using relative weights for each and then the cumulative wealth index scores were log-transformed and divided into quintiles of socio-economic status: SES  based on the percentile distribution.For all the members of the selected households, information regarding last three episodes of ailments that forced them to seek some healthcare services within last two months was collected. Occurrence, perceived severity and healthcare-seeking behavior  regarding specific NCDs like: acid peptic disorder (APD) or peptic ulcer disorder (PUD), chronic obstructive pulmonary disease (COPD), hypertension (HTN), diabetes mellitus (DM), anemia and osteoarthritis (OA) as well as communicable diseases like: gastroenteritis, respiratory tract infection (RTI), typhoid and skin infections were also collected.Thus between October 2013 and July 2014, 43999 individuals (with approximately 8% non-response) were recruited from 10107 households  and collected data were analyzed using Statistical Analysis System (SAS) version 9.3.2. Distribution of the socio-demographic characteristics, morbidity pattern and healthcare-seeking were determined by conducting descriptive analyses using survey frequency procedure to determine overall and stratified frequencies, proportions and corresponding 95% confidence intervals (95%CI). Bivariate and multivariate logistic regression analyses were next conducted to determine unadjusted (OR) and adjusted  odds ratios (AOR) as the measures of association (with corresponding 95%CIs) between study variables. Multinomial logistic regressions  were useAmong 43999 subjects, majorities were aged 18–40 yrs , male , Hindu , general caste  and educated up to secondary level . For 38.82% (n = 17080). Maximum adult education in the household was also up to secondary level, 95.73% (n = 42122) stayed in own house, 39.60% (n = 15888) were in sedentary work and 62.60% (n = 27543) lived in rural areas. Only 5.31% (n = 2336) were drinking safe water, 50.32% (n = 22140) had to bring drinking water from outside, 95.06% (n = 41825) were not doing any water treatment at home, 29.08% (n = 12794) were using gas/electricity for cooking, 27.20% (n = 11961) were living in pacca houses. Electricity was the source of lighting at home for 88.87% (n = 39098), regarding toilet use 30.63% (n = 13475) had good sanitary practices and overall 19.44% (n = 8553) belonged to upper SES. Overall and stratified  socio-demographic distribution are presented in Regarding the distribution of self-perceived most recent (within past 2 month) morbidity, 44.09% (n = 19399) did not suffer from any such recently while for 17.28% (n = 7605), 13.48% (n = 5929) and 6.25% (n = 2749) residents the most recent morbidity was related to respiratory, gastrointestinal and musculoskeletal system respectively. Among the most recent ailments, NCDs were 50.92% (n = 12031), 53.16% (n = 13074) episodes were treated by non-qualified practitioners, 34.02% (n = 8368) by qualified practitioner from private sector and only 12.82% (n = 3153) by qualified practitioner from Govt. sector. Non-qualified practitioners were treating more communicable diseases compared to NCDs [57.52% (n = 7194) vs. 42.48% (n = 5313)]. Based on last three healthcare-seeking episodes, among specific ailments (suffered or not), 19.01% (n = 6734) suffered from RTI, 8.18% (n = 2554) had PUD/APD, 6.45% (n = 1977) experienced gastroenteritis while 3.60% (n = 1070) had some skin problems. Among subjects visiting nonqualified practitioners, only 16.85% (n = 1551) perceived their ailments as severe while this fraction for private sector qualified practitioners, was 40.85% (n = 1829). 41–60 = 2.01(1.82–2.23), AOR>60 = 2.86(2.41–3.39)], COPD , HTN , DM , OA , gastroenteritis  and RTI .Association of socio-demographics with morbidity and healthcare-seeking are presented in Tables Compared to males, females had higher odds of suffering from APD [AOR = 1.60(1.45–1.77)], HTN [AOR = 1.53(1.28–1.83)], anemia [AOR = 16.26(10.75–24.59)] and OA [AOR = 2.58(2.07–3.22)] and lower odds for COPD [AOR = 0.59(0.48–0.73)] and DM [AOR = 0.73(0.57–0.92)]. Muslims suffered less from APD [AOR = 0.77(0.69–0.87)] and gastroenteritis [AOR = 0.86(0.74–0.99)] but more from DM [AOR = 1.40(1.06–1.85)], typhoid [AOR = 1.80(1.31–2.46)] and skin infections [AOR = 1.25(1.06–1.49)] than Hindus. With reference to general, backward castes suffered less from APD [AOR = 0.74(0.67–0.81)], HTN [AOR = 0.82(0.69–0.97)] and anemia [AOR = 0.77(0.60–0.98)] but more from typhoid [AOR = 1.93(1.40–2.67)].Higher Secondary = 0.57(0.47–0.70), AOR≥Graduation = 0.57(0.46–0.70)], COPD , anemia [AOR≥Graduation = 0.48(0.26–0.87)], OA , gastroenteritis  and RTI .Compared to illiterates, higher familial education was associated with lower likelihood of APD [AORHard workers (reference = Sedentary) were more prone to APD [AOR = 1.45(1.24–1.71)] and anemia [AOR = 1.89(1.17–3.04)] but less vulnerable to COPD [AOR = 0.53(0.40–0.69)] and HTN [AOR = 0.60(0.46–0.77)]. Rural residents, compared to urban, were less likely to have HTN [AOR = 0.54(0.43–0.67)] but more prone to OA [AOR = 1.47(1.15–1.87)], gastroenteritis [AOR = 1.76(1.50–2.07)], typhoid [AOR = 2.85(1.86–4.38)], RTI [AOR = 1.27(1.16–1.38)] and skin infection [AOR = 1.45(1.19–1.77)].Upper middle = 0.64(0.44–0.92), AORUpper = 0.59(0.40–0.88)], gastroenteritis [AORUpper = 0.72(0.60–0.86)], typhoid [AORUpper = 0.63(0.41–0.99)], RTI  and skin infections . Higher SES also seemed to be associated with higher odds of having HTN  and DM . , AOR>60 years = 4.25(3.61–5.00)], familial education , sanitation level regarding toilet use practices  and SES . Perception of severity was lower among hard-workers  and rural residents . ]. 5–18 = 2.51(2.22–2.83)], and older residents were less likely  to suffer from communicable diseases (reference = NCD). Compared to respective reference groups, females [AOR = 0.72(0.67–0.77)], residents having higher familial education [AOR = 0.71(0.62–0.83)] and higher SES [AOR = 0.84(0.75–0.92)] had lower likelihood of communicable diseases. Muslims [AOR = 1.18(1.09–1.28)], persons belonging to backward [AOR = 1.15(1.08–1.24)] caste, those who had higher individual education [AOR≥Graduation = 1.38(1.13–1.69)] and rural [AOR = 1.47(1.36–1.60)] residents suffered more from communicable diseases. , AORGovt = 0.80(0.68–0.95)], females [AORGovt = 0.80(0.73–0.88)], Muslim religion , backward caste [AORGovt = 0.93(0.91–0.96)], physically demanding occupation  and rural residence  were associated with lower likelihood of visiting qualified practitioners . Age > 40 years , higher individual [for higher secondary: AORPrivate = 1.42(1.19–1.69) and for ≥ Graduation: AORPrivate = 1.30(1.06–1.59)] and familial education [for higher secondary: AORPrivate = 1.26(1.13–1.41) and for ≥ Graduation: AORPrivate = 1.40(1.22–1.62)], better sanitary practices [for average practice: AORPrivate = 1.17(1.07–1.28) and for good practice: AORPrivate = 1.58(1.42–1.75)] and higher SES [for Upper middle: AORPrivate = 1.59(1.43–1.77) and for Upper: AORPrivate = 1.51(1.35–1.69)] were associated with higher odds of seeking care from qualified  practitioners. , AORGovt = 0.36(0.31–0.43)], OA , gastroenteritis , RTI , skin infections [AORPrivate = 0.65(0.55–0.77)]. Those who had COPD , HTN , DM , typhoid and NCDs  were more likely to visit qualified practitioners. Higher self-perceived disease severity  was also positively associated with visiting qualified practitioners.  of the participants suffered from some recent morbidity while respiratory, gastrointestinal and musculoskeletal diseases were most common. This observed burden of self-perceived morbidity was considerably higher than previously reported values (ranged between 27% and 48%) in similar settings.–29 StudiMore than half of the ailments were treated by non-qualified practitioners, which raised a few concerns. Only about 13% visited qualified physicians from Govt. sector. The scenario seemed similar to that of other parts of India, Vietnam and Bangladesh ,28,32 buAmong specific ailments, RTI was perceived to be the commonest, followed by APD, gastroenteritis and skin problem. Contrary to some other study, perceived burden of HTN and DM were found to be relatively lower. May be sWhile more than two third subjects considered their ailments as less severe, those who perceived the severity, visited qualified doctors especially in private sector. The perceived severity probably helped them to overcome the potential barriers  in better healthcare-seeking.,35,38,39Corroborating with prior observation in similar settings elsewhere, children and adolescents were less likely to suffer from NCDs like APD, COPD, HTN, DM, anemia and OA but more from RTI, gastroenteritis and skin infection.,35,36,40Similar to some previous observation, females had higher likelihood of having APD, anemia and OA but less likely to suffer from COPD and DM –28 but gSupporting some prior evidences  and contOccupation with hard work was associated with higher odds of APD and anemia but lower odds of COPD and HTN. Physical exertion, work environment and appropriate nutrition probably were the key factors. Negative association between physical activity and HTN was well-established in prior studies.Rural residents compared to urban were less prone to HTN  but they had higher likelihood of having OA, gastroenteritis, typhoid, RTI and skin infection most likely due to lifestyle related factors, less awareness, poor hygiene and inappropriate sanitation. Urban preponderance of HTN was also reported previously  althoughDrinking safer water was associated with higher perceived burden of HTN and DM. Subjects having better sanitary practices regarding toilet use were also suffering more from APD, HTN, DM and OA. Health awareness and knowledge as probably a confounder here that positively influenced both better practices  and improved perception. Reverse causation might also be a possibility (being diagnosed with the disease resulted in better sanitation and hygiene). Drinking safer water and practicing better sanitation regarding toilet use seemed to be also associated with lower likelihood of suffering from gastroenteritis, typhoid, RTI and skin infections.Alike prior studies, we also found that, residents having comparatively higher SES were less likely to suffer from anemia, gastroenteritis, typhoid, RTI and skin infections ,29 but sPerceived severity of ailments was higher among those with higher age, better familial education, improved sanitation and upper SES and lower among hard-workers and rural residents had. Higher severity of self-perceived morbidity among elderly was also reported previously. Thus perCompared to those aged between 18–40 years, 5–18 years age group were more likely, and older residents were less likely to suffer from communicable diseases than NCDs. Female gender, better familial education and higher SES were negatively associated with risk of communicable diseases. Muslim religion, backward caste, higher individual education and rural residents had higher odds of suffering from communicable diseases.Socio-demographic predictors of Healthcare-seeking behavior in our study were quite similar to those reported from other parts of the world as well as India with some variations. While elderly subjects commonly visited qualified private and govt. sector physicians, older chSubjects suffering from NCDs were more likely to visit qualified practitioners especially the private sector. Alike soSelf-perceived severity of ailments were positively associated with odds of visiting qualified practitioners more so in private sector and this finding also supported prior evidences.,36,40 ThDespite efficient sampling design, use of detailed questionnaire and robust analyses, our study had certain limitations. Like any other cross-sectional study, causal interpretation of the observed associations is not recommended. Due to the potential vulnerability to temporal ambiguity by design, some of our observations might have suffered from reverse causation. Although self-perceived morbidity and severity are currently being considered an efficient parameter for the estimation of health needs in communities worldwide, keeping the lower literacy and potential lack of awareness in mind, the reported self-perceived morbidity pattern should only be interpreted as perceived health need of the community, not the prevalence. Residual confounding due to variables not included in our analyses could also be an issue. Information bias due to misclassification of self-reported information should always be kept in mind, especially due to the potential for differential recall. But we do not consider those to be serious issues here because we only dealt with the recent ailments, hence recall period was short and in majority of cases, medical records were consulted. Although results of our study should be extrapolated beyond the study sample with caution, still we are not worried about the generalizability of our results due to the representative nature of our study sample and very low (<8%) non-response.In this poor-resource setting, most important predictor for healthcare-seeking was the perception regarding severity and nature of ailments, while age, gender, caste, religion, familial education, SES, residential area, sanitation and hygiene influenced the morbidity pattern and relevant healthcare-seeking. Keeping the high burden of self-perceived morbidity in mind, interventions to improve physical health, awareness and care-seeking practices targeting children, elderly, females, backward castes, minority groups, illiterates, rural residents and those having lower SES, poor sanitary practices and inadequate access to safe drinking water were required urgently. Simultaneously, efforts to improve the healthcare service delivery might consider implementation of intervention targeting improvement of knowledge and practice among non-qualified practitioners in poor-resource settings where seeking healthcare services from these practitioners seemed to be a common occurrence.


Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.Among the 3989 predicted proteins from atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.The results indicate that this real-time PCR method targeting the  Mycobacterium genus is constituted of several pathogenic species, including the M. tuberculosis complex (MTC) responsible for tuberculosis , M. leprae responsible for leprosy, and non-tuberculous mycobacteria (NTM), which are environmental potentially pathogenic species causing mycobacteriosis [rrs, gyrA, gyrB, hsp65, recA, rpoB, sodA genes and 16S-23S internal transcribed spacer (ITS) genes, to detect and/or identify mycobacteria species by sequence analysis [Mycobacterium genus in clinical and environmental samples, several studies have proposed targeting different loci of the 16S rRNA gene [gyrB [rpoB[hsp65[Corynebacterium, Nocardia, Rhodococcus, Mycobacterium (CNM) group [Mycobacterium genus targets with high levels of specificity and sensitivity that will be useful for studying mycobacteria in their habitat.teriosis . Detectiteriosis . Identifanalysis . In ordeRNA gene , or othene [gyrB , rpoB[19poB[hsp65. NeverthpoB[hsp65. Indeed,poB[hsp65 which alM) group  and whicM. tuberculosis H37Rv), the number of mycobacterial sequences has considerably increased due to advances in sequencing capacity and the appearance of high throughput sequencing techniques [M. tuberculosis and M. bovis species), two strains of M. leprae, and eleven species and subspecies of pathogenic (P) and non-pathogenic (NP) NTM: M. abscessus (P), M. avium (P), M. avium subsp. paratuberculosis (P), M. gilvum (NP), M. marinum (P), M. smegmatis (NP), Mycobacterium sp. JLS (NP), Mycobacterium sp. KMS (NP), Mycobacterium sp. MCS (NP), M. ulcerans (P), M. vanbaalenii (NP), [M. intracellulare, M. kansasii, M. parascrofulaceum). This increasing number of completely sequenced mycobacterial genomes led to the development of the MycoHit software, which permits gene- and protein-level comparisons across mycobacteria species, [As new mycobacterial sequences are added into genetic databases, our knowledge of mycobacterial genomes is increasing and this may help to design new primers and probes that will be both specific and sensitive. Since the whole sequencing of the first mycobacterial genome in 1998  by Sangechniques . Today, ii (NP), -26. Morespecies, . This sospecies, . HoweverMycobacterium spp.. We compared in silico proteins of whole mycobacterial genomes with those of non-mycobacterial genomes using the MycoHit software, in order to find conserved sequences among mycobacteria that will not be shared with non-mycobacterial species. Based on the screening results a primer pair and a probe targeting the atpE gene were designed and tested by real-time PCR. This novel target proved to be totally specific and sensitive. It also offers the advantage of targeting a gene present as a single copy in the genome. Thus this new real-time PCR method appears promising for water quality survey, and should be useful for studying the ecology of mycobacteria in aquatic, terrestrial and urban environments.In this paper, we used this tool for screening sensitive and specific targets of rrs gene and ITS , and according to our strategy of genome comparison  code for proteins which present similar conformations in non-mycobacterial studied genomes , and also for the 12 non-mycobacterial genomes studied .Excluding n Figure  most of M. tuberculosis H37Rv genome , and less than 50% similarity levels in comparison with genomes (n = 12) of the other CNM group genera. As a result, among the 3989 predicted proteins of M. tuberculosis H37Rv genome , hypothetical PE or PPE family proteins (loci Rv0285 and Rv3022c), proteins coded by esxG, esxH and esxR genes in M. tuberculosis H37Rv , and proteins such as a lipoprotein coding by lppM gene (locus Rv2172c), an oxidoreductase (locus Rv0197), and a small secreted protein (locus Rv0236A).Among the 3989 predicted proteins of e Figure , about 5e Figure B displaycmaA1 gene, lipoprotein coding by lppM gene, as well as PE, PPE and proteins coded by esx genes esxG, esxH and esxR, were highly conserved in studies MTC species (tuberculosis and bovis) but very polymorphous in the 14 NTM species studied , the oxidoreductase (locus Rv0197), and the small secreted protein locus Rv0236A), are the less polymorphous among the 14 NTM species studied  whereas quantification limits were estimated at about 100 genome equivalents. In the positive collection all 31 mycobacteria species were positively detected by the real-time PCR method. This collection includes NTM species, leprae species and MTC species as tuberculosis and bovis  , and evaR Figure A. DetectatpE gene, shows a vast diversity of mycobacteria concentration, ranging from 104 to 106 ge/L in water column and neuston samples, and 105 to 106 ge/g DW (dry weight) in sediment samples. Comparison with the previously published methods targeting 16S rRNA [Mycobacteria quantification in lake samples by real-time PCR targeting 16S rRNA  shows a gyrA, gyrB, hsp65, recA, rpoB, and sodA genes are appropriate for identification purposes [gyrB [rpoB[hsp65[Helicobacter pylori show positive amplification with several Mycobacterium specific primer pairs [atpE real-time PCR method that we propose is just as specific, but more sensitive than the previously proposed rrs real-time PCR method which cannot detect some mycobacterial species [Although purposes ,4, our res [gyrB , rpoB[19poB[hsp65 genes, hpoB[hsp65. For exaer pairs . Prospec species .M. tuberculosis H37Rv, sorting proteins according to similarity requests and listing candidate proteins . Only the aptE gene could be used to design primers and a probe for mycobacteria detection. Concerning the other genes, the sequence polymorphism among NTM species did not allow designing molecular targets for Mycobacterium spp. detection. However, these genes could be of immunological or pathogenic importance. Indeed, PE and PPE family proteins represent 0.9 to 4.2% of the genome coding capacity of several mycobacteria [esx gene clusters, which encode ATP dependent specific secretion system [esx gene clusters are very small and polymorphous among genomes of the 11 NTM species compared  is exclusively conserved in the genomes of the 17 mycobacterial species studied , which codes for the ATP synthase protein subunit C. These results also showed that our strategy of target design based on MycoHit software , slow mycobacteria growth, clumping of mycobacterial cells, high hydrophobicity of mycobacteria and contamination of culture media by other fast growing environmental microorganisms [rganisms .atpE with previously described method targeting 16S rRNA, [atpE gene presents two major advantages over the method targeting rrs gene. First, the new method detects all the tested mycobacterial strains, while the method targeting rrs gene cannot detect isolates of M. celatum, M. heckeshornense, and M. leprae[atpE gene is present in a single copy in the Mycobacterium genomes, while the 16S rRNA gene is present either in 1 or 2 copies in the genome [Comparison of the method targeting 6S rRNA, , showed M. leprae. Second,e genome . When coin vitro as positive controls whereas more than 150 mycobacterial species have been described so far [atpE real-time PCR method using a large representative collection of mycobacterial species , including members of MTC (n = 2), M. leprae species (n = 1), slow growing NTM (n = 13), and rapid growing NTM (n = 15). Given the broad diversity of mycobacterial species we have tested in this study, we expect the method to be applicable to all species within the Mycobacterium genus. In addition, it is the first time that a sensitive and specific molecular target has been identified based on an in silico comparison of 16 mycobacterial (13 species) and 12 non-mycobacterial genomes (4 closely related species).One of the limitations of this study is that only 31 mycobacterial species were tested Archaea. Using these databases, MycoHit, or other new software, may then be used to design new primers for real-time PCR detection or quantification, for in situ hybridization and other molecular tools. With this approach we were able to design primer pairs and a probe that target specific mycobacterial atpE gene, and could be used to detect and quantify very specifically mycobacteria in environmental samples. Although the atpE gene may not be appropriate for microdiversity studies, it appeared to be very useful for specific detection of the genus Mycobacterium in environmental samples. More generally, genome comparison used here showed its utility to identify specific genera’s targets, and could be used to identify specific proteins for antimicrobial design as previously emphasized [In conclusion, although our strategy did not take into account non-coding regions, such as insertion sequences, repetitive units, non-functional RNA, and structural ribosomal RNAs, the comparison of whole bacterial genomes for design of specific primers is a promising approach not only for mycobacteria but also for other cultured bacterial or archaeal groups for which whole sequenced genomes are accumulating in databases. Metagenomic libraries from environmental samples which are increasingly performed in microbial ecology studies  could alphasized .M. tuberculosis genes, presenting homologue genes in other mycobacterial genomes, and not presenting homologue genes in non-mycobacteria genomes, we used the MycoHit software version 14.17  and performed an alignment search with Stand Alone tblastn algorithm as previously described [M. tuberculosis H37Rv has been used as a reference of the Mycobacterium genus, because it is the most historically described mycobacterial genome [M. tuberculosis H37Rv, corresponding to the query sequences used in order to search for matches in the genomic DNA of other organisms , allowed identification of the conserved mycobacterial proteins presenting no homology in non-mycobacterial genomes  using the accession numbers: M. abscessus ATCC 19977 (CU458896.1) (P), M. avium 104 (CP000479.1) (P), M. avium subsp. paratuberculosis K10 (AE016958.1) (P), M. bovis subsp. bovis AF2122/97 (BX248333.1) (P), M. gilvum PYR-GCK (CP000656.1) (NP), M. marinum M (CP000854.1) (P), M. smegmatis MC2 155 (CP000480.1) (NP), Mycobacterium sp. JLS (CP000580.1) (NP), Mycobacterium sp. KMS (CP000518.1) (NP), Mycobacterium sp. MCS (CP000384.1) (NP), M. tuberculosis CDC1551 (AE000516.2) (P), M. tuberculosis H37Ra (CP000611.1) (NP), M. tuberculosis H37Rv (AL123456.2) (P), M. tuberculosis KZN 1435 (CP001658.1) (P), M. ulcerans Agy99 (CP000325.1) (P), and M. vanbaalenii PYR-1 (CP000511.1) (P). In order to avoid data lost during genome comparisons performed by MycoHit software, we have chosen to ignore some mycobacterial genomes. Since the number of coding proteins is much lower compared to other mycobacterial species, M. leprae Br4923 (FM211192.1) (P), and M. leprae TN (AL450380.1) (P) were ignored in the analysis  [M. bovis BCG Pasteur 1173P2 (AM408590.1) (NP) and M. bovis BCG Tokyo 172 (AP010918.1) (NP) were also not taken into account, because these vicinal genomes present mutations [M. intracellulare ATCC 13950 (ABIN00000000) (P), M. kansasii ATCC 12478 (ACBV00000000) (P) and M. parascrofulaceum BAA-614 (ADNV00000000) (P) were also not used during MycoHit proceedings, because their genomes were still not assembled at the moment we performed the first screening step of our analysis. Nevertheless, the genomes of M. leprae, M. bovis BCG, M. intracellulare, M. kansasii and M. parascrofulaceum were used during alignment of nucleic sequences of the most conserved proteins in mycobacterial genomes.In order to perform comparisons of pathogenic (P) and non-pathogenic (NP) mycobacterial genomes with 122/97 BX8333.1 (PMC2 155) ,35. GenoCorynebacterium aurimucosum ATCC 700975 (CP001601.1), C. diphtheriae NCTC 13129 (BX248353.1), C. efficiens YS-314 (BA000035.2), C. glutamicum ATCC 13032 (BX927147.1), C. jeikeium K411 (NC_007164), C. kroppenstedtii DSM 44385 (CP001620.1), C. urealyticum DSM 7109 (AM942444.1), Nocardia farcinica IFM 10152 (AP006618.1), Nocardioides sp. JS614 (CP000509.1), Rhodococcus erythropolis PR4 (AP008957.1), R. jostii RHA1 (CP000431.1), and R. opacus B4 (AP011115.1).We selected non-mycobacterial genomes of species from the CNM group using the following accession numbers: In order to check the homology of the selected mycobacterial sequences, the protein and DNA sequences of these selected proteins were aligned using the ClustalW multiple alignment of the BioEdit software 7.0.9.0 with 1000 bootstraps . Primer et al. protocol [M. avium, M. fortuitum, M. intracellulare and M. gordonae . Specificity and sensitivity were estimated against 30 non-mycobacteria (negative) strains and 31 mycobacteria (positive), respectively. The collection contained reference and environmental strains of mycobacteria, as well as, strains of the closely related CNM group, and other non-actinobacteria strains isolated from the environment [leprae species (n = 1), as well as species of slow growing NTM (n = 13), and rapid growing NTM (n = 15). TaqMan® real-time PCR were performed in duplicate using an ABI7500 real-time PCR system (Applied Biosystems), a Lifetech 7500 software version 2.0.6 (Applied Biosystems) and TaqMan fast virus 1-STEP Master Mix with 6-carboxy-X-rhodamine (ROX) (Applied Biosystems). The TaqMan® probes were labeled (Eurogentec) with the fluorescent dyes 6-carboxyfluorescein (5′ end) and Black Hole Quencher (3′ end). All reactions were performed in a 25 μl reaction mixture volume (2.5 μl of DNA) with 500 nM of forward primer, 500 nM of reverse primer, 50 nM of probe and 5 mM of MgCl2. Reverse transcriptase was inactivated immediately  according to the manufacturer instruction, and real-time PCR consisted in 40 cycles of denaturation (95°C for 3 s), annealing and extension (both steps at 60°C for 30 s). Determinations of cycle threshold were performed by setting the instrument’s threshold line at 0.02 ∆Rn units .Reproducibility, sensitivity and specificity of the new real-time PCR method were estimated using DNA from a previously described microorganism collection, and according to Radomski protocol . Reproduprotocol  were estironment . Mycobacet al. [et al. [In order to compare the new real-time PCR method to the culture method, 26 tap water distribution points in Paris (France) were sampled between April 2011 and July 2011, corresponding to 90 samples. Briefly, one liter of tap water was sampled in sterile plastic bottle, then centrifuged at 5000 × g for 2h and finally re-suspended in 1 ml of water. Mycobacteria density was estimated by culture (Method A) in all these samples following the procedure previously described by Le Dantec et al. . In para [et al.   between Mycobacterium tuberculosis H37Rv (AL123456.2) proteins and proteins of targeted mycobacterial genomes and proteins of non-targeted genomes. Targeted mycobacterial genomes include M. tuberculosis H37Ra (CP000611.1), M. tuberculosis CDC 1551 (AE000516.2), M. tuberculosis KZN 1435 (CP001658.1), M. bovis AF2122/97 (BX248333.1), M. ulcerans Agy99 (CP000325.1), M. marinum M (CP000854.1), M. avium 104 (CP000479.1), M. paratuberculosis K10 (AE016958.1), M. smegmatis MC2 155 (CP000480.1), M. abscessus ATCC 19977 (CU458896.1), M. gilvum PYG-GCK (CP000656.1), M. vanbaalenii PYR-1 (CP000511.1), Mycobacterium sp. JLS (CP000580.1), Mycobacterium sp. KMS (CP000518.1), Mycobacterium sp. MCS (CP000384.1), and non-targeted genomes include Corynebacterium aurimucosum ATCC 700975 (CP001601.1), C. diphteriae NCTC 13129 (BX248353.1), C. efficiens YS-314 (BA000035.2), C. glutamicum ATCC 13032 (BX927147.1), C. jeikeium K411 (NC_007164), C. kroppenstedtii DSM 44385 (CP001620.1), C. urealyticum DSM 7109 (AM942444.1), Nocardia farcinica IFM 10152 (AP006618.1), Nocardioides sp. JS614 (CP000509.1), Rhodococcus erythropolis PR4 (AP008957.1), R. jostii RHA1 (CP000431.1) and R. opacus B4 (AP011115.1).Click here for fileProtein sequence alignment of conserved proteins in mycobacterial genomes. Sequences are from genomes of M. abscessus ATCC 19977 (CU458896.1), M. avium 104 (CP000479.1), M. avium subsp. paratuberculosis K10 (AE016958.1), M. bovis subsp. bovis AF2122/97 (BX248333.1), M. bovis BCG Pasteur 1173P2 (AM408590.1), M. bovis BCG Tokyo 172 (AP010918.1), M. gilvum PYR-GCK (CP000656.1), M. intracellulare ATCC 13950 (ABIN00000000), M. kansasii ATCC 12478 (ACBV00000000), M. leprae Br4923 (FM211192.1), M. leprae TN (AL450380.1), M. marinum M (CP000854.1), M. parascrofulaceum BAA-614 (ADNV00000000), M. smegmatis MC2 155 (CP000480.1), Mycobacterium sp. JLS (CP000580.1), Mycobacterium sp. KMS (CP000518.1), Mycobacterium sp. MCS (CP000384.1), M. tuberculosis CDC1551 (AE000516.2), M. tuberculosis H37Ra (CP000611.1), M. tuberculosis H37Rv (AL123456.2), M. tuberculosis KZN 1435 (CP001658.1), M. ulcerans Agy99 (CP000325.1) and M. vanbaalenii PYR-1 (CP000511.1).Click here for fileDNA sequence alignment of conserved proteins in mycobacterial genomes. Sequences are from genomes of M. abscessus ATCC 19977 (CU458896.1), M. avium 104 (CP000479.1), M. avium subsp. paratuberculosis K10 (AE016958.1), M. bovis subsp. bovis AF2122/97 (BX248333.1), M. bovis BCG Pasteur 1173P2 (AM408590.1), M. bovis BCG Tokyo 172 (AP010918.1), M. gilvum PYR-GCK (CP000656.1), M. intracellulare ATCC 13950 (ABIN00000000), M. kansasii ATCC 12478 (ACBV00000000), M. leprae Br4923 (FM211192.1), M. leprae TN (AL450380.1), M. marinum M (CP000854.1), M. parascrofulaceum BAA-614 (ADNV00000000), M. smegmatis MC2 155 (CP000480.1), Mycobacterium sp. JLS (CP000580.1), Mycobacterium sp. KMS (CP000518.1), Mycobacterium sp. MCS (CP000384.1), M. tuberculosis CDC1551 (AE000516.2), M. tuberculosis H37Ra (CP000611.1), M. tuberculosis H37Rv (AL123456.2), M. tuberculosis KZN 1435 (CP001658.1), M. ulcerans Agy99 (CP000325.1) and M. vanbaalenii PYR-1 (CP000511.1).Click here for file


T) was obtained in China from crickets  living in cropland deserted for approximately 10 years. The isolated bacteria were Gram-negative, facultatively anaerobic, oxidase-negative rods. A preliminary analysis of the 16S rRNA gene sequence indicated that the strain belongs to either the genus Erwinia or Pantoea. Analysis of multilocus sequence typing based on concatenated partial atpD, gyrB and infB gene sequences and physiological and biochemical characteristics indicated that the strain belonged to the genus Erwinia, as member of a new species as it was distinct from other known Erwinia species. Further analysis of the 16S rRNA gene showed SCU-B244T to have 94.71% identity to the closest species of that genus, Erwinia oleae (DSM 23398T), which is below the threshold of 97% used to discriminate bacterial species. DNA-DNA hybridization results (5.78±2.52%) between SCU-B244T and Erwinia oleae (DSM 23398T) confirmed that SCU-B244T and Erwinia oleae (DSM 23398T) represent different species combined with average nucleotide identity values which range from 72.42% to 74.41. The DNA G+C content of SCU-B244T was 55.32 mol%, which also differs from that of Erwinia oleae (54.7 to 54.9 mol%). The polyphasic taxonomic approach used here confirmed that the strain belongs to the Erwinia group and represents a novel species. The name Erwinia teleogrylli sp. nov. is proposed for this novel taxon, for which the type strain is SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T).A bacterial isolate (SCU-B244 Our aim was to explore the relationship between pesticide resistance and symbionts. Among 274 isolates cultured from Teleogryllus occipitalis, 27 strains of genera Lysinibacillus, Pseudomonas, Sphingobacterium, Exiguobacterium and Staphylococcus could evidently degrade chlorpyrifos, a common insecticide used in this field for many years. One isolate (SCU-B244T) that could degrade chlorpyrifos was cultured on TSA (tryptone soy agar) medium in August 2012 and could not be identified to the species level. A polyphasic taxonomic approach was used to investigate the strain, with the results suggesting that SCU-B244T represents a novel species of the genus Erwinia.In a recent study, we focused on the culturable strains associated with crickets  was used as reference strain in this study.Three crickets  were added to 100 mL sterile 0.85% (w/v) NaCl solution in a 250 mL flask and shaken at 220 rpm for 30 min. The supernatant which contained bacteria was plated onto TSA plates  and subsequently incubated at 37°C for 5 days. Glycerol stock at -80°C was adopted for long term preservation of the isolates. Exponential phase cells cultured in TSB  medium with shaking  at 37°C were harvested for DNA G+C content and ANI analysis. Phase contrast and transmission electron microscopy were used to examine cellular morphology and motility after growth on TSA medium at 37°C for 24 h. Gram staining was performed as described by Gerhardt T, PCR amplification, primers used and DNA sequencing conditions of 16S rRNA gene were performed as previously described [www.ezbiocloud.net/eztaxon) by comparison with 16S rRNA gene sequence data. A neighbour-joining phylogenetic tree was constructed using the method of Saitou and Nei [DNA extraction from strain SCU-B244escribed  , with the highest similarity scores being 96.1% to 94.1%. Neighbour-joining and maximum-likelihood phylogenetic trees were constructed using the methods described above.The results of 16S rRNA gene sequence alignment on the EzTaxon server revealed that strain SCU-B244T, other neighbour-joining and maximum-likelihood phylogenetic trees based on 16S rRNA gene sequences were constructed, including the strain SCU-B244T and all culturable type strains of the genera Erwinia (Candidatus Erwinia dacicola) and Pantoea, based on the same methods described above.To refine the taxonomic position of SCU-B244atpD, gyrB, infB and rpoB gene sequences enables the differentiation of the phylogenetically related genera Erwinia, Pantoea and Tatumella. A good congruence has previously been observed between DNA-DNA hybridization and MLSA [atpD, gyrB, and infB, were amplified and sequenced with the primers described by Brady et al. [atpD, gyrB and infB gene sequences were constructed using the same method as described above. Strains from genera Erwinia, Pantoea and Tatumella were used in MLSA analysis, including SCU-B244T. The partial sequence of atpD, gyrB and infB gene of related strains were obtained from GenBank and the accession numbers are indicated on the figures.Multilocus sequence analysis (MLSA) of concatenated partial and MLSA –16. In ty et al. . NeighboT and Erwinia oleae DSM 23398T was conducted as described by De Ley et al. [260/280 = 1.8 to 1.9) was extracted using the Omega Bacterial DNA kit (E.Z.N.A.®)DNA-DNA hybridization between SCU-B244y et al.  with hybT was determined by whole genome sequencing data.The DNA G+C mol% content of strain SCU-B244et al.[API 20E, API 50CHE acid production tests and API ZYM enzymatic characteristics test (BioMérieux) were performed according to the manufacturer’s instructions. BIOLOG GN2 carbon-source utilization analysis was conducted according to the instruction manual. Cellular fatty acids analysis was also performed. Biomass for fatty acid analysis was prepared by scraping growth from TSA plates after 24h incubation at 37°C. Lipids were extracted using the method of Folch et al.. Total let al., The comT, Next Generation Sequencing (Illumina Miseq) was conducted at Majorbio Inc. and the data was used to calculate the Average Nucleotide Identity (ANI). DNA extraction method and purity were described above. ANI is a similarity measure between two genome sequences and it correlates well with DNA-DNA hybridization values. [www.ezbiocloud.net/ezgenome/browse_db). We used Orthologous Average Nucleotide Identity Tool  to calculate orthologous ANI values. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession LLXO00000000. The version described in this paper is version LLXO01000000.For the genome sequencing of SCU-B244 values.  A value  values.  Genome dTeleogryllus occipitalis and a bacterial isolate (SCU-B244T) is herein described. Initial microbiological characterization of the strain revealed that the cells were Gram-negative, oxidase-negative, rod-shaped, catalase-positive and facultatively anaerobic, suggesting that the strain belongs to the family Enterobacteriaceae [In total 274 isolates belonging to 29 genera were cultured from eriaceae .T and strains of the genera Erwinia and Pantoea cluster together. The diagram shows the phylogenetic relationship between SCU-B244T and genera Erwinia and Pantoea.Neighbour-joining  and maxiErwinia and Pantoea, indicated that strain SCU-B244T belongs to the genus Erwinia and is most closely related to Erwinia oleae (DSM 23398T), which is consistent with the results of the initial phenotypic analysis.Further neighbour-joining  and maxiatpD, gyrB and infB gene sequences, strain SCU-B244T and Erwinia oleae (DSM 23398T) cluster together on a single branch, consistent with the previous results, suggesting strain SCU-B244T is most closely related to Erwinia oleae (DSM 23398T).In neighbour-joining  and maxiT with species of genera Erwinia and Pantoea were 95.43% (Erwinia psidii LMG 7039T) and 95.42%  respectively, phylogenic closest strain was Erwinia oleae (DSM 23398T) and the identity was 94.71%, which is lower than the 97% threshold that has been established to discriminate species. Strains showing less than 97% 16S rRNA gene identity are unlikely to have more than 60 to 70% DNA—DNA relatedness [T is a novel species. The DNA—DNA relatedness between SCU-B244T and Erwinia oleae (DSM 23398T) was 5.79 ± 2.52%. The value is mean of six hybridizations ± SD, which is significantly lower than the 70% value considered to be the threshold for the delineation of bacterial species [T and related species are listed in The highest 16S rRNA gene identity of SCU-B244leae DSM 398T and T was 55.32 mol% which could be discriminated from Erwinia oleae (DSM 23398T), which has a G+C content of 54.7 to 54.9 mol%.The DNA G+C content of strain SCU-B244T strain can be discriminated from each recognized species of the genus Erwinia by at least three characteristics, and from the phylogenetically most closely related species, Erwinia oleae, by 10 API 50CHE characteristics (API 20E and API 50CHE (BioMérieux) tests were also carried out and the results were compared to related strains . The reseristics .API ZYM enzymatic characteristics and BIOLOG GN2 carbon-source utilization results are presented as supplementary data  Tables.T and related species of genera Erwinia and Pantoea is shown in T contained C16:0 (28.2%), Summed feature 4 (12.4%), C11:0 3-OH (11.6%), Summed feature 7 (10.5%), Summed feature 3 (10.0%), C14:0 (6.4%) as the major fatty acids, while strain DSM 23398T contained C16:0 (38.8%), Summed feature 4 (32.7%), Summed feature 7 (9.1%), C12:0 (7.7%), C16:0 Δ9 cyclo (4.6%) as the major fatty acids (14:0 2-OH (3.7%) and C18:0 (3.7%) were detected from strain SCU-B244T while those were not detected from strain DSM 23398T. Fatty acid composition of SCU-B244T and reference strains data from Erwinia and Pantoea showing that most of the compositions were in the range of Erwinia and Pantoea [T belongs to group Erwinia and Pantoea.Fatty acid composition analysis of SCU-B244ty acids . Summed  Pantoea , which iT is a member of genus Erwinia. DNA-DNA hybridization results, ANI values and phenotypic data obtained in this study can separate this isolate from related species. In conclusion the bacterial strain isolated from a cricket (genus Teleogryllus) represents a novel species for which the name Erwinia teleogrylli sp. nov. is proposed. Strain SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T) is the type strain.Based on the genotypic data , it is clear that SCU-B244Erwinia teleogrylli .The colonies are milky white, circular and convex with entire margins. Cells are short Gram-negative, facultatively anaerobic rods that are oxidase-negative, catalase-positive and able to grow in 6% (w/v) NaCl. Growth pH range is 6 to 9, with optimum growth at 7. Nitrate is reduced to nitrite.2S and indole production, acid production from sorbitol and sucrose. According to API 50CHE tests (BioMérieux), acid is formed from glycerol, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, D-mannitol, D-sorbitol, methyl-αD-glucopyranoside, N-acetylglucosamine, arbutin, esculin ferric citrate, salicin, D-cellobiose, D-maltose, D-melibiose, D-trehalose, gentiobiose, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate. No acid is produced from erythritol, D-arabinose, L-xylose, D-ardonitol, methyl-β D-xylopyranoside, dulcitol, inositol, methyl-αD-mannopyranoside, amygdalin, D-lactose, D-saccharose, inulin, D-melezitose, D-raffinose, amidon, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol. Results obtained with API ZYM tests (BioMérieux) gave positive enzymatic activity results for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase, naphthol-AS-B1-phosphohydrolase, β-galactosidase, β- glucosidase and N-acetyl-β- glucosaminidase, and negative results for lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase.Results obtained with API 20E tests (BioMérieux) gave positive results for acid production from citrate, glucose, mannitol, rhamnose, melibiose, amygdalin and arabinose, and negative results for β-galactosidase, gelatinase, urease, arginine digydrolase, lysine decarboxylase and ornithine decarboxylase activity, HThe following substrates were utilized according to BIOLOG GN2 carbon-source utilization analysis: dextrin, N-acetyl-D-glucosamine, L-arabinose, D-fructose, D-galactose, α-D-glucose, maltose, D-mannitol, D-mannose, D-melibiose, β-methyl-D-glucoside, D-psicose, L-rhamnose, D-trehalose, turanose, pyruvic acid methyl ester, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, p-hydroxy phenylacetic acid, α-keto glutaric acid, propionic acid, D-saccharic acid, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-proline, L-serine, inosine, uridine, thymidine, glycerol, D,L-α-glycerol phosphate, glucose-1-phosphate, glucose-6-phosphate.16:0 (28.2%), Summed feature 4 (12.4%), C11:0 3-OH (11.6%), Summed feature 7 (10.5%) and Summed feature 3 (10.0%), with other minor components, including C14:0 (6.4%), C17:0 Δ9 cyclo (4.9%), C12:0 (4.3%), C14:0 2-OH (3.7%), iso-C18:0 (3.6%), C18:0 14-methyl (1.9%), C14:1 Δ11 (1.1%), C8:0 2-CH2CH3 (0.6%), C15:0 (0.5%). The DNA G+C content is 55.32 mol%.Major fatty acids are CT (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T), was isolated from crickets (genus Teleogryllus) sampled in Chengdu , Sichuan Province, China.The type strain, SCU-B244S1 Certification(PDF)Click here for additional data file.S2 Certification(PDF)Click here for additional data file.S1 FigErwinia teleogrylli sp. nov. and the first 66 hit strains at the EzTaxon server within the family Enterobacteriaceae. Bar, 0.5% nucleotide substitutions. Numbers at branching points are bootstrap percentage values based on 1000 replications. Only values >50% are shown.The diagram shows the phylogenetic relationship between (TIF)Click here for additional data file.S2 FigErwinia teleogrylli sp. nov. and the first 66 hit strains at the EzTaxon server within the family Enterobacteriaceae. Bar, 0.5% nucleotide substitutions. Numbers at branching points are bootstrap percentage values based on 1000 replications. Only values >50% are shown.The diagram shows the phylogenetic relationship between (TIF)Click here for additional data file.S3 FigErwinia teleogrylli sp. nov. and taxa related type species of the genera Erwinia and Pantoea except Candidatus Erwinia dacicola. Escherichia coli ATCC 11775T was used as the outgroup. Bar, 0.5% nucleotide substitutions. Numbers at branching points are bootstrap percentage values based on 1000 replications. Only values >50% are shown.The diagram shows the phylogenetic relationship between (TIF)Click here for additional data file.S4 FigErwinia teleogrylli sp. nov. and taxa related species of genera Erwinia, Pantoea and Tatumella. Bar, 0.5% nucleotide substitutions. Numbers at branching points are bootstrap percentage values based on 1000 replications. Only values >50% are shown.The diagram shows phylogenetic relationship between (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


There are a number of errors in the legends for Figs. 3, 4, and 5. The complete, correct legends for Figs. 3, 4, and 5 are:Figure 3. Wallerian degeneration in mice lacking GD3s: tissue infiltration by activated macrophages and Schwann cell proliferation. A-B; D-E: Longitudinal sections of lesioned sciatic nerves from adult WT and GD3s KO mice at 5 days after crush lesioning immunolabeled for NF-200  or F4–80  and imaged at the distal nerve stump. The nuclei were counterstained with DAPI. C and F: Histograms indicating the number of NF-200 fragments (C) and active macrophages (cells positive for F4–80) at the distal nerve stump (F). G-H; J-K: Longitudinal sections of lesioned sciatic nerves from WT and GD3s KO mice imaged at the distal stump 7 days after crush lesioning and immunolabeled for p-histone H3 (G-H) or double immunolabeled for Ki-67 and GFAP (J-K). The nuclei were counterstained with DAPI. I and L: Histograms indicating the number of cells positive for phistone H3 (I) or KI-67/GFAP (L) at the distal stump in each group of mice. M-O: Optical slices obtained by confocal microscopy from transversal sections of wildtype uninjured (M), wildtype injured (N) or GD3s Ko injured (O) sciatic nerves immunolabeled for GFAP at distal stump, 5 days after crush lesion. Bars: A-B, G-H = 100 mm; and D-E, J-K = 50 mm; M-O = 20 mm. Statistics: Mann-Whitney, ns, p>0.05. doi: 10.1371/journal.pone.0108919.g003Figure 4. Committed nerve regeneration in adult mice lacking GD3s is restored by administration of exogenous GD3 in vivo and in vitro. A: Longitudinal sections of sciatic nerves proximally or 1 mm or 3 mm distally immunolabeled for GAP-43 at 21 days after crush lesioning. B: Histogram indicating the axonal density in the regenerating nerves from WT, GD3s KO and GD3-treated GD3s KO mice. C: Images of P1 mouse DRG explants seeded on PDL/laminin coverslips. The DRG samples from WT, GD3s KO and GD3s KO exogenously treated with GD3 ganglioside were incubated for 5 days in vitro. GD3 was added on day 2 of the incubation. Low-magnification images of DRGs immunolabeled for Tuj-1. D: Histogram quantifying neurite growth. E: High-magnification images of neurites immunolabeled for R24  or CD-60b . The nuclei were counterstained with DAPI (white) Bars: A = 100 mm; C = 500 mm; and E = 20 mm. Statistics: ANOVA ***p<0.001; *p<0.01. doi: 10.1371/journal.pone.0108919.g004Figure 5. Integrin-β1 expression but not calcium influx is modified in neurons lacking GD3s. A-C: Images of integrin-β1 obtained by apotome microscopy of mice neonate (P1) DRGs from WT (A), GD3s KO (B) and GD3s KO with exogenous GD3 (C). Samples were cultured for 5 days. A′–C′: High-magnification optical sections of neurites (DIC) labeled for integrin-β1. A″-C″: High-magnification optical sections of neurites (DIC) double labeled for integrin-β1 and CD60-b (9-O-ac. GD3). Yellow dots indicate colocalization of the two markers. D: DIC image of the field shown in C, illustrating DRG (lower right) and neurites extended. E, E′: Histogram of quantitative analysis of the number of integrin- β1 (E) or 9-O-Ac. GD3 (E′) clusters along extended neurites. F and J: P1 postnatal DRGs were dissected, cleaned, dissociated and cultured for 48 h in the presence of 50 ng/ml NGF. Cell cultures from both WT (F) and GD3s KO (J) mice show typical neurons with extensive neurites and flat Schwann cells. The same fields are shown under fluorescence . Further, H and L show the same microscope field under fura-2 fluorescence, in SCCI experiments. Typical responses are shown for 4 cells in WT (I) or in GD3s KO mice (M) when stimulated with 50 mM KCl  or 100 mM ATP . As shown in WT , cells #4 and #7 are neurons (with large cell bodies), whereas cells 14 and 10 are flat, typical of Schwann glia. The same is observed for GD3s KO cells , where cells #17 and #10 are neurons, and cells #28 and #39 are glia. N, O: Histograms indicating maximum calcium influx when stimulated by KCl  or ATP . Bars: A-D = 500 mm; A′-C′, A′-C″ = 100 mm F-H, J-L = 20 mm. Statistics: ns, p = 0.760 Mann-Whitney; ***p<0.0001, *p<0.001 ANOVA. doi: 10.1371/journal.pone.0108919.g005


P. aeruginosa an important pathogen causing lower respiratory tract infections (LRTI) both in HIV and non-HIV population. Molecular characterization of Pseudomonas spp. helps in better understanding of their clonal distribution among these patient populations. Our study aims to discriminate and generate highly specific fingerprints using 16S-rDNA PCR and amplified ribosomal DNA restriction analysis (ARDRA) techniques.Pseudomonas spp. were subjected to 16SrDNA PCR using universal primers and amplicons digested with Hae III, Alu I and Rsa I for ARDRA analysis. Nucleic acid size confirmation of the digested amplicons was done using MultiNA Bioanalyser. Phylogenetic tree was constructed by maximum parsimonious method using MEGA 4.0. Representative isolates from the major clones were sequenced and submitted to genbank for accession numbers and genetic relatedness was identified by UPGMA using NTSYS 1.80 software. Mean genetic distance (GD) and intraspecies mean GD were calculated.Seventy-two isolates  of P. aeruginosa, P.putida, P.stutzeri, Alcaligenes feacalis strains by BLASTN analysis. The overall mean GD and intra-species GD of the various species was as follows: P. aeruginosa : 4.664, 16.663, 7.536, 0.733, 0.096, 2.402 and 0.221, 3.487, 2.010, 0.029, 2.728, 0.011;P.putida:11.904, 7.3 and 1.064, 1.371; P.stutzeri (JF303641) is 2.732, 0.036; Alcaligenes feacalis (JF303642) : 30.248, 5.377 respectively.Based on ARDRA banding pattern, 14 groups and 10 clones were obtained from the 72 pseudomonas isolates with the following accession numbers: JF279962-64, JF303639-45. Sequences showed 97-100% similarity with known Pseudomonas spp causing LRTI among HIV and Non HIV patients.The highly variable mean GD values amongst the isolates from different and within the same species indicates high genetic diversity among


Nucleic Acids Res. 2014 Dec 16;42(22):13488–99. doi: 10.1093/nar/gku1097Streptococcus pneumoniae strain ST556, as shown in Table - M. Spn556I - M. Spn556II 8TTYG)- M. Spn556III 7TATC).In this article, the authors have incorrectly designated three DNA methyltransferases in In addition, the recognition sequence of the type I system encoded by MYY570-MMY572 in Figure 3A should be AAG(N)8TTYG.


Deep-sea fungi, the fungi that inhabit the sea and the sediment at depths of over 1000 m below the surface, have become an important source of industrial, agricultural, and nutraceutical compounds based on their diversities in both structure and function. Since the first study of deep-sea fungi in the Atlantic Ocean at a depth of 4450 m was conducted approximately 50 years ago, hundreds of isolates of deep-sea fungi have been reported based on culture-dependent methods. To date more than 180 bioactive secondary metabolites derived from deep-sea fungi have been documented in the literature. These include compounds with anticancer, antimicrobial, antifungal, antiprotozoal, and antiviral activities. In this review, we summarize the structures and bioactivities of these metabolites to provide help for novel drug development. Chromocleista sp. was described by Park et al. chromen-4-one (96), together with six known compounds (97–100) -4(97–100) , were isectively .101)  , was isoectively .102) and dihydrooxosorbiquinol (103)  against P388, A549, HL60, BEL-7402, and K562 cell lines [Two new bisorbicillinoids, named oxosorbiquinol (ol (103) , have be104–106), and the known breviones (107–110) (Penicillium sp. and display strong cytotoxic effects against MCF-7 cells (IC50: 7.44–28.4 μM). Compound 106 exhibits cytotoxic activity against A549 cells with IC50 of 32.5 μM [111–113)  [111–113) show 25.2%–44.9% cytotoxicity against HeLa at 10 μg/mL. Particularly, compound 111 displays a very strong cytotoxicity to HIV-1 replication in C8166 cells with an EC50 of 14.7 μM [Three new breviane spiroditerpenoids, breviones I–K (107–110)  have bee 32.5 μM . Similar111–113) , have be. (2009) . These c 14.7 μM .d-Pro-d-Phe) (114), cyclo(d-Tyr-d-Pro) (115), phenethyl 5-oxo-l-prolinate (116), cyclo(l-Ile-l-Pro) (117), cyclo(l-Leu-l-Pro) (118), and 3β,5α,9α-trihydroxy--ergosta-7,22-dien-6-one (119)  , have be120), and a known compound, cholesta-7,22-diene-3b,5a,6b-triol (121)  , were idlymerase .122), ferrineoaspergillin (123), aflatoxin (124), and (11S)-hydroxyl aspergillic acid (125) (Aspergillus sp. 16-02-1. These compounds possess certain cytotoxicity against human cancer cell lines K562 (33.6%–43.6%), HL-60 (24.1%–53.3%), HeLa (18.8%–45.4%), and BGC-823 (36.2%–51.2%) at the concentration of 100 μg/mL [New aspergillic acid (id (125)  have bee126)  [A known antibacterial compound, xanthocillin X (126) , was iso2 μg/mL) .127) (Penicillium sp. F00120. It inhibits in vitro proliferation of mouse melanoma (B16), human melanoma (A375), and human cervical carcinoma (HeLa) cell lines with IC50 of 0.08, 0.06, and 0.12 mM, respectively [One new sesquiterpene quinone, named penicilliumin A (127) , was isoectively .128), oxisterigmatocystin B (129), and oxisterigmatocystin C (130), together with one known compound, 5-methoxysterigmatocystin (131)  , have beectively .132) and 6-deoxy-5a,6-didehydrogliotoxin (133), and five known metabolites (134–138) , whereas compounds 136–138 containing a disulfide or tetrasulfide bond show potential inhibitory activity against histone methyltransferase (HMT) G9a (IC50: 2.1–6.4 μM) [Seven gliotoxin-related compounds including two new metabolites, bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin (134–138)  have bee–6.4 μM) .Antimicrobials are the most important drugs to protect human beings from infective diseases. Deep-sea fungi are one of the potential pools for screening antimicrobial metabolites, which can be developed into new drugs.139–143), together with six known analogues (144–148) (Emericella sp. SCSIO 05240. All of them show weak growth inhibition against bacteria. The inhibition zone of compounds 139 and 141 against Escherichia coli (ATCC 29922), Klebsiella pneumonia (ATCC 13883), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Acinetobacter baumanii (ATCC 19606), and Aeromonas hydrophila (ATCC 7966) is 1–3 mm in diameter. Moreover, compound 143 displays broad antifungal activities (3–4 mm in diameter) against Fusarium sp., Penicillium sp., Aspergillus niger, Rhizoctonia solani, Fusarium oxysporium f. sp. niveum, and Fusarium oxysporium f. sp. cucumeris [Four new prenylxanthones, emerixanthones A–D 144–148) , were is , were i149–163) (Spiromastix sp. These compounds exhibit significant growth inhibition (MIC: 0.1–8.0 μg/mL) against Gram-positive bacteria including Staphylococcus aureus, Bacillus thuringiensis, and Bacillus subtilis. In addition, compounds 154–158 display potential inhibitory effects on methicillin-resistant bacterial strains of Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). Moreover, compound 158 inhibits the growth of the vancomycin-resistant bacteria of Enterococcus faecalis and Enterococcus faecium (VRE) [Fifteen new depsidone-based analogues, named spiromastixones A–O (149–163) , have beum (VRE) .et al. (2013) [164)  against Staphylococcus aureus, Bacillus subtilis, Vibrio sp. 385, Vibrio sp. 333, and Vibrio sp. 1758 [Liu . (2013)  have rep3) [164)  that wassp. 1758 .165)  , has bees aureus .et al. (2014) [Acremonium implicatum DFFSCS001 (AI001), Aspergillus westerdijkiae DFFSCS013 (AW013), Alternaria tenuissima DFFSCS003 (AT003), Cladosporium cladosporioides DFFSCS016 (CC016), Cladosporium sphaerospermum DFFSCS019 (CS019), Engyodontium album DFFSCS021 (EA021), Geomyces vinaceus DFFSCS022 (GV022), and Tritirachium sp. DFFSCS034 (TS034). These fungal species were isolated from sediments of the South China Sea [Loktanella hongkongensis and Micrococcus luteus, and one marine pathogenic bacterium. Based on bioassay-guided isolation technique, they have isolated five compounds (166–170)   have evahina Sea , and alm166–170)  from the.9 μg/mL .171)  , has beef 276 μM .et al. (2014) [172), and five known diketopiperazine derivatives, diketopiperazine V (173), brevianamide Q (174), brevianamide R (175), brevianamide K (176), and brevianamide E (177) , 53.7% (174), 46.2% (175), 61.4% (176), and 19.3% (177) at 13.9 μM, respectively [Kong . (2014)  have rep E (177) , which wectively .178 (Aspergillus westerdijkiae SCSIO 05233 and shows strong antifouling activity with an EC50 of 8.8 mg/mL [Compound 178  has been.8 mg/mL .p-hydroxyphenopyrrozin (179)   , with a 79 , withnd (180)  that wasalbicans .181) and penicitrinone A (182)  at 100 μM [Two citrinin type compounds, phenol A acid ( A (182) , with ant 100 μM .The fungi in deep-sea environments are very diverse and abundant, making them a versatile reservoir of metabolites with both new structures and bioactivities that can be of potential use, acting as leading compounds to synthesize new modern medicine. Although the research on deep-sea fungi is not as up-to-date as the research on fungi in other environments such as terrestrial soil, fresh water, and shallow marine areas due to difficulties in both sample collection and fungal cultivation methods, more and more fungi have been cultivated from the deep sea based on culture-dependent methods. These deep-sea fungi can provide a potential source for natural bioactive product screening and new drug discovery. Up to now, more than 180 new and/or bioactive secondary metabolites from deep-sea fungi with broad bioactivities, such as anticancer, antimicrobial, antifungal, anti-larval settlement, and antiviral, have been described in the literature. Most of the investigated bioactive compounds exhibit cytotoxic activity, then antimicrobial activity. These bioactive compounds not only help deep-sea fungi to defend themselves against predators in the natural ecosystem, but also have the potential of becoming treatments for human diseases and probes for new biological targets. Work to isolate fungi from deep-sea environments and characterize their bioactive metabolites is underway and is of increased importance due to the urgent need for new drugs to overcome emerging and drug-resistant diseases.


Headache is a very common health problem among adolescents, causing school and social disability . This isThree hundred and seventy-six adolescents aged 11-15 years, living in the Pavia province , were recruited for this cross-sectional school-based study. They were assessed about their headache using a medical history questionnaire; headache diagnosis was made according to the new classification system of the International Headache Society (ICDH-3 Beta). Headache-related disability was assessed by means of PedMIDAS (Pediatric Migraine Disability Assessment Score).Of the 376 students enrolled 91 24.2%) had headache: 60.44% girls (n=55) and 39.56% boys (n=36). At the first diagnosis, the prevalence of any migraine was 28.6% (n=26), any tension-type headache (TTH) 60.4% (n=55), any medication-overuse headache 3.3% (n=3) and “unclassifiable” headache was 7.7% (n=7) .None declared.


BMS-663068 is a prodrug of BMS-626529, an attachment inhibitor that binds directly to HIV-1 gp120, preventing initial viral attachment and entry into the host CD4+ T-cell. AI438011 is an ongoing, Phase IIb, randomized, active-controlled trial investigating the safety, efficacy and dose–response of BMS-663068 vs. atazanavir/ritonavir (ATV/r) in treatment-experienced (TE), HIV-1-positive subjects. At Week 24, response rates across the BMS-663068 arms were consistent with ATV/r.50 100 nM) were randomized equally to four BMS-663068 arms  and a control arm (ATV/r 300/100 mg QD), with tenofovir disoproxil fumarate (TDF) + raltegravir (RAL). The complete safety profile through Week 24 is reported.Antiretroviral TE subjects  with susceptibility to all study drugs (including BMS-626529 ICn=196)). In the ATV/r arm, Grade 2–4 drug-related AEs occurred in 14/51 (27.5%) subjects and were mostly secondary to gastrointestinal and/or hepatobiliary disorders. Serious adverse events (SAEs) occurred in 13/200 (6.5%) and 5/51 (9.8%) subjects receiving BMS-663068 and ATV/r, respectively; most were secondary to infections and none were related to study drugs. The most common AE reported for BMS-663068 was headache , occurring in 5/51 (10%) subjects in the ATV/r arm; in the BMS-663068 arms, this was not dose-related. There were no deaths.In total, 251 subjects were treated . No BMS-663068-related adverse events (AEs) led to discontinuation. Grade 2–4 drug-related AEs occurred in 17/200 (8.5%) subjects across the BMS-633068 arms; however, these events were mostly single instances and no dose-relationship was seen. Similarly, no noticeable trend for Grade 3–4 laboratory abnormalities was seen and Grade 3–4 hematologic changes and liver chemistry elevations were uncommon (neutropenia, 2.5%; AST/ALT elevations, 1% (BMS-663068 was generally well tolerated across all arms, with no related SAEs or AEs leading to discontinuation and no dose-related safety signals. There were no trends for Grade 2–4 AEs or clinical laboratory abnormalities. These results support continued development of BMS-663068.Previously submitted at IDWeek, Philadelphia, PA, 8 October 2014.


HIV-infected patients treated with Highly Active Antiretroviral Therapy (HAART) may be predisposed to hypertriglyceridemia, which gives rise to a highly atherogenic lipid profile known as atherogenic dyslipidemia (AD). We propose that genetic variability leaves some HIV-infected patients more predisposed to AD than others , 2.This was a cross-sectional, observational study conducted in 468 antiretroviral-treated HIV-infected patients attending at the outpatient clinic of a tertiary hospital over a 6-month period, who were classified as normolipidemic (n=173) or presenting with AD (triglycerides: 1.7 mmol/L and HDLc < 1.02 [men] or 1.28 mmol/L [women]) (n=148). Polymorphisms were identified in the APOA5, APOC3, LPL, CETP, HL, MTP, APOE, LRP5 and VLDLR genes.Atherogenic dyslipidemia was detected in 31% of patients, most of whom were men (77%). This group was also older and had higher levels of remnant lipoprotein cholesterol (RLPc) than normolipidemic patients. The polymorphisms rs328 in LPL, rs708272 in CETP and rs1800588 in HL were 10–40% significantly more frequent in normolipidemic patients. At least 1 of these polymorphisms was detected in 90% of normolipidemic patients; in AD patients, the percentage decreased to 75% (p=0.003). This effect was dependent on both the allele and the dose of HAART and independent of the regimen administered. The protective combination showed a trend towards higher HDLc (1.13 [0.40] vs 1.24 [0.23] mmol/L), lower triglycerides (2.23 [2.34] vs 1.89 [1.24] mmol/L) and lower RLPc (16.41 [11.42] vs 12.99 [11.69] mmol/L).Polymorphisms in LPL, CETP and HL protect HIV-infected patients from developing AD in a dose-dependent manner .


Kabuki syndrome (KS) is a multiple congenital anomalies/intellectual disability syndrome characterized by developmental delay, specific facial features, skeletal and visceral abnormalities. This syndrome is caused by mutations in MLL2 and KDM6A gene. Autoimmune abnormalities such as idiopathic thrombocytopenic purpura, hemolytic anemia, thyroiditis, and vitiligo have been described very rarely in patients with KS ,2,3. HerCase report: A female patient aged 7 7/12 years presented with short stature. There was no consanguinity between her parents. She was born as a term neonate weighing 2100 g as the product of a twin pregnancy and had no perinatal complications. On physical examination, the patient was noted to have large and low-set ears, broad and arched eyebrows, elongated palpebral fissures with eversion of the lateral third of the lower eyelid, as well as high and narrow palate. She also had other phenotypic malformations such as numerous vitiligo lesions of different sizes in the neck, brachydactyly, prominent fetal finger pads, and hyperlaxity in her joints. Her height was 116.3 cm (-1.85 standard deviation score [SDS]) and weight was 21.7 kg (-0.78 SDS). She had sensorineural hearing loss  and moderate mental retardation . She had a normal female karyotype . A clinical diagnosis of KS was considered.4) was 0.42 ng/dL (reference range: 0.91-1.92), Anti-microsomal antibody: 450.6 U/mL (reference range: 0-9), anti-thyroglobulin antibody: 2766 U/mL (reference range: 0-4)]. Thyroid ultrasonography demonstrated a rough pattern, consistent with thyroiditis. Levothyroxine (50 µg/day) replacement therapy induced a euthyroid state (TSH: 3.65 mIU/L and fT4: 1.15 ng/dL).Additionally, laboratory findings revealed autoimmune thyroiditis [thyroid-stimulating hormone (TSH): 242 mIU/L (reference range: 0.55-6.7) and free thyroxine (fTThe main causes of KS are point mutations with large intragenic deletions and duplications of the histone methyl transferase MLL2 gene ,4,5. We In several patients, KS was reported to be associated with autoimmune abnormalities such as idiopathic thrombocytopenic purpura, hemolytic anemia, thyroiditis, and vitiligo ,5. The aPeer-review: Internal peer-reviewed.


Current semi-quantitative cardiovascular magnetic resonance (CMR) techniques are of limited diagnostic value in patients with suspected myocarditis. T1- and T2-mapping CMR are promising novel quantitative approaches to assess myocardial injury. This study evaluated the performance of T1 and T2 mapping CMR to identify patients with myocarditis in comparison with conventional CMR techniques.This study included 104 patients with myocarditis and 26 controls, who underwent CMR at 1.5 Tesla. The CMR protocol included conventional sequences to assess myocardial edema (T2w-STIR), Early- (T1w-TSE) and Late-Gadolinium-Enhancement (PSIR). T1 quantification was performed using the modified Look-Locker inversion-recovery (MOLLI) sequence before and 15 minutes after administration of 0.075 mmol/kg Gadolinium-BOPTA. T2 quantification was performed using a free-breathing, navigator-gated multi-echo sequence. T1, T2 and extracellular volume (ECV) maps were calculated with a dedicated plug-in written for the OsiriX software. The diagnostic performance was compared between conventional parameters and global myocardial T1 (native and post-contrast), ECV and T2.The ROC areas-under-the-curve (AUC) to discriminate between patients with myocarditis and controls were 0.58 (p = 0.19) for the signal-intensity ratio myocardium/skeletal muscle on T2w-STIR, 0.67 (p = 0.01) for early myocardial enhancement on T1w-TSE and 0.80 (p < 0.0001) for presence of Late-Gadolinium-Enhancement, respectively. Furthermore, the AUC to discriminate between patients with myocarditis and controls were 0.77 (p < 0.0001) for native global myocardial T1, 0.59 (p = 0.14) for post-contrast global myocardial T1, 0.85 (p < 0.0001) for global myocardial ECV and 0.79 (p = 0.0001) for global myocardial T2, respectively.T1 and T2 mapping provide a superior performance to identify patients with myocarditis compared to conventional CMR techniques. In particular, the estimation of global myocardial ECV by T1 mapping improves the diagnostic value of CMR in patients with clinically suspected myocarditis.Marija-Orlovic-Foundation.


The correct name is Christiane Rollenhagen. The correct citation is: Rollenhagen C, Macura SL, Lathrop MJ, Mackenzie TA, Doncel GF, Asin SN (2015) Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues. PLoS ONE 10(6): e0131919. doi:


To evaluate whole body diffusion-weighted MR imaging (WB-DWI MRI) for detection, staging and operability assessment in recurrent ovarian cancer compared with CT.Fifty-one women suspected for recurrent ovarian cancer underwent 3-Tesla WB-DWI/MRI using 2 b-values (b=0-1000 s/mm²), T2- and contrast T1-weighted sequences in addition to CT. WB-DWI/MRI and CT were compared for per-patient detection of recurrence, per-site detection of disease extent including peritoneal, serosal, retroperitoneal, periportal and distant metastases and for detecting disease extent according to institutional operability criteria. Imaging findings were correlated with surgical/pathological findings or imaging follow-up for at least 6 months.According to the reference standard, recurrence was confirmed in 48/51 patients. WB-DWI MRI showed 94% accuracy for detecting recurrence, versus 78% for CT. Per-site analysis showed significantly higher sensitivity of WB-DWI MRI over CT for assessing disease extent of the peritoneum, small bowel and colon mesentery and serosa , retroperitoneal suprarenal lymphadenopathies and periportal lesions (both p=0.031). Following institutional operability criteria, WB-DWI/MRI showed better sensitivity for detection of disease extent compromising operability; mesenteric root infiltration (p=0.008), carcinomatosis of small bowel (p=0.002) and colon (p=0.016), high volumetric peritoneal disease load (p=0.004) and irresectable distant metastases (p=0.016). WB-DWI MRI correctly predicted complete cytoreduction in 93% patients undergoing cytoreductive surgery versus 40% for CT.WB-DWI MRI showed higher accuracy compared with CT for recurrence detection while improving the sensitivity for staging and operability assessment of disease extent. WB-DWI MRI may be most valuable to select patients for surgical resection.


Background. Plasmid-mediated quinolone resistance (PMQR) has received considerable attention recently. Data analysis in Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER) revealed 75% of the Enterobacteriaceae isolates to be ciprofloxacin-resistant in 2012. Few reports regarding the prevalence of PMQR are available from India. Hence, the present study was carried out to ascertain the prevalence of PMQR genes among clinical isolates of ciprofloxacin-resistant Enterobacteriaceae in JIPMER.Methods. The study included 642 ciprofloxacin-resistant clinical Enterobacteriaceae isolates. JIPMER hospital’s annual consumption data for fluoroquinolones were retrieved from the Department of Pharmacy. The test isolates were screened for the presence of qnr A, B, D, S and aac(6′)-Ib-cr genes. PMQR-positive isolates alone were tested for the presence of class I (intI1) and class II (intI2) integrons. Randomly selected PCR amplicons were sequenced and analysed using MEGA software. A total of 30 PMQR strains chosen at random were assessed for the transferability of the PMQR genes.Results. A majority of the strains exhibited high MIC values with 106 strains exhibiting MIC values >256 µg/mL. The aac(6′)-Ib-cr gene had the highest prevalence at 64% (414) while, qnrB and qnrS genes were present in 15% (97) and 10% (64) of the isolates respectively. None of the strains were positive for qnrA and qnrD. All PMQR-positive isolates were screened for class I (intI1) and class II (intI2) integrons. Class I integron was found to be predominant among the test isolates with a few of them carrying both the classes of integrons. Transferability of PMQR genes to transconjugants was identified.Conclusion. The incidence of PMQR genes in the tertiary-care setup of the JIPMER hospital was found to be high which could be probably due to the increased prescription of fluoroquinolones. Thus, there is a need for rational usage of fluoroquinolones. The ATCC strain E. coli 25922 served as the quality control in the antimicrobial susceptibility test.A total of 642 clinical isolates belonging to the family Enterobacteriaceae resistant to ciprofloxacin by Kirby-Bauer disc diffusion method subsequently confirmed by agar dilution MIC were part of the study. Only one positive culture per patient was included. Standard methods were followed for isolation and identification of the bacteria from clinical specimens like blood, pus, CSF, etc. . E. coliE. coli ATCC 25922 was included as the quality control. The lowest concentration of antibiotic at which the growth of bacteria had been completely inhibited was recorded as the MIC.The discs for amikacin (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), ciprofloxacin (5 µg) and gentamicin (10 µg), meropenem (10 µg) were prepared in-house using antibiotics in pure power form  whereas cefoperazone-sulbactam disc was procured from Microxpress Tulip Diagnostics Pvt. Ltd, India. In particular cases, bacteria were also tested for imipenem  and piperacillin-tazobactam . The antibiotic susceptibility of the test isolates was interpreted as per CLSI M100-S25. Similarly, Minimum Inhibitory Concentration (MIC) values were determined according to CLSI M100-S25 guidelines for ciprofloxacin alone by agar dilution method using Muller Hinton agar procured from Himedia Laboratories, India. JIPMER hospital’s annual prescribing data for fluoroquinolones were retrieved from Department of Pharmacy, JIPMER.intI1) and class II (intI2) integrons. Electrophoresis and staining analysed the PCR products with ethidium bromide.DNA templates were prepared from the overnight inoculum of test strains grown on Nutrient HiVeg™ Agar  resuspended in MiliQ water after three rounds of washing. Crude template DNA was prepared by boiling lysis method . The reahttp://blast.ncbi.nlm.nih.gov/Blast.cgi) against the GenBank database of the National Center for Biotechnology Information. The phylogeny of the randomly sequenced aac(6′)-Ib-cr amplicons were determined based on the nucleotide substitutions per site, i.e., the number of ’nucleotide substitutions’ within the given sequence divided by the length of the sequence using MEGA software  were randomly selected for assessing the transferability of the PMQR genes following a previously described method (E. coli (J53) AziR (sodium azide-resistant) as the recipient strain. Transconjugants were selected on MacConkey agar containing sodium azide (100 µg/mL) and ciprofloxacin (0.5 µg/mL) and confirmed based on the results of biochemical and antimicrobial susceptibility tests carried out for transconjugants, recipient and donor bacterial cells. Screening of the transconjugants by PCR assay determined the transferability of PMQR genes.PMQR-positive strains numbering 30 -Ib-cr. The qnrB and qnrS genes were present in 97 (15%) and 64 (10%) isolates respectively. None of the strains were positive for qnrA & qnrD, indicating the absence of these qnr alleles among the clinical isolates included in the study (aac(6′)-Ib-cr, qnrB and qnrS among the clinical isolates was found to be 64.49, 15.1 and 9.96 respectively. The overall prevalence of aac(6′)-Ib-cr, qnrB and qnrS genes among the clinical Enterobacteriaceae strains isolated from JIPMER hospital could probably fall in the range of 60.72–68.12, 12.5–18.04 and 7.82–12.47 respectively.Remarkably, out of the 642 strains, as many as 414 (64.5%) harboured he study . The proE. coli had the maximum frequency of aac(6’)-Ib-cr(197) and qnrB(43) genes. E. coli constituted almost half of the total aac(6′)-Ib-cr positive isolates. On the other hand, the frequency of the qnrS gene was highest among K. pneumoniae isolates (32) with Klebsiella spp. altogether accounting for more than half of the total qnrS gene identified (47). The most interesting fact was that PMQR genes were more predominant among E. coli, Klebsiella spp., and Enterobacter spp. The occurrence of qnr alleles among Citrobacter spp., Serratia spp., Proteus mirabilis and Providencia spp. were almost negligible with Proteus mirabilis alone carrying the qnrB allele -Ib-cr gene. None of the isolates harboured qnrB and qnrS simultaneously. E. coli, Klebsiella spp., Enterobacter spp. and Proteus mirabilis were the organisms carrying multiple PMQR genes but, the association of qnrS with aac(6′)-Ib-cr was seen only in E. coli and K. pneumoniae. The presence of aac(6′)-Ib-cr and qnrS genes were associated with high MIC values (≥64 µg/mL) and these associations were statistically significant with p-values <0.0000001 and <0.006261 respectively.The majority of the strains were found to carry one of the PMQR genes. But 7% of the isolates were found positive for multiple PMQR genes. All these isolates carried either Out of 528 PMQR positive isolates 212 were found to carry class I integron whereas, 95 isolates were found to carry class 2 integron and 47 isolates were positive for both the classes of integrons. However, we must admit that the study neither included the integron sequence analysis nor screened the integron-positive isolates for the presence of contiguous resistance gene cassettes.aac(6′)-Ib-cr, four were positive for qnrB, and two were positive for qnrS genes. It is interesting to note that one particular transconjugant was found positive for both aac(6′)-Ib-cr and qnrB.Of the 30 PMQR positive strains included in the conjugation only 18 transconjugants were successfully achieved. Among the transconjugants 11 were positive for aac(6′) Ib-cr, seven qnrB and four qnrS strains were sequenced but none them had novel mutations within the nucleotide sequence. We have submitted nucleotide sequences of eight aac(6′) Ib-cr, two qnrB and one qnrS genes to GenBank and accession numbers assigned are: KR080535, KR080536, KR080537, KR080538, KR080539, KR080540, KR080541 and KR080543 for aac(6′)-Ib-cr, KR080544 & KR080545 for qnrB and KR080546 for qnrS. All the identified PMQR genes were found to be closely related based on the pair-wise distance matrix value. The overall distance matrix for aac(6′)-Ib-cr was found to be 2.642 whereas the pair-wise distance matrix for the qnrB gene sequences was found out be 1.255. The study did not attempt to identify variants of qnr genes. The pair-wise distance matrix of the aac(6′)-Ib-cr gene has been summarised as percentages, i.e., the number of nucleotide substitution per 100 nucleotide in the A total of 25 qnr determinants are qnrA, qnrB and qnrS -Ib-cr genes among our isolates was found to be high. The prevalence of aac(6′)-Ib-cr in previously published reports ranged between 7% and 40% . Therefore, there was a significant association between increased MIC and the presence of PMQR genes, opening up the possibility that PMQR genes could have contributed to high MICs among the test isolates. But, we must agree that the efflux pump activities of these test isolates were not elucidated and their QRDR mutation profile was also not identified. Thus, this particular finding of the study is inconclusive as to how far PMQR genes have contributed to the increase in MIC of a strain against ciprofloxacin. With future investigation of these clinical isolates for QRDR mutations and efflux mechanisms, the prominence of PMQR in fluoroquinolone resistance can be elucidated.It is known that QRDR mutations induce high-level MICs while, PMQR genes induce low-level MICs . HoweverIn the present study, we have elucidated the prevalence of the plasmid-mediated quinolone resistance genes among clinical Enterobacteriaceae isolates recovered from a tertiary care hospital in Puducherry, India. Resistance to fluoroquinolones has predominantly increased with a majority of the isolates exhibiting high MIC values. Therefore, to combat this increased fluoroquinolone resistance it would be appropriate to use fluoroquinolones rationally for treating gram-negative infections. However, the significant finding of our study is that the prevalence of PMQR genes in JIPMER hospital is very high.qnr gene are on prevalence rates from around the world and reports on mechanistic aspects at the molecular level are very few. Future research should focus more the molecular mechanism of the PMQR genes and its encoded proteins.Moreover, the majority of the literatures on 10.7717/peerj.1995/supp-1Data S1Click here for additional data file.10.7717/peerj.1995/supp-2Data S2Click here for additional data file.10.7717/peerj.1995/supp-3Supplemental Information 1Click here for additional data file.10.7717/peerj.1995/supp-4Supplemental Information 2Click here for additional data file.10.7717/peerj.1995/supp-5Supplemental Information 3Click here for additional data file.10.7717/peerj.1995/supp-6Supplemental Information 4Click here for additional data file.10.7717/peerj.1995/supp-7Supplemental Information 5Click here for additional data file.10.7717/peerj.1995/supp-8Supplemental Information 6Click here for additional data file.10.7717/peerj.1995/supp-9Supplemental Information 7Click here for additional data file.10.7717/peerj.1995/supp-10Supplemental Information 8Click here for additional data file.10.7717/peerj.1995/supp-11Supplemental Information 9Click here for additional data file.10.7717/peerj.1995/supp-12Supplemental Information 10Click here for additional data file.10.7717/peerj.1995/supp-13Supplemental Information 11Click here for additional data file.10.7717/peerj.1995/supp-14Supplemental Information 12Click here for additional data file.10.7717/peerj.1995/supp-15Supplemental Information 13Click here for additional data file.10.7717/peerj.1995/supp-16Supplemental Information 14Click here for additional data file.10.7717/peerj.1995/supp-17Supplemental Information 15Click here for additional data file.10.7717/peerj.1995/supp-18Supplemental Information 16Click here for additional data file.10.7717/peerj.1995/supp-19Supplemental Information 17Click here for additional data file.10.7717/peerj.1995/supp-20Supplemental Information 18Click here for additional data file.10.7717/peerj.1995/supp-21Supplemental Information 19Click here for additional data file.10.7717/peerj.1995/supp-22Supplemental Information 20Click here for additional data file.10.7717/peerj.1995/supp-23Supplemental Information 21Click here for additional data file.10.7717/peerj.1995/supp-24Supplemental Information 22Click here for additional data file.10.7717/peerj.1995/supp-25Supplemental Information 23Click here for additional data file.10.7717/peerj.1995/supp-26Supplemental Information 24Click here for additional data file.10.7717/peerj.1995/supp-27Supplemental Information 25Click here for additional data file.10.7717/peerj.1995/supp-28Supplemental Information 26Click here for additional data file.10.7717/peerj.1995/supp-29Supplemental Information 27Click here for additional data file.10.7717/peerj.1995/supp-30Supplemental Information 28Click here for additional data file.10.7717/peerj.1995/supp-31Supplemental Information 29Click here for additional data file.10.7717/peerj.1995/supp-32Supplemental Information 30Click here for additional data file.10.7717/peerj.1995/supp-33Supplemental Information 31Click here for additional data file.10.7717/peerj.1995/supp-34Supplemental Information 32Click here for additional data file.10.7717/peerj.1995/supp-35Supplemental Information 33Click here for additional data file.10.7717/peerj.1995/supp-36Supplemental Information 34Click here for additional data file.10.7717/peerj.1995/supp-37Supplemental Information 35Click here for additional data file.10.7717/peerj.1995/supp-38Supplemental Information 36Click here for additional data file.10.7717/peerj.1995/supp-39Supplemental Information 37Click here for additional data file.10.7717/peerj.1995/supp-40Supplemental Information 38Click here for additional data file.10.7717/peerj.1995/supp-41Supplemental Information 39Click here for additional data file.10.7717/peerj.1995/supp-42Supplemental Information 40Click here for additional data file.10.7717/peerj.1995/supp-43Supplemental Information 41Click here for additional data file.10.7717/peerj.1995/supp-44Supplemental Information 42Click here for additional data file.10.7717/peerj.1995/supp-45Supplemental Information 43Click here for additional data file.10.7717/peerj.1995/supp-46Supplemental Information 44Click here for additional data file.10.7717/peerj.1995/supp-47Supplemental Information 45Click here for additional data file.10.7717/peerj.1995/supp-48Supplemental Information 46Click here for additional data file.10.7717/peerj.1995/supp-49Supplemental Information 47Click here for additional data file.10.7717/peerj.1995/supp-50Supplemental Information 48Click here for additional data file.10.7717/peerj.1995/supp-51Supplemental Information 49Click here for additional data file.10.7717/peerj.1995/supp-52Supplemental Information 50Click here for additional data file.


Phytophthora infestans and Solanum nigrum, S. villosum and S. scabrum. European J of Plant Pathol 120(3): 233–240. doi: 10.5580/e27. The correct reference 41 is: Rohani ER, Ismanizan I, Noor NM (2012) Somatic embryogenesis of mangosteen. Plant Cell Tiss Org Cult 110(2): 251–259. doi: 10.1007/s11240-012-0147-4.References 40 and 41 are incorrectly switched. The correct reference 40 is: Lebrecka R (2008) Host-pathogen interaction between