We previously developed a mass spectrometry-based method, dynamic organellar maps, for the determination of protein subcellular localization and identification of translocation events in comparative experiments. The use of metabolic labeling for quantification (stable isotope labeling by amino acids in cell culture [SILAC]) renders the method best suited to cells grown in culture. Here, we have adapted the workflow to both label-free quantification (LFQ) and chemical labeling/multiplexing strategies (tandem mass tagging [TMT]). Both methods are highly effective for the generation of organellar maps and capture of protein translocations. Furthermore, application of label-free organellar mapping to acutely isolated mouse primary neurons provided subcellular localization and copy-number information for over 8,000 proteins, allowing a detailed analysis of organellar organization. Our study extends the scope of dynamic organellar maps to any cell type or tissue and also to high-throughput screening.  •High-resolution organellar maps with label-free quantification (LFQ)•High-throughput organellar maps with TMT-based multiplexing•Deep mapping of EGF-induced protein localization changes with SILAC, LFQ, and TMT•A quantitative spatial proteome from mouse primary neurons Dynamic organellar maps previously provided a proteomic method for capturing protein subcellular localization changes in cultured cells. Itzhak et al. have now adapted the approach to a universal format, extending the method to all cell types. Application to primary mouse neurons provides spatial and quantitative information for more than 8,000 proteins. Spatial proteomics is an emerging field that promises to chart the location of all proteins within cells, allowing a systems view of cellular organization . We haveThe principle of subcellular proteomic profiling is to partially separate organelles by biochemical means and then to quantify the distributions of proteins across the differentially enriched subfractions. Organelle-specific profiles are derived from the distributions of known marker proteins, enabling subcellular assignment of proteins without known location. Importantly, complete isolation of individual organelles is not required; overlapping profiles can be de-convoluted and resolved by subsequent cluster analysis, provided they are sufficiently different. In the original dynamic organellar maps workflow, cell lysate is separated by differential centrifugation into six fractions . Each ofMap performance for the different quantification strategies was assessed with two MS protocols, a “fast” method that minimizes measuring time and a “deep” method that maximizes protein coverage. These reflect run parameters we anticipate will be employed by users. The MS measurement requirements for SILAC and LFQ5 were identical , and substantially lower for TMT  because of the multiplexing of samples.It was expected that the LFQ implementation would be most challenging because of the noisier quantification relative to SILAC or TMT A; hence,Organellar classification using the combined profiles of several SILAC maps enhances performance . To inveFor in-depth performance analysis of maps generated with the different quantification methods, the predictions for individual organellar clusters were evaluated. We calculated recall (the proportion of marker proteins correctly assigned to the cluster) and precision . A perfectly resolved cluster includes all relevant marker proteins and no markers from any other clusters . The harmonic mean of recall and precision, the F1 score, provides a single metric of cluster performance. A comparison of the different methods revealed that some clusters perform well irrespective of the MS acquisition method G; these Organellar predictions of non-marker proteins were stratified into four confidence classes based on SVM scores . Marker prediction accuracies within each class served as a proxy for the prediction accuracy of non-markers H. SILAC Finally, it was evaluated to what extent the organellar assignments made with the different methods agree. Concordance was calculated as the proportion of proteins with identical predictions between two quantification methods. For each comparison, the SILAC (deep) set was used as reference. Importantly, only non-maker predictions were included in the analysis. Baseline concordance was very high in all cases 84%–87%; B. A striWe next investigated the suitability of TMT and LFQ maps to capture induced protein translocations. For optimum comparison, an identical set of samples, comprising three replicate experiments of control cells or cells stimulated with epidermal growth factor (EGF) for 20 min, was analyzed with all three methods using both fast and deep protocols. These samples were used previously to follow endocytic uptake of activated EGF receptor (EGFR) but were analyzed only with the fast SILAC protocol . Here, aTo identify proteins that show subcellular movement upon EGF treatment, an improved version of our previously developed outlier test was applied . This coAlthough all three approaches successfully identified major translocations, they differed in the number of detected minor movements . Here, SKey metrics and characteristics for static and dynamic applications of each method are summarized in The successful implementation of LFQ organellar maps opened the possibility to investigate the spatial proteome of primary cells. To test this, we prepared acutely isolated neurons from embryonic mice (sacrificed at embryonic day 15 [E15]). At this stage of development, neurons show relatively little neurite arborization, which facilitates their isolation . In totaIn addition to the organellar localization data, our analysis also provided information on the global distribution across the membrane, nuclear, and cytosolic fractions for over 6,000 proteins. These included 1,120 proteins classified as mostly nuclear, 1,471 as mostly cytosolic, and 528 as nuclear and cytosolic . FinallyThe combined knowledge of protein abundance and subcellular localization data allows the reconstruction of cellular anatomy, as we have shown previously for HeLa cells . We prepAt the qualitative proteome level, 78%  of all proteins detected in the neurons were also expressed in HeLa cells  with the corresponding HeLa cell organelles E–6I. As Here we have established that SILAC, LFQ, and TMT are all highly effective for generating dynamic organellar maps through fractionation profiling, widely extending the scope of this method  were housed in a specific pathogen-free (SPF) facility with a 12:12 hr light/dark cycle and food and water available ad libitum. All animal experiments were performed in compliance with institutional policies approved by the government of upper Bavaria. For preparation of cortical neurons from embryonic mice (E15), the procedure described in Cell lysis and subcellular fractionation were performed as reported previously  and as sMass spectrometric analysis of LFQ and SILAC samples was performed with a Q Exactive HF , as described previously . For samRaw files were processed with MaxQuant version 1.5  using thEach map experiment generated an abundance distribution profile across the subcellular fractions for every quantified protein; typically, several thousand proteins were profiled in an experiment. To allow cluster analysis, established marker proteins of various subcellular compartments were then identified from a previously defined set . For unsThe detection of protein translocations followed the procedure established in To achieve FDR control, data from a previous “mock” experiment  were useStatistical analyses, data transformation, and filtering were performed in Perseus , Prism 6Copy numbers per cell, protein concentrations, and cell volumes were estimated with the proteomic ruler approach , implemehttp://www.MapOfTheCell.org).We have improved the web interface for our database of human subcellular localization predictions (Conceptualization, D.N.I., C.D., M.P.W., and G.H.H.B.; Methodology, D.N.I., C.D., A.M., J.C., M.P.W., and G.H.H.B.; Formal Analysis, D.N.I. and G.H.H.B.; Investigation, D.N.I., C.D., R.A., and J.W.; Writing, D.N.I. and G.H.H.B.; Visualization, D.N.I. and G.H.H.B.; Website, S.T.; Supervision, M.P.W. and G.H.H.B.


Plinia trunciflora is a Brazilian native fruit tree from theMyrtaceae family, also known as jaboticaba. This species has great potential byits fruit production. Due to the high content of essential oils in their leavesand of anthocyanins in the fruits, there is also an increasing interest by thepharmaceutical industry. Nevertheless, there are few studies focusing on itsmolecular biology and genetic characterization. We herein report the completechloroplast (cp) genome of P. trunciflora using high-throughputsequencing and compare it to other previously sequenced Myrtaceae genomes. Thecp genome of P. trunciflora is 159,512 bp in size, comprisinginverted repeats of 26,414 bp and single-copy regions of 88,097 bp (LSC) and18,587 bp (SSC). The genome contains 111 single-copy genes . Phylogenetic analysis using 57 cp protein-codinggenes demonstrated that P. trunciflora, Eugenia uniflora andAcca sellowiana form a cluster with closer relationship toSyzygium cumini than with Eucalyptus. Thecomplete cp sequence reported here can be used in evolutionary and populationgenetics studies, contributing to resolve the complex taxonomy of this speciesand fill the gap in genetic characterization. Plinia trunciflora (O.Berg) Kausel, synonym Myrciariatrunciflora O.Berg, is a native Brazilian tree that belongs to theMyrtaceae family and is widely distributed in the southern and southeastern areas ofBrazil  Berg (synonym M. cauliflora (Mart.) O.Berg), P.jaboticaba (Vell.) Berg (synonym M. jaboticaba O.Berg) andP. trunciflora are endemic to Brazil. All of these species producea similar grape-like edible fruit, known as jaboticaba, which presents a sweetjelly-like white pulp covered by a purple peel. Jaboticaba (P.trunciflora) has attracted attention because of its significant levels ofphenolic compounds associated with health benefits, such as antidepressant andantioxidant effects and the prevention of neurodegenerative diseases and diabetes (P. trunciflora) is largely consumedfresh or used to make jellies, juices, wines, spirits and vinegar  genome is a circular molecule of double-stranded DNA that consistsof four distinct regions, a large and a small single copy region  separated by two inverted repeat regions (IRa and IRb). Despite the highdegree of conservation in its structure, gene content and organization, the presence ofmutations, duplications and rearrangements of genes make it an attractive option forphylogenetic studies : 29°51′52″; longitude (W): 50°53′53″) were used toextract total DNA by the CTAB method (TableS1) using BWA software with two mismatches allowed from 56 species belonging to Malvids (EurosidsII) (TableS3) were used with Vitis viniferaserving as outgroup. Nucleotide sequences were aligned using MUSCLE available in MEGAversion 6.0 (http://tree.bio.ed.ac.uk/software/figtree/).In this study, young leaves from a B method . DNA qua allowed. The obtsion 6.0  with 5,0sion 6.0  was usedP.truncliflora nuclear genome were filtered against 42 Myrtaceae cp genomes.The 8,912,157 obtained reads were de novo assembled into non-redundantcontigs and singletons covering about 99% of the genome . Two final large scaffolds were obtained and joined intoa cp circular genome using Sanger sequencing. The complete cp genome of P.trunciflora is 159,512 bp in size and was submitted to GenBank (accessionnumber: KU318111). The size is similar to that of other Myrtaceae species  is the largest in Myrtaceae, e.g. S.cumini presents 14,469 bp and the same is observed in E.globulus and E. grandis. The size of the intergenic spacerlocated between the IRa/LSC border and the first gene of LSC in P.trunciflora is more similar to Eucalyptus species than itscloser species E. uniflora , S. cumini (855bp), E. globulus (855 bp) and E. grandis (853 bp). Thesame size (678 bp) for this gene is found in Arabidopsis thaliana. Theeffective size of the coding sequence is confirmed by the presence of a thymine inposition 53,811 bp in the cp genome from P. trunciflora that creates astop codon and makes this gene shorter than in other Myrtaceae.The whole cp genome analysis revealed that the cp genomes of  cumini, . Despiteuniflora . The comP. trunciflora, E. uniflora andAcca sellowiana form a single cluster of Neotropical Myrtaceae, andthat this clade has a shorter genetic distance with S. cumini than tothe Australian Myrtaceae clade  ofMyrtaceae species showed that Eucalyptus, Syzygium,Eugenia and Myrciaria (synonym ofPlinia) form a distinct clade that is consistent withcharacteristics of the pollen (Plinia could beparaphyletic in relation to Eugenia and Acca, inagreement with the embryo morphology and studies using cp regions that placedPlinia, Myrciaria andSiphoneugena as the emerging “Plinia group” (Our phylogeny includes the sister relationship of the orders Brassicales, Malvales andSapindales and the orders Geraniales and Myrtales. All these results agree with previousstudies based on multiple genes or complete cp genomes (ae clade .Additioe pollen , Plinia Plinia trunciflora genome represents the first complete cp genomesequence for the genus Plinia and shows a set of features that could befurther explored for population and phylogenetic studies within this group. Moreover,these data increase the genetic and genomic resources available in Myrtaceae by adding anew strategy of organelle genome assembly.The


Plasmodium falciparum and P. vivax, RDTs are unable to differentiate mixed infection with uncommon parasite species, i.e., P. ovale and P. malariae [Plasmodium in peripheral blood of a young boy from a remote community health centre (CHC), Darbha, in Bastar district of Chhattisgarh state, India  of the Indian Council of Medical Research (ICMR) was established at Darbha CHC of Bastar district in 2015 to provide prompt diagnosis and treatment of malaria in difficult and conflict-affected areas, as the villages in Darbha CHC are inaccessible and this region has been under continuous attack by the Maoists. There is no public transport available in the villages because of thick forest. People visit the hospital when they come to the market to purchase their daily needs.Plasmodium species identification using standard protocol [Plasmodium, with restriction sites for enzymes XhoI and BamHI. The full length of 18S rRNA gene was amplified with Phusion High Fidelity DNA Polymerase  and cloned into pBluescript SK (+) vector through restriction and ligation using DH5α competent cells. Amplification of the partial 18S rRNA gene was also performed using vector-specific M13 forward primer and Plasmodium species-specific reverse primer from ligated product. Sequencing of 18S rRNA from clones and ligated product was done by Di-deoxy chain termination method using 3130xl genetic analyser . Genes specific to each species, P. falciparum dihydropteroate synthase gene (Pf-dhps), P. vivax dihydropteroate synthase gene (Pv-dhps), P. malariae merozoite surface protein 1 gene (Pm-msp1), and P. ovale reticulocyte binding protein gene (Po-rbp) were also amplified and sequenced [Symptomatic patients were screened for malaria by bivalent RDT, SD Bioline Malaria Antigen P.f./P.v. , and microscopy. A finger-prick blood sample was collected from patients after taking written informed consent. Patients having doubtful parasite species were identified by molecular methods. Genomic DNA was isolated and polymerase chain reaction (PCR) was carried out for protocol . Universequenced , 11.This study protocol for collection of blood samples from patients with malaria infection was approved by the Institutional Ethics Committee of NIRTH, Jabalpur (Madhya Pradesh), India.A 12-year-old boy from a very remote village attended the Darbha CHC hospital with the complaint of repeated history of fever with no other complications.3, differential leukocyte count (DLC) = neutrophils (52%), lymphocyte (45%), eosinophils (2%), monocyte (1%), and serum creatinine = 0.5 mg/dL. The bivalent RDT showed the presence of P. falciparum only. Subsequently, peripheral blood smear was found positive for P. falciparum along with some doubtful structures, which a microscopist was unable to identify. He was given oral artesunate plus sulphadoxine/pyrimethamine (AS+SP). As fever was not coming down and Hb had lowered to <8 gm/dL, the patient was referred to district hospital where he was treated with intravenous antimalarial (quinine 20 mg/kg body weight on admission followed by maintenance dose of 10 mg/kg 8 hourly) along with other supportive treatment, as per the duty physician. However, he did not take the complete treatment and left hospital against medical advice. Follow-up of the patient was not possible due to inaccessibility of the area.At the time of admission, he gave history of 4 days’ fever and his body temperature was recorded as 100.8°F. His pulse rate was 101/min and his respiratory rate was 28/m. The laboratory findings of blood test at the time of admission were Hb≤10 gm/dL, blood glucose = 88 mg/dL, total leukocyte count (TLC) = 4,000/mmP. vivax and P. falciparum were highest (19%), followed by P. falciparum, P. vivax, and P. malariae (2.5%); P. falciparum and P. malariae (1.3%); and only 1 case with all 4 species (P. falciparum causes the most severe symptoms, i.e., severe anaemia, cerebral malaria, multiorgan failure, and death [P. vivax and P. ovale, although responsible for mild infection, may persist within the liver as hypnozoites, causing relapses even after treatment with blood schizonticides [P. malariae is also mild, may persist in the human population at very low density, and may cause renal failure [P. falciparum and P. vivax affected the clinical outcome in patients [P. malariae and P. ovale are largely underestimated by microscopy [In this study, 23% mixed infections were found with 2 or more species out of 160 cases of doubtful identification . Molecul species . All thend death . P. vivanticides . P. mala failure . The ava failure , Sri Lan failure , and Van failure  revealed highest %, followpatients . It is wcroscopy . The low highest %, followcroscopy . This stPlasmodium species was found, which is very rare, and this is the first report of such a case in India.A case of mixed infection of 4 Microscopy is the gold standard for malaria diagnosis, but in case of mixed infection, 1 or more species may be missed, especially in case of low parasitemia.The study showed high proportion of mixed infection by molecular methods.High proportion of mixed infections signifies the need of adequate training of health staff involved in malaria diagnosis, which is prerequisite for malaria elimination.


Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.We present here the genome sequence of the attenuated  Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a chronic disease that affects small ruminants worldwide. C. pseudotuberculosis is a Gram-positive bacterium that belongs to the Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus (CMNR) group  group . Here, wal signs . It was in sheep .http://www.bioinformatics.babraham.ac.uk/projects/fastqc), and de novo assembly was performed using Newbler 2.9 . The assembly process produced seven contigs with an N50 value of 367,637 bp. The contigs were oriented and positioned based on an optical map. The optical mapping system measures the lengths of DNA fragments after digestion with restriction enzymes. This high-resolution technique can generate ordered maps of whole genomes and can also be used in the discrimination of closely related bacterial strains  was used. Repetitive regions were mapped with the CLC Genomics Workbench 7.0 software from Qiagen, USA (CLC bio), using as a reference the genome of C. pseudotuberculosis strain 1002. The gap-filling process was done with SIMBA (http://ufmg-simba.sourceforge.net), CLC Genomics Workbench 7.0, and in-house scripts. Automatic annotation was performed by transferring information from a curated database using in-house scripts. Genes encoding tRNAs, rRNAs, and some coding sequences (CDSs) that were absent following the transfer by in-house scripts were predicted using RAST (http://rast.nmpdr.org). All CDSs were manually curated using the Artemis software (http://www.uniprot.org).The genome was sequenced using the Ion Torrent Personal Genome Machine (PGM) system, a 200-bp-fragment library kit, and a coverage of 110-fold. The quality of the reads was analyzed using the FastQC software , 49 tRNAs, and 24 pseudogenes.The complete genome of CP015100.The complete genome sequence has been deposited in GenBank under the accession no.


To the Editor: The genus Burkholderia, first proposed in 1992, has now expanded to consist of many species  and Brucella melitensis (94% probability). Because of the possible B. melitensis infection, all excess specimens and agar plates were sealed and discarded after being autoclaved. Later DNA amplification and sequencing of the bacterial 16S rDNA identified the isolates previously identified as P. fluorescens and B. melitensis as Mesorhizobium amorphae and Burkholderia fungorum (isolate BF370), respectively. The 16S rDNA sequences of BF370 (GenBank accession no. LN868266) displayed 99% similarity to B. fungorum strain DBT1 (GenBank accession no. HM113360).Arthroscopy indicated that the anterior cruciate ligament graft was intact. However, the synovial fluid was turbid, and synovial tissue infection was suspected. A synovial tissue biopsy was sent to the microbiology laboratory for culture. Test results for HIV, hepatitis B, and hepatitis C infection were negative. Fungal culture was also negative. After the tissue biopsy sample was incubated on blood agar for 48 h at an ambient temperature of 37°C, 2 different colonies grew; both showed big, yellow colonies of gram-negative rods. Vitek 2  identified the isolates as Burkholderia cepacia , gltB , gyrB , recA , lepA , phaC , and trpB . Intravenous ampicillin/sulbactam  was administered to the patient from November 29, 2013, through December 13, 2013. No pus was observed at the wound, and the patient was afebrile. He did not report discomfort, and physiotherapy commenced on day 8 after surgery.Disk-diffusion tests performed according to the Clinical and Laboratory Standards Institute interpretive standards for B. melitensis raised concern for possible laboratory transmission of this pathogen (Brucella spp. have been reported to cause joint infections (B. fungorum. This different finding suggests that commercial phenotypic identification system could sometimes be unreliable. The isolation of B. fungorum from the synovial tissue of this patient, however, is similar to an earlier case of leg tissue infection in a girl (B. fungorum had first been recovered from vaginal secretions and cerebrospinal spinal fluid of 2 women (Initial identification of the isolate as B. fungorum was detected in a granuloma specimen from a 26-year-old patient (B. fungorum infection remained unclear, recent cases (,Recently, B. fungorum during his initial injury, perhaps from the environment. The concurrent isolation of M. amorphae, a plant bacterium (B. fungorum to act as a slowly replicating opportunistic pathogen associated with tissue injuries. The slow replication rate of B. fungorum presumably mimics the growth characteristic of Burkholderia mallei, essential for the bacterium’s survival, proliferation, and evasion of host adaptive immune responses (We suspect that the patient reported here acquired


Zoothamnium Bory de St. Vincent, 1824, isolated from shallow-water sunken wood in the North Adriatic Sea (Mediterranean Sea), proposed as Zoothamnium ignavum sp. nov. We found this ciliate species to be associated with a novel genus of bacteria, here proposed as “Candidatus Navis piranensis” gen. nov., sp. nov. The descriptions of host and symbiont species are based on morphological and ultrastructural studies, the SSU rRNA sequences, and in situ hybridization with symbiont-specific probes. The host is characterized by alternate microzooids on alternate branches arising from a long, common stalk with an adhesive disc. Three different types of zooids are present: microzooids with a bulgy oral side, roundish to ellipsoid macrozooids, and terminal zooids ellipsoid when dividing or bulgy when undividing. The oral ciliature of the microzooids runs 1¼ turns in a clockwise direction around the peristomial disc when viewed from inside the cell and runs into the infundibulum, where it makes another ¾ turn. The ciliature consists of a paroral membrane (haplokinety), three adoral membranelles (polykineties), and one stomatogenic kinety . One circular row of barren kinetosomes is present aborally . Phylogenetic analyses placed Z. ignavum sp. nov. within the clade II of the polyphyletic family Zoothamniidae (Oligohymenophorea). The ectosymbiont was found to occur in two different morphotypes, as rods with pointed ends and coccoid rods. It forms a monophyletic group with two uncultured Gammaproteobacteria within an unclassified group of Gammaproteobacteria, and is only distantly related to the ectosymbiont of the closely related peritrich Z. niveum  Ehrenberg, 1838.Symbioses between ciliate hosts and prokaryote or unicellular eukaryote symbionts are widespread. Here, we report on a novel ciliate species within the genus  Chlorella (Trebouxiophyceae) is the most common genus of unicellular algae that lives in symbiosis with freshwater protists, such as ciliates and amoebae .The Latin noun Z. ignavum sp. nov. falls into the class of Gammaproteobacteria, forming a cluster with two uncultured and unclassified Gammaproteobacteria isolated from environmental samples rather than with other ecto- or endosymbionts  were similar, indicating that no additional bacteria were present in the bacterial coat on the surface of the colonies, except for the most proximal parts of the stalk. These are apparently overgrown by various unspecific prokaryotes (data not shown). The specific ectosymbionts were found on the stalk, branches, terminal zooids as well as on the macro- and microzooids . Collections in spring 2014 and summer 2015 with temperatures between 17.2 and 31.5°C indicate that this symbiosis tolerates very different temperatures. Whether they occur also in fall and especially in winter at lower temperatures needs to be further investigated.Here, we describe a novel Zoothamnium ignavum sp. nov. was found on wood pieces co-occurring with Z. niveum and its thiotrophic gammaproteobacterium “Ca. Thiobios zoothamnicoli” [Z. niveum was found on the strongly degraded parts of the wood where a strong smell of sulfide was noticeable, Z. ignavum sp. nov. was encountered on less degraded or intact parts of the wood. Occasionally, also free-living white bacteria, most likely sulfide-oxidizing bacteria could be observed  being the closest relative (96.7% sequence similarity). Another Z. alternans population from the USA, however, is rather distantly related to the Qingdao population (96.7% sequence similarity) within clade II [Z. alternans populations represent different species. Therefore, a revision of the current classification of Z. alternans with detailed morphological comparisons of populations from different geographic locations is necessary.The genus  pattern . Besidesormation . In addiclade II . This maZoothamnium was revealed during various molecular DNA analyses of the small subunit of the ribosomal RNA and the internal transcribed spacer (ITS) regions ITS1 and ITS2 [Zoothamnium, species belonging to clade II comprise various symbiotic associations with bacteria: Z. niveum with the thiotrophic bacterium “Ca. Thiobios zoothamnicoli” [Z. pelagicum with unknown bacteria assumed to be cyanobacteria [Z. ignavum sp. nov. with “Ca. Navis piranensis” gen. nov., sp. nov. (this study). For Z. alternans it is unclear whether it is associated with bacteria or not [The polyphyly of and ITS2 –57,59–61mnicoli” –37, Z. pbacteria ,88,89,91a or not ,44.Z. entzi Stiller, 1946, Z. thiophilum Stiller, 1946, Z. perlatum Stiller, 1946, and Z. urceolatum Stiller, 1946, being partially or fully covered by the sulfur-oxidizing bacterium Thiothrix Winogradsky, 1888 [Zoothamnium seems to exhibit some kind of tolerance to epigrowth. Also, the nematode family Desmodoridae Filipjev, 1922 is known for microbial fouling [Various kinds of unspecific epigrowth have been observed in other representatives of this genus –48, inclky, 1888 ,50. Cons fouling ,92.Z. ignavum sp. nov. involves a single ectosymbiont species. The colony is rather fragmentarily covered by the symbiont, with certain zooids being fully covered by a monolayer or even a multilayer of symbionts, but others, particularly most recently formed cells on the distal end of the colonies being completely aposymbiotic. In the closely related peritrich Z. niveum, the ectosymbiont forms a strict monolayer, covering the whole colony except for the most basal part of the colony [Z. ignavum sp. nov. the bacterial layer is highly variable, indicating that the growth of host and symbiont are not well coordinated in this symbiosis.The 16S rRNA gene sequence analyses and microscopic studies presented in this work revealed that the symbiosis of e colony . In ordee colony . In Z. iRiftia pachyptila (Polychaeta), which is rod-shaped but changes by terminal differentiation into larger cocci, showing transitional stages between the two morphotypes [Z. niveum coccoid rods are restricted to the oral side of the host’s microzooids and rods are found on all other parts of the host [Ca. Navis piranensis” gen. nov., sp. nov.We observed two different morphologies of the ectosymbiont, rods with pointed ends and cocci with SEM. Coccoid shaped symbionts were especially found on the oral side of the microzooids. Morphological polymorphism is found widespread in symbiotic bacteria, e.g., the thiotrophic endosymbiont of the tubeworm photypes ,94. Simithe host . These cthe host . In the Z. ignavum sp. nov. ectosymbiont forms a monophyletic group with uncultured bacteria isolated from environmental samples rather than with other symbionts. BLASTn analysis revealed that a free-living bacterium isolated from the Tao Dam hot spring in Thailand is the closest relative of “Ca. Navis piranensis” gen. nov., sp. nov. (92.1% sequence identity). Other free-living bacteria included in the phylogenetic tree represent uncultured bacteria from different shallow water to deep-sea habitats  from deep-sea hydrothermal vents in the Indian Ocean [Zoothamnium niveum [Zoothamnium niveum has a cosmopolitan distribution and was reported on or near decaying organic material in tropical to temperate waters [Crysomallon squamiferum endosymbiont revealed the variety of its metabolic capabilities, including hydrogen oxidation and assimilatory ammonification next to sulfur-compound oxidation. Further, pseudogenized genes in the endosymbiont genome included putative ABC transporters of organic compounds and putative sugar phosphotransferase transport systems, suggesting that the symbiont had an ability to grow heterotrophically until recently [Ca. Navis piranensis” gen. nov., sp. nov. remain to be further studied.The newly identified habitats . Symbiotan Ocean ,97 and tm niveum –37. Zoote waters ,98–100. recently . The metS1 Figa–c) colony, d–f) microzooid, g–i) oral ciliature. (1): stalk with branches and spasmoneme, (2): stalk without branches and with spasmoneme, (3): stalk without branches and spasmoneme, pm: paroral membrane (haplokinety), am1-3: adoral membranelles 1–3 (polykineties), in: infundibulum, man: macronucleus, s: stomatogenic kinety. b, e: modified from ; h: reus(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.


Objective:B. contaminans was cultured from respiratory secretions and liquid docusate (Colace) in a Neurosurgical Intensive Care Unit (NICU) patient with community-acquired Legionnaire’s disease but not from another bottle given to the patient. Unexpectedly, C. pelliculosa was cultured from two bottles, but not the B. contaminans bottle or respiratory secretions. Methods:B. cepacia, later identified as B. contaminans, was cultured from a bottle of liquid docusate (Colace) dispensed to a non-cystic fibrosis patient. His respiratory secretions were colonized with B. contaminans. Results: Eradication of B. contaminans colonization in the patient’s respiratory secretions was attempted. With levofloxacin, B. contaminans developed multidrug resistance (MDR). Subsequent TMP-SMX therapy did not result in further MDR. Nine other ICU patients were given docusate from the same lot, but there were no other B. contaminans isolates. Conclusion:B. contaminans colonization of respiratory secretion may be difficult to eliminate. The significance of C. pelliculosa cultured from liquid docusate (Colace) remains to be elucidated. In this case, it appeared that B. contaminans may have inhibited the growth of C. pelliculosa in the same bottle. Others should be alerted to the possibility that C. pelliculosa may be present in B. contaminans–contaminated lots of liquid docusate (Colace). B. cepacia or B. cepacia complex (BCC) colonization or infection may be acquired from a variety of contaminated aqueous sources e.g., disinfectants, mouthwash, nebulizers, intravenous solutions/medications, moisturizing cream and bacteremias, pneumonias, and urinary tract infections have rarely been reported [B. cepacia endocarditis, septic arthritis, and sepsis “B. cepacia syndrome” have been reported due to contaminated medical devices or liquid medications [Nosocomial reported ,2,3,4. Rications ,9,10,11.B. cepacia outbreak was reported by the Centers for Disease Control (CDC) and, as of 18 July 2016, included 49 confirmed cases from five states. The multistate B. cepacia outbreaks were found to be associated with contaminated liquid docusate (Colace). Cases have occurred in patients without cystic fibrosis involving critically ill patients on ventilators primarily in intensive care units (ICUs).In June 2016, an ongoing multistate B. contaminans colonization of respiratory secretions after receiving contaminated liquid docusate (Colace) in a critically ill patient with Legionnaire’s disease.We report a case of Legionella sp. titers that continued to increase over two weeks, and he was switched to levofloxacin to complete therapy. On HD #7 and HD #8 his respiratory secretions were Gram-stained and cultured. The Gram stain showed some white blood cells (WBCs) and cultures were reported positive for aerobic Gram-negative bacilli (GNB). Two days later, the GNB isolated was identified as B. cepacia. The unusual isolate was reported to Infection Control (IC) who informed the hospital epidemiologist of the CDC alert regarding the ongoing multistate outbreak of B. cepacia colonization/infection in ventilated and critically ill patients.A 28-year-old male sustained a motor vehicle accident with severe head trauma, was admitted to the neurosurgical intensive care unit (NICU) and intubated. On hospital day (HD) #3 a chest film showed a new right lower lobe infiltrate. In addition to his chest film infiltrates, extra-pulmonary findings suggested a non-zoonotic atypical community-acquired pneumonia (CAP), most likely Legionnaire’s disease. Accordingly, he was treated empirically with doxycycline for presumed Legionnaire’s disease. The diagnosis of Legionnaires disease was confirmed by serial elevated B. cepacia) was sent to the New York State Department of Health (NYS DOH) for speciation . All unused liquid docusate (Colace) lots were returned to the manufacturer by the pharmacy department.Immediately, the hospital epidemiologist advised the pharmacy department not to dispense any further liquid docusate (Colace) to any critically ill, ICU, organ transplant, hemodialysis, or oncology patients. The index case was dispensed liquid docusate (Colace) from two bottles (bottles #1 and #2: opened/used). A third bottle (bottle #3: unopened/unused) was intended for but not given to the patient. The pharmacy department recalled the three bottles related to the index case which were transported to the microbiology laboratory for culture. The index case isolate . The pharmacy department investigation revealed that at the time of the B. cepacia (B. contaminans) index case, nine other ICU patients had also received liquid docusate (Colace), but B.cepacia (B. contaminans) was not isolated from any of these or any other ICU patients. The liquid docusate (Colace) bottles used for these nine patients were discarded, and were not available for culture.The index case had serial Gram stains and cultures of his respiratory secretions to determine the duration of B.cepacia pneumonia, but chest films remained clear without infiltrates.Serial chest films showed complete resolution of the patient’s community-acquired Legionnaire’s disease, as well as normalization of the non-specific laboratory abnormalities associated with his Legionnaire’s disease, e.g., hypophosphatemia, elevated ferritin, elevated creatinine phosphokinase CPK. He remained afebrile after doxycycline therapy, and levofloxacin was used to complete therapy of Legionnaire’s disease. During the remainder of his NICU stay, serial chest films were done to detect potential B. contaminans susceptible to levofloxacin. Serial Gram stains and cultures of his respiratory secretions were obtained to monitor possible antibiotic resistance and to assess the effectiveness of levofloxacin on decreasing the intensity and duration of B.contaminans carriage.Repeat cultures of his respiratory secretions revealed heavy growth of B. contaminans, and bottle #2 (the other used/opened bottle) grew (heavy growth) C. pelliculosa. Bottle #3 (unopened/used) also grew C.pelliculosa. C. pelluosa cultured from liquid docusate (Colace) bottles  was never cultured from the patient’s respiratory secretions. C. pelliculosa was identified using API20C AUX bio Merieux Vitek, Inc., Hazelwood, MO, USA.These concerns had important Infection Control and Antibiotic Stewardship Program (ASP) implications. Liquid docusate (Colace), from the two bottles (bottles #1 and #2) given to the patient and the third bottle (bottle #3) intended for the patient (unused/closed), but not dispensed, were cultured. Bottle #1 (opened/unused) grew abundant B.contaminans spread to other patients in the ICU and the hospital. The prolonged persistence of increasingly multidrug-resistant (MDR) B. contaminans in the respiratory secretions of the index case was concerning. From an infectious disease standpoint, colonization is not usually treated. From an ASP perspective, treatment of colonization usually fails, and in the process, the organism not only persists but may become resistant or more MDR.NICU staff was educated by Infection Control on the importance of strict contact precautions to prevent B. contaminans colonization from the patient’s respiratory secretions with trimethprim-sulfamethoxazole (TMP-SMX). TMP-SMX was selected because it was the one of the few antibiotics remaining that was effective against this strain of B.contaminans and because of its ability to penetrate into respiratory secretions well. Although his strain of B. contaminans remained susceptible to TMP-SMX, TMP-SMX (10 mg/kg/day) failed to eliminate B. contaminans colonization from his respiratory secretions. However, the concentration (numbers) of B. contaminans in his respiratory secretions decreased over time, minimizing the potential for spread to other NICU patients.Because of the potential for nosocomial spread of MDR GNB in the NICU , it was decided to try to eliminate B.contaminans isolates in our 600-bed university-affiliated teaching hospital. The patient was eventually transferred to a chronic care facility for rehabilitation of his neurologic deficits.Since the index case, ongoing Infection Control and microbiology laboratory surveillance has revealed no further B. contaminans has been isolated from soil, water, and contaminated medications [B. contaminans has been reported in isolated cases, but only rarely in hospital outbreaks. BCC species are well-known opportunistic pathogens in cystic fibrosis (CF) patients [B. mulitvorans and B. cenocepacia. However, over the past decade, B. contaminans has emerged in the most frequent BCC species. BCC species differ in their clinical manifestations and preferred hosts. B. multivorans and B. cenocepacia have been the most frequent species colonizing/infecting CF patients [B. contaminans has emerged as a bona fide pathogen causing bacteremia or necrotizing pneumonia in critically ill non-cystic fibrosis patients [B. contaminans may gain access to respiratory secretions or the bloodstream in a variety of ways, e.g., B. contaminans–contaminated fluids. Unlike B. cenocepacia and B.multivorans,B.contaminans is relatively more susceptible to antibiotics, but may become MDR. During treatment, an important characteristic of BCC species is their ability to colonize/persist in fluids including respiratory secretions. In non-CF patients, B. contaminans isolated from blood or respiratory secretions should alert Infectious Control and clinicians to the possibility of a common source of BCC-contaminated fluids, particularly in ventilated or critically ill patients. BCC-contaminated fluids include mouthwash, chlorhexidine, saline solutions, nasal sprays, inhaled medications, heparin and intravenous solutions. BCC also has the potential to present as a pseudoinfection, i.e., pseudobacteremia [First identified as a separate BCC species in 2009 by DNA testing, ications ,16,17,18patients ,15,16,17patients ,14. BCC patients ,20. B. ccteremia .In hospitalized patients, colonization of body fluids with MDR GNB is ordinarily contained by Infectious Control contact precautions and usually not “treated” with antibiotics. Since BCC species are difficult to eradicate from respiratory secretions, prolonged antibiotic therapy often not only fails to eliminate the organism from secretions, but the strain often becomes more resistant over time.B. contaminans, like other BCC species, is a fastidious GNB. In the clinical microbiology laboratory, two days are required for the identification of BCC. DNA testing is required for B. contaminans speciation. Limiting the spread of MDR GNB, e.g., B. contaminans in respiratory secretions of critically ill patients, is an ongoing Infectious Control challenge. Although the usual manifestation of B. contaminans is colonization rather than infection, there have been reports of bacteremia, sepsis, and necrotizing pneumonia due to this organism in non-CF critically ill or immunosuppressed patients.B. contaminans depends on selecting an antibiotic with a high degree of inherent activity against the organism and the antibiotic should have a “low resistance” potential [B. contaminans carriage from secretions, resistance potential is an important therapeutic consideration. The antibiotic selected should also possess the requisite physiochemical and pharmacokinetic properties that permit penetration into respiratory secretions in therapeutic concentrations, i.e., antibiotics with a high volume of distribution (dV) > 0.7 L/kg. Taking these factors into account, the preferred antibiotics for B. contaminans eradication in respiratory secretions include levofloxacin, minocycline, and TMP-SMX. Other antibiotics that initially may be reported as susceptible for B. contaminans, e.g., ceftazidime, have a “high resistance potential” and are prone to develop resistance. In addition, ceftazidime’s low dV < 0.7 L/kg predicts its poor penetration into respiratory secretions. Other things being equal, subtherapeutic tissue concentrations predispose the development of MDR GNB [B. contaminans from the patient’s respiratory secretions. However, during levofloxacin therapy, B. contaminans persisted in his respiratory secretions and became more resistant. TMP-SMX was given in an attempt to decrease/eliminate B. contaminans from his respiratory secretions in order to minimize the potential for nosocomial spread. Effective Infection Control contact precautions to contain the MDR GNB in the NICU were repeatedly stressed to the staff. During TMP-SMX therapy, there was neither further loss of susceptibility nor increased resistance. TMP-SMX decreased the concentration of B. contaminans in his respiratory secretions and he never developed B. contaminans pneumonia or bacteremia. However, TMP-SMX was unable to eradicate the carriage of B. contaminans from his respiratory secretions  of liquid docusate (Colace). Not only is C. pelliculosa an extremely rare Candida sp., it was isolated from two of three liquid docusate (Colace) bottles, some of which was administered to the patient (from bottle #2). C. pelliculosa was also isolated from bottle #3 (unused/unopened) of liquid docusate (Colace). It is noteworthy that some B. contaminans strains, e.g., MS 14, produce a potent anti-fungal glycopeptide, occidiofungin. Interestingly, C. pelliculosa did not grow from bottle #1 containing B. contaminans. However, bottles #2 and #3 both grew C. pelliculosa, but B. contaminans did not grow from either bottle #2 or bottle #3.Another intriguing microbiologic aspect of this case was the significance of C. pelliculosa is a yeast and is the asexually reproducing anamorph of Wickerhamomyces anomalus  found in soil, grain, water, sewage, and especially fermenting fruit. C. pelliculosa is important in flavor enhancement, food processing, dairy fermentation and waste water treatment. C. pelliculosa has been found to be associated with the malaria vector Anopheles stephensi in Asia [C. pelliculosa has been used as a biocontrol agent due to its ability to produce potent mycotoxins, e.g., occidiofungin [C. pelliculosa has been reported to cause keratitis in corneal transplants, arthritis, meningitis (in HIV), and acute pancreatitis in bone marrow and solid organ transplants, as well as nosocomial infections in neonatal and pediatric ICUs. Risk factors associated with C. pelliculosa infection include central venous catheters (CVCs) and total parenteral nutrition (TPN). In sickle cell patients, C. pelliculosa may cause urinary tract infections or fungemia in critically ill or immunocompromised patients [ in Asia ,20. In riofungin ,25. C. ppatients ,20.B. contaminans may have also been contaminated with C. pelliculosa. C. pelliculosa–contaminated liquid docusate (Colace) bottles may have inhibited the growth of B. contaminans. We suggest that hospitals with outbreaks of B. cepacia/B. contaminans involving contaminated lots of liquid docusate (Colace) should also be alert to the potential of C. pelliculosa as well as B. contaminans intrinsic contamination. Bottles contaminated with C. pelliculosa may have also contained B. contaminans which may not have grown if the strain of B. contaminans involved produced occidiofungin and inhibited the growth of B. contaminans.It is interesting to speculate that some lots of liquid docusate (Colace) contaminated with B. cepacia (B. contaminans in this case) outbreaks. Secondly, B. contaminans is an “unusual” nosocomial isolate that may cause colonization or infection in non-CF, particularly critically ill or ventilated patients. Thirdly, as a fastidious aerobic GNB, B. contaminans requires two days for microbiologic identification as BCC. Fourth, while not highly virulent or invasive, B. contaminans may persist in aqueous environments, e.g., the patient’s secretions or body fluids as well as a variety of aqueous solutions, such as liquid docusate (Colace) in this instance. Fifth, patient exposure to C. pelliculosa from bottle #2 of the liquid docusate (Colace) did not result in the colonization of his respiratory secretions. Sixth, GNB colonization is difficult to eliminate even with antibiotics highly active against the organism, even when the antibiotic penetrates well into secretions, e.g., levofloxacin, TMP-SMX. Seventh, strict Infection Control contact precautions remain the cornerstone of containing MDR GNB, e.g., B. contaminans colonization and spread in the ICU and the hospital. Lastly, following the CDC alert, stopping further patient exposure was rapidly achieved by conducting epidemiologic and microbiologic investigations, and instituting strict Infection Control containment measures. These patient-protective measures were possible due to the rapid response of effective leadership and the coordination of several key individuals and hospital departments, e.g., the hospital epidemiologist, the Infection Control department, the NICU nursing staff, the medical microbiology laboratory, the pharmacy department, and the Infectious Disease Division.Firstly, the CDC was critical in alerting us to the ongoing multistate B. contaminans (bottle #1) and the other that grew C.pelliculosa (bottle #2), but only B. contaminans colonized the patient’s respiratory secretions. Could potential additional exposure to C. pelliculosa–containing liquid docusate (Colace) (bottle #3: unopened/unused) have resulted in C. pelliculosa colonization? As the multistate outbreak continues, Infection Control, medical microbiology and infectious disease personnel should also be alerted to the growth-suppression potential of C. pelliculosa in bottles of liquid docusate (Colace) containing B. contaminans.As in all such cases, some unanswered questions remain. In our experience, the index patient was exposed to two contaminated liquid docusate (Colace) bottles, i.e., one that grew


Pseudomonas aeruginosa and Acinetobacter species are common causes of nosocomial infections worldwide. Recently we reported the occurrence of carbapenem resistant Enterobacteriaceae, P. aeruginosa and Acinetobacter species at Mulago National Referral Hospital in Kampala, Uganda, but the isolates were not analyzed for genetic relatedness. Herein we report the intra-species genotypic diversity among P. aeruginosa and Acinetobacter baumannii isolated from hospitalized patients and the environment at Mulago Hospital, using repetitive elements-based PCR (Rep-PCR) genotyping.Multidrug resistant P. aeruginosa and 7 (0.95%) A. baumannii. Similarly, 100 samples from the hospital environment were processed yielding 33 (33%) P. aeruginosa and 13 (13%) A. baumannii. Altogether, 62 non-repetitive isolates were studied (42 P. aeruginosa and 20 A. baumannii), of which 38% (16/42) P. aeruginosa and 40% (8/20) A. baumannii were multidrug resistant . Carbapenem resistance prevalence was 33 and 21% for P. aeruginosa from patients and the environment, respectively, while it was 14 and 86% for A. baumannii from patients and environment, respectively. Cluster analysis of the Rep-PCR fingerprints revealed a high level of genetic diversity among the isolates within each species as few isolates were clustered (at 100% level of similarity). More to this, the clustered isolates revealed a complex nature of multidrug resistant P. aeruginosa and A. baumannii clones circulating at Mulago Hospital. Importantly, certain isolates from the environment and patients were clustered, implying that hospitalized patients at Mulago were probably infected with strains from the environment.A total of 736 specimens from hospitalized patients were processed for culture and sensitivity testing yielding 9 (1.2%) P. aeruginosa and A. baumannii is high at Mulago Hospital but carbapenem resistance prevalence remains relatively low in isolates from hospitalized patients. Importantly, the prevalence of carbapenem resistance in isolates from the environment is high implying the infection control practices at the hospital might be inadequate.The prevalence of multidrug resistant The online version of this article (doi:10.1186/s13104-017-2612-y) contains supplementary material, which is available to authorized users. Pseudomonas aeruginosa and Acinetobacter species are ubiquitous in hospital environments worldwide, and they are frequently implicated among the commonest causes of nosocomial infections that are difficult to treat. This is attributed to the fact that these organisms possess inherent drug resistance mechanisms , and they are also capable of acquiring additional resistance mechanisms to multiple classes of antimicrobials  through horizontal gene transfer [P. aeruginosa and Acinetobacter species. However, the emergence of carbapenemase producing strains, majority of which are multidrug resistant, is threatening the use of carbapenems in the management of such infections [transfer –3. Carbafections , 5.P. aeruginosa and Acinetobacter were first described in the 1990s and many carbapenemase producing Gram-negative species are increasingly being reported worldwide with increasing rates [P. aeruginosa and Acinetobacter baumannii at Mulago National Referral Hospital in Kampala, Uganda [P. aeruginosa and A. baumannii isolated from hospitalized patients and the environment at Mulago Hospital, using repetitive elements-based PCR (Rep-PCR) genotyping. Cluster analysis revealed a high level of genetic diversity as few isolates were clustered at ≥85% level of similarity. More to this, the few clustered isolates revealed a complex nature of multidrug resistant P. aeruginosa and A. baumannii clones circulating at Mulago Hospital.Carbapenemase producing ng rates –7. Of cong rates , 8–11 po, Uganda . HoweverP. aeruginosa and 7 (0.95%) A. baumannii . P. aeruginosa was isolated from tracheal aspirates (3), urine (2), pus (2), ear swab (1) and sputum (1), while A. baumannii was isolated from sputum (3), ear swabs (2), and tracheal aspirates (2). On the other hand, 100 samples from the hospital environment  that were processed yielded 33 (33%) P. aeruginosa and 13 (13%) A. baumannii. Altogether, 62 non-repetitive isolates were characterized (42 P. aeruginosa and 20 A. baumannii).Between September 2012 and October 2013, a total of 736 specimens from hospitalized patients were processed for culture and sensitivity testing yielding 9 (1.2%) P. aeruginosa; amikacin 15%, gentamicin 35%, meropenem 5%, ceftazidime 30%, cefepime 5%, aztreonam 40%, piperacillin/tazobactam 70%, ciprofloxacin 40%, and trimethoprim/sulfamethoxazole (SXT) 60% for A. baumannii. All P. aeruginosa isolates were susceptible to meropenem while three isolates from patients and seven from the environment were imipenem resistant, Table The antimicrobial susceptibility patterns among the isolates are shown in Tables P. aeruginosa from patients and the environment, respectively, while it was 14 and 86% for A. baumannii from patients and environment, respectively. Furthermore, all the imipenem resistant P. aeruginosa isolates were also resistant to aztreonam while 90% were resistant to piperacillin/tazobactam. On the other hand, all carbapenem resistant A. baumannii were resistant to piperacillin/tazobactam while 57% were aztreonam resistant. Of note, SXT-susceptible, carbapenem resistant A. baumannii from patients and the environment was noted, Table P. aeruginosa and 40% (8/20) of A. baumannii isolates were multidrug resistant .Overall, 67% of the carbapenem resistant isolates were positive with the imipenem/EDTA double disk synergy test, implying the isolates were metallo-beta-lactamase producers, Additional file Genotypic diversity within each species was investigated using Rep-PCR genotyping. Rep-PCRs produced 0–15 consistent polymorphic bands per isolate of sizes 250–9400 bp (BOXAIR-PCR), 90–8000 bp (ERIC-PCR), and 100–6557 bp (REP-PCR), Additional file A. baumannii isolates were not clustered  generated fingerprints that were clustered at 100% similarity scale, implying a low level of genetic relatedness but a complex nature of multidrug resistant P. aeruginosa clones could be circulating at Mulago Hospital given the diverse antimicrobial susceptibility profiles generated by the clustering isolates, Additional file For s 10 of 4, 24% genP. aeruginosa fingerprints was 0.9829 (BOXAIR-PCR), 0.9756 (ERIC-PCR), and 0.9791 (REP-PCR), indicating that the three genotyping methods successfully discriminated P. aeruginosa but BOXAIR-PCR and REP-PCR performed better than ERIC-PCR. For A. baumannii, Simpson’s index of discrimination was 0.9632 (BOXAIR-PCR), 0.9608 (ERIC-PCR), and 0.9474 (REP-PCR) indicating that the three methods also successfully discriminated the isolates but BOXAIR-PCR and ERIC-PCR performed better.The Simpson’s index of discrimination (D) for P. aeruginosa and A. baumannii at Mulago Hospital to be high but comparable to rates reported by Pitout et al. in Kenya [In this study, we found the prevalence of multidrug resistant in Kenya , Lee et in Kenya  and Gu ein Kenya . Additioin Kenya . This coin Kenya . Importain Kenya , 17 and Acinetobacter species that were SXT-susceptible were noted. Generally, SXT is not very active against Acinetobacter but it was recommended where there are no other options [Furthermore, carbapenem resistance prevalence in this study remains low in isolates from patients in accordance with recent findings at Mulago , 10, 18, options , e.g. foAlthough the metallo-beta-lactamases were associated with imipenem resistance in this study,  it is important to note that carbapenemases are not the only mechanisms of acquired resistance to carbapenems . Other mP. aeruginosa strains that are resistant to aztreonam have been reported. This is not surprising in that production of a metallo-beta-lactamase does not exclude presence of intrinsic resistance mechanisms in P. aeruginosa, e.g. up-regulated efflux pumps or outer membrane impermeability [P. aeruginosa and Acinetobacter strains that are resistant to aztreonam have been reported by researchers worldwide [P. aeruginosa isolated from hospital environments [P. aeruginosa isolates from sewage exhibited decreased susceptibility to aztreonam while 50% of the clinical isolates were resistant to this drug [Additionally, it is widely accepted that aztreonam susceptibility combined with resistance to carbapenems and other beta-lactams is a “classic” metallo-beta-lactamase phenotype . Howevereability  outlinedorldwide . Taken tronments . In a stronments  on isolahis drug .P. aeruginosa and Acinetobacter strains at Mulago. However, we could have missed detecting ESBLs and AmpC producing isolates as the double disc synergy test we used is not very efficient at detecting these enzymes particularly when ESBL activity is low, leading to wide zones of inhibition around cephalosporin/aztreonam discs [AmpC and ESBL genes are transmissible via horizontal gene transfer mechanisms and are associated with multidrug resistance, AmpC and ESBL screening in organisms other than Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis in hospitals where ESBLs and AmpC are encountered is considered an important component of infection control programmes [P. aeruginosa and Acinetobacter species is necessary.In a recent study at Mulago National Referral Hospital, more than three quarters of the Enterobacteriaceae were found to be ESBL-producers . Althougam discs . As the ogrammes . TherefoP. aeruginosa and A. baumannii circulating at Mulago Hospital as the isolates exhibited diverse antimicrobial susceptibility patterns. Further studies with a larger number of isolates are necessary to obtain a better picture of the circulating clones at Mulago Hospital. Other researchers who used Rep-PCR genotyping also reported a high level of genetic diversity among P. aeruginosa isolated from a hospital in Belo Horizonte, Brazil [P. aeruginosa, supported by lack of evidence for a widespread cystic fibrosis transmissible clone, has been reported [Generally, cluster analysis in this study revealed a low level of genetic relatedness among the isolates. The few clustered isolates point to likely occurrence of complex clones of multidrug resistant , Brazil . Moreovereported .P. aeruginosa and Acinetobacter species colonizing hospital settings in high income countries are characterized with clonality, the picture might be different in low-income settings characterized with overcrowding of patients, lack of adequate infection control and proper hygiene. Indeed, hospitals in Uganda are overcrowded with clients from all over the country hence, maintaining environmental hygiene becomes tedious [my five moments for hand hygiene’ designed for healthcare environments describes recommended levels of bed spacing and occupancy that are normally feasible in high-income countries. Overcrowding of healthcare facilities in low-income countries is often worsened by minimal infection control practices as evidenced by low standards of hand hygiene among health care workers [On the other hand, the high genetic diversity among isolates in this study might also point to either a high host diversity from where the bacteria were isolated, or lack of infection control practices. As the only national referral hospital in Uganda, Mulago admits patients from the whole country characterized by variation in geographic landscapes, human populations and human development index (HDI) . Althoug tedious . More to workers . With lo workers .P. aeruginosa and A. baumannii is high at Mulago Hospital. Carbapenem resistance prevalence remains low in isolates from hospitalized patients but high in the environment, possibly due to inadequate infection control practices. As isolates from patients and environment were clustered, hospitalized patients are at high risk of infection with multidrug resistant strains from the environment. Although the repetitive sequence elements on which Rep-PCR genotyping is based are highly conserved throughout the prokaryotic kingdom [P. aeruginosa and Acinetobacter species and other bacterial species [The prevalence of multidrug resistant  kingdom  and have species –35, we rThis was a follow-up surveillance study conducted at Mulago Hospital in Kampala Uganda, following the detection of carbapenem resistant Gram negative bacteria at the hospital in the period 2007–2009 , 10. A tP. aeruginosa from other lactose non-fermenting Gram-negative rods. Acinetobacter species were presumptively identified based on negative motility, catalase, oxidase and glucose fermentation tests, and inability to grow under aerobic conditions.The sample collection and processing procedure was described in Kateete et al. . BrieflyblaOXA-51 gene intrinsic to A. baumannii [P. aeruginosa to species level, and to determine the antimicrobial susceptibility profiles of both A. baumannii and P. aeruginosa isolates, minimum inhibitory concentrations (MICs) were performed with the ‘Phoenix™ Automated Microbiology System’ according to the manufacturer’s guidelines with minor modifications for Gram negative bacteria as described elsewhere [A. baumannii isolates, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quality control and maintenance for the ‘Phoenix™ Automated Microbiology System’ were performed as recommended by the manufacturer using reference strains of E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Phoenix™ default MIC-breakpoints were described in Kateete et al. [To confirm Acinetobacter isolates to species level, the ‘Phoenix™ Automated Microbiology System’  was used; in addition, PCR-amplification followed by DNA sequencing of the species-specific region of the aumannii  was perflsewhere , 37. In K. pneumoniae ATCC 700603 and E. coli ATCC 25922 as indicator strains [K. pneumoniae strains ATCC BAA-1705 and ATCC BAA-1706 served as positive and negative controls, respectively; strain BAA-1705 possesses a K. pneumoniae carbapenemase KPC-2 that is highly active against cephamycins, carbapenems, and to a substrate range of ESBLs [All the carbapenem-resistant isolates were tested for metallo-β-lactamase activity as bacteria producing these enzymes are characterized by rapid spread worldwide . Assays  strains , 38, 39. strains , 39. K. of ESBLs . Positivof ESBLs , 39, 41.Isolates that exhibited resistance to extended-spectrum cephalosporins (ceftazidime and aztreonam) were subjected to the double disk synergy test to detect production of ESBLs as described elsewhere , by placP. aeruginosa and A. baumannii. Chromosomal DNA used as templates in PCRs was extracted by the Cetyltrimethyl Ammonium Bromide (CTAB) method [HindIII digest DNA ladder  (New England Biolabs Inc.). REP-, ERIC-, and BOXAIR-PCR profiles were visualized under ultraviolet (UV) light after staining the gels with ethidium bromide for 30 min. Images were captured and stored as TIFF files using BioDoc-it Imaging system .Genotyping was performed with Rep-PCR assays . Initially, we followed the procedure described by Syrmis et al.  to optim) method , 43 and ) method , 44, witDNA fingerprints were analyzed with the BioNumerics software version 4  as described elsewhere , 45, 46.K. pneumoniae DSMZ 9377, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. We optimized Rep-PCR assays using the procedure described for Yersinia [P. aeruginosa and A. baumannii. Genotyping was performed at least twice in separate assays to ensure reproducibility of the patterns; BOXAIR-PCR, ERIC-PCR, and REP-PCRs were repeated for five unrelated isolates of P. aeruginosa and A. baumannii. PCRs and electrophoresis were performed in two separate trials starting from the same DNA preparation, using the same PCR reagents. None of the isolates showed qualitative differences in the fingerprints. We also investigated the effect of the DNA preparation method using chromosomal DNA extracted with two in-house methods of CTAB and boiling; although CTAB-prepared DNA consistently yielded fingerprints with stronger band intensities, no significant difference in band sizes was detected suggesting stability of our Rep-PCR fingerprints.Controls for Rep-PCR genotyping included known well characterized strains Yersinia  and adapAdditional file 1: Table S1. Antimicrobial resistance profiles of P. aeruginosa and A. baumannii.Additional file 2: Table S2. Antimicrobial resistance profiles of isolates that were clustered at 100% similarity scale. The diverse antimicrobial susceptibility profiles exhibited by the clustered isolates reveal a complex nature of multidrug resistant P. aeruginosa and A. baumannii clones circulating at Mulago Hospital.Additional file 3: Figure S1. Representative image showing agarose gel electrophoresis of P. aeruginosa and A. baumannii PCR fingerprints following BOXAIR-PCR genotyping. Panels A and B, lanes 18-42, P. aeruginosa fingerprints; panel B, lanes 43-51, A. baumannii fingerprints; lane M, DNA size marker that was reconstituted by mixing 50 base pair-DNA ladder (50-1350 bp size range) with λ DNA-HindIII digest-DNA ladder . Carbapenem-resistant isolates are indicated in red font.Additional file 4: Figure S2. Dendrogram based on Dice’s coefficient of similarity using the UPGMA method implemented by the Bionumerix program showing relationships between A. baumannii isolates according to ERIC-PCR genotyping. (E), isolate from hospital environment; (H), isolate from hospitalized patient. The susceptibility profiles (extreme right panel) for carbapenem-resistant isolates are indicated in brown font; AK, Amikacin; CN, Gentamicin; IMP, Imipenem; MEM, Meropenem; CAZ, Ceftazidime; FEP, Cefepime; ATM, Aztreonam; TZP, Piperacillin/tazobactam; CIP, Ciprofloxacin; SXT, Trimethoprim/sulfamethoxazole. bMultidrug-resistance pattern; cSXT-susceptible carbapenem-resistant A. baumannii.Additional file 5: Figure S3. Dendrogram based on Dice’s coefficient of similarity using the UPGMA method implemented by the Bionumerix program showing relationships between P. aeruginosa isolates according to ERIC-PCR genotyping. (E), isolate from hospital environment; (H), isolate from hospitalized patient. The susceptibility profiles (extreme right panel) for carbapenem-resistant isolates are indicated in brown font; AK, Amikacin; CN, Gentamicin; IMP, Imipenem; MEM, Meropenem; CAZ, Ceftazidime; FEP, Cefepime; ATM, Aztreonam; TZP, Piperacillin/tazobactam; CIP, Ciprofloxacin. bMultidrug-resistance pattern.Additional file 6: Figure S4. Dendrogram based on Dice’s coefficient of similarity using the UPGMA method implemented by the Bionumerix program showing relationships between P. aeruginosa isolates according to REP-PCR genotyping. (E), isolate from hospital environment; (H), isolate from hospitalized patient. The susceptibility profiles (extreme right panel) for carbapenem-resistant isolates are indicated in brown font; AK, Amikacin; CN, Gentamicin; IMP, Imipenem; MEM, Meropenem; CAZ, Ceftazidime; FEP, Cefepime; ATM, Aztreonam; TZP, Piperacillin/tazobactam; CIP, Ciprofloxacin. bMultidrug-resistance pattern.


Abutilon indicum, Hibiscus sabdariffa, Sida acuta and Sida rhombifolia are ethnomedicinal plants of Malvaceae, commonly used in Indian traditional system of medicines. Traditionally these plants were used in the form of extracts/powder/paste by tribal populations of India for treating common ailments like cough and cold, fever, stomach, kidney and liver disorders, pains, inflammations, wounds, etc. The present review is an overview of phytochemistry and ethnopharmacological studies that support many of the traditional ethnomedicinal uses of these plants. Many phytoconstituents have been isolated from the four ethnomedicinal plants and some of them have shown pharmacological activities that have been demonstrated by in vivo and/or in vitro experiments. Ethnomedicinal uses, supported by scientific evidences is essential for ensuring safe and effective utilization of herbal medicines.The ethnomedicinal values of plants form the basis of the herbal drug industry. India has contributed its knowledge of traditional system medicines (Ayurveda and Siddha) to develop herbal medicines with negligible side effects. The World Health Organization has also recognized the benefits of drugs developed from natural products.  Herbal drugs from ethnomedicinal plants have gained considerable importance in the recent past not only in India but also around the world . TraditiAbutilon indicum, Gossypium herbaceum, Hibiscus mutabilis, Hibiscus sabdariffa, Hibiscus rosa-sinensis, Sida acuta, Sidacordifolia, Sida rhombifolia and several others anthracene-induced preneoplastic lesions in a mouse mammary organ culture model [Terpenes are hydrocarbons of plant origin with general formula  is represented in A. indicum: anti-asthmatic, increased fertility and anti-estrogenic; H. sabdariffa: anti-hyperammonemic, anti-hypertensive and anti-mutagenic; S. acuta: abortifacient). The ethnopharmacological activities of extracts/isolates of various plant parts of A.indicum, H. sabdariffa, S. acuta and S. rhombifolia are detailed in A summary of ethnopharmacological activities (based on scientific investigations) reported among the Malvaceae members /roots of S. rhombifolia suppress carrageenan induced oedema in rats and the effect is comparable to ibuprofene [H. sabdariffa has demonstrated anti-inflammatory activity and anti-nociceptive activities by xylene-induced ear oedema and acetic acid writhing test in rat models [S. acuta showed analgesic effect in wistar mice model [S. acuta [All four plant species exhibit anti-inflammatory and analgesic activities . Ethanoluprofene  and indouprofene ,223 respt models . Aqueousce model . DiabeteS. acuta .A. indicum reduces insulin resistance in rodents by peroxisome proliferator activated receptor-gamma (PPAR-γ) agonist activity and enhancing glucose utilization [A. indicum promoted insulin production in moderately diabetic rats [H. sabdariffa is shown to prevent streptozotocin-induced liver injury in diabetic rats [S. acuta, the leaf extract exhibited hypoglycaemic and hypolipidaemic effectson alloxan-induced diabetic rats [S. rhombifolia decreased blood glucose and hence demonstrated anti-hyperglycaemic effect in streptozotocin-induced diabetic rats [Butanol extract of WP of lization . Also, tlization . Aqueoustic rats . The caltic rats . In S. atic rats . Methanotic rats .H. sabdariffa showed antibacterial effect against several bacteria including oral cavity bacteria, Streptococcus aureus and Micrococcus lutens [H. sabdariffa. It has been suggested that EtOH extract and protocatecchuic acid can be useful in food industry for preventing microbial contaminants [S. acuta [S. rhombifolia exhibited antimicrobial activity  and XO inhibition assays [S. rhombifolia, antioxidant activity in MeOH extract of leaves [The antioxidant activity of extracts . In H. s of leaf ,159, cal of leaf  and flow of leaf  showed a of leaf ,234. Fur of leaf , increas of leaf . Acetonen assays ,202. In f leaves  and EtOHf leaves .A. indicum exhibited cardioprotective effect against isopropanol- induced myocardial infarction in male rats [A. indicum caused lowering of elevated cholesterol and triglyceride in Triton WR1339 administered rats [H. sabdariffa to hypertensive rats helped in reversing cardiac hypertrophy [S. acuta reduce heart beat rate and blood flow in cardiac vessels [S. rhombifolia has significant cardioprotective effect on isoproterenol induced myocardial necrosis in rats [Ethanolic extract of roots of ale rats . Oral adred rats . In anotred rats . Adminisertrophy . Extractertrophy . Methano vessels . Ethanol in rats .A. indicum [H. sabdariffa exhibited hepatoprotective effect on thioacetamide induced hepatotoxicity in rats [S. acuta significantly decreased bilirubin, SGPT and SGOT values in paracetamol-induced hepatotoxicity in rats [S. rhombifolia has hepatoprotective effect against CCl4-induced hepatotoxicity in rats [Paracetamol and carbon tetrachloride-induced hepatotoxicity in rats could be reversed by aqueous extract of  indicum . Anthocy in rats . Root ex in rats . Powdere in rats .A. indicum showed nephroprotective effect in gentamicin-induced acute renal failure in rats [H. sabdariffa significantly decreases the effect of adenine-induced chronic kidney disease (CKD) in rats. Infusion in the form of tea is taken in many parts of the world and therefore its nephroprotective effect adds to its dietary value [S. acuta have shown to inhibit kidney stone  growth [S. rhombifolia, in vivo experiments have shown that leaf extract has nephroprotective effect in gentamicininduced nephrotoxicity in rats by decreasing urea and creatine in urine along with an increase of renal antioxidants [Ethanolic extract of roots of  in rats . Aqueousry value . In vitr) growth . In S. roxidants .A. indicum was tested for anti-anxiety property on rats at a dose of 400 mg/kg [H. sabdariffa exhibited anxiolytic effect in animal models using EPM test. Increased anxiolytic and sedative effect was also observed with repeated administration of the extract doses [H. sabdariffa was administered to patients with hypertension. The anti-hypertensive effect was then compared with control group who were given catopril. The results showed no difference in anti-hypertensive activity between experimental and control group indicating effectiveness of calyx extract to reverse hypertension [S. acuta and EtOH extract of WP of S. rhombifolia to mice in EPM experiment showed anxiolytic effect on mice [Anxiety and hypertension are often treated together. Alcoholic leaf extract of 00 mg/kg . The Elect doses . In humartension . Adminst on mice ,230. The on mice .A. indicum exhibited cytotoxic activity on U87MG human glioblastoma cells. Maximum activity was observed in a sub fraction of chloroform extract, which yielded four different components inpurified form through repeated chromatography. These components were methyl caffeate, syringic acid, trans-p-courmarate and pinellic acid. Methyl caffeate was found to be relatively more active [A. indicum has been shown to reduce the growth and viability of Schizosaccharomyces pombe cells and the active compound responsible for cytotoxicity was identified to be phytol [H. sabdariffa showed cytotoxic effect in brine shrimo lethality assay [S. acuta and S. rhombifolia, in vitro studies demonstrated cytotoxic activity in extracts of WP, AP, and/or leaf [S. acuta showed significant cytotoxicity in mouse hepatoma cells [S. acuta showed strong cytotoxicity to TRAIL (Tumor necrosis factor Related Apoptosis-Inducing Ligand)-sensitive human gastric adenocarcinoma cells [Petroleum ether, methanol, chloroform and ethyl acetate fractions of e active . The hyde phytol . Aqueousty assay . In S. a/or leaf ,203. Thrma cells . Anotherma cells .A. indicum showed anti-proliferative activity on cancer cell line by inducing the gene of apoptosis- activating factor (Apaf-1) through a network of various proteins [A. indicum induces apoptosis in colon cancer cells [H. sabdariffa have anticancerous effects. Calyx extract has shown chemopreventive effect on human gastric carcinoma [H. sabdariffa has cytostatic effect on multiple myeloma cells and oral squamous cell carcinoma, thus indicating antitumor activity [S. acuta showed strong activity in overcoming TRAIL-resistance in human gastric adenocarcinoma (AGS) cells [The ethanolic leaf extract of proteins . Gold naer cells . Aqueousarcinoma . Aqueousactivity . CryptolS) cells .A. indicum exhibited significant anti-diarrheal activity in castor oil-induced and prostaglandin E2-induced diarrhoea in rats compared to the standard drug loperamide [H. sabdariffa also exhibits anti-diarrheal activity in castor oil-induced diarrhoea in rats [S. rhombifolia could treat diarrhoea in castor oil-induced diarrhoea in mice models [Aqueous and MeOH extract of leaf of peramide . Calyx e in rats . Methanoe models .A. indicum were orally administered to experimental mice, an immune-stimulating effect was observed in the animals [H. sabdariffa showed higher immune-stimulatory effect in comparison to the drug levamisole. The extract stimulated production of interleukin-10 and lowered the production of TNF-α in mouse model [When aqueous and EtOH extracts of leaf of  animals . Aqueousse model . The extA. indicum exhibited anticonvulsant activity against pentylene tetrazole (PTZ) and Maximal Electro Shock (MES) induced convulsion in Wistar rats. An oral dose of extract (100 mg/kg and 400 mg/kg) could protect the rats against induced convulsions [of H. sabdariffa, aqueous extract of calyx prevent lipid-peroxidation in pro-oxidant induced lipid peroxidation in rat brain cells, thus suggesting a neuroprotective role. This activity could be attributed to high phenolic content resulting in strong antioxidant properties of the extract [S. acuta, leaf and stem extracts have anticonvulsant effects on pentylene tetrazole (PTZ)- induced seizures in mice [S. acuta showed neuroprotective effect in cerebral cells of experimental rats [The aqueous and EtOH leaf extracts of vulsions . Furthervulsions . In case extract . In S. a in mice . Ethanoltal rats .A. indicum showed significant antiulcer activity in asprin plus pyrolus-induced, ethanol-induced and acetic acid-induced ulcers in rat models. The treatment with extract indicated higher gastroprotective activity when compared to famotidine [H. sabdariffa in indomethacin-induced gastric ulcer showed antiulcer effect in Wistar Albino rat models [S. acuta in rats models, indicated significant antiulcer activity compared to reference ulcer drug famotidine [Leaf extract of motidine . Oral adt models . In vivomotidine .A. indicum could inhibit the activity of enzymes present in the venom of Echis carinatus  [S. acuta can neutralize the venom of Bothraps atrax [Methanolic leaf extract of d viper) . In vitrps atrax .A. indicum has been tested in vitro for anti-arthritic activity which showed a dose dependant effect on protein denaturation, membrane stabilization and inhibition of proteinases. The herbal extract exhibited more potent analgesic activity than acetyl salicylic acid, a well-established analgesic drug, for arthritis [S. rhombifolia exhibited significant anti-arthritic effect in vivo, using adjuvant-induced arthritis in rat model [The plant extract of rthritis . Ethanolat model .H. sabdariffa and leaf of S. acuta have antipyretic effect on yeast-induced fever in rats [H. saddariffa inhibits the formation of interleukin, interferons and tumor necrosis factor-α, which are produced during fever.Ethanolic extract of calyx of  in rats ,188. It H. sabdariffa inhibit low density lipoprotein (LDL) oxidation and slow down the progression of atherosclerosis by preventing lipid accumulation in rabbits fed with high cholesterol diet [Anthocyanin rich extracts from rol diet .H. sabdariffa shows antispasmodic effect in muscle preparations, e.g., rabbit aortic strip, rat uterus and rat diaphragm [S. rhombifolia, n-hexane extract of whole plant showed has strong anti-cholinesterase activity, thus suggesting its anticholinergic role [Aqueous extract of calyx of iaphragm . In S. rgic role .S. acuta, dichloromethane and ethylacetate fractions of WP extract showed antigout activity in XO inhibitory assay [S. rhombifolia, the flavonoid fraction from extract of AP showed significant antigout effect by XO activity inhibition.In ry assay . In S. rS. acuta contain cryptolepine which shows antiplasmodic effect against Plasmodium falciparum [S. rhombifolia showed antiplasmodial activity against Plasmodium bergheii infection in mice [Aerial parts of lciparum . Methano in mice .A. indicum showed mast cell stabilization in egg albumin- induced mast cell degranulation, in rat peritoneum. In the same study, anti-inflammatory effect was observed in carageenan-induced rat paw oedema model. It was suggested that bronchial asthma could be treated because of mast cell stabilization and anti-inflammatory effects of the plant extract [Methanolic extract of aerial parts of  extract .S. acuta showed significant anti-implantation activity in pregnant rats up to 7 days after conception [Ethanolic leaf extract of nception .A. indicum were tested for uterotropic and uterine peroxidise activities in ovariectomized rats and a negative correlation was established between these parameters and the plant extract. The plant extract could significantly reduce the activity of these enzymes and uterotropic response in estradiol treated rats [Methanolic plant extracts of ted rats .H. sabdariffa to ammonium chloride-induced, hyperammonemic rats, reduces ammonia, urea, uric acid, creatinine and non-protein nitrogen to normal levels in blood, indicating its anti-hyperammonemic effect [Oral administration of alcoholic extract of c effect .H. sabdariffa showed chemoprotective effects in cyclophosphamide- induced DNA damage in male Wistar rats, thus indicating anti-mutagenic activity against chemical (cyclophosphamide)- induced carcinogenesis [Aqueous extract of calyx of ogenesis .H. sabdariffa [clinical and H37Rv of Mycobacterium tuberculosis. Root extract of S. rhombifolia showed effectiveness against the standard strain M. tuberculosis H37Rv [S. rhombifolia is effective against a strain of M. tuberculosis which was resistant to streptomycin, isoniazid, rifampicin and ethambutol, thus emphasizing its antitubercular activity [Methanolic extract of WP of bdariffa  showed iis H37Rv . The ethactivity .H. sabdariffa showedin vitroantiviral effect against virus extract consisting of Hep-2 cells [S. acuta showed antiviral activity against Herpes simplex virus in virus-induced cytopathic assay [The leaf ethanolic extract of -2 cells . Methanoic assay .H. sabdariffa could relax aortic rings of muscles of hypertension-induced rats, thus demonstrating a vasorelaxant effect [S.rhombifolia showed vasorelaxant activity in rat mesenteric arterial rings. Cryptolepinone, the compound isolated from the alkaloid fraction is shown to have vasorelaxant activity [Aqueous extract of petals and the crude MeOH extract of calyces of t effect ,181. Theactivity . The alkactivity .A. indicum was measured in Swiss mice. Administration of either of the above plant material did not show any significant effect on body weight [A. indicum leaves was measured in Swiss albino mice. These were found to be safe at dose of 4000 mg/kg and 2000 mg/kg respectively and did not show mortality in mice [H. sabdariffa calyces [H. sabdariffa for 90-day were examined in albino rats. A dose of 2000 mg/kg caused the death of the animals which was preceded by a severe weight loss [H. sabdariffa by single and long-term oral administration in rats. The results indicated that the single oral administration of extract in the amount of 5000 mg/kg body weight does not produce acute toxicity.Toxicity testing is done to obtain information on the biological activity and mechanism of action of the drug. The information generated by the test is used to assess safety of the drug. Toxicity of any compound is measured in the terms of LD50 which is standard measure of toxicity of a substance that is sufficient to kill half of the sample population of a test animal. Acute toxicity of dried powder of aerial parts as well as fresh juice of leaves of y weight . Acute o in mice . In mice calyces . Effectsght loss . Sireeraght loss  measuredS. acuta, mice were injected with aqueous acetone extract of dried plant material at dose of 1; 2; 2.5; 3; 4; 5 and 6 g/kg and the LD50 values of 3.2 g/kg was determined, suggesting that the extract has negligible level of toxicity when administered orally [S. acuta was suggested to be toxic when administered at a dose of 200 mg/kg to Wistar rats [S. rhombifolia was measured using Albino wistar rats. The animals exhibited slight changes in general behaviour but did not expressed changes in their physio-pathological activities [For determining acute toxicity test of d orally .While ettar rats . Acute ttivities .A. indicum, H. sabdariffa, S. acuta and S. rhombifolia) by Indian tribal populations; and (ii) their use as ingredients in Indian traditional medicines. The pharmacological activities of the extracts and isolates of these plants that have been investigated, can be correlated with the traditional ethnomedicinal uses, detailed in S. acuta contain good quantity of crytolepine and quindoline whereas these two compounds are absent from aerial parts of S. rhombifolia. It is also observed that many ethnopharmacological activities  are common to all four plants  can help in assessing the presence of that compound in the rest of the plants. Some of the phytoconstituents are common between two or more of the four Malvaceae members, but their reported ethnopharmacological effects are different . This suggests that the phytochemicals in separate sets of conditions can exhibit different pharmacological activities which may be due to complex interaction of the phytochemicals in the cells/body of the organisms. Therefore, further studies may be undertaken to understand the exact mechanism of action of different phytoconstituents showing various pharmacological activities, by taking cue from existing scientific investigations.This review presents scientific investigations thatjustify (i) the use of plant extracts of 4 Malvaceae members (ants see . Since a


Helicobacter pylori-related gastritis with endoscopically proven gooseflesh skin-like nodularity in the gastric antrum. Although an association between nodular gastritis and gastric malignancies has been suggested, there is neither a treatment strategy nor a treatment guideline for this condition because of its relative rarity. We have recently experienced two cases of diffuse-type nodular gastritis invading both the antrum and corpus of the stomach with atypical findings that required specific treatments in two young females. The first patient was diagnosed with a suspicious low grade gastric mucosa associated lymphoid tissue (MALT) lymphoma lesion on a diffuse-type nodular gastritis, and was cured by H. pylori eradication. The second patient was diagnosed with a signet cell type gastric cancer on a diffuse-type nodular gastritis, and was cured by surgical resection. When considering the nature and significance of these gastric lesions, a link between nodular gastritis and gastric malignancy should be considered, especially in young women who have diffuse-type nodular gastritis involving both the antrum and corpus of the stomach.Nodular gastritis is a  Helicobacter pylori (H. pylori) infection in children [H. pylori infection is higher than the Western countries, nodular gastritis is reported up to 2.9% in adults [Nodular gastritis is a gooseflesh or chicken skin appearing gastritis on the endoscopic finding. This special antral gastritis is characterized by an unusual military pattern with prominent lymphoid follicles in a biopsy specimen . Althougchildren , it is rn adults . Interesn adults , 4, wherH. pylori infection is known as a cause of several gastric diseases such as peptic ulcer disease, mucosa associated lymphoid tissue (MALT) lymphoma, and gastric cancer with accumulating evidences that chronic inflammation induce gene mutations, gene amplifications, and DNA dislocations in the affected cells [ed cells . Notablyed cells . HoweverH. pylori-infected lesion suggestive of low grade gastric MALT lymphoma  and was taking iron supplement intermediately. She denied any past medical history or family history on gastrointestinal neoplasm. On arrival, her laboratory examination showed IDA with a serum hemoglobin level of 9.1 g/dL , hematocrit 28.6% , ferrum 22 µg/dL , total iron binding capacity 391 µg/dL , and ferritin 5.33 ng/mL . The fecal occult blood test showed negative finding. On the upper gastrointestinal endoscopic examination, diffuse mucosal nodularity with a cobblestone appearance was noticed . Biopsy lymphoma . PolymerH. pylori eradication was carried out, and follow up endoscopic examination was carried out after 4 weeks of therapy. Cobblestone appearance indicating diffuse-type nodular gastritis was still noticed from the antrum to lower corpus (H. pylori infection nor a gastric MALT lymphoma in all the biopsied specimens from the antrum and corpus. Finally, the diagnosis was given as a nodular gastritis without an evidence of gastric MALT lymphoma-mimicking lesion after a successful H. pylori eradication.r corpus . HistopaAfter three months of regular iron intake, IDA was no longer detected. The blood tests showed serum hemoglobin level of 12.4 g/dL, hematocrit 36.0%, ferrum 174 µg/dL, total iron binding capacity 313 µg/dL, and ferritin 30.94 ng/mL. Follow up endoscopic examination with multiple biopsies showed an improvement of nodular gastritis . The patA 30 years old Korean female visited our health promotion center for a routine check-up. She had neither a remarkable past medical history nor a family history. She did not take any medication, although she felt intermittent upper epigastric discomfort recently. No abnormal finding was noticed on her initial blood test. On the upper gastrointestinal endoscopic examination, a 3 cm sized ulcerated lesion was detected . BiopsieThe tumor was resected by subtotal gastrectomy  with mulH. pylori infection can lead to several gastric malignancies [H. pylori-associated gastritis, and that lymphoproliferative pangastritis with a lymphoepithelial lesion would be a temporal change in H. pylori-induced B cell proliferation leading to a gastric MALT lymphoma [gnancies , 6. Althlymphoma , 7. In aH. pylori infection leading to a severe form of nodular gastritis [H. pylori infection is lower in women than in men before menopause [The cause of female predominance in nodular gastritis is uncertain, although there might be a gender specific immunity affecting the outcome of astritis , 4. Indeenopause . HoweverH. pylori eradication therapy is usually indicated for gastric dysplastic lesions based on the Correa hypothesis [H. pylori eradication [H. pylori eradication [H. pylori eradication, number and size of the lymphoid follicles decrease significantly when compared to the groups without H. pylori eradication or failed eradication [H. pylori eradication, but resolves slowly taking more than three months to regress, as noticed in our first case. In summary, H. pylori eradication therapy may decrease the risk for MALT lymphoma in patients with nodular gastritis.Chemoprevention by pothesis . In addidication . Howeverdication , 10. Whedication . Indeed,dication . It seemHerein, we reported two cases of diffuse-type nodular gastritis with significant gastric lesions in young women. Based on our cases, there might be a link between nodular gastritis and gastric malignancy especially in young women with diffuse-type nodular gastritis.