Phytomedicine19 (2012) 1270–1284
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Phytomedicine
j o u r n a l h o m e p a g e:w w w.e l s e v i e r.d e/p h y m e
d
Review
Endophytic fungi—The treasure chest of antibacterial substances
Nataˇs a Radi′c a,b,?,BorutˇStrukelj c,d
a Celica,Biomedical Center,Technology Park24,Ljubljana,Slovenia
b Laboratory for Neuroendocrinology-Molecular Cell Physiology,Institute of Pathophysiology,Faculty of Medicine,University of Ljubljana,Zaloska4,1000Ljubljana,Slovenia
c Department of Pharmaceutical Biology,Faculty of Pharmacy,University of Ljubljana,Aˇs kerˇc eva cesta7,Ljubljana,Slovenia
d Department of Biotechnology,Joˇz efˇS tefan Institute,Jamova39,Ljubljana,Slovenia
a r t i c l e i n f o
Keywords:
Endophytic fungi
Antibacterial
Metabolites
Bacterial resistance a b s t r a c t
Over more than20years,the endophytic fungi have been explored as“biofactories”of novel bioactive substances,and they have not disappointed.Among the extracts and pure substances obtained from the culture broths or fungal biomass,some have exerted antibacterial activity ranging from moderate to powerful when tested on the bacterial strains resistant to the antibiotics currently in use.In this article we review the accumulated data on endophytic fungi isolated from plants that produce metabolites with antibacterial activity against human pathogenic bacteria.
? 2012 Elsevier GmbH. All rights reserved.
Contents
Introduction (1270)
Antibacterial substances isolated from endophytic fungi (1271)
Endophytic fungi producing metabolites effective against Helicobacter pylori (1281)
Endophytic fungi as producers of the same metabolites as the host plant (1282)
Discussion (1282)
Con?ict of interest (1283)
Acknowledgments (1283)
References (1283)
Introduction
According to the World Health Organization(WHO)Global Bur-den of Disease report in the year2004,cardiovascular diseases were the leading cause of death in the world,particularly among women. Infectious and parasitic diseases were the next leading cause,caus-ing15.6%of all deaths in women and16.7%in men,while cancers occupy the third place on the list(WHO,World Health Organization 2008).The World Health Day2011campaign,launched by the WHO is offering a strategy to safeguard the existing antibiotics for future generations and contain the spread of antimicrobial resis-tance.In the battle against the ever-increasing multidrug resistance of human pathogenic bacteria,we urgently need new alternatives to the currently available broad-spectrum antibiotics.Bacterial species recently named as the“ESKAPE”pathogens:Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter ?Corresponding author at:Celica,Biomedical Center,Technology Park24, Ljubljana,Slovenia.Tel.:+38640252279.
E-mail address:natasaradic@https://www.doczj.com/doc/7511492464.html,(N.Radi′c).baumanii,Pseudomonas aeruginosa,and Enterobacter species cause the majority of hospital infections and effectively“escape”the effects of antibacterial drugs(Rice2008).Resistance has increased in both Gram-positive and Gram-negative pathogens,and poses a serious threat to the successful treatment of infectious diseases. Another alarming fact is that the number of new antibacterial drugs that make it through the complete development process and ultimately receive approval has decreased over the past25 years.Boucher et al.report a75%decrease in systemic antibacteri-als approved by the FDA from1983through2007,with evidence of continued decrease in approvals between the years2003and2007 (Boucher et al.2009).Modern medicine was built on a reliance on antibiotics,but we are now heading towards a world without them, which is why there are more and more initiatives to raise awareness of this problem.European Federation of Pharmaceutical Industries and Associations,the voice of the research-based pharmaceutical industry in Europe,welcomed and supported the launch by the Innovative Medicines Initiative(IMI)a D223,7million program to tackle antimicrobial resistance and to speed up the development of new antibiotics(EFPIA,European Federation of Pharmaceutical Industries and Associations2011).
0944-7113/$–see front matter? 2012 Elsevier GmbH. All rights reserved. https://www.doczj.com/doc/7511492464.html,/10.1016/j.phymed.2012.09.007
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So,where do we look for new antibiotics?Whenever a new niche of biodiversity is discovered and accessed,new natural products are found.The realization that there is a large,and mostly unex-plored,group of fungi living inside higher plants(endophytic fungi) led to focused discovery efforts in both industrial and academic laboratories.Plant endophytic fungi are fungal microorganisms which spend all or part of their lifecycle inter-and/or intracel-lularly colonizing healthy tissues of their host plants,typically causing no apparent disease symptoms(Tan and Zou2001).Plant endophytic fungi,have the special ability to produce the same or similar compounds originated from their host plants,as well as a great number of diverse bioactive compounds,which have been implicated in the protection of its host against pathogens and herbi-vores(Wicklow et al.2005).These structurally diverse molecules have potential therapeutic value which is why people’s interest in screening endophytic fungi for discovery of novel metabolites,and more speci?cally novel antibiotics,has increased.The natural prod-uct that is mostly“to blame”for the recognition of plant endophytic fungi as an important source of natural bioactive products is pacli-taxel(taxol)due to the discovery of the taxol producing endophytic fungus Taxomyces andreanae in1993(Stierle et al.1993).
Endophytic fungi have been found in each plant species exam-ined,and it is estimated that there are over one million endophytic fungi existing in nature(Petrini1991).Since the?rst endophytic fungus was identi?ed,a lot of attention has been given to the potential of exploitation of these fungi for the production of novel antibiotics.This niche should be meticulously investigated and used as a base for sustainable research and development of new antibacterial substances that can both respond to current antimi-crobial resistance and anticipate evolving resistance.This paper reviews the data concerning the research of new antibacterial substances against human pathogenic bacteria produced by endo-phytic fungi isolated from plants.
The discovery of new species of endophytic fungi continues as well as the discovery of novel antibacterial substances.The rea-soning behind host plant selection has mostly been to investigate plants that are used in traditional medicine for the treatment of infections and identify the endophytic fungi found in different parts of those plants.The success of discovering naturally occur-ring therapeutic agents depends on bioassay-guided fractionation and puri?cation procedures.Fractionation of the culture broth and mycelium extract leads to the isolation of the metabolite respon-sible for the antibacterial activity.In the following text we review the results of antibacterial testing of crude extracts and puri?ed substances obtained from different endophytic fungi.We try to list them in alphabetical order of the genus of the tested fungi as far as possible.Presented in Table1are endophytic fungal strains, host plant from which they were isolated,the host plant’s habitat, type of extract or secondary metabolite with antibacterial activ-ity,strains of human pathogenic bacteria and the method used for antibacterial testing and the literature references.
Antibacterial substances isolated from endophytic fungi The number of secondary metabolites produced by endophytic fungi is larger than that of any other endophytic microorganism class.This might partially be a consequence of the high frequency of isolation of endophytic fungi from plants(Zhang et al.2006). Due to the same reason,some fungal genera seem to have a higher frequency of isolation and therefore a relatively greater chance of discovering an antibacterial substance produced by its belonging species.
The discovery of an antibacterial effect of a crude extract of the culture broth or the mycelium is the?rst of the steps needed for the discovery of a new antibiotic.It often happens that the individual substances comprising a crude extract do not have a potent antibacterial activity themselves,but act synergistically in a mixture.The identi?cation and structure elucidation of the most potent metabolite is essential in the development of a new antibi-otic that would potentially be used in therapy.
Two new10-oxo-10H-phenaleno[1,2,3-de]chromene-2-carboxylic acids,xanalteric acids I and II(Fig.1),and11known secondary metabolites were obtained from extracts of the endo-phytic fungus Alternaria sp.,isolated from the mangrove plant Sonneratia alba collected in China(Kjer et al.2009).The two new compounds xanalteric acids I and II showed weak antibacterial activity against Staphylococcus aureus with MIC values of250 and125?g/ml,respectively.Altenusin(Fig.1)exhibited broad antimicrobial activity against several resistant pathogens with MIC values in the range of31.25–125?g/ml.
In a more recent study of antimicrobial activity of crude extracts from mangrove fungal endophytes Buatong et al.(2011)tested a total of385extracts from150fungal endophytes with an antimi-crobial screening test(a colorimetric microdilution method).They isolated endophytic fungi from leaves and branches12mangrove species(Aegiceras corniculatum,Avicennia alba,Avicennia of?cinalis, Bruguiera gymnorrhiza,Bruguiera parvi?ora,Lumnitzera littorea, Rhizophora apiculata,Rhizophora mucronata,Sonneratia caseolaris, Scyphiphora hydrophyllacea,Xylocarpus granatum and Xylocarpus moluccensis)collected from mangrove areas in the south of Thailand in Satun,Songkhla,Surat Thani and Trang Provinces.They prepared crude ethyl acetate extracts from the culture broth and ethyl acetate and hexane extracts from the fungal mycelia and determined their MIC and minimal bactericidal concentrations(MBC)against human pathogenic bacteria.Ninety-two isolates produced inhibitory com-pounds.Most of the extracts(28–32%)inhibited S.aureus(MIC/MBC 4–200/64–200?g/ml).Only two extracts inhibited P.aeruginosa (MIC/MBC200/>200?g/ml)and none of the extracts inhibited E.coli.The most active fungal extracts were from six genera, Acremonium,Diaporthe,Hypoxylon,Pestalotiopsis,Phomopsis,and Xylaria.Phomopsis sp.MA194isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of8–32?g/ml against Gram-positive bacteria.
Chromatographic separation of extracts of cultures grown in liq-uid or on solid rice media of the endophytic fungi Ampelomyces sp.isolated from the medicinal plant Urospermum picroides yielded 14natural products that were identi?ed based on their1H and 13C NMR as well as mass spectra and comparison with previously published data.3-O-methylalaternin,obtained from the extracts of Ampelomyces sp.grown in liquid culture,and altersolanol A, form the fungus grown on solid rice medium,both displayed antimicrobial activity against the Gram-positive pathogens.3-O-methylalaternin showed activity with a MIC of12.5?g/ml against Staphylococcus epidermidis,S.aureus,Enterococcus faecalis.Alter-solanol A featured a MIC value of12.5?g/ml against S.epidermidis and E.faecalis,and25?g/ml against S.aureus(Aly et al.2008).An earlier report by Yagi et al.had already revealed that altersolanol A inhibits the growth of Gram-positive bacteria and Pseudomonas aeruginosa IFO3080when tested using the broth dilution method (Yagi et al.1993).
Aspergillus sp.HAB10R12was isolated from the root of Garcinia scortechinii,a small tree distributed throughout Malaysia which is often used by local people for peptic ulcer and postpartum care (Ramasamy et al.2010).Xanthones isolated from the host plant G.scortechinii have been found previously to inhibit methicillin-resistant Staphylococcus aureus(MRSA)(Sukpondma et al.2005). The antimicrobial activity of the crude ethyl acetate extract of the culture agar was tested against Bacillus subtilis,Escherichia coli, Micrococcus luteus and S.aureus using a disc diffusion method. Extract of the HAB10R12was able to signi?cantly(p<0.05)inhibit B.subtilis(24mm)and S.aureus(23mm),better than M.luteus
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trukelj /Phytomedicine 19 (2012) 1270–
1284Fig.1.Structural formulas of some of the antibacterial substances produced by endophytic fungi.
(18mm).The positive controls (ampicillin,ceftriaxone,cephalexin
and gentamicin)showed inhibitory activity as expected,resulting in inhibition zones with diameters ranging from 11to 25mm.The antibacterial effect of HAB10R12was similar to that of the con-trol antibiotics when tested against M.luteus and S.aureus and signi?cantly (p <0.05)greater than gentamicin against B.subtilis and E.coli ,and cephalexin against B.subtilis .
Kharwar et al.reported a highly functionalized naphthaquinone javanicin (Fig.1),with promising antibacterial activity,from an endophytic Chloridium sp.that was isolated from the surface treated root tissues of Azadirachta indica A.Juss (Kharwar et al.2009).In their antibacterial test,javanicin was active against E.coli and Bacillus sp.at a higher MIC value of 40?g/ml.The bacte-ria that were the most sensitive to javanicin (2?g/ml)were P.aeruginosa and P.?uorescens .This could be an indicator of the selective antibacterial activity of javanicin,but it should be con-?rmed with additional testing.
Arivudainambi et al.have isolated a new endophytic fungus Col-letotrichum gloeosporioides from the medicinal plant Vitex negundo
L.and tree different extracts (hexane,ethyl acetate and methanol)were screened for their antibacterial activity against methicillin-,penicillin-and/or vancomycin-resistant clinical strains of S.aureus (Arivudainambi et al.2011).The results of the disc diffusion method showed that methanol extract had an effective antimicrobial activ-ity against all tested bacteria.The methanol extract produced a maximum inhibition zone of 21.6mm against S.aureus ,19.6mm against B.subtilis ,18.3mm against E.coli and 18.6mm against P.aeruginosa .In contrast,the hexane extract had no inhibitory effect against all the tested organisms.The ethyl acetate extract exhibited moderate antimicrobial activity against all the tested microorganisms.More importantly,they also tested the extracts on 10different clinical isolates of resistant S.aureus .The hexane and ethyl acetate fungal extracts had no antibacterial activity against multidrug-resistant S.aureus strains.But the methanol extract of C.gloeosporioides showed an effective antibacterial activity against S.aureus strains.A maximum inhibition zone of 20mm was observed against S.aureus strain 9which was resistant to vancomycin,methi-cillin and penicillin.
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It is also fairly common to?nd the same endophytic fungi in different host plant species.Colletotrichum gloeosporioides is an example of this.The same endophytic fungus was isolated from the stem of Artemisia mongolica(Fisch.ex Bess.)Nakai.Antimicro-bial bioassay revealed that colletotric acid(Fig.1),isolated from the culture liquid,was inhibitory to the bacteria B.subtilis,S.aureus, and Sarcina lutea with minimal inhibitory concentrations(MICs)of 25,50,and50?g/ml,respectively(the MICs of ampicillin against these microorganisms:0.05,0.5,and0.01?g/ml)(Zou et al.2000). What is interesting is that colletotric acid was puri?ed from the ethyl acetate extract of the culture broth,and Arivudainambi et al. also reported moderate inhibitory activity of the ethyl acetate extract.
Similarly,Lu et al.reported that the metabolites of endophytic fungus Colletotrichum sp.isolated form Artemisia annua had strong antimicrobial activity against the bacteria B.subtilis,S.aureus,S. lutea and Pseudomonas sp.(Lu et al.2000).With the use of a combi-nation of spectroscopic methods(IR,MS,1H and13C NMR)they elu-cidated the structure of tree new metabolites:6-isoprenylindole-3-carboxylic acid;3b,5a-dihydroxy-6b-acetoxy-ergosta-7,22-diene and3b,5a-dihydroxy-6b-phenylacetyloxy-ergosta-7,22-diene and also isolated tree more substances with known structures:3b-hydroxy-ergosta-5-ene;3-oxo-ergosta-4,6,8(14),22-tetraene and 3b-hydroxy-5a,8a-epidioxy-ergosta-6,22-diene.All of them exhib-ited antimicrobial activities with MICs ranging from25to75?g/ml.
Endophytic fungus from the genus Colletotrichum was isolated among others from healthy tissues of Lippia sidoides,a medic-inal plant used as an antiseptic in the northeast of Brazil(de Siqueira et al.2011).From480fragments of leaves and stems of L.sidoides,a total of203endophytic fungi were isolated,represent-ing14species belonging to the groups Ascomycota,Coelomycetes and Hyphomycetes.Colletotrichum gloeosporioides was the most frequently isolated,followed by Alternaria alternata,Guignardia bid-welli and Phomopsis archeri.All the isolated fungal strains were submitted to an antimicrobial assay on solid medium(Ichikawa et al.1971).The endophytic fungi with antimicrobial activity were limited to four species:A.alternata,P.archeri,C.gloeosporioides and Drechslera dematioidea with inhibition zones ranging from17to 25mm against S.aureus and Bacillus subtilis.
In the investigation of the endophytic fungi associated with medicinal plants Dendrobium devonianum and D.thyrsi?orum col-lected in Vietnam30endophytic fungi were isolated from100 tissue segments(50segments from stem and root each)of D.devo-nianum,while23isolates were gained from D.thyrsi?orum.The antimicrobial activity of all53endophytic fungi was evaluated against six human pathogens(Xing et al.2011).Antimicrobial activ-ity of the ethanol extract of the fungal fermentation broth at a concentration of100?g/disk was tested with the agar diffusion method against S.aureus,E.coli,and B.subtilis.Epicoccum from root of D.thyrsi?orum exhibited an inhibitory activity against S.aureus, E.coli,and B.subtilis.The emphasis was put on the ethanol crude extract of Epicoccum nigrum that displayed the strongest antagonis-tic effect on S.aureus even superior to ampicillin.Four isolates in D. devonianum exhibited antagonistic effects against more than one pathogenic microorganism out of which Phoma showed greatest inhibitory activity against E.coli.Seven endophytes from D.thyr-si?orum were active.Phoma from root of D.thyrsi?orum exhibited strongest activity against B.subtilis and slightly inhibited S.aureus as well.Fusarium tricinctum from D.thyrsi?orum showed antagonis-tic actions against E.coli and B.subtilis.The antibacterial potential of Epicoccum sp.was noted back in1978when Baute et al.stud-ied a strain751-5of Epicoccum nigrum,which was isolated from atmosphere in1958at the Centre de Recherches de Bioclimatolo-gie of Pau(France).It should be noted that in this case the strain of E.nigrum was not endophytic,but nonetheless,the research group successfully isolated epicorazine A and B as the active antibacterial substances from the chloroformic extract of the culture broth (Baute et al.1978;Def?eux et al.1978a,b).
Adding to the already broad palette of structural diversity of metabolites produced by Fusarium endophytes,Shu et al.have iso-lated two antibacterial cerebrosides,designated cerebroside1and 2,from chloroform–methanol(1:1)extract of Fusarium sp.IFB-121, an endophytic fungus in Quercus variabilis.The cerebrosides were strongly active against B.subtilis,E.coli,and P.?uorescens,with the MICs of cerebroside1being7.8,3.9,and7.8?g/ml,and that of cere-broside2being3.9,3.9,and1.9?g/ml,respectively.The MICs of the amikacin that was used as a positive reference against the three bacteria were0.45,3.9,and3.9?g/ml,respectively(Shu et al.2004).
Investigations on the antimicrobial activities of endophytic fungi in Dendrobium loddigesii Rolfe were carried out by Chen et al. (2010b).They isolated48fungal cultures from120healthy sam-ples of D.loddigesii and grouped them into18identi?ed genera,of which Fusarium and Acremonium represented21of the total iso-lates and were the dominant genera in the plants.Chaetomella, Cladosporium,Nigrospora,Pyrenochaeta,Sirodesmium,and Thielavia were found in Dendrobium for the?rst time.The ethyl acetate extract of the culture?ltrate of17(35.4%)isolates,all obtained from roots,showed antimicrobial activity against one or more of the human pathogenic microbes.Surprisingly,none of the tested extracts exhibited inhibitory activity against E.coli.When tested on S.aureus,the most active were the strains designated F. solani2and Bionectria sp2.,with the diameter of inhibition zone of22.7±0.7mm and24.0±0.6mm,respectively.Accounting for almost half of the active isolates,Fusarium was the predominant genus in antimicrobial isolates.
In a study by Sim et al.a total of24endophytic fungi that com-prised10different genera were successfully recovered from the isolation process,which included four isolates from Garcinia man-gostana,and20from Garcinia parvifolia,selected randomly from the fruit farm of Sungai Rengit Village,Johor,Malaysia(Sim et al. 2010).The antibacterial activity of the?ltered broth suspension of all the isolated endophytic fungi was measured using a well diffusion method.Eleven isolates(45.8%)displayed antimicrobial activity against at least one test microorganism with inhibition zones that ranged from5to12mm.Two isolates,56GP and190GP, identi?ed as Fusarium equiseti and Guignardia vaccinii,respectively, possessed the most antibacterial activities.
Dragon’s blood is a deep red resin,which has been used as a famous traditional medicine since ancient times in many countries. In China,the red resin of Dracaena cambodiana Pierre ex Gagnep. (Agavaceae)is used as the main source of Chinese dragon’s blood. The other very important source is Aquilaria sinensis(Lour.)Gilg. (Thymelaeceae).Gong and Guo studied the endophytic fungi of these two plants and obtained300isolates.172were from Dracaena cambodiana and128from Aquilaria sinensis.21isolates showed antimicrobial activity,however,none of these isolates was active against E.coli,and only15were active against B.subtilis and/or S. aureus.Out of those15,nine isolates were a Fusarium sp.and active against B.subtilis with the inhibition zone diameter in the range of 10.33–22.00mm.The isolate DC-2–32,identi?ed as Fusarium sp.1, exhibited strong antibacterial activity with a22mm inhibition zone diameter to B.subtilis,and should be considered for further inves-tigation.Moreover,some of the isolates exhibited broad spectrum antimicrobial activity and the inhibition zones ranged from7to 27mm.The active isolates were identi?ed to17taxa.Just as in the study by Chen et al.(2010b),in this one also Fusarium spp.was the most dominant genera in two plants and showed the most potent antimicrobial activity(Gong and Guo2009).
The same research group investigated samples containing Dragon’s blood from D.cambodiana and D.cochinchinensis and a total of49fungal isolates were obtained,of which43iso-lates belonged to18genera,and another six were unidenti?ed
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fungi.Fusarium was again the dominant genus isolated from D. cambodiana,comprising14isolates out of26(Cui et al.2011). Twenty isolates displayed antimicrobial activity against at least one pathogenic microorganism.The inhibition zones ranged from 8.0mm to30.9mm.Among all the isolates,14.3%,16.3%and18.4% of endophytic fungi inhibited E.coli,B.subtilis and S.aureus,respec-tively.Isolate YNDC07exhibited signi?cant antibacterial activity against S.aureus with a diameter of the inhibition zone greater than17mm.Isolates YNDC05and YNDC11also exhibited strong activities against B.subtilis and E.coli,respectively.
In Indonesia,the indigenous communities have been using Garcinia mangostana plant for the treatment of various infectious diseases.Guided by the possibility that the endophytic fungi resid-ing within part of plants could also produce metabolites similar to the activity of their respective hosts,a screening of the antibacterial activity of endophytic fungi isolated from surface sterilized leaves and small branches of Garcinia mangostana was conducted(Radji et al.2011).During this study,24fungal isolates were recovered and the crude ethyl acetate extracts of all the endophytic fungi were tested for their antibacterial activity against S.aureus,B.subtilis, E.coli,P.aeruginosa,S.typhi and M.luteus.Antibacterial activity was determined using the disc diffusion method.Out of24isolates,10 isolates could inhibit some tested human pathogenic bacteria used in this study.Each of them displayed antimicrobial activity against at least one test microorganism with inhibition zones that ranged from6.5to14.7mm.More than half of the active isolates inhibited strains of Gram-positive bacteria better than Gram-negative bacte-ria.The strongest antibacterial activity against selected bacteria was displayed by isolate RGM-02.The inhibition zones against B. subtilis,S.aureus,and M.luteus were14.7,12.9and13.5mm,respec-tively.Surprisingly,the ethyl acetate extract from isolate RGM-02 had MIC values of25?g/ml against M.luteus and25?g/ml against S.aureus,which was only two and four times higher than that of amoxicillin(MIC12.5and6.25?g/ml),respectively.Based on18S ribosome RNA sequence analysis,the isolate RGM-02was identi?ed as Microdiplodia hawaiiensis CZ315.
Krohn et al.have reported the discovery of several new aromatic,hydrogenated,and structurally unique ring-extended xanthones from different endophytic fungi.Three new metabolites, microsphaeropsones A–C with a unique oxepino[2,3-b]chromen-6-one(ring-enlarged xanthone)skeleton,citreorosein and an enone(oxidized microsphaeropsone A)were isolated from the endophytic fungus Microsphaeropsis species.From Microsphaerop-sis species,large amounts of fusidienol A and known aromatic xanthones were isolated and from Microdiplodia sp.3,4-dihydroglobosuxanthone A was puri?ed.Preliminary studies by agar diffusion assay showed that those metabolites have antibac-terial activity against E.coli(Krohn et al.2009).
In2004,fusicoccane diterpenes,named periconicins A and B (Fig.1),were isolated from an endophytic fungus Periconia sp.,col-lected from small branches of Taxus cuspidata(Kim et al.2004). They were puri?ed from the ethyl acetate extracts of the broth, which were active in the antibacterial assays.Periconicin A exhib-ited signi?cant antibacterial activity against B.subtilis,S.aureus, Klebsiella pneumoniae and Salmonella typhimurium with MIC in the range of3.12–12.5?g/ml,in comparison to gentamicin,with the MIC in the range of1.56–12.5?g/ml.Periconicin B exhibited mod-est antibacterial activity against the same strains of bacteria with MIC in the range of25–50?g/ml.Both periconicins A and B were inactive against E.coli.
Phomodione,an usnic acid derivative,was isolated from culture broth of a Phoma species,discovered as an endophyte on a Guinea plant(Saurauia scaberrinae).In addition to phomodione,known compounds with antibiotic activity,usnic acid and cercosporamide, were also found in the culture medium.Phomodione exhibited a MIC of1.6?g/ml against S.aureus using the disk diffusion assay.None of the compounds was effective against E.coli at500?g or lower,but MICs of all three compounds were approximately the same on S.aureus,indicating that these compounds may be much more effective against gram positive bacteria(Hoffman et al.2008).
Rodrigues et al.have studied the endophytic fungi Guignar-dia,Pestalotiopsis guepinii and Phomopsis sp.isolated from Spondias mombin L.(Anacardiaceae),which has been used in Brazil in tra-ditional medicine because of its antimicrobial properties.Only the culture broth ethyl acetate extract of Guignardia was active against S.aureus and E.coli(Rodrigues et al.2000).HPLC analysis showed that this crude extract contained at least?ve metabolites,and in following research they were able to isolate guignardic acid,a new type of secondary metabolite,but have not reported on its individ-ual antibacterial activity(Rodrigues-Heerklotz et al.2001).Later, in2004,Corrado and Rodrigues examined the crude extract of cul-tures of13fungal strains identi?ed as Phomopsis sp.and isolated as endophytes from the leaves of Aspidosperma tomentosum and twigs of Spondias mombin for their antibacterial activities.The screening was conducted using the bioautographic TLC agar-overlay tech-nique against bacteria(E.coli,P.aeruginosa,S.aureus).Four strains isolated from Aspidosperma tomentosum designated:IOC4240,IOC 4239,IOC4242and IOC4243had a4–5mm zone inhibition against E.coli and P.aeruginosa,but none against S.aureus.Two of the strains from Spondias mombin,named IOC4236and IOC4235,were active against E.coli and S.aureus while the third,IOC4237was able to inhibit the growth of P.aeruginosa.
Phomopsichalasin(Fig.1),a metabolite from an endophytic Pho-mopsis sp.,represents the?rst cytochalasin-type compound with a three-ring system replacing the cytochalasin macrolide ring.This metabolite exhibited antibacterial activity in disk diffusion assays (at a concentration of4?g/disk)against B.subtilis(12-mm zone of inhibition)and S.aureus(8-mm zone of inhibition)(Horn et al. 1995).
Dai et al.screened the endophytic fungi from four medicinal plants of the family Euphorbiaceae(Sapium sebiferum,Euphorbia pekinensis,E.hetioscopia and Bischo?a polycarpam)and detected the antibacterial activity of these strains(Dai et al.2006).Their results indicated that11strains of a total of43belonged to Alternaria spp., Fusarium spp.,Chaetomium spp.,Coniothyium spp.and Phomopsis spp.and showed antibacterial activity against tested bacteria such as S.aureus and B.subtilis.
Phomol(Fig.1),a novel antibiotic,was isolated from the fer-mentation broth of Phomopsis sp.strain E02018in the course of a screening of endophytic fungi from the medicinal plant Erythrina crista-galli(Weber et al.2004).In the serial dilution assay it showed only moderate antibacterial activity against Arthrobacter citreus, Corynebacterium insidiosum and Pseudomonas?uorescens and it was not active against E.coli or B.subtilis.
As part of a program on the isolation of biologically active compounds the research group of prof.Krohn has investigated a number of metabolites from Phomopsis sp.(internal strain no. 8966),isolated from the plant Notobasis syriaca(Hussain et al. 2011).The culture medium of the endophyte was extracted and fractionated with silica gel column chromatography and TLC to give the following pure compounds:phomosine K,a new phomosine derivative and six known compounds:phomosine A,phenylalanine amide,2-hydroxymethyl-4?,5?,6?-trihydroxycyclohex-2-en,(?)-phyllostine,(+)-epiepoxydon,and(+)-epoxydon monoacetate. Preliminary studies showed that phomosine A had strong antibac-terial activity the MICs against E.coli and Bacillus megaterium were determined to be12.5?g/ml.
The same group has also isolated an another interesting group of secondary metabolites with antibacterial activity from a differ-ent Phomopsis sp.isolated from Cistus salvifolius(internal strain 7852)(Hussain et al.2012).The mycelium was extracted with ethyl acetate and the crude extract further fractionated on a silica gel
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column to yield a crude mixture containing pyrenocines J-M.Fur-ther silica column chromatography or preparative TLC gave the pure compounds.Solutions of individual pyrenocines in acetone were tested using the agar diffusion test for antibacterial activity against E.coli and B.megaterium.Against E.coli all of the tested pyrenocines showed moderate antibacterial activity with zone of inhibition radiuses ranging from5to10mm.Penicillin and tetra-cycline were used as positive controls and had zones of inhibition with much higher radiuses(14and18mm,respectively against E.coli).Against B.megaterium none of the pyrenocines showed com-plete inhibition of growth,though partial inhibition of growth was evident.
Zao et al.have investigated the plant Paris polyphylla var.yun-nanensis(Franch)Hand.-Mazz.(Trilliaceae)that has been used in traditional Chinese medicine(TCM)for treatment of injuries from falls,fractures,contusions,bleeding and immunity disorders(Zhao et al.2010;Zhao and Zhou2008).The crude n-butanol extracts of sixteen endophytic fungi isolated from the rhizomes of Paris poly-phylla var.yunnanensis were preliminarily investigated for their antibacterial activity by the agar well diffusion assay and a mod-i?ed broth dilution test.Eight endophytic fungi(i.e.Ppf1,Ppf2, Ppf4,Ppf8,Ppf9,Ppf10,Ppf14and Ppf15)showed strong antibac-terial activity against the test bacteria(B.subtilis,S.haemolyticus, E.coli).MIC values of the extracts were between0.0625and 2mg/ml.The endophytes with better antibacterial activity were identi?ed by morphological characters and internal transcribed spacer(ITS)rRNA gene sequence analysis(Zhao and Zhou2008). After determining the antibacterial activity of the crude extract, they proceeded to the bioassay-guided fractionation to determine the antimicrobial components produced by the endophytic fun-gus Pichia guilliermondii Ppf9.They identi?ed four compounds as follows:ergosta-5,7,22-trienol;5?,8?-epidioxyergosta-6,22-dien-3?-ol;ergosta-7,22-dien-3?,5?,6?-triol,and helvolic acid(Zhao et al.2010).The antimicrobial activities of these compounds were further evaluated by micro dilution colorimetric and spore ger-mination assays.The antimicrobial activity assay indicated that helvolic acid(Fig.1)could be the main antimicrobial component in endophytic fungus P.guilliermondii Ppf9,as this compound exhib-ited the strongest antibacterial activity on E.coli,B.subtilis,S. aureus and S.haemolyticus,with MIC values of3.13,3.13,50and
6.25?g/ml,respectively.Its antibacterial activity was close to or
a little stronger than that of streptomycin sulfate(the positive control).Among the three isolated steroids5?,8?-epidioxyergosta-6,22-dien-3?-ol exhibited relatively strong antimicrobial activity and the authors speculated that the peroxide bridge between C-5 and C-8positions may be crucial for the antimicrobial activity.
A strain PRE-5was isolated from Panax notoginseng,collected in Yunnan Province,China,and identi?ed as Trichoderma ovalisporum. This strain showed antibacterial activity,which is why the culture broth of the strain was further characterized and the antibacterial activity of the individual compounds from this fungal strain tested (Dang et al.2010).Five compounds were isolated from the culture broth:koninginin A(Fig.1),(E)-2,3-dihydroxypropyl octadec-9-enoate,shikimic acid,cytosine ribonucleoside and a compound considered to be adenine ribonucleoside.Strain PRE-5showed obvious antibacterial activity against S.aureus,B.cereus,M.luteus, and E.coli.In the paper-disc diffusion antimicrobial assay,shikimic acid showed moderate antibacterial activity against S.aureus,B. cereus,M.luteus and E.coli with diameter of the resulting bacteria-free zone10mm,9mm,7mm and11mm,respectively.The rest of the isolated compounds did not show any obvious activity against the tested strains.
Many bioactive compounds,including antifungal agents,have been isolated from the genus Xylaria residing in different plant hosts,The bioactive compound isolated from the culture extracts of the endophytic fungus Xylaria sp.YX-28isolated from Ginkgo biloba L.was identi?ed as7-amino-4-methylcoumarin(Liu et al.2008).Its antimicrobial activity was determined by the agar-well diffusion method and the MICs were determined.The compound showed strong antibacterial activity against S.aureus(MIC16?g/ml),E.coli (MIC10?g/ml),Salmonella typhia(MIC20?g/ml),S.typhimurium (MIC15?g/ml),Salmonella enteritidis(MIC8.5?g/ml),Aeromonas hydrophila(MIC4?g/ml),Yersinia sp.(MIC12.5?g/ml),Vibrio anguillarum(MIC25?g/ml),Shigella sp.(MIC6.3?g/ml),and Vibrio parahaemolyticus(MIC12.5?g/ml).Most importantly,the obtained MICs were comparable or even lower than those obtained for the positive control antibiotics ampicillin,gentamicin or tetra-cycline.Another isolate of the endophyte Xylaria sp.NCY2from Torreya jackii was described by Hu et al.(2010)to produce seven novel polyketides:1-(xylarenone A)xylariate A,xylarioic acid B,xylariolide A,xylariolide B,xylariolide C,methyl xylariate C, and xylariolide D and additionally the known taiwapyrone.In an antibacterial assay these metabolites inhibited the growth of E.coli, B.subtilis,and S.aureus with MIC values above10?g/ml.
Some of the discovered endophytes remain unidenti?ed.As part of a project exploring the endophytic fungi of Costa Rica,fungal extracts were screened for antibiotic activity against drug-resistant strains of S.aureus and E.faecalis.Guanacastepene A,a diterpene with a novel guanacastepane carbon skeleton,was isolated from an unidenti?ed endophytic fungus from the branch of Daphnopsis americana by Brady et al.in2000and its structure was determined by X-ray crystallography(Brady et al.2000).In agar diffusion assays run on bacterial lawns,pure guanacastepene A showed antibi-otic activity against methicillin-sensitive and-resistant S.aureus and vancomycin-resistant E.faecalis.Against MRSA100?g of gua-nacastepene A or vancomycin produced11and17mm zones of growth inhibition,respectively.While vancomycin was ineffective against VREF,guanacastepene A produced a9mm zone of growth inhibition.In2001,several additional guanacastepenes B-O have been isolated from the same fungus and structurally characterized (Brady et al.2001).A setback in the exploitation of this metabo-lite as a new antibacterial agent is the haemolytic activity against human erythrocytes(Singh et al.2000).Its promising pro?le against MRSA and VREF has stimulated an intensive world-wide search for synthetic and naturally occurring analogues.
An unidenti?ed ascomycete fungus was isolated from Melilo-tus dentatus(Hussain et al.2009),and the ethyl acetate extract of the culture broth was chromatographed on silica gel to give four pthalides(7-hydroxyphthalide,4-hydroxyphthalide, 5-methoxy-7-hydroxyphthalide,5,7-dihydroxyphthalide),two isocoumarins((3R,4R)-cis-4-hydroxymellein and(3R,4R)-cis-4-hydroxy-5-methylmellein)and two steroids(ergosterol and 5?,8?-epidioxyergosterol).4-Hydroxyphthalide and5-methoxy-7-hydroxyphthalide were active against E.coli.
In a study of endophytic fungi from Malaysian plant species, a total of300endophytes were isolated from43plants(Hazalin et al.2009).Ardisia colorata was found to host the highest number of endophytes(14isolates),followed by Molineria latifolia(13iso-lates)and Zingerberaceae sp.,KT43(13isolates).The ethyl acetate extracts of the ground mycelium and culture agar of all the isolated endophytes were tested against B.subtilis,M.luteus,S.aureus,E.coli and P.aeruginosa.Out of the300isolates tested,only24displayed antibacterial activity against at least one test microorganism with inhibition zones of7–8mm and none of the isolates were as potent as gentamicin sulfate.
Endophytic fungi producing metabolites effective against Helicobacter pylori
Part of the research is also focused on the search of new antibac-terial agents for the therapy of Helicobacter pylori infections.Ma
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et al.have investigated the methanol extract of the solid-substrate culture of Rhizoctonia sp.found in Cynodon dactylon(Poaceae)(Ma et al.2004).With fractionations directed by anti-Helicobacter pylori test,four compounds were characterized from the extract:rhizoc-tonic acid(Fig.1),monomethylsulochrin(Fig.1),ergosterol and 3b,5a,6b-trihydroxyergosta-7,22-diene.All four metabolites were subjected to a more detailed in vitro assessment of their antibac-terial action against?ve clinically isolated and one reference H. pylori strain.The MICs of the compounds against all of the?ve clinical and a reference strain were in the range from10.0to 30.0?g/ml while the MIC of ampicillin used as the positive con-trol against these strains was2.0?g/ml(Ma et al.2004).Their search for a more potent product continued and a year later,the same research group isolated32endophytic fungi from the same medicinal herb(Li et al.2005).The endophytic fungi were then grown in in vitro culture,and the ethyl acetate extracts of the cultures were examined for anti-H.pylori activity.As a result,a total of16endophyte culture extracts were identi?ed as having potent anti-H.pylori activities.Subsequently,a detailed bioassay-guided fractionation of the extract of the most active endophyte (strain number:CY725)identi?ed as Aspergillus sp.,was performed. Four anti-H.pylori secondary metabolites were identi?ed to be helvolic acid,monomethylsulochrin,ergosterol and3b-hydroxy-5a,8a-epidioxy-ergosta-6,22-diene with corresponding MICs of8.0, 10.0,20.0and30.0?g/ml,respectively.The MIC of ampicillin co-assayed as a reference drug against H.pylori was2.0?g/ml.Fur-thermore,preliminary examination of the antimicrobial spectrum of helvolic acid,the most active anti-H.pylori metabolite charac-terized from the endophyte culture,showed that it was inhibitory to the growth of Sarcina lutea,and S.aureus with MICs of15.0and 20.0?g/ml,respectively.
Interestingly,the most recent report from the same research group again features monomethylsulochrin and rhizoctonic acid, as well as a new fungal toxin named guignasul?de,with very similar MIC values as the compounds responsible for the anti-H. pylori activity of a methanol extract.This time the methanol extract was prepared from the solid-substrate culture of Guignardia sp. IFB-E028isolated from healthy leaves of Hopea hainanensis Mer-rill&Chun(Dipterocarpaceae)(Wang et al.2010).The isolated metabolites were moderately inhibitory on the growth of H.pylori with the corresponding MIC values of28.9±0.1,60.2±0.4,and 42.9±0.5?M.For comparison,the MIC of ampicillin co-assayed as a positive reference was5.4±0.2?M.After recalculating,the MICs of monomethylsulochrin and rhizoctonic acid are around10 and20?g/ml,respectively,which is consistent with their previ-ous report.Although the isolated substances do have antibacterial activity,their MICs are higher than that of ampicillin.Perhaps,with some effort from pharmaceutical chemists,certain structural mod-i?cation would be able to improve their antibacterial potency. Endophytic fungi as producers of the same metabolites as
the host plant
Some endophytes can produce the same rare and important bioactive compounds originally characteristic of the host plant (Tan and Zou2001).This ability is of great importance in that it provides an alternative strategy for reducing the need to harvest slow-growing and possibly rare plants and also help to preserve the world’s ever diminishing biodiversity.Moreover,the production of a high value phytochemical by exploiting a microbial source is eas-ier and more economical and it leads to increased availability and the reduced market price of the product(Strobel et al.2004).One of the most frequently mentioned is the taxol producing endophytic fungus,but examples of endophytic fungi producing substances with antibacterial activity,originally found in host plants have also been discovered.
Kusari et al.have isolated the endophytic fungus designated INFU/Hp/KF/34B and later identi?ed as Thielavia subthermophila from Hypericum perforatum and studied the production of hyper-icin,a naphthodianthrone derivative,and its precursor,emodin (Kusari et al.2008,2009).Both compounds demonstrated antimi-crobial activity against several bacteria and fungi,including S. aureus ssp.aureus,K.pneumoniae ssp.ozaenae,P.aeruginosa, Salmonella enterica ssp.enteric,and E.coli(Kusari et al.2008).
Eucommia ulmoides Oliver is a traditional medicinal plant used in China,and it is one of the main sources of chlorogenic acid. This natural medicinal resource is in short supply because of the over-collection of the wild plant,which is now protected in China Chen et al.reasoned that by isolating an endophytic fungus from Eucommia ulmoides which would be able to produce the same secondary metabolite as its host plant,they would protect the plant from extinction and?nd an alternative way to produce its active constituents to satisfy the demand.They carried out a study to iso-late endophytic fungi from Eucommia ulmoides Oliver and to?nd out whether any of these fungi can produce chlorogenic acid(Chen et al.2010a).
Twenty-nine endophytic fungal strains were isolated on the basis of morphology,and they were divided into six groups(N, B,S,C,A,E).Due to the antimicrobial activity of chlorogenic acid they discriminated the isolated endophytic fungi as potential pro-ducers of chlorogenic acid based on the antibacterial activity of their extracts.Most of them were positive for antibacterial activity and were analyzed by HPLC,GC–MS,and LC–MS.Chromatographic analysis indicated that strain B5might be able to produce chloro-genic acid,although the yield was relatively low and was not quite suitable for its production on an industrial scale.
Discussion
The results reviewed in this article provide the information about the strains of endophytic fungi whose crude extracts should be studied in further detail.Antibacterial activity is most frequently detected when testing cultures of species belong to genera Fusa-rium,Phomopsis and Phoma.Unfortunately this cannot be assigned to its superior antibacterial activity but rather to the higher fre-quency of isolation of these fungi in comparison to the rest.Studies in which extract prepared using different solvents are tested show that crude extracts obtained with methanol,ethanol and most often ethyl acetate have antibacterial activity,whereas extracts prepared with nonpolar solvents display no antibacterial activ-ity.The comparison of the intensity of the antibacterial activity between different studies is very dif?cult,due to the fact that no uniform test strains are used.Perhaps it makes more sense to use clinical isolates and always include positive control to prove the relevancy of the results.
It is important to note that these fungi are capable of producing antibacterial substances in a laboratory and more effort is needed to isolate and purify them in order to determine their structure and mechanism of action.In cases where these natural products have been identi?ed through biological assays,they should be con-sidered as leads,which become candidates for drug development (Molinari2009).More often than not however,this is where the research comes to a halt,leaving the potential unused.A wide range of additional data about the lead compound needs to be obtained, one of them being the overall toxicity of the substance as well as possible chemical modi?cations of the natural compounds in order to obtain a more effective antibacterial agent.
The number of antibacterial substances obtained from endo-phytic fungi is not as large as those obtained from different
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endophytic bacterial strains,among which Streptomyces sp.seem to be the most productive.There are quite a large number of pub-lished articles,not included in this review,dealing with structurally novel metabolites produced by endophytic fungi whose antibacte-rial potential has not been tested yet.This is also a goal for the future:to perform a comprehensive screening of the antibacte-rial activity of those metabolites against clinical strains of resistant bacteria.
While the estimated?gure of1.5million of all fungal species (Hawksworth2001)is commonly used,it is estimated that there might be1million of endophytic fungal species.This seems rea-sonable if each individual higher plant,of approximately250,000 different plant species on Earth,hosts an average of four endo-phytes(Ganley et al.2004).In the past century,however,only about100,000fungal species including endophytic fungus were described(Ganley et al.2004).Due to the fact that endophytic fungi are south for in plants which have established use in traditional medicine or that grow in areas of great biodiversity,it is obvious that a great deal of potentially useful metabolites await to be discovered.Focusing on the investigation of endophytic fungal diversity,relationships between endophytic fungi and their host plants,seeking for natural bioactive compounds they produce,and improving the productivity of some potential candidates by taking advantage of genetic engineering and microbial fermentation pro-cesses might lead to the discovery of the much needed antibiotic for the treatment of infections caused by multidrug-resistant bacteria. It is clear that we still have a very long way to go until the?nal goal is achieved.However,it is encouraging to know that we are on the right track.
Con?ict of interest
No con?ict to disclose.
Acknowledgments
The authors would especially like to thank the Department of Pharmaceutical Biology,Faculty of Pharmacy,University of Lju-bljana where both authors were employed during the writing of this article.
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胸腔穿刺操作流程 WEIHUA system office room 【WEIHUA 16H-WEIHUA WEIHUA8Q8-
技能培训与考核方案-胸腔穿刺操作流程 普宁人民心内科王晓群 一、培训计划与安排 1.培训对象10人 2.培训与考核教官:心内科所有医生 3.进修生培训：6月18-20日 4.学员考核：6月21日下午，开放五组模拟同时考核，两次完成考核，一下午学员全部完成考核 二、考核标准 1、向患者家属说明目的意义及操作过程中的注意事项，签协议书； ? 2、核对患者床号、姓名、性别、年龄，嘱患者排尿； ? 3、了解病变部位（阅胸片、视触叩听） 4、准备胸穿包、无菌手套、3%碘酒、75%酒精、棉签、胶布、2%利多卡因、5ml注射器、标本收集试管及 容器、污物桶； ? 5、体位：取反椅坐位或取半卧位； ?
6、术者洗手，戴口罩、帽子； ? 7、穿刺点选择：抽气选第二肋间，锁骨中线处，胸腔积液取肩胛下角线7～9肋间，腋中线第6～7肋间（可 用龙胆紫在穿刺点皮肤上作标记），若遇到有包裹性积液或特殊情况时可通过床边B超定位穿刺点。 8、术者带无菌手套常规消毒局部皮肤、铺巾、局部麻醉；? 9、术者穿刺前先将血管钳夹闭穿刺针的胶管，左手固定穿刺部位皮肤，右手持针经麻醉处之肋骨上缘垂直 缓慢刺入，当针头抵抗感突然消失时，表示已达胸膜腔，接上50ml针筒，松开血管钳抽取胸腔内积液液， （如多次抽取，应于脱下针筒前将血管钳夹住以防空气进入胸腔）； 10、助手协助固定针头，避免针头摆动；抽液毕拔针，针眼涂上3%碘酒并盖纱布稍用力压迫片刻，胶布固 定。 ? 11、整理用品，填写检验单并送检。术后严密观察并做好记录；整理用物，填写检验单并送检。 ?
胸膜腔穿刺术操作规范 【意义】 胸膜腔穿刺术,简称胸穿,就是指对有胸腔积液(或气胸)的患者,为了诊断与治疗疾病的需要而通过胸腔穿刺抽取积液或气体的一种技术。【术前准备】 1、操作前应向患者说明穿刺目的,消除顾虑,签同意书; 2、询问有无药物过敏史; 3、穿刺前清洁穿刺部位,嘱患者排尿; 4、对精神紧张者,可于术前半小时给予地西泮10mg肌注。嘱咐患者在操作过程中,避免深呼吸与咳嗽,如有任何不适及时提出。 5、器械准备,包括无菌胸腔穿刺包、无菌手套、消毒用品、麻醉药品、胶布等。 6、术前检查,再次核对适应证,查瞧有无禁忌症。 【操作程序】 1、穿戴工作服、帽、口罩,洗净双手。 2、向病人解释穿刺目的,对于过分精神紧张者,可于术前半小时给予地西泮10mg以镇静镇痛。 3、准备消毒器械及穿刺包。 4、扶病人坐位,面向椅背,两前臂置于椅背上,前额伏于手臂上。 5、一般选择肩胛下角线或腋后线7-8 肋间作为穿刺点(必要时结合X 线及超声波检查确定,并用在皮肤上做标志)。 6、常规消毒,戴无菌手套,覆盖消毒洞巾。 7、常规胸膜腔穿刺术 a、检查器械,注意穿刺针就是否通畅,胶管就是否漏气及破损。 b、以2%利多卡因局部逐层浸润麻醉(注意穿刺点应选在下一肋骨的上缘)。 c、用血管钳夹住穿刺针后面的胶管,使之不漏气。 d、左手固定穿刺部位皮肤,右手持穿刺针沿麻醉部位经肋骨上缘垂直缓慢刺人,当有突破感时停止。 e、接上注射器后,再松开止血钳(此时助手用止血钳固定穿刺针防止针摆动及刺入肺脏)。
f、注射器抽满后再次用血管钳夹闭胶管才能取下注射器。 g、将抽出液注入弯盘及专门准备的容器中。 h、抽完液后拔出穿刺针,再次消毒皮肤,覆盖无菌纱布。稍用力压迫片刻,用胶布固定。 8、中心静脉导管行胸腔穿刺置管引流术 a.2％利多卡因逐层浸润麻醉至胸膜腔。 b、持蓝空针(y 型)沿麻醉点进针至回吸有胸水流出。 c、通过蓝空针针芯置入导引钢丝(弯头朝前)深度弯头超过蓝空针针尖即可,不要过深以免导丝打折,固定导丝不动拔出蓝空针。 d、沿导引钢丝置入扩皮器扩皮,注意力度适当,不要采取暴力,拔出扩皮器。 e、沿导丝置入静脉留置软管,深度 10－15厘米均可,胶贴固定,引流管外接负压引流袋。 f、每次放液或注药完毕后一定要用肝素封管 9、将抽出液送化验、记量。 10、术后嘱病人静卧。告诉病人有不适立即通知工作人员。 11、整理物品。 【适应症】 1.诊断性穿刺:对原因未明的胸腔积液,作胸水涂片、培养、细胞及生化学检查,从而确定胸腔积液的性质,以进一步明确疾病的诊断。 2.治疗 a、减轻胸腔大量积液、气胸引起的压迫症状 b、抽取脓液治疗脓胸 c、向胸腔内注射药物。 【禁忌症】 多脏器功能衰竭者禁忌胸膜腔穿刺;出血性疾病及体质衰竭、病情危重,难以耐受操作者应慎重。 【注意事项】 1.操作前应向患者说明穿刺目的,消除顾虑; 2.操作中应密切观察患者的反应,如有头晕、面色苍白、出汗、心悸、胸部压迫感或剧痛、昏厥等胸膜过敏反应;或出现连续性咳嗽、气短、
胸腔穿刺术操作规范 【题干】 患者男性，26岁，胸闷气短2周，查体及胸片等检查示双侧胸腔中等量积液，其性质不明，现需作诊断性穿刺，请你施行胸腔穿刺术，并抽取50ml胸水送检。 【操作前准备】 拿病历板。报告操作开始。核对医嘱，熟悉患者病情，明确胸穿适应症，排除禁忌症，与患者及家属充分沟通并签署知情同意书（向评委展示知情书）。嘱患者做好准备。 卫生手消毒（速干手消毒剂揉搓双手，七步洗手法，保持指尖朝下，总时间不少于15秒）。戴帽子。戴口罩（外科口罩，单独包装，系带式，有颜色面朝外，罩住鼻、口及下巴，下方带系于颈后，上方带系于头顶中部）。 准备物品：胸腔穿刺包1个（内有铺巾1个，洞巾1个，弯钳l 个，无齿镊l个，12号及16号胸穿针各1个，弯盘1个，方纱布3块，含棉球消毒杯1个，治疗碗1个），碘伏1瓶，棉签1包，无菌纱布1包，医用胶布1卷，砂轮1个，2%利多卡因针5ml，无菌手套2副，5ml及50ml注射器各1个，医用标记笔1支，试管架1个，试管3-4个。 操作者立于患者拟穿刺侧，物品及治疗车置于操作者右手。您好，请问是xx先生么？核对腕带，患者信息无误。我是您的主管大夫，根据病情需要，现在要为您进行胸腔穿刺术，请您配合，不用紧张。
【摆体位、定穿刺点】 卫生手消毒。 协助患者摆体位：患者反向骑跨于靠背椅上，上肢屈肘交叉置于椅背，前额伏于前臂上。 请您在操作过程中保持这个体位。现在为您做背部叩诊检查。卷起患者上衣，充分暴露背部。自上而下、左右对称进行叩诊。选择浊音最明显处（或实音处）为穿刺点，本次操作选取左/右肩胛下角线第8肋间为穿刺点，记号笔标记。 卫生手消毒。 【开包、消毒、铺巾】 拿起穿刺包，观察包外灭菌指示胶带。穿刺包消毒合格，在有效期内，包布完好无破损，可以使用。打开穿刺包外包布。抽出无齿镊，持镊打开穿刺包内包布。查看灭菌指示卡，灭菌指示卡变色达标，灭菌合格。摆开穿刺物品，检查是否齐全。穿刺物品齐全。 无齿镊夹出消毒杯。助手述碘伏在有效期内，可以使用。向消毒杯内注入碘伏。 对患者说现在开始消毒，可能会感觉有点儿凉，请不要用手触碰已消毒区域。镊子夹持碘伏棉球，镊子尖端不得超过持镊手指水平（两把消毒镊时，交替传递棉球），以穿刺点为中心环形消毒，直径不小于15cm，至少消毒2遍，第二次消毒范围不超过第一次范围。用后棉球弃于治疗车下层或地上的医疗废物桶内，镊子及消毒杯放于治疗车下层。
呼吸系统疾病护理常规 一、呼吸系统疾病一般护理常规 1、严密观察病情。注意体温、脉搏、呼吸、血压、神志等生命体征的变化。有否感 染性疾病所致全身毒性反应如畏寒、发热、乏力、食欲减退、体重减轻、衰竭等； 以及本系统疾病的局部表现如咳嗽、咳痰、咳血、哮喘、胸痛等。 ２、恢复期可下床适当活动，危重患者应绝对卧床休息。 ３、给予高蛋白、高热量、多维生素易消化饮食。高热和危重患者，可给流质或半流质饮食。 ４、病室空气要流通，每日通风两次，每次１５～３０分钟，但避免对流。空气消毒每日１次，每月一次监测空气污染情况和消毒效果。 ５、当患者需进行支气管造影、纤维支气管镜窥视、胸腔穿刺、胸腔测压抽气、胸膜活检等检查时做好术前准备、术中配合、术后护理。 ６、呼吸困难者应给予氧气吸入。护士必须掌握给氧的方法（如持续或间歇给氧的流量、给氧器材的选择）。 ７、结合临床，了解肺功能检查和血气分析的临床意义。发现异常及时通知医生。８、呼吸衰竭患者如出现兴奋、烦躁、谵妄时应慎用镇静药，禁用吗啡和地西泮等巴比妥类药，以防抑制呼吸中枢。 ９、留取痰液、脓液、血液标本时按常规操作。取样要新鲜，送检要及时，标本容器要清洁干燥。 １０、做好卫生宣教工作，积极宣传预防呼吸系统疾病的措施。指导患者进行体育锻炼，阐明吸烟对人体的危害，劝告患者注意保暖预防感冒。 １１、备好一切抢救物品和药物。
二、急性上呼吸道感染的护理常规 病情观察 １、注意体温的变化及呼吸形态。 ２、注意有无并发症症状，如头痛，耳鸣，脓涕等。 护理措施 １、保持室内空气新鲜，每日通风２次，每次15-30分钟。 ２、保证病人适当休息，病情较重或年老者应卧床休息。 ３、多饮水，饮水量视病人体温，出汗及气候情况而异，给予清淡，易消化，含丰富的维生素，高热量，高蛋白的饮食。 ４、体温超过38.5摄氏度给予物理降温。高热时按医嘱使用解热止痛片。观察降温后的效果。出汗多的病人要及时更换衣物，做好皮肤的清洁护理。 ５、寒战时，要注意保暖。 ６、按医嘱用药。 健康指导 １、注意呼吸道隔离，预防交叉感染。 ２、保持充足的营养、休息、锻炼，增加机体抵抗力。 ３、忌烟。 ４、坚持冷水洗脸，提高机体对寒冷的适应能力。
胸腔穿刺的护理 胸腔穿刺术常用于：（1）检查胸腔积液的性质，有无特殊细胞及病原体，以确定诊断；（2）抽出胸腔积液或积气，减轻压迫症状；（3）向胸腔内注射药物进行治疗。 病情观察： 1. 呼吸困难 2. 胸痛，胸闷，气短 3. 原发病的表现（肿瘤.肺炎.心衰.肺结核.肝脓肿） 术前护理: 避免咳嗽、深呼吸、转动身体以免穿破肺泡而引起气胸；对精神过于紧张者，可于术前静卧15-30分钟，紧张或激动的患者必要时给予镇静剂，或进行对症处理。帮助患者解除紧张、忧虑和恐惧，从而保持良好的心理平衡，准备与查对备齐穿刺术所需的各种用物，严格执行“三查七对”。 术中护理： 1.穿刺中护理严格无菌操作，消毒范围必须超过洞巾边缘2 cm以上，避免洞巾移位，造 成污染。指导患者掌握正确配合操作的方法，穿刺中勿咳嗽，咳嗽时大口哈气，或咀嚼水果。胸膜活检及胸水常规检查者，标本取出后迅速送检，以便得出准确结果。持续放液者应注意观察医师抽液情况，随时调节针头位置，确保抽液顺利。 2.抽液抽气量每次胸腔穿刺时,抽吸速度不能过快、过多，以防止纵隔移位发生意外。诊断 性抽液50~100 ml即可，一般首次不超过600 ml，以后每次不超过1000 ml，注意无菌操作并防止空气进入胸腔。 3.病情观察要注意生命体征变化,患者有无头晕、心悸、胸闷、面色苍白、出汗、刺激性干 咳、甚至晕倒等胸膜反应。如有上述症状时应立即停止抽液,拔出穿刺针，用无菌纱布压穿刺部位，嘱患者平卧，予低流量吸氧2~5 l/min，心电监护。如果患者症状不缓解，予皮下注射0.1%肾上腺素0.3~0.5 mg，并做好记录。 术后护理： 1. 穿刺后护理穿刺完毕局部覆盖无菌纱布并按压几分钟，协助患者卧床休息，及时观察病 情，监测生命体征的变化。观察局部有无渗血、渗液，疼痛剧烈者给予盐酸哌替啶镇痛。 2. 监测生命体征平稳6h后取半卧位，有利于呼吸和引流，保持胸腔引流管通畅，定时挤 压引流管，观察和记录引流量及颜色。 3. 穿刺完毕,嘱患者静卧, 24 h后方可洗澡。监测穿刺后的反应，观察患者脉搏和呼吸状况， 注意血胸、气胸、肺水肿等并发症。注意穿刺部位，如出现红、肿、热、痛、体温升高等应及时汇报医生。 健康指导： 1. 指导病人有意识地使用控制呼吸的技巧，如进行缓慢的腹式呼吸，并每天监督指导病人 于餐前及睡前进行有效的咳嗽运动，每次15-30min 2. 给予舒适的体位，抬高床头，半卧或健侧卧位，以利呼吸,遵医嘱给氧2-4L/min，氧浓度 35%-40%，并保持输氧装置通畅。 3 鼓励病人积极排痰，保持呼吸道通畅，以利呼吸。 4. 鼓励病人下床活动，增加肺活量，以防肺功能丧失。
1胸膜腔穿刺术主要作用 ① 取胸腔积液进行一般性状检测、化学检测、显微镜监测和细菌学检测，明确积液的性质，寻找引起积液的病因； ② 抽出胸膜腔的积液和积气，减轻液体和气体对肺组织的压迫，使肺组织复张，缓解病人的呼吸困难等症状； ③ 抽吸胸膜腔的脓液，进行胸腔冲洗，治疗脓胸； ④ 胸膜腔给药，可胸腔注人抗生素或者抗癌药物。 2适应症 1、诊断性：原因未明的胸腔积液，可作诊断性穿刺，作胸水涂片、培养、细胞学和生化学检查以明确病因，并可检查肺部情况。 2、治疗性：通过抽液、抽气或胸腔减压治疗单侧或双侧胸腔大量积液、积气产生的压迫、呼吸困难等症状；向胸腔内注射药物（抗肿瘤药或促进胸膜粘连药 物等）。 3禁忌证 1﹒体质衰弱、病情危重难以耐受穿刺术者。 2. 对麻醉药过敏。 3﹒凝血功能障碍, 严重出血倾向,患者在未纠正前不宜穿刺。 4﹒有精神疾病或不合作者。 5﹒疑为胸腔包虫病患者，穿刺可引起感染扩散，不宜穿刺。 6﹒穿刺部位或附近有感染。 4术前准备 1．了解、熟悉病人病情。 2．与病人家属谈话，交代检查目的、大致过程、可能出现的并发症等，并签字。3．器械准备：胸腔穿刺包、无菌胸腔引流管及引流瓶、皮肤消毒剂、麻醉药、无菌棉球、手套、洞巾、注射器、纱布及胶布。
器械准备 5操作步骤 体位 患者取坐位面向背椅，两前臂置于椅背上，前额伏于前臂上。不能起床患者可取半坐位，患者前臂上举抱于枕部。 选择穿刺点 选在胸部叩诊实音最明显部位进行，胸液较多时一般常取肩胛线或腋后线第7-8肋间；有时也选腋中线第6-7肋间或腋前线第5肋间为穿刺点。包裹性积液可结合X线或超声检查确定，穿刺点用蘸甲紫（龙胆紫）的棉签或其他标记笔在皮肤上标记。 操作程序 （1）常规消毒皮肤：以穿刺点为中心进行消毒，直径15厘米左右，两次。（2）打开一次性使用胸腔穿刺包，戴无菌手套，覆盖消毒洞巾，检查胸腔穿刺包内物品，注意胸穿针与抽液用注射器连接后检查是否通畅，同时检查是否有漏气情况。 （3）助手协助检查并打开2%利多卡因安瓶，术者以5ml,注射器抽取2%利多卡因2-3ml,在穿刺部位由表皮至胸膜壁层进行局部侵润麻醉。如穿刺点为肩胛线或腋后线，肋间沿下位肋骨上缘进麻醉针，如穿刺点位腋中线或腋前线则取两肋之间进针。 （4）将胸穿针与抽液用注射器连接，并关闭两者之间的开关保证闭合紧密不漏气。术者以一手示指与中指固定穿刺部位皮肤，另一只手持穿刺针沿麻醉处缓缓刺入，当针锋抵抗感突感消失时，打开开关使其与胸腔相通，进行抽液。助手用止血钳（或胸穿包的备用钳）协助固定穿刺针，以防刺入过深损伤肺组
胸腔积液病人护理常规 任何原因使胸液形成过多或吸收过少，均可导致胸液异常积聚，称为胸腔积液。 1、护理评估和观察要点 1．1意识、面容、表情、营养状况及精神变化。 1．2评估胸部体征，胸痛的性质、持续时间及胸膜反应的表现。 1．3评估咳嗽、咳痰、呼吸困难的程度与性质。 1．4口唇、指（跖端）皮肤颜色、呼吸频率、节律、深度、发绀状态、体位、血氧饱和度、动脉血气分析（必要时）、肺功能（必要时）及体温、脉搏、血压的情况。 1．5评估恶性胸腔积液病人是否伴有消瘦、贫血貌、恶液质、锁骨上淋巴结肿大。 1．6日常活动的耐受水平。 1．7生命体征、有无感染的症状和体征。 2、护理问题 2．1气体交换受损 2．2疼痛 2. 3营养失调 3、护理措施 3．1执行呼吸系统疾病病人一般护理常规。 3．2保持室内空气清新、通风良好，温度、湿度适宜。 3．3患侧位或半坐位。
3．4高热量、高蛋白、高维生素饮食。 3．5观察意识及生命体征变化，胸痛的性质、持续时间；营养状况、精神状态。 3．6协助医生进行胸腔穿刺术。 3．6．1术前心理护理，消除病人紧张。 3．6．2严格无菌操作。 3．6．3观察有无头昏、心慌、出汗、面色苍白等胸膜反应，若出现胸膜反应立即拔出穿刺针，使病人平卧，必要时皮下注射肾上腺素。 3．6．4每次抽液不超过1000ml。 3．6．5术后观察呼吸、心率、胸痛情况，早期发现和防止气胸。 3．7使用抗痨治疗者观察有无耳鸣、肝肾功能等损害。 3．8保持皮肤、口腔清洁。 3．9心理护理安慰病人，消除紧张心理。 3．10健康教育戒烟，注意保暖，防止上呼吸道感染。加强营养及身体锻炼，提高身体抵抗力，定期复查血常规、按时按量足月服用抗结核药，定期复查肝肾功能。 4、健康指导要点 4．1戒烟，避免各种诱发因素如劳累、受凉、呼吸道感染、情绪激动等。 4．2加强营养及身体锻炼，提高身体抵抗力。 4．3按时按量足月服用抗结核药，定期复查肝肾功能。 4．4采取高蛋白、高维生素、易消化饮食，忌辛辣产气性食物。 5、护理评价
第二章菌类植物习题 一、名词解释： 1、菌核：菌核是由拟薄壁组织和疏丝组织形成的一种休眠体， 既是真菌贮藏养分的器官，又是真菌用以度过不良环境的休 眠机构。 2、分生孢子：真菌的一种外生的无性生殖细胞。由分生孢子囊 梗的顶端或侧面产生的一种不动孢子，借气流或动物传播。 3、担子果：由次生菌丝体形成子实体，称担子果（三生菌丝体）—课件上的解释 高等单子菌的子实体称担子果—课本上的解释 4、菌丝体：真菌的营养体除个别种类为单细胞外，绝大多数由 分支的丝状体构成，组成一个菌体的丝状体称为菌丝体。（具气生菌丝（匍匐枝）、假根） 5、菌丝组织体（营养变态）：许多真菌在生活史的某些阶段在环 境条件不良或繁殖的时候，菌丝互相密结，菌丝体变态成菌丝组织体。（它有两种组织：拟薄壁组织和疏丝组织） 二、判断题 1、细菌是微小的单细胞原核有机体，有明显的细胞壁，除少数细菌外皆为异养。( √) 2、真菌既不含叶绿素，也没有质体，是典型的异养生
物。( √) 3、绝大多数真菌生活史中无核相交替和世代交替。( ×) - 4、根霉的营养菌丝有横隔。( ×) - 5、青霉产生孢囊孢子进行无性生殖。( ×) - 6、子囊菌在形成子囊孢子之前要经过锁状联合过程。( ×)- 7、子囊果和担子果都是子实体。(√) 8、担子菌亚门的所有真菌均形成担子果。( ×)- 9、担子菌的锁状联合和子囊菌的钩状体相似。( √) 10、菌类植物是一群低等异养的真核生物。( ×)- 三、填空题 1、细菌形态上有球状、杆状、螺旋状和丝状4种类型。 2、多数细菌在一定条件下，细胞壁的周围包被着1层粘性的薄膜，称为荚膜，由多糖类物质组成。 3、某些细菌生长到一定的阶段，失水浓缩，形成1个圆形或椭圆形的内生孢子，称为芽孢。 4、某些低等真菌细胞壁的成分为纤维素，高等真菌细胞壁的主要成分为几丁质。 5、某些真菌在环境不良或繁殖时，菌丝体可以发生变态形成菌丝组织体，常见的有根状菌索、菌核和子座。 6、囊孢子和担孢子都是单倍核相孢子；从形成方式上区分，子囊孢子是内生孢子，而担孢子是外生孢子。 7、子囊菌的子实体又称为子囊果，而而高等担子菌的子实体又称为担子果。 8、根霉有性生殖时产生的有性孢子是二倍体的接合孢子。蘑菇的有性孢子是在担子上产生的单倍体的担孢子。 9、子囊菌有性过程形成子囊和子囊孢子；担子菌有性过程形成担子和担孢子。 、担孢子、典型的双核菌丝体和常具特殊的锁状联合是担子菌亚门的3个明显特征。 11、担子菌的双核菌丝，具有一种特殊的细胞分裂方式，称锁状联合。 12、根霉属于藻菌藻纲，青霉属于子囊菌纲，冬虫夏草子囊菌纲，香菇属于担子菌（无隔担子亚纲）纲。 四、选择题
考生姓名：所在科室：评分： 患者姓名：住院号：病案号：床号： 评分项目评分细则满分扣分 准备阶段(20分) 准备用物 检查用品是否齐备：腹穿包、安尔碘、2%利多 卡因注射液、注射器、棉签、胶布、无菌敷料 5 着装穿戴工作服、戴口罩、戴帽 5 病人准备 向病人解释穿刺目的、消除紧张感、并嘱其排 空小便 5 病人（模型）坐在靠背椅上，或平卧、半卧、 稍左侧卧位均可 5 操作阶段(80分) 消毒铺巾 (20分) 定位：应以叩浊部为准；选择左下腹部脐与髂 前上棘连线中外1/3交点为穿刺点 8 常规消毒术区皮肤，直径15cm 5 戴无菌手套 2 覆盖并固定无菌洞巾 5 麻醉穿刺 (40分) 检查器械，注意穿刺针是否通畅，胶管是否漏 气及破损 5 2％利多卡因局部腹膜壁层逐层浸润麻醉（皮肤 至腹膜） 5 用血管钳夹闭穿刺针后面的胶管，使之不漏气 5 左手食指和中指固定穿刺部位的皮肤、针头 3 右手将穿刺针在麻醉处垂直刺入腹壁 3 当进针阻力突然消失时，应用止血钳固定穿刺 针针体 4 接上注射器后，再松开止血钳 5 注射器抽满后用血管钳夹闭胶管，取下注射器 5 将抽出液注入弯盘及专门准备的容器中 5 穿刺结束 (20分) 抽完液后拔出穿刺针，覆盖无菌纱布。稍用力 压迫片刻 5 用胶布固定覆盖术口，大量放液后患者缚以多 头腹带 5 将抽出液送化验、记量（诊断性取液取50ml， 放液一次通常为<3000ml） 5 术后嘱病人静卧。告诉病人有不适立即通知工 作人员 3 整理物品 2
考生姓名：所在科室：评分： 患者姓名：住院号：病案号：床号：评分项目评分细则满分扣分 准备阶段(20分) 准备用 物 检查用品是否齐备：胸穿包、安尔碘、2%利多 卡因注射液、注射器、棉签、胶布、无菌敷料 5 着装穿戴工作服、戴口罩、戴帽 5 病人准 备 向病人解释穿刺目的、消除紧张感 5 病人（模型）取坐位，面向椅背，两前臂置于 椅背上，前额伏于手臂上；不能起床者取半坐 卧位，前臂上举抱于枕部。 5 操作 阶段 (80分) 消毒铺 巾(20 分) 定位：选择肩胛下角线或腋后线7-8 肋间作为 穿刺点；或腋中线第6、7肋间隙作为穿刺点； 包裹性积液，可根据X线或B超引导进行穿刺； 气胸穿刺点常取患侧锁骨中线第2肋间隙。 8 常规消毒术区皮肤，直径15cm 5 戴无菌手套 2 覆盖并固定无菌洞巾 5 麻醉穿刺(40分) 检查器械，注意穿刺针是否通畅，胶管是否漏 气及破损 5 2％利多卡因局部逐层浸润麻醉 （注意穿刺点应选在下一肋骨的上缘） 5 用血管钳夹住穿刺针后面的胶管，使之不漏气 5 左手固定穿刺部位皮肤 4 右手持穿刺针沿麻醉部位经肋骨上缘垂直缓慢 刺入 4 当有突破感时停止 2 接上注射器后，再松开止血钳（诊断性抽液 50-100ml；减压抽取首次不超600ml，以后每 次不超1000ml） 5 注射器抽满后用血管钳夹闭胶管，取下注射器 5 将抽出液注入弯盘及专门准备的容器 5 穿刺结束(20分) 抽完液后拔出穿刺针，覆盖无菌纱布。稍用力 压迫片刻 5 用胶布固定覆盖术口 5 将抽出液送化验、记量 5 术后嘱病人静卧（态度和蔼）。告诉病人有不适 立即通知工作人员 3 整理物品 2
胸膜腔穿刺术适应症禁忌症及注意事 项 1
1胸膜腔穿刺术主要作用 ① 取胸腔积液进行一般性状检测、化学检测、显微镜监测和细菌学检测，明确积液的性质，寻找引起积液的病因； ② 抽出胸膜腔的积液和积气，减轻液体和气体对肺组织的压迫，使肺组织复张，缓解病人的呼吸困难等症状； ③ 抽吸胸膜腔的脓液，进行胸腔冲洗，治疗脓胸； ④ 胸膜腔给药，可胸腔注人抗生素或者抗癌药物。 2适应症 1、诊断性：原因未明的胸腔积液，可作诊断性穿刺，作胸水涂片、培养、细胞学和生化学检查以明确病因，并可检查肺部情况。 2、治疗性：经过抽液、抽气或胸腔减压治疗单侧或双侧胸腔大量积液、积气产生的压迫、呼吸困难等症状；向胸腔内注射药物（抗肿瘤药或促进胸膜粘连药物等）。 3禁忌证 1﹒体质衰弱、病情危重难以耐受穿刺术者。 2. 对麻醉药过敏。
3﹒凝血功能障碍, 严重出血倾向,患者在未纠正前不宜穿刺。 4﹒有精神疾病或不合作者。 5﹒疑为胸腔包虫病患者，穿刺可引起感染扩散，不宜穿刺。 6﹒穿刺部位或附近有感染。 4术前准备 1．了解、熟悉病人病情。 2．与病人家属谈话，交代检查目的、大致过程、可能出现的并发症等，并签字。 3．器械准备：胸腔穿刺包、无菌胸腔引流管及引流瓶、皮肤消毒剂、麻醉药、无菌棉球、手套、洞巾、注射器、纱布及胶布。 器械准备 5操作步骤 体位
患者取坐位面向背椅，两前臂置于椅背上，前额伏于前臂上。不能起床患者可取半坐位，患者前臂上举抱于枕部。 选择穿刺点 选在胸部叩诊实音最明显部位进行，胸液较多时一般常取肩胛线或腋后线第7-8肋间；有时也选腋中线第6-7肋间或腋前线第5肋间为穿刺点。包裹性积液可结合X线或超声检查确定，穿刺点用蘸甲紫（龙胆紫）的棉签或其它标记笔在皮肤上标记。 操作程序 （1）常规消毒皮肤：以穿刺点为中心进行消毒，直径15厘米左右，两次。 （2）打开一次性使用胸腔穿刺包，戴无菌手套，覆盖消毒洞巾，检查胸腔穿刺包内物品，注意胸穿针与抽液用注射器连接后检查是否通畅，同时检查是否有漏气情况。 （3）助手协助检查并打开2%利多卡因安瓶，术者以5ml,注射器抽取2%利多卡因2-3ml,在穿刺部位由表皮至胸膜壁层进行局部侵润麻醉。如穿刺点为肩胛线或腋后线，肋间沿下位肋骨上缘进麻醉针，如穿刺点位腋中线或腋前线则取两肋之间进针。
菌类植物复习题 一、名词解释 1．寄生腐生专性腐生专性寄生兼性寄生兼性腐生 2．单主寄生转主寄生 3．无隔菌丝有隔菌丝 4．初生菌丝体次生菌丝体三生菌丝体 5．菌丝体菌组织体子实体 6．外菌幕内菌幕菌环菌托 二、判断与改错（对者打“＋”，错者打“－”，并改正） 1．所有的细菌均具鞭毛。( ) 2．细菌是微小的单细胞原核有机体，有明显的细胞壁，除少数细菌外皆为异养。( ) 3．放线菌可看作是细菌和真菌间的过渡类型。( ) 4．粘菌在繁殖时期产生一团裸露、无壁、多核、能变形运动和吞食食物的原生质团。( ) 5．真菌既不含叶绿素，也没有质体，是典型的异养生物。( ) 6．真菌的光合作用产物主要是肝糖。( ) 7．真菌有些种类细胞中含有叶绿素等光合色素，故呈现不同的颜色。( ) 8．绝大多数真菌生活史中无核相交替和世代交替。( ) 9．水霉有性生殖时精囊中放出的精子在水中游近卵囊，与卵囊中的卵结合形成卵孢子。( ) 10．根霉的营养菌丝有横隔。( ) 11．青霉产生孢囊孢子进行无性生殖。( ) 12．子囊菌在形成子囊孢子之前要经过锁状联合过程。( )
13．子囊果和担子果都是子实体。( ) 14．担子菌亚门的所有真菌均形成担子果。( ) 15．酵母形成子囊果。( ) 16．赤霉菌分生孢子有两种：大型分生孢子新月形，小型分生孢子卵形。( ) 17．担子菌的双核菌丝与子囊菌的产囊丝来源相同，都是经过有性结合后产生的双核体。( ) 18．担子菌的锁状联合和子囊菌的钩状体相似。( ) 19．担子果的菌丝体为次生菌丝体。( ) 20．孢囊孢子是在孢子囊内形成的游动孢子。( ) 21．菌类植物是一群低等异养的真核生物。( ) 22．无隔菌丝为初生菌丝，有隔菌丝为次生菌丝。( ) 23．伞菌子实体菌柄上都具菌托和菌环。( ) 三、填空 １．多数细菌在一定条件下，细胞壁的周围包被着１层粘性的薄膜，称为———，由———物质组成，有———作用。 ２．发网菌的生活史分为———阶段和———阶段。 ３．某些低等真菌细胞壁的成分为———，高等真菌细胞壁的主要成分为———。４．某些真菌在环境不良或繁殖时，菌丝体可以发生变态形成———，常见的有———、————。
胸腔穿刺术的护理配合 【概念】 胸腔穿刺术是自胸膜腔内抽取积液或积气的操作,常用于检查胸腔积液的性质、抽液减压或通过穿刺胸膜腔内给药。 【适应症】 1.胸腔积液性质不明者，抽取积液检查,协助病因诊断。 2.胸腔内大量积液或积气者,排除积液或积气，以缓解压迫症状，避免胸膜粘连增厚。 3.脓胸抽脓灌洗治疗，或恶性胸腔积液需胸腔内注入药物者。 【评估】 1.操作环境是否安全、安静。 2.患儿意识状态及合作能力、凝血功能及血小板情况。 3.患儿穿刺局部皮肤有无破溃、结节、皮疹等。 【术前准备】 1.物品准备：胸腔穿刺包、无菌手套、帽子、口罩、2%利多卡因、2ml、5ml、20ml、50ml注射器各2支、肾上腺素1支、生理盐水，必要时备无菌水封瓶。 2.心理护理：向患儿及家长解释穿刺目的、操作步骤，取得信任，协助患儿克服恐惧心理，消除家长顾虑。 3.术前指导年长儿练习穿刺体位，并告知患儿在操作过程中保持穿刺体位，不要随意活动，避免咳嗽或深呼吸，以免损伤胸膜或肺组织。必要时给予镇静药。【术中配合】 1.根据病变部位，协助患儿取合适体位。抽液时，协助患儿取坐位，面向椅背，两手前臂平放于椅背上，前额伏于前臂上。抽气时，协助患儿半卧位。 2.协助医生常规皮肤消毒，局麻。 3.协助医生标记穿刺点，一般胸腔积液的穿刺点在肩胛线或腋后线第7-8肋间隙或腋前线第5肋间隙。气胸的穿刺点在患侧锁骨中线第2肋间隙或腋前线第4-5肋间隙。 4.穿刺过程中密切观察患儿有无面色苍白、咳嗽、冷汗等反应，注意询问有无头晕、心悸、疼痛等不适感。
5.穿刺处给予无菌敷料覆盖保护，胸腔闭式引流时协助连接胸腔闭式引流瓶，标记置管时间。 【术后护理】 1.记录穿刺时间、抽液抽气量、胸腔积液的颜色以及患儿在术中的状态。 2.监测患儿生命体征的变化，尤其是脉搏呼吸，注意观察有无气胸、血胸、肺水肿等并发症的发生。 3.观察穿刺部位，如出现红、肿、热、痛、体温升高或液体溢出等，及时通知医生处理，保持穿刺部位敷料干燥。 4.嘱患儿静卧休息，鼓励患儿深呼吸，促进肺膨胀。 【并发症的观察及处理】 1.胸膜反应：抽吸时，若患儿突然感觉头晕、心悸、冷汗、面色苍白、脉细、四肢发凉，提示可能出现胸膜反应。立即停止抽吸，取平卧位，遵医嘱予药物应用。 2.气胸：若患儿出现突发性胸痛，继而胸闷、呼吸困难、刺激性咳嗽等提示气胸，应立即配合医生在锁骨中线第2或第3肋间隙或腋中线乳头水平处行胸腔穿刺抽气急救，然后采用胸腔闭式引流。 3.血胸：若患儿出现面色苍白、脉搏细速、呼吸急促、血压逐步下降等低血容量休克症状考虑血胸,小量积血不必穿刺抽吸，可自行吸收；中、大量血胸配合医生行胸腔闭式引流，以促进肺膨胀，改善呼吸；并遵医嘱及时补充血容量，防止低血容量性休克，必要时开胸探查，止血。 【参考文献】 1.江载芳,申昆玲，沈颖.诸福棠实用儿科学. 北京：人民卫生出版社.2017 2.赵艳伟.呼吸内科护理工作指南. 北京：人民卫生出版社.2016 3.尤黎明,吴瑛.内科护理学. 北京：人民卫生出版社.2017 4.张素.呼吸科护士规范操作指南. 北京：中国医药科技出版社.2017 5.李乐之,路潜.外科护理学.北京：人民卫生出版社.2017
菌类是个庞大的家族，它无处不在。现在，已知的菌类大约有10多万种。菌类植物结构简单，没有根、茎、叶等器官，一般不具有叶绿素等色素，大多营异养生活。菌类植物可分为粘菌门和真菌门两类彼此并无亲缘关系的生物。其中粘菌是介于动物和真菌之间的生物。它在营养期为裸露的、无细胞壁、多核的原生质团，称变形体（与变形虫相似）。但在繁殖期，它可产生具纤维素细胞壁的孢子，又具真菌的性状。 中文学名：菌类植物 门：通常分为黏菌门和真菌 门 种： 菌类植物约有7.2万 种 分布区域：几乎到处都有分布 目录 简介 生长形态 分类 木耳 银耳 灵芝 猴头菌 香菇 平菇 草菇 竹荪(sūn) 马勃 舞茸 门分类介绍 粘菌门 真菌门 食用菌类 食用菌的营养价值 食用菌的药效 菌类在自然界中的作用 关于各种菌类的介绍 世界四大食用菌 菌类与植物 可作美味佳肴的菌类 怎样辨别毒菇 药用价值 营养价值 教您如何完美保存香菇菌类简介 生长形态 分类
银耳 灵芝 猴头菌 香菇 平菇 草菇 竹荪(sūn) 马勃 舞茸 门分类介绍 粘菌门 真菌门 食用菌类 食用菌的营养价值 食用菌的药效 菌类在自然界中的作用 关于各种菌类的介绍 世界四大食用菌 菌类与植物 可作美味佳肴的菌类 怎样辨别毒菇 药用价值 营养价值 教您如何完美保存香菇菌类 展开 编辑本段简介 菌类（Fungi） 人们可以食用的大型真菌的总称。具体指大型真菌中，能形成具有胶质或肉质的子实体或浚河组织，并能食用或药用的菌类。 此类族群包括酵母菌、霉、伞菌和霉菌，绝大部分属于担子菌亚门，只有少数属于子曼菌亚门。它们缺乏叶绿素，也没有有机植物根茎叶的构造。它们存在的证据可追溯到距今约四亿两千万年前，但古生物学家认为它们应该出现地更早。 菌类，一大类不含叶绿素、不能进行光合作用、异养的低等类植物。其中包括细菌、粘菌和真菌三个门类。其共同特征是：植物体没有根、茎、叶的分化，不含叶绿素等光合色素（极少数光合细菌除外），不能进行光合作用，腐生生活或寄生生活，即异养生活。生殖器
胸腔穿刺术操作规程及评分标准第三节内科各种相关操作 一、胸腔穿刺术 1.1 胸腔穿刺术操作规程 1.1.1 要求:1)掌握胸腔穿刺术的适应症、禁忌症。2)掌握胸腔穿刺术的操作方法。3)掌握胸腔穿刺术的注意事项。4)掌握胸腔各种不同胸腔穿刺。 1.1.2 操作规程 1)操作前准备 (1)明确需要胸腔穿刺的临床情况(适应症)①诊断性穿刺，以确定积液的性质。②穿刺抽液或抽气以减轻对肺脏压迫，或抽吸脓液治疗脓胸。③胸腔内注射药物或人工气胸治疗。 (2)判断患者是否可以进行胸腔穿刺:①相对禁忌症:出
血性疾病及体质衰弱、病情危重，难于耐受操作者应慎用。 ②绝对禁忌症:胸腔穿刺术原则上无绝对禁忌症。 (3)不同胸穿的适用情况(判断要行何种胸穿):①诊断性胸穿:胸腔积液量少，仅为明确胸水性质，无需放液减压时可选择。②胸穿抽液:中-大量胸腔积液，为明确胸水性质及放液减压，接触患者呼吸困难症状时可行胸穿抽液。 ③胸穿抽气:大于30%且小于50%压缩体积的闭合性气胸，或气胸急诊状态时可行胸穿抽气。④胸腔内给药:结核性渗出性胸膜炎或肺癌胸膜转移的患者可以根据病情选择胸腔内给药。 (4)与病人和家属沟通，签署穿刺同意书(祥见附件各种穿刺同意书):告知可能的并发症:气胸、出血、感染、损伤周围组织、血管、神经、胸膜反应、药物过敏、手术不成功、麻醉意外、心脑血管意外、其他不可意料的意外。 (5)准备用物:穿刺包、手套、消毒液、消毒器械、5ml注射器、50ml注射器、2%利多卡因注射液、可待因片、胶带、多余胸水容器、弯盘等
2)操作过程 (1)与病人沟通:介绍自己，核对姓名、性别、床号等，询问有无药物(特别是局麻药)过敏史，同时嘱咐患者操作前注意事项(是否排尿等)。 (2)再次确认患者的病情、体征:测量脉搏和血压、再次胸部重点体查，查看X线片和检查报告(B超)，确认需要的操作无误。 (3)选择合适的体位，确定穿刺点:①患者体位:患者取坐位，面向椅背，双手前臂平放于椅背上，前额伏于前臂上，不能起床者，可取半卧位，患侧前 臂置于枕部。②穿刺点选择:A.诊断性穿刺、胸腔穿刺抽液、胸腔给药:先进 行胸部叩诊，选择实音明显的部位进行穿刺，穿刺点可用甲紫在皮肤上作标记。常选择肩胛下角线7~9肋间或者腋后线7~8肋间(坐位)，腋中线6~7肋 间或腋前线5~6肋间(半卧位)。B.包裹性胸腔积液，可结
1．患者取斜坡卧位。手术部位应依体征、X线胸片或超声检查确定，并在胸壁作标记。常规皮肤消毒，术者戴无菌手套，铺无菌巾，局麻。 2．首先用注射器作胸膜腔穿刺，以确定最低引流位置。作皮肤切口，用直钳分开各肌层(必要时切开)，最后分开肋间肌进入胸膜腔(壁层胸膜应注入足量局部麻醉剂)，置入较大橡胶管。引流管伸入胸腔之长度一般不超过4～5cm，以缝线固定引流管于胸壁皮肤上，末端连接无菌水封瓶。 [1]1．肋间插管法：(1)病人取半坐位或平卧位，如以引流液体为主，则患侧可抬高30度-45度。以1%普鲁卡因 20ml，先作插管处皮肤、皮下及肌层浸润；至少有一半麻醉药注射在胸膜外(注射针在抽得气体或液体时，为胸膜腔内，针头稍退出在不能抽得气体或液体处，即为胸膜外)。(2)选择一根适当的引流管(引流气体则口径可稍小，引流浓液的口径宜大些)，引流管一端剪成弧形，距顶端1cm，再开一侧孔。根据注射麻醉剂针头进入胸膜腔的距离。可了解患者胸壁的厚度。在引流管侧孔远端，在以胸壁厚度加1cm处，以丝线作标记，即引流管应插入胸膜腔之深度(丝线平皮肤处)。(3)一切准备好之后，于皮肤浸润麻醉处切开1.5-2.0cm，以血管钳分离皮下组织、肌层，直至胸膜腔，并扩大胸膜上的裂口。以血管钳夹住引流管弧形端，经切口插入胸膜腔。将引流管与水封瓶连接。观察有无气体或液体溢出。如果引流通畅，
将引流管调整至适当深度(即丝线标记处)，即可缝合皮肤切口，并固定引流管，以免滑脱。切口以消毒纱布覆盖，并以胶布固定，引流管必须垂直于皮肤，以免造成皮肤压迫性坏死。(4)水封瓶为一广口玻璃瓶，以橡胶瓶塞密封瓶口，瓶塞上穿过长、短各一两根玻璃管。长玻璃管一端，应与胸腔引流管连接，另一端应在瓶内水面下2cm。引流瓶应较胸膜腔低50-60cm。瓶内应放置消毒盐水或冷开水，放入水后应作标记。根据引流瓶外的刻度(标记)，可以随时观察及记录引流量。每日应更换引流瓶内消毒水一次。引流管必须保持通畅。若引流管通畅，则长玻璃管内液面，随病人呼吸而上下波动。液面波动停止，则表示引流管已被堵塞，或肺已完全膨胀。经常挤压胸腔引流管，是一保证引流通畅的有效方法。引流过程中，应严观察患侧呼吸音，和必要时作胸部X线检查，了解引流后肺膨胀情况。若引流后未达到肺完全膨胀，应即时更换引流部位。引流液体的性质和量，应详细记录，随时根据情况，作相应检查，如细菌培养及药敏；乳糜定性等，然后作进一步处理。引流气体者，停止排气24小时后；胸腔引，流液24小时内少于100ml，则可拔除胸腔引流管。拔管时，应先清洁皮肤，及引流管近皮肤段，剪断固定丝线后，嘱病人深呼气后摒住，以8层凡士林油纱布堵塞伤口，迅速拔出引流管，并以宽胶布封贴敷料，以免拔管后，外界空气漏入，再造成气胸。(5)也可采用有侧臂的套管针，引流管的
胸腔穿刺术（2） 【适应症】 1、诊断性穿刺，以确定积液的性质。 2、穿刺抽液或抽气，以减轻对肺的压迫或抽吸脓液治疗脓胸。 3、胸腔内注射药物或人工气胸治疗。 【禁忌症】 出血性疾病及体质衰弱、病情危重，难于耐受操作者应慎用。 【操作步骤】 一、准备工作 1、向患者及家属说明穿刺的目的、意义，操作过程及并发症，消除其顾虑及精神紧张。取得同意并签协议。 2、询问药物过敏史者，必要时需做皮肤试验或用2%利多卡因局部麻醉。 3、核对患者床号、姓名、性别、年龄。 4、物品准备：胸腔穿刺包、无菌手套、治疗盘（弯盘、2%利多卡因、0.5%碘伏、棉签、胶带）血压计、听诊器、标准容器、椅子。如需胸腔内注药，应准备好所需药品。 5、衣帽整齐、戴口罩、规范洗手。 6、穿刺前测脉搏、血压，并观察病情变化。 二、操作方法 1、摆体位：患者取坐位面向椅背，两前臂置于椅背上，前额伏于前臂（不能起床者，可取半坐卧位，患者前臂上举抱于枕部）。 2、选择穿刺点：先进行胸腔叩诊，选择胸部叩诊实音最明显部位穿刺，穿刺点可用甲紫在皮肤上作标记，一般常取肩胛线或腋后线第7- 8肋间；也可选腋中线第6-7 肋间或腋前线第5肋间。包裹性胸腔积液，可结合X线及超声波定位进行穿刺。气胸抽气减压：穿刺部位一般选取患侧锁骨中线第2肋间或腋中线4-5肋间。
3、常规消毒：以穿刺点为中心用碘伏消毒3遍，直径约15厘米。打开穿刺包，确认灭菌标识及有效期，检查包内物品是否完善，戴无菌手套：打开手套包，取出手套，左手捏住手套反折处，右手对准手套5指插入戴好。已戴手套的右手，除拇指外4指插入另一手套反折处，左手顺势戴好手套。铺无菌洞巾。 4、局麻：检查并抽取2%利多卡因，在穿刺点皮内打一皮丘自皮肤至壁层胸膜进行局部麻醉。 5、穿刺：左手固定穿刺部位皮肤，右手持穿刺针以垂直皮肤的方向缓慢刺入，有明显突破感时接50ml注射器，松开止血钳，缓慢抽取胸腔积液。 6、用止血钳夹住穿刺针后的橡皮管，拔出穿刺针，覆盖无菌纱布，消毒穿刺部位，纱布加压覆盖，胶布固定。 7、术后口述再次测血压，并交代注意事项。 【注意事项】 1、向患者解释穿刺目的，消除紧张情绪，必要时给予镇静药 2、操作中应密切观察患者的反应，如发生连续咳嗽或出现头晕、出汗、心悸等胸膜反应时，应立即停止抽液，让病人平卧，观察血压、脉搏，必要时皮下注射0.1%肾上腺素0.3-0.5ml，并注意观察其它并发症，如血胸、气胸、穿刺口出血，胸壁蜂窝组织炎、脓胸、空气栓塞等。 3、抽液不可过多过快。①诊断性抽液50-100 ml，最多不超1000 m l ②脓胸要抽净③细胞学检查至少抽100 m l 4、严格无菌操作，操作中要防止空气进入胸腔，始终保持胸腔负压。 5、避免在第9肋间以下穿刺，以免穿透膈肌损伤腹腔脏器。 【问答】 1、胸腔穿刺的目的是什么？ （1）诊断性穿刺：通过穿刺液化验及病理检查，确定积液的性质或病因。 （2）治疗性穿刺：通过抽液或抽气，减轻胸腔内压迫；胸腔内注入药物治疗脓胸、胸膜炎、人工气胸治疗等。
胸腔积液 一、定义 任何原因导致胸膜腔内出现过多的液体称胸腔积液，俗称胸水。我们常说胸腔积液，实际上是胸膜腔积液。正常人胸膜腔内有3ml～15ml液体，在呼吸运动时起润滑作用，但胸膜腔中的积液量并非固定不变。即使是正常人，每24小时亦有500ml～1000ml的液体形成与吸收。胸膜腔内液体自毛细血管的静脉端再吸收，其余的液体由淋巴系统回收至血液，滤过与吸收处于动态平衡。若由于全身或局部病变破坏了此种动态平衡，致使胸膜腔内液体形成过快或吸收过缓，临床产生胸腔积液。按照胸腔积液的特点分类，可以将胸腔积液分为漏出液、渗出液（浆液性或血性）、脓胸、血胸、乳糜胸。 脓胸是指由各种病原微生物引起的胸膜腔感染，伴有胸腔渗出液。 二、病因 （一）按照病因分类可以有： 1.感染性疾病 胸膜炎（结核病、各类感染）、膈下炎症肺结核、各类肺感染、肺结核。 2.循环系统疾患 上腔静脉受阻、充血性心力衰竭、缩窄性心包炎。 3.肿瘤 恶性肿瘤、胸膜间皮瘤。 4.肺梗死 5.血管瘤破裂、胸导管受阻 6.低蛋白血症、肾病综合征、肝硬化 7.其他疾患 黏液性水肿、药物过敏、放射反应、风湿热、系统性红斑狼疮、胸部手术后、气胸、胸腔穿刺术后继发化脓性感染外伤、气胸（伴胸膜粘连带撕裂）、外伤致胸导管破裂、丝虫病。 胸腔积液以渗出性胸膜炎最为常见。肿瘤（如肺癌、乳腺癌、淋巴瘤等）
累及胸膜，使其表面通透性增加，或淋巴引流受阻，或伴有阻塞性肺炎累及胸膜，均可引起渗出性胸腔积液。偶因胸导管受阻，形成乳糜胸。如心包受累而产生心包积液，或因上腔静脉受阻而使血管内静水压升高，或因恶性肿瘤所致营养不良性低蛋白血症，胸腔积液可为漏出液。 三、临床表现 结核性胸膜炎多见于青年人，常有发热。中老年人出现胸腔积液，应提高警惕，可能是恶性病变。 炎性积液多为渗出性，常伴有胸痛及发热。由心力衰竭所致胸腔积液为漏出液。肝脓肿所伴右侧胸腔积液可为反应性胸膜炎，亦可为脓胸。 积液量少于0.3升时症状多不明显；若超过0.5升，患者可感到胸闷。医生在给患者进行体格检查时，会发现局部叩击呈浊音，呼吸的声音减低。积液量多时，两层胸膜隔开，不再随呼吸摩擦，胸痛亦渐缓解，但呼吸困难会逐渐加剧。若积液进一步增大，使纵隔脏器受压，患者会出现明显的心悸及呼吸困难。 四、检查 （一）外观 漏出液透明清亮，静置不凝固，比重1.018。脓性胸液若为大肠杆菌或厌氧菌感染常有臭味。血性胸液呈程度不同的洗肉水样或静脉血样；乳状胸液为乳糜胸；若胸液呈巧克力色应考虑阿米巴肝脓肿破溃入胸腔的可能；黑色胸液可能为曲菌感染。 （二）细胞 正常胸液中有少量间皮细胞或淋巴细胞，胸膜炎症时，胸液中可见各种炎症细胞及增生与退化的间皮细胞。漏出液细胞数常少于100×106/L，以淋巴细胞与间皮细胞为主。渗出液的白细胞常超过500×106/L。脓胸时白细胞多达1000×106/L以上。中性粒细胞增多时提示为急性炎症；淋巴细胞为主则多为结核性或恶性；寄生虫感染或结缔组织病时嗜酸性粒细胞常增多。胸液中红细胞超过5×109/L时，可呈淡红色，多由恶性肿瘤或结核所致。胸腔穿刺损伤血管亦可引起血性胸液，应谨慎鉴别。红细胞超过100×109/L时应考虑创伤、肿瘤或肺梗