Ubururu bwa metilene kuri biofilm: ibyo ubushakashatsi bwo muri laboratwari bukoresha urumuri bupima

Biofilm si itsinda ry’ingirabuzimafatizo zireremba mu mazi. Ni imiterere yubatse: ingirabuzimafatizo zifashe ku buso, zikikijwe n’urusobe rw’ibinyabutabire ubwazo zikora rugizwe na polisakaride, poroteyine na DNA iri hanze y’ingirabuzimafatizo, kandi urugero rwa ogisijeni n’intungamubiri rukagenda ruhinduka kuva hanze kugera imbere. Iyo miterere ni yo ituma ikinyabutabire cyica bagiteri zireremba mu mazi mu minota mike gishobora kunanirwa kuri izo bagiteri zimaze gukura zigakora biofilm. Umwanzuro uwo ari wo wose ku bururu bwa metilene na biofilm ugomba gusomwa hitawe kuri iyo miterere, kuko urwo rusobe ari rwo rugena ingano y’irangi igera ku ngirabuzimafatizo n’ingano y’urumuri igera ku irangi.

Iri sesengura rikoresha inyandiko eshatu gusa. Imwe itanga amakuru y’ibanze. Ebyiri ni igerageza ryo muri laboratwari. Gutandukanya izo nshingano ni byo bigize ubu buryo bwose, kandi birinda ikosa rikunze kugaragara cyane muri iyi ngingo: kuvuga ibyavuye mu isahani ya laboratwari nk’aho ari ibimenyetso by’uko umuntu yavurwa ubwandu.

Inyandiko y’amakuru y’ibanze ivuga ubutabire, si ukwica mikorobe

Inyandiko ya kera muri izo eshatu ni isesengura risobanura iterambere ry’ibinyabutabire byo mu bwoko bwa phenothiazinium bikangurwa n’urumuri, ryasohotse mu 2005. Rikurikirana ubururu bwa metilene kuva bukiri irangi mu kinyejana cya 19 kugeza bubaye ikinyabutabire cy’ibanze mu guteza imbere kuvura kanseri hakoreshejwe urumuri, nyuma no mu gukoresha ibinyabutabire bikangurwa n’urumuri mu kurwanya mikorobe. Umusanzu waryo ni ugusobanura isano iri hagati y’imiterere y’ikinyabutabire n’ibikorwa byacyo: kongera amatsinda ya metile ku gice cyacyo gitanga ibara byongera ubushobozi bwo kwivanga n’ibinure, ingano ya ogisijeni yo mu mimerere ya singlet ikorwa, n’ubushobozi bwo kwica bagiteri hakoreshejwe urumuri, bikagabanya kandi umuvuduko wo guhinduka, binyuze mu kwakira elegitoroni, imiterere ya leuco idakora cyane. Rinavuga neza ikibazo kiri muri uru rwego. Inyandiko nyinshi zakoresheje amarangi asanzwe agurishwa, ariko guteza imbere ibinyabutabire bikomoka kuri ayo marangi byari bikiri bike.

Koresha iryo sesengura mu gice cy’uburyo ibikorwa bibaho mu ishusho wiyubakamo yo gusobanukirwa iyi ngingo: urumuri rukangura irangi, imimerere ya triplet igakorana na ogisijeni binyuze mu guhererekanya ingufu kwa Type II kugira ngo ikore ogisijeni yo mu mimerere ya singlet, cyangwa mu guhererekanya elegitoroni kwa Type I kugira ngo ikore radikali. Icyo gitero gikoresha uburyo bwinshi ni cyo gituma uburyo busanzwe mikorobe zikoresha mu kwihanganira imiti budakora neza iyo zicwa hakoreshejwe ibinyabutabire bikangurwa n’urumuri. Ntukoreshe iyo nyandiko nk’isoko ry’igabanuka ripimwe muri log. Nta na rimwe yapimye. Ubutabire bukorwa n’urumuri busobanura neza ubwo buryo bw’ibikorwa buri mu cyegeranyo cy’ibimenyetso ku buvuzi bukoresha ibinyabutabire bikangurwa n’urumuri, naho amategeko yerekeye uburebure bw’umuraba agena niba itara runaka rishobora kubukangura asobanurwa mu ngingo ivuga uko ubururu bwa metilene bwinjiza urumuri rutukura.

Igerageza ribiri kuri biofilm, uburyo bubiri butandukanye bwo gusobanura ibisubizo

Inyandiko zombi z’igerageza zipima igikorwa cy’ubururu bwa metilene bukangurwa n’urumuri kuri biofilm zo mu kanwa hakoreshejwe 660 nm, ariko hafi ya byose bisigaye biratandukanye. Amatsinda ya mikorobe atandukanye, igihe zimaze zikura gitandukanye, uburyo irangi riteguye butandukanye, urugero rw’urumuri rutandukanye, kandi cyane cyane ibipimo by’ibisubizo bitandukanye. Iryo tandukaniro ni ryo ngingo y’iyi nyandiko, kandi ni na ryo rigaragazwa mu mbonerahamwe y’umwimerere igereranya ubwo bushakashatsi iri hasi.

Iya mbere ni ubushakashatsi bwo muri laboratwari kuri biofilm ikuze yo munsi y’ishinya irimo amoko menshi ya mikorobe, bwasohotse mu 2024. Amoko mirongo itatu n’atatu yarerewe hamwe ku duti twa polistirene mu gikoresho cya Calgary Biofilm Device, agaburirwa ahatari ogisijeni. Igitunga mikorobe cyasimbuwe nyuma y’amasaha 72, maze zikomeza gukura kugeza ku minsi 7 yari yateganyijwe. Hakoreshejwe ubururu bwa metilene butaboshywe ku kindi kintu ku rugero rwa 0.01 ku ijana, hamwe na LED itukura ya 660 nm, itanga hafi 330 mW kuri cm kare, mu minota 5, iri ku ntera ya 2 mm. Kubara ibyo hamwe bitanga hafi 99 J kuri cm kare. Igerageza ryateguwe neza kugira ngo buri kintu n’imikoranire yabyo bipimwe, rigira amatsinda ane: iryo kugereranyaho ritagize icyo rikorerwa, irangi ridafite urumuri, urumuri rudafite irangi, n’irangi rifatanijwe n’urumuri.

Iya kabiri ni ubushakashatsi bwo muri laboratwari kuri biofilm yo mu kanwa igizwe na mikorobe zibanza kuhatura, bwasohotse mu 2021. Amoko ane ya Streptococcus yarerewe hamwe mu masaha 24 mu masahani afite utwobo 96, harimo sukoroze ya 1 ku ijana. Ikinyabutabire gikangurwa n’urumuri cyari ubururu bwa metilene buhujwe n’uduce duto cyane twa beta sikolodekisitirine, ku rugero rwa 4.65 micromolar. Ubu buryo bwo gutegura irangi bwari bugamije kurinyuza mu nzitizi za biofilm. Amasoko y’urumuri yombi yatangaga urumuri rutukura rwa 660 nm mu gihe cy’amasegonda 90, ariko ingano y’urumuri yakoreshejwe yaratandukanye cyane: 320 J kuri cm kare mu itsinda rya laser na 8.1 J kuri cm kare mu itsinda rya LED. Amatsinda yo kugereranyaho yarimo irangi ridafite urumuri, laser yonyine, LED yonyine, amazi arimo umunyu nk’itsinda ritagamije kugira igikorwa ku mikorobe, na korulegisidine ya 0.2 ku ijana nk’igipimo cyo kugereranyaho mu kurwanya mikorobe.

Itsinda ry’amoko 33 ryakuze iminsi 7 ahatari ogisijeni na biofilm y’amoko 4 abanza kuhatura yakuze amasaha 24 ni uburyo butandukanye bwo gupima. Imibare ikurikira ntishobora gushyirwa hamwe ngo ikurwemo impuzandengo.

Ibyo buri bushakashatsi bwapimye koko

Mu bushakashatsi kuri biofilm ikuze, ibipimo by’ibisubizo byari imikorere y’ingirabuzimafatizo ipimwe hakurikijwe kwakira elegitoroni kwa TTC, igasomwa kuri 485 nm, n’ubwinshi bwa bagiteri bupimwe hakoreshejwe ihuzwa rya DNA na DNA mu buryo bwa checkerboard, hamwe n’udupimisho twa DNA tw’amoko 33. Umubare muto washoboraga gutahurwa wari hafi ingirabuzimafatizo 10,000. Nta kubara mikorobe zikiri nzima hakoreshejwe kuzirera ku masahani, nta gupima ingano y’ibinyabuzima hakoreshejwe crystal violet, nta mashusho atandukanya ibizima n’ibyapfuye, kandi nta gukurikirana niba byongeye gukura. LED yonyine yagabanyije imikorere y’ingirabuzimafatizo hafi 50 ku ijana ugereranyije n’amatsinda atakoreshejweho LED, naho irangi rifatanijwe na LED rigabanya hafi 55 ku ijana. Mu isesengura ry’imibare, irangi ryonyine ryashyizwe mu itsinda rimwe n’iryo kugereranyaho ritagize icyo rikorerwa. Ku mibare yakomotse kuri DNA, irangi ryonyine, LED yonyine n’irangi rifatanijwe na LED byose byari munsi y’itsinda ryo kugereranyaho ku buryo bufite agaciro mu isesengura ry’imibare, ariko nta tandukaniro rifite agaciro ryari hagati y’ayo matsinda atatu. Abanditsi bavuga umwanzuro mu buryo butaziguye: ibyo byakoreshejwe ntibyashenye biofilm yari imaze kwiyubaka, ifashe ku buso kandi igizwe na mikorobe z’amoko menshi. Biofilm ikuze yo mu bice bikikije amenyo ikeneye gusenywa hakoreshejwe uburyo bwa mekanike, naho ibinyabutabire bikangurwa n’urumuri bikaba, nibura, uburyo bwunganira gusa.

Mu bushakashatsi kuri mikorobe zibanza kuhatura, igipimo cy’ibisubizo cyari mikorobe zikiri nzima zabonetse nyuma yo kuzirera ku bitunga mikorobe byatoranyaga izashakishwaga, umubare wazo ukagaragazwa ku gipimo cya logaritimi. Itsinda ry’irangi ridafite urumuri ntiryatandukanye n’iry’amazi arimo umunyu. Amatsinda yombi yakoresheje irangi ryakanguriwe n’urumuri, ari yo laser ifatanijwe n’irangi ryafungiwemo n’utwo duce duto na LED ifatanijwe n’irangi ryafungiwemo, yagabanyije umubare wa mikorobe zikiri nzima kugera kuri hafi 4 log ugereranyije n’itsinda ryo kugereranyaho ritagamije kugira igikorwa ku mikorobe. Kuri icyo gipimo, iryo ni igabanuka ry’inshuro 10,000 cyangwa 99.99 ku ijana. Nta na rimwe muri ayo matsinda ryatandukanye na korulegisidine mu isesengura ry’imibare. Ibisubizo by’urumuri rwonyine n’iby’irangi ridafite urumuri ntibyatangajwe nk’imibare ya log itandukanye kandi yizewe. Bityo incamake idakabya ni iyo inyandiko ishyigikira: irangi ryafungiwemo n’utwo duce duto kandi rigakangurwa n’urumuri ryatanze iryo gabanuka, irangi ridafite urumuri ntiryabikoze, kandi ingano y’igabanuka yari ihwanye n’iy’umuti wo kwica mikorobe wafashwe nk’igipimo cyo kugereranyaho muri iyi biofilm yari ikiri nto.

Nta nyandiko n’imwe yapimye kongera gukura nyuma y’ibyakoreshejwe, ingano y’ibinyabuzima yasigaye cyangwa imiterere y’urusobe rubikikije. Igabanuka rya CFU nta cyo rivuga ku rusobe rusigaye. Kugabanuka kw’imikorere y’ingirabuzimafatizo nta cyo kuvuga ku zarokotse. Umubare ushingiye kuri DNA ntushobora kuvuga ibikiri bizima.

Kugereranya uburyo bwo gupima n’ibipimo by’ibisubizo

Iyi mbonerahamwe ni yo gikoresho cyo kugereranya kiri muri iyi ngingo. Soma umurongo utambitse kugira ngo urebe icyo ubushakashatsi bumwe bugaragaza, hanyuma usome inkingi umanuka kugira ngo urebe ibibazo ubushakashatsi bwombi budasubiza.

Amatandukaniro atatu akwiriye gushimangirwa. Irya mbere, mu buryo bwa biofilm ikuze, itsinda ry’urumuri rwonyine risobanura hafi igikorwa cyose ku mikorere y’ingirabuzimafatizo. Bityo umubare wa 55 ku ijana ntugaragaza kwica mikorobe bitewe n’irangi. Irya kabiri, mu buryo bwa biofilm ikiri nto, irangi ryateguwe ariko ntirihabwe urumuri nta gikorwa rigira, kandi igikorwa kigaragara gusa iyo hari urumuri; ibyo ni ikimenyetso cyo gukangurwa n’urumuri. Irya gatatu, igabanuka rya 80 ku ijana mu mibare yakomotse kuri DNA ryaba hafi 0.7 log mu mibare gusa iyo ikipimwa cyaba ari umubare wa mikorobe zikiri nzima. Ariko si cyo gipimwa, bityo ntirigomba na rimwe kuvugwa nk’iyicwa rya mikorobe ripimwe muri log. Amakuru afitanye isano na biofilm zo mu menyo akusanyirijwe mu bushakashatsi kuri biofilm zo mu kanwa no ku menyo, naho uburyo rusange bwo gutandukanya uburyo bw’igerageza, inzira zo gutanga ibinyabutabire n’ibipimo by’ibisubizo mu bushakashatsi butandukanye busobanurwa mu gitabo cy’uburyo bukoreshwa muri laboratwari.

Impamvu ubushakashatsi kuri biofilm bukeneye amategeko yihariye yo kubusoma

Ibintu bine ni byo bigena niba igisubizo ku bururu bwa metilene na biofilm hari icyo kivuze kirenze isahani cyabonewemo.

Igihe biofilm imaze n’urusobe ruyikikije biza ku mwanya wa mbere. Biofilm zimaze igihe ziba zibyibushye kurushaho, zifite ihuriro ryinshi hagati y’ibice byazo, kandi irangi rikazinjiramo bigoranye. Abanditsi bo mu 2021 bagaragaza ko kwinjira kw’irangi no kudakwirakwira neza k’urumuri muri biofilm yubatse ari byo nzitizi nyamukuru. Uburyo bwo gutwara irangi, nko kurifungira muri sikolodekisitirine, ni ukugerageza gukemura iyo nzitizi nyine. Ni yo mpamvu uburyo irangi riteguye ari ikintu gihindurwa mu igerageza, atari akantu gato gasobanura gusa uko ryakozwe.

Kubara ingano yakoreshejwe biza ku mwanya wa kabiri. Ingano y’ingufu z’urumuri kuri buri buso aha iratandukanye hafi inshuro mirongo ine, kuva kuri 8.1 kugeza kuri 320 J kuri cm kare, ku burebure bumwe bw’umuraba. Naho imbaraga z’urumuri kuri buri buso ziri hagati ya 90 mW na W nyinshi kuri cm kare. Uburebure bumwe bw’umuraba ntibuvuga ko ingano y’urumuri yakiriwe ari imwe, kandi nta bushakashatsi muri ubu bwombi buhindura ingano yakoreshejwe mu buryo buhoraho kandi buhagije bwo gushushanya umurongo ugaragaza uko igisubizo gihinduka.

Amatsinda yo kugereranyaho aza ku mwanya wa gatatu. Ubushakashatsi budafite itsinda ry’irangi ridafite urumuri ntibushobora gukuraho ko irangi ubwaryo ryaba rifite uburozi. Ubushakashatsi budafite itsinda ry’urumuri rwonyine ntibushobora kwemeza ko igikorwa runaka cyatewe no gukangurwa n’urumuri. Inyandiko zombi zirimo ayo matsinda yombi, kandi ibyo ni byiza. Inyandiko kuri biofilm ikuze yerekana impamvu itsinda ry’urumuri rwonyine ari ngombwa: iyo ritaza kubaho, igisubizo ku mikorere y’ingirabuzimafatizo cyari gusomwa nk’igikorwa cy’irangi.

Ibipimo by’ibisubizo biza ku mwanya wa kane. Kuba mikorobe zikiri nzima, imikorere y’ingirabuzimafatizo, ubwinshi bwa DNA, ingano y’ibinyabuzima, imiterere no kongera gukura ni imyanzuro itandatu itandukanye. Nta nyandiko hano ipima ibirenze bibiri. By’umwihariko, kongera gukura ntikwapimwe na hamwe, bityo gukomeza kw’icyo gikorwa ntikwapimwe aho kuba kwaragaragajwe.

Ibyo ibi bitagaragaza ku kuvura ubwandu

Nta na bumwe muri ubu buryo bwateguwe burimo itembera ry’amaraso, ingirabuzimafatizo z’ubwirinzi bw’umubiri, gutemba kw’amacandwe, gusimburana kw’amatembabuzi yo mu mwanya uri hagati y’iryinyo n’ishinya, cyangwa gukuraho ibifashe hakoreshejwe uburyo bwa mekanike. Ingano y’irangi mu kobo k’isahani si ingano iba mu ngirangingo nyuma yo kuritanga mu muntu hakoreshejwe inzira iyo ari yo yose. Gukoresha urumuri rimwe gusa mu minota 5 cyangwa mu masegonda 90 kuri biofilm yo muri laboratwari ikiri nto cyangwa n’iyamaze gukura si gahunda y’ubuvuzi. Abanditsi kuri biofilm ikuze ubwabo barabivuga, bashyira uburyo bukoresha ibinyabutabire bikangurwa n’urumuri mu rwego rw’uburyo bushobora kunganira gukuraho biofilm hakoreshejwe uburyo bwa mekanike, aho kuba uburyo bwonyine bwo kuyirandura.

Incamake nyayo irasobanutse kandi igarukira ku byagaragajwe. Ubururu bwa metilene bukangurwa n’urumuri bushobora kugabanya imikorere y’ingirabuzimafatizo n’ubwinshi bupimwe hakoreshejwe DNA mu itsinda rikuze rya mikorobe zo mu kanwa ryarezwe muri laboratwari, bitarishenye. Muri ubwo buryo bw’igerageza, urumuri rwonyine ni rwo rugira uruhare runini mu kugabanya imikorere y’ingirabuzimafatizo. Irangi ritwarwa na sikolodekisitirine kandi rigakangurwa n’urumuri rishobora kugabanya umubare wa mikorobe zikiri nzima ku gipimo cy’inshuro nyinshi za cumi muri biofilm ikiri nto ya streptococci yo muri laboratwari, ku rugero rugereranywa n’urwa korulegisidine muri ubwo buryo bumwe. Niba kimwe muri ibyo byagaragaye gishobora no kubaho mu bwandu bw’umuntu biterwa n’uburyo ikinyabutabire kigezwa aho kigomba gukorera, uko urumuri ruhagera, ogisijeni, imiterere y’umubiri n’ibipimo by’ibisubizo byo kwa muganga iri gerageza ritigeze ritegurirwa gupima.