黑膠蟲(chóng)污染

1、Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant,abstract,Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and pote

2、ntially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks移動(dòng)的黑點(diǎn)observed in multiple c

3、ell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies分離菌. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequ

4、encing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics.Attempts to decontaminate the eukaryotic真核

5、cell culture used multiple antibiotics at different concentrations. The contaminating chromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics cipro

6、floxacin and piperacillin.環(huán)丙沙星、哌拉西林,,In the case of non-commercially經(jīng)濟(jì) available lines, all attempts areusually taken to clean up the culture by selectively killing thecontamination without harming the cell line. Often

7、 the culprits首因ofculture contamination is mycoplasma支原體, intracellular bacteria thatcan be almost undetectable in culture. Approximately 28% of 460human cell lines surveyed contained mycoplasma [1]. Numerouscommercia

8、l kits試劑盒are available to identify mycoplasma contamina-tion [2,3] and antibiotic solutions are commercially available to ridcultures of them. Additional culture contamination includes otherbacteria, mold, and fungi,U

9、nknown bacterial contamination can be transient. Contami-nation is common with poor aseptic technique and can be devas-tating in a research setting [3]. While many undesired organismsmay steal nutrients from cell line

10、s in culture, they may also prey onthe cells themselves. Predatory bacteria have been shown to feedon other bacteria [5,6], particularly in a limited nutrient environ-ment[7]. Experimental results mayalso be altered d

11、ue to unwantedactivation of cells. Different cellular functions, including thosetriggered by Toll-like receptors, can be activated by a variety ofbacterial components. Several online science blogs discuss a cell cultu

12、re contaminatethat looks like black specks in the cell culture (http://www.cellculturechat.com/i.html/T3470/H3448.html; http://www.protocol-online.org/forums/index.php?s¼bc4fecd655ee6898191ab6ed43f5a5a7&show

13、topic¼9373&pid¼30730&st¼0). The researcherson these blogs describe black dots that fidget or swim and lookmore like rods than dots. Our laboratory has experienced sporadic cell culture contaminat

14、ion fitting this description. Here we reportthe identification of a bacterial cell culture contaminate, a memberof the Achromobacter genus, matching anecdotal descriptions ofcell culture contaminants of multiple eukar

15、yotic cell lines. Inaddition, we provide information regarding antibiotic resistanceand treatment effectiveness.,Antibiotics and doses tested to treat Achromobacter contaminated cells.Antibiotic Dose ( m g/ml)Gentam

16、icin慶大500–2000Amikacin 50–2500Tobramycin妥布霉素50–600Imipenem 2–64Erythromycin 25–500Piperacillin哌拉西林0.5–1000Choramphenicol氯霉素50–500Ciprofloxacin環(huán)丙沙星10–750Plasmocin 25–62.5清除支原體污染,Antibiotics were added to the compl

17、ete media which was replaced every 3–5 days with fresh media and antibiotics.,,Achromobacter can survive and multiply in water and moist soil.Sources have been identified as untreated well water, swimmingpools, dialysi

18、s fluids, distilled water, deionized water, tap water,disinfectants, water systems, ventilators, humidifiers, IV fluids, andinfant formula,未經(jīng)處理的水，游泳池、透析液、蒸餾水、去離子水、自來(lái)水，消毒劑、水系統(tǒng)、風(fēng)機(jī)、加濕器、靜脈輸液，和嬰兒配方奶粉,,Initial attempts to ly

19、se裂解the culture contamination were done inthe eukaryotic cell culture supernatant. Boiling (5–10 min at 95.C)and sonicating聲波降解標(biāo)本(15–30 s at output power 4) did not result in available DNA for PCR. It is possible that

20、the large amount of protein present in eukaryotic真核culture media, primarily from the FBS, could have protected the bacterial cells. DNA was finally isolated using a commercial kit from Promega from isolated colonies grow

21、n on blood agar瓊脂.,,The PCR products, about1400 base pairs in size, were isolated by ethanol乙醇precipitation沉淀and sent for sequencing. After the sequencing, the resulting sequences were checked for similarity to other kno

22、wn sequences using NCBI’s BLAST and the Ribosomal Database Project (RDP). Sequence 1 shared 99% sequence identity with Alcaligenes and Achromobacter 16S rDNA gene sequences, as,,Members of the Achromobacter and Alcaligen

23、esgenera have reclassified to and from these two genera, includingAchromobacter (Alcaligenes) xylosoxidans and Achromobacter (Alca-ligenes) denitrificans [22,23]. It is unclear whether the similarity toboth members o

24、f the Achromobacter and Alcaligenes genera is dueto uncertainty in the nomenclature of previously submittedsequences or due to the actual sequence of the contaminatingbacteria. RDP, a web-based program containing only

25、 16S rDNAsequences, was utilized to confirm the contaminating bacteria’sgenus. Results using SeqMatch from the RDP identified thesequences as belonging to the genus Achromobacter. Phylogenetictrees based on the BLAST

26、and RDP results grouped the bacteriawithpredominantly Achromobacter 16S rDNA sequences (data notshown). Sequences 2, 3, and 4 share 99% sequence identity withsequence 1,,Achromobacter 革蘭氏陰性 ，氧化酶陽(yáng)性，有鞭毛，在37–42 C，pH為6.5

27、–8.5生長(zhǎng)良好In our This contaminant was cultured from FBS and NCS, tap water,distilled water, the water bath (in the water and colony material onthe metal), phospho-buffered saline, and tris-buffered saline. FBSand NCS w

28、ere suspected as the primary, but not only, sources ofcontamination for several reasons. First, cells thawed from cryo-storage (stored in 95% FBS or NCS and 5% dimethyl sulfoxide),which had been previously uncontamina

29、ted, contained the bacteriaonce thawed解凍. Second, multiple sources of FBS and NCS appeared tobe contaminated with the rod-like棒狀motile structures, presumed tobe bacteria, when samples were examined under a microscope.

30、Third, this contaminant morphology and motility has beenobserved in cells obtained from other labs using NCS from the samesource.,,Cultures of Achromobacter were visualized using microscopy.Cultures were mixed with a

31、n equal volume of agarose and placedon a slide. Fig. 1A shows many bacterial cells while 1B shows,,IMCE大腸腺瘤, YAMC小鼠結(jié)腸上皮, MC38小鼠結(jié)腸癌,小鼠胚胎成纖維3T3-L1,,individual cells. Cells were approximately 4–5 m m and were rod-Shaped.On

32、e member of the genus, Achromobacter xylosoxidans木糖has been found to be the causative agent病原體in multiple cases of septicemia敗血癥, mainly in immunocompromised individuals, and sometimes due to contaminated medical supplie

33、s [25–30]. Achromobacter is becoming a serious threat to the health of individuals with cystic fibrosis囊性纖維化.,,Eukaryotic cells were killed at various concentrations of antibi-otics, depending on cell type. For example,

34、 a ciprofloxacinconcentration greater than 60 m g/ml killed both MC38 and YAMCcells. A combination of two antibiotics, piperacillin and cipro-floxacin哌拉西林、環(huán)丙沙星, at a concentration of 10 m g/ml each, was best at selec-

35、tively killing the Achromobacter contaminant without appearing to harm the eukaryotic cells. Achromobacter have been previously been shown to be susceptible to antibiotics administered together. A. xylosoxidans is resist

36、ant to ciprofloxacin, but susceptible to a combination of piperacillin and tazobactam,,Inthis study, isolation of an unknown contaminantwas revealedas member of the genus Achromobacter. The bacteria in this genuscan su

37、rvive in a wide range of environments including tap water,the water bath, FBS, NCS, and many common laboratory solutions.In addition, commercial cells were found to contain this contami-nant. It is important that rese

38、archers utilizing cell culture forexperiments become aware of this multi-drug resistant bacterialcontaminant. Selective killing of these bacteria in eukaryotic cellculture was successful only with a combination of two

39、 antibiotics,piperacillin and ciprofloxacin, both at a concentration of 10 m g/mleach. This report presents the novel identification of this cellculture contaminant and a possible way to remove the swimmingblack dots