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 potentially limitless sample size. Reliable results cannot be obtained, however, whe
2、n cultures contain contamination. This report discusses the observation and identification of mobile black specks移动的黑点observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies分离菌. The 16S rDNA gene was PCR amplified using primers that will
3、 amplify the gene from many genera, and then sequenced. Sequencing 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cell culture used mul
4、tiple antibiotics at different concentrations. The contaminating chromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin.环丙沙星、哌拉西林,In the case of non-commercially经济 available lines, all attempts a
5、reusually taken to clean up the culture by selectively killing thecontamination without harming the cell line. Often the culprits首因ofculture contamination is mycoplasma支原体, intracellular bacteria thatcan be almost undetectable in culture. Approximately 28% of 460human cell lines surveyed contained m
6、ycoplasma 1. Numerouscommercial kits试剂盒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,Unknown bacterial contamination can be transient. Contam
7、i-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 lines in culture, they may also prey onthe cells themselves. Predatory bacteria have been shown to feedon other bacteria 5,6, particularly in a
8、limited nutrient environ-ment7. Experimental results mayalso be altered due 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 culture contami
9、natethat looks like black specks in the cell culture (http:/ http:/www.protocol-online.org/forums/index.php?sbc4fecd655ee6898191ab6ed43f5a5a7&showtopic9373&pid30730&st0). The researcherson these blogs describe black dots that fidget or swim and lookmore like rods than dots. Our laboratory has experi
10、enced sporadic cell culture contamination 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 eukaryotic cell lines. Inaddition, we provide info
11、rmation regarding antibiotic resistanceand treatment effectiveness.,Antibiotics and doses tested to treat Achromobacter contaminated cells.Antibiotic Dose ( m g/ml)Gentamicin庆大5002000Amikacin 502500Tobramycin妥布霉素50600Imipenem 264Erythromycin 25500Piperacillin哌拉西林0.51000Choramphenicol氯霉素50500Ciproflo
12、xacin环丙沙星10750Plasmocin 2562.5清除支原体污染,Antibiotics were added to the complete media which was replaced every 35 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, dialysis fluids, d
13、istilled water, deionized water, tap water,disinfectants, water systems, ventilators, humidifiers, IV fluids, andinfant formula,未经处理的水，游泳池、透析液、蒸馏水、去离子水、自来水，消毒剂、水系统、风机、加湿器、静脉输液，和婴儿配方奶粉,Initial attempts to lyse裂解the culture contamination were done inthe eukaryotic cell culture supernatant. Boiling (51
14、0 min at 95.C)and sonicating声波降解标本(1530 s at output power 4) did not result in available DNA for PCR. It is possible that 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 ki
15、t from Promega from isolated colonies grown on blood agar琼脂.,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 known sequences using NCBIs BLAST and the
16、Ribosomal Database Project (RDP). Sequence 1 shared 99% sequence identity with Alcaligenes and Achromobacter 16S rDNA gene sequences, as,Members of the Achromobacter and Alcaligenesgenera have reclassified to and from these two genera, includingAchromobacter (Alcaligenes) xylosoxidans and Achromobac
17、ter (Alca-ligenes) denitrificans 22,23. It is unclear whether the similarity toboth members of 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 conta
18、ining only 16S rDNAsequences, was utilized to confirm the contaminating bacteriasgenus. Results using SeqMatch from the RDP identified thesequences as belonging to the genus Achromobacter. Phylogenetictrees based on the BLASTand RDP results grouped the bacteriawithpredominantly Achromobacter 16S rDN
19、A sequences (data notshown). Sequences 2, 3, and 4 share 99% sequence identity withsequence 1,Achromobacter 革兰氏阴性 ，氧化酶阳性，有鞭毛，在3742 C，pH为6.58.5生长良好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-b
20、uffered saline, and tris-buffered saline. FBSand NCS were 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 uncontaminated, contained the bacteriaon
21、ce thawed解冻. Second, multiple sources of FBS and NCS appeared tobe contaminated with the rod-like棒状motile structures, presumed tobe bacteria, when samples were examined under a microscope.Third, this contaminant morphology and motility has beenobserved in cells obtained from other labs using NCS fro
22、m the samesource.,Cultures of Achromobacter were visualized using microscopy.Cultures were mixed with an equal volume of agarose and placedon a slide. Fig. 1A shows many bacterial cells while 1B shows,IMCE大肠腺瘤, YAMC小鼠结肠上皮, MC38小鼠结肠癌,小鼠胚胎成纤维3T3-L1,individual cells. Cells were approximately 45 m m and
23、 were rod-Shaped.One member of the genus, Achromobacter xylosoxidans木糖has been found to be the causative agent病原体in multiple cases of septicemia败血症, mainly in immunocompromised individuals, and sometimes due to contaminated medical supplies 2530. Achromobacter is becoming a serious threat to the hea
24、lth of individuals with cystic fibrosis囊性纤维化.,Eukaryotic cells were killed at various concentrations of antibi-otics, depending on cell type. For example, a ciprofloxacinconcentration greater than 60 m g/ml killed both MC38 and YAMCcells. A combination of two antibiotics, piperacillin and cipro-flox
25、acin哌拉西林、环丙沙星, at a concentration of 10 m g/ml each, was best at selec-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 resistant to ci
26、profloxacin, 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 survive in a wide range of environments including tap water,the water bath, FBS, NCS, and many
27、 common laboratory solutions.In addition, commercial cells were found to contain this contami-nant. It is important that researchers 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 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 from cell culture.,
