耐性细菌的分离鉴定及重金属污染修复初步研究

清远市是中国最大的电子废弃物拆解基地之一，小作坊生产模式已经进行了20多年，大量无法回收的电子废料和处理残渣等被倾倒在田地、沟渠和山谷中，致使周边土壤长期受到重金属Cd、Cu、Pb污染。近年来国内外对拆解区周边的土壤重金属污染现状分析、健康风险评价等相关报道较多，但针对电子废弃物复合重金属污染生物修复技术的研究却并不多见。通过富集、驯化、分离，从清远市电子废弃物拆解区污染土壤中得到4种耐性菌株，经菌落形态、扫描电镜分析以及16S rDNA技术鉴定得出，菌株HS-01、JH-02、YB-03、JY-04分别为海水芽孢八叠球菌（Sporosarcina aquimarina）、佐吕间湖生芽孢八叠球菌（Sporosarcina saromensis）、巨大芽孢杆菌（Bacillus megaterium）、甲基营养型芽孢杆菌（Bacillus methylotrophicus）。由生长特性实验得到，细菌HS-01与JY-04生长周期相同，在0～8 h为调整期，8～12 h为对数期，12～24 h为稳定期，24～32 h为衰亡期。而细菌JH-02与YB-03生长周期相同，在0～4 h为调整期，4～12 h为对数期，12～24 h为稳定期，24～32 h为衰亡期。细菌HS-01、JH-02、JY-04的最适温度与pH分别为30℃和8，而YB-03则为35℃与7.5。生物吸附实验结果显示，随着重金属离子质量浓度的升高，4种耐受细菌对重金属离子的吸附量也逐渐升高，但吸附量增长率以及吸附率却逐渐降低，这4种细菌对Cd2＋、Cu2＋、Pb2＋最大吸附量分别达到了2.25、2.05、2.28、2.25 mg，8.19、4.95、8.53、11.78 mg和10.84、10.59、7.66、9.02 mg。最大吸附率则分别达到了94.4、99.2、100、93.3%，86.1、90.8、88.6、87.3%和88.9、82.2、81.2、86.7%。其中细菌HS-01、YB-03、JY-04吸附Cd2＋能力较强，而细菌JY-04对Cu2＋以及HS-01、JH-02对Pb2＋吸附能力最强。上述结果显示了4种耐性细菌均具有较好的修复复合重金属污染水体的应用潜力，但对于电子废弃物复合重金属污染土壤的修复机理和效率还有待进一步研究。

EDTA与耐性细菌对黑麦草吸收复合污染红壤中铅镉的影响

为比较耐性细菌与EDTA两种方法强化植物富集重金属效果的优劣,以黑麦草(Lolium perenne L.)为供试植物,利用土培试验和室内分析,比较EDTA与耐性细菌(Rhodococcus baikonurensis,编号为J6)对黑麦草吸收重金属复合污染土壤中铅(Pb)、镉(Cd)的影响。试验采用双因素完全随机区组设计,因素一为Cd、Pb污染浓度,设置6个水平,各个水平的Cd、Pb浓度分别为0、0 mg·kg^(-1);5、50 mg·kg^(-1);10、100 mg·kg^(-1);20、200 mg·kg^(-1);50、400 mg·kg^(-1);80、800 mg·kg^(-1),并依次以代号A1、A2、A3、A4、A5、A6表示。因素二为分别添加EDTA、接种J6菌,以空白为对照。结果表明:在A1、A2条件下,添加J6菌可促进黑麦草生长,提高生物量。接种J6菌对黑麦草吸收Pb、Cd具有促进作用,在不同浓度的重金属处理条件下,接菌后的黑麦草地上部Cd含量呈提高趋势,最高达27%、地下部Pb、Cd含量也分别提高17%~64%、5%~23%;而添加EDTA后的黑麦草仅有地下部Pb含量提高40%(A5)、60%(A6)。接种J6菌可降低根际土的Cd、Pb全量,同时提高有效态Cd、Pb含量。接种J6菌也可提高黑麦草Cd的转运系数。总体而言,接种J6菌可促进黑麦草对重金属复合污染土壤中的Cd、Pb的吸收,其效果优于添加螯合剂EDTA。

耐性细菌与活化剂对土壤镉生物有效性的影响

为探讨耐性细菌与活化剂对镉(Cd)污染土壤Cd生物有效性的影响,采用盆栽试验方法,研究了耐性细菌与活化剂(有机肥、EDTA、黄腐酸、柠檬酸)对黑麦草吸收Cd的影响。结果表明:赤红壤和棕色石灰土中菌剂50 mL/盆(J1)、菌剂200 mL/盆(J2)、有机肥40 g/盆(Y1)、有机肥80 g/盆(Y2)、菌剂50 m L/盆+有机肥40 g/盆(JY1)、菌剂100 mL/盆+有机肥80 g/盆(JY2)、黄腐酸5 g/盆(H1)、黄腐酸50 g/盆(H2)处理的黑麦草总生物量较对照(自来水)提高了22.89%~65.93%,EDTA和柠檬酸使黑麦草总生物量降低。赤红壤中,菌剂与活化剂处理的黑麦草地上部、地下部Cd含量较对照分别提高了8.28%~66.45%、10.92%~74.17%;棕色石灰土中,菌剂与活化剂处理的黑麦草地上部、地下部Cd含量较对照分别提高10.88%~70.97%、10.10%~51.82%。赤红壤和棕色石灰土中,J1、J2、Y1、Y2、JY1、JY2、E1(EDTA 5 mmol/盆)、E2(EDTA 15mmol/盆)处理的黑麦草地上部Cd吸收量较对照提高了6.93%~126.45%。黑麦草对Cd吸收量越大,土壤中弱酸可溶态和残渣态Cd越少,可还原态、可氧化态Cd越多。综上,耐Cd菌剂与活化剂可提高Cd生物有效性、提高黑麦草对Cd污染土壤的生物修复效果,具有较好的应用推广前景。

镉耐性固定细菌的筛选及其对不同品种小麦镉吸收的阻控效应

为筛选重金属耐性固定细菌并探索其阻控小麦对重金属的积累效应,从根际土壤筛选镉耐性固定细菌,研究细菌吸附特性以及细菌对两种小麦Cd含量、富集系数和转移系数的影响。结果表明:筛选到一株镉耐性固定细菌菌株YM4,经16S rDNA序列分析鉴定为假单胞菌(Pseudomonas sp.)。YM4能耐受225 mg·L-1高浓度Cd2+,在含50 mg·L-1Cd2+培养基中对Cd的去除率达到58.9%。YM4对Cd的去除以胞外吸附为主。红外光谱分析发现,细胞壁上参与吸附或结合Cd的官能基团主要有─OH、─NH、羧基和磷酸基团等。小麦盆栽试验中,YM4显著降低小麦地上部(茎叶、麦壳)Cd积累量(11.31%~46.05%和10.12%~42.22%),使籽粒Cd积累量明显下降(26.79%~62.24%,红麦;25.27%~28.99%,白麦)。YM4能显著降低两种小麦茎叶、籽粒和麦壳的Cd富集系数以及籽粒的Cd转移系数。研究表明,镉耐性固定细菌Pseudomonas sp. YM4能降低小麦籽粒Cd含量,阻控小麦对Cd的吸收积累,对中轻度Cd污染土壤的粮食安全生产具有潜在应用价值。

Valence variation of arsenic in bioleaching process of arsenic-bearing gold ore

The concentration and variational trend of As3 +and As 5+,the bacterial resistance for the As 3+and As 5+and converting conditions from As3 +to As 5+were analyzed.The additive was used to prompt the bacterial leaching efficiency by changing valence state of arsenic.The results show that the concentration of As 3+ is larger than that of As 5+ in the lag phase.The concentration of As 3+ decreases in the log phase,and is lower than that of As5 +.HQ-0211 typed bacteria express better resistance for As 3+and As 5+and remain growing when the concentrations of As3 +and As 5+are above 6.0 g/L and 12.0 g/L,respectively.It is found that Fe 3+cannot oxidize As3 +singly as strong oxidant in the leaching system,but can cooperate with pyrite or chalcopyrite to do that.The oxidation of As 3+ is prompted with addition of H2O2.The bacterial activity is improved in favor of bacterial leaching efficiency.NaClO restrains the bacterial growth to depress leaching efficiency because of the chloric compounds affecting bacterial activity.

Research Progress in Phage

With excessive utilization of antibiotics in recent years,bacterial drug resistance problem is serious increasingly,and it is more and more difficult to develop anti-infective drug,while it does not have these problems to use phage controlling disease.Phage is a kind of prokaryotic virus,widely exists in the nature and includes bacteriophage,cyanophage and actinophage.Due to its potential of replacing antibiotics to treat disease,phage receives more and more attention.In this paper,based on development status of phage research at home and abroad,discovery process,naming method and classification basis of phage are introduced comprehensively,and advantages and limitations of phage applying in prevention and control of bacterial diseases are analyzed.We introduce application status of phage in human medicine,prevention and control of diseases for terrestrial animals and aquaculture,and the effects of phage in sewage treatment,prevention and control of microbial contamination of food and detection technology,and point out the shortages of phage in the above application.Meanwhile,we also discuss application prospects of phage in disease prevention and control,environmental protection and food safety.

Conjugation with Acridines Turns Nuclear Localization Sequence into Highly Active Antimicrobial Peptide

The emergence of multidrug-resistant bacteria creates an urgent need for alternative antibiotics with new mechanisms of action. In this study, we synthesized a novel type of antimicrobial agent, Ac r_3-NLS, by conjugating hydrophobic acridines to the N-terminus of a nuclear localization sequence(NLS), a short cationic peptide. To further improve the antimicrobial activity of our agent, dimeric(Acr_3-NLS)_2 was simultaneously synthesized by joining two monomeric Acr_3-NLS together via a disulfide linker. Our results show that Acr_3-NLS and especially(Acr_3-NLS)_2 display signifi cant antimicrobial activity against gramnegative and gram-positive bacteria compared to that of the NLS. Subsequently, the results derived from the study on the mechanism of action demonstrate that Acr_3-NLS and(Acr_3-NLS)_2 can kill bacteria by membrane disruption and DNA binding. The double targets—cell membrane and intracellular DNA—will reduce the risk of bacteria developing resistance to Acr_3-NLS and(Acr_3-NLS)_2. Overall, this study provides a novel strategy to design highly eff ective antimicrobial agents with a dual mode of action for infection treatment.

Antagonistic Activity, Antimicrobial Susceptibility and Potential Virulence Factors of Enterococcus faecalis

Enterococcus faecalis isolates （87） were phenotypically and genotypically identified and subsequently subjected to the antagonism test and antimicrobial susceptibility. The lipolitic, hemolytic and DNAse activities were identified along with the genes gelE, cylL, cylS, ccf, cpd and cob that, encode virulence determinants. Thirty seven percent of isolates inhibited Listeria monocytogenes （CERELA）, Listeria innocuous （CERELA）, Staphylococcus aureus （ATCC25932）, Lactococcus lactis （IL1403）, Micrococcus luteus （ATCC 10240） and Enterococcusfaecalis （ATCC29212）. All strains were sensitive to the ampicillin antibiotic, but 47% were resistant to at least one antimicrobial agent and 6% of isolates presented multidrug resistance. Ninety seven percent of isolates contained the gelE gene, but 77% of these isolates showed gelatinase activity. Presence of cylL and cylS genes was observed in 25% of the isolates, but only 5% presented hemolytic activity. None isolates showed lipase and DNAse activities. Eight percent of isolates contained the ccf gene and 2% showed the presence of the cpd and cob genes. The ability to inhibit pathogenic bacteria, low resistance to antibiotics and absence of virulence factors make some of Enterococcusfaecalis strains characterized in the present study promising for exploitation in other applications such as probiotics in aquaculture.

贵金属纳米材料的抑菌机制

贵金属纳米材料(Noble metal-based nanomaterials, NMNs)具有广谱抗菌性。NMNs可通过纳米穿孔、破坏膜稳定性、诱导活性氧、与DNA等生物大分子结合等方式杀灭细菌。同时,在NMNs选择压力下,细菌进化出了抵抗NMNs损害的能力。细菌对NMNs的耐性可通过膜电位排斥、分泌吸附蛋白、激活抗氧化损伤相关酶、外排有毒颗粒以及群体效应等途径得以表现。本文一方面总结了NMNs对细菌的抑制机制,另一方面探讨了细菌对NMNs产生耐性的方式、原因及目前研究的不足,以期为合理开发并应用抑菌贵金属纳米材料提供科学依据。

Selective inactivation of Gram-positive bacteria in vitro and in vivo through metabolic labelling

Bacterial infections are grave threats to human health,particularly those caused by the most common Grampositive bacteria.The massive administration of broad-spectrum antibiotics to treat various bacterial infections has led to the evolution and spread of drug resistance.As a universal antimicrobial technique unapt to induce drug resistance,photothermal therapy(PTT)is attracting extensive attention in recent years.However,its unspecific killing capability and side effects towards adjacent mammalian cells severely impede the practical applications.Herein,we proposed a metabolic engineering strategy to selectively inactivate Gram-positive bacteria by PTT.A bioorthogonal photothermal agent was prepared by the conjugation of IR-780 iodide and dibenzocyclooctyne(IR780-DBCO).Upon pre-metabolizing with 3-azido-D-alanine,Gram-positive bacteria rather than Gramnegative ones,such as Staphylococcus aureus and vancomycinresistant Enterococcus faecalis(VRE),could be specifically tied up by the explosive IR780-DBCO via copper-free click chemistry.Thereafter,they spontaneously detonated under 15 min near-infrared light irradiation and inactivated nearly 100% Gram-positive bacteria in vitro.Moreover,superbug VRE-induced infection was significantly inhibited by this approach in a mouse skin wound model.This metabolic labelling-based photothermal ablation strategy specific to Gram-positive microbes would stimulate the development of precise antibacterial candidates for preclinical applications.

Occurrence and Fate of Antibiotics in the Aqueous Environment and Their Removal by Constructed Wetlands in China： A review

Overuse of antibiotics has become a serious ecological problem worldwide. There is growing concern that antibiotics are losing their effectiveness due to an increased antibiotic resistance in bacteria. During the last twenty years, consumption of antibiotics has increased rapidly in China, which has been cited as one of the world＇s worst abusers of antibiotics. This review summarizes the current state of antibiotic contamination in China＇s three major rivers （the Yangtze River, Yellow River, and Pearl River） and illustrates the occurrence and fate of antibiotics in conventional municipal wastewater treatment plants （VC-VVTPs）. The analytical data indicate that traditional W-WTPs cannot completely remove these concerned pharmaceuticals, as seen in the large difference between the distribution coefficient （Kd） and the uneven removal efficiency of various types of antibiotics. Although constructed wetlands （CWs） offer a potential way to remove these antibiotics from water supplies, knowledge of their mechanisms is limited. There are four main factors affecting the performance of CWs used for the treatment of antibiotics in water supplies, the types and configurations of CWs, hydraulic load rates, substrates, and plants and microorganisms. Further researches focusing on these factors are needed to improve the removal efficiency of antibiotics in CWs.

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration （MIC） of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick＇s laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antirnicrobial agents, and guide clinical treatment.

Rapid eradication of antibiotic-resistant bacteria and biofilms by MXene and near-infrared light through photothermal ablation

With the development and rising of antimicrobial resistance,rapid and effective killings of bacteria are urgently needed,especially for antibiotic-resistant bacteria and bacterial biofilms that are usually hard to be treated with conventional antibiotics.Here,a rapid and broad-spectrum antibacterial strategy is demonstrated through photothermal ablation with MXene and light.Ti3C2 MXenes,when combined with 808 nm light,show significant antibacterial effects in just 20 min.The antibacterial strategy is effective to 15 bacterial species tested,including methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococci(VRE).In addition,the rapid antibacterial strategy works for MRSA biofilms,by damaging the structures as well as killing bacteria in biofilms.Furthermore,the investigation of the antibacterial mechanisms shows that Ti3C2 with light kills bacteria mainly physically through inserting/contact and photothermal effect.This work broadens the potential applications of MXene and provides a way to eradicate bacteria and biofilms physically,without the likelihood of resistance development.

New strategy for reversing biofilm-associated antibiotic resistance through ferrocene-substituted carborane ruthenium(Ⅱ)-arene complex

Bacterial biofilms are inherently resistant to antimicrobial agents and are difficult to eradicate with conventional antimicrobial agents, resulting in many persistent and chronic bacterial infections. In this contribution, a new strategy for reversing the biofilm-associated antibiotic resistance has been explored by induction of a carborane ruthenium(II)-arene complex (FcRuSB). Our results demonstrate that the FcRuSB could be utilized as an inducer to efficiently reverse the biofilm-associated antibiotic resistance of multidrug-resistant (MDR) clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa. The induced effect of FcRuSB is correlated with a considerable decrease in the expression of extracellular matrix proteins (EMP) of the two strains. The considerable decrease of the EMP of induced cells, resulting in the reduction of adherence and biofilm formation ability of the two types of MDR pathogens, and then can cause significantly enhanced sensitivity of them to antibiotics.

Discussion on research methods of bacterial resistant mutation mechanisms under selective culture——uncertainty analysis of data from the Luria-Delbrück fluctuation experiment

Whether bacterial drug-resistance is drug-induced or results from rapid propagation of random spontaneous mutations in the flora prior to exposure, remains a long-term key issue concerned and debated in both genetics and medicinal fields. In a pio-neering study, Luria and Delbruck exposed E. coli to T1 phage, to investigate whether the number of resistant colonies fol- lowed the Poisson distribution. They deduced that the development of resistant colonies is independent of phage presence. Similar results have since been obtained on solid medium containing antibacterial agents. Luria and Delbruck＇s conclusions were long considered a gold standard for analyzing drug resistance mutations. More recently, the concept of adaptive mutation has triggered controversy over this approach. Microbiological observation shows that, following exposure to drugs of various concentrations, drug-resistant cells emerge and multiply depending on the time course, and show a process function, incon-sistent with the definition of Poisson distribution （which assumes not only that resistance is independent of drug quantity but follows no specific time course）. At the same time, since cells tend to aggregate after division rather than separating, colonies growing on drug plates arise from the multiplication of resistant bacteria cells of various initial population sizes. Thus, statisti-cal analysis based on equivalence of initial populations will yield erroneous results. In this paper, 310 data from the Lu- ria-DelbNck fluctuation experiment were reanalyzed from this perspective. In most cases, a high-end abnormal value, resulting from the non-synchronous variation of the two above-mentioned time variables, was observed. Therefore, the mean value cannot be regarded as an unbiased expectation estimate. The ratio between mean value and variance was similarly incompara-ble, because two different sampling methods were used. In fact, the Luria-Delbrtick data appear to follow an aggregated, rather than Poisson distribution. In stmnnary, the statistical analysis of Luria and Delbruck is insufficient to describe rules of resistant mutant development and multiplication. Correction of this historical misunderstanding will enable new insight into bacterial resistance mechanisms.