Tolerance of pesticides and antibiotic resistance in bacteria isolated from wastewater-irrigated soil

A total of 64 bacterial isolates (40 Pseudomonas spp., 12 Azotobacter and 12 Rhizobium spp.) were characterized on the basis of morphological, cultural and biochemical characteristics. All the isolates were tested for their tolerance to the pesticides endosulfan, carbofuran, and malathion. 12.5% of the Pseudomonas isolates from soil tolerated concentrations of 1600 g malathion ml whereas 7.5% of isolates tolerated the same concentration of carbofuran. However, Pseudomonas isolates demonstrated a tolerance limit to endosulfan at a concentration of 800 g/ml. Asymbiotic N₂-fixers (Azotobacter) and symbiotic N₂-fixers (Rhizobium spp.) were also able to tolerate concentrations of pesticides up to 1600 g/ml. All the isolates were further tested for their antibiotic susceptibility against seven different antibiotics, nalidixic acid, cloxacillin, chloramphenicol, tetracycline, amoxycillin, methicillin, doxycycline. 100% of the Pseudomonas isolates were resistant to cloxacillin and 57.5% were resistant to methicillin. 7.5% of the isolates exhibited multiple resistance to five different antibiotics in three different combinations whereas 25% of the isolates showed multiple resistance to four different antibiotics in seven different combinations. Some of the resistant isolates were also screened for plasmid DNA and found to harbour a single plasmid.     

Antibiotic resistance and survival of faecal coliforms in activated sludge system in a semi-arid region (Beni Mellal,Morocco)

The activated sludge process is one of the biological treatment methods used in many countries to reduce the high levels of organic and mineral pollutants and pathogenic micro-organisms present in wastewater. The present work was undertaken to study the dynamic and antibiotic-resistance of faecal coliforms (FC) in the activated sludge system of Beni Mellal. This work has also as objective the study of the survival of FC, protozoan cysts, helminth eggs and FC antibiotic resistance in the sludge dehydrated in drying beds in order to know if the agricultural usage of sludge presents any problems to public health. The activated sludge treatment of Beni Mellal resulted in an average reduction of FC and faecal streptococci of 90.75 and 91.06%, respectively. The overall resistance (resistance to at least one antibiotic) of 111 FC strains isolated from the system was 72.07%. This treatment system did not increase the incidence of FC antibiotic resistance in treated wastewaters. The antibiotic resistance of FC was found to be similar in both raw (71.05%) and treated sewage (77.77%). High levels of antibiotic resistance were towards streptomycin (54.05%), ampicillin (42.34%), amoxicillin (42.34%) and amoxicillin–clavulanic acid (31.53%). The treatment of sludge in drying beds appeared to be efficient in eliminating pathogenic micro-organisms: FC, protozoan cysts and helminth eggs. Moreover, the FC antibiotic resistance did not change over time in sludge-drying bed. According to the standard norms, agricultural utilization of this sludge cannot be excluded. However, it is important to study in the receptor environment the survival and the behaviour of antibiotic-resistant FC present in sludge and water.     

Metal and antibiotic resistance traits in Bradyrhizobium sp. (cajanus) isolated from soil receiving oil refinery wastewater

Forty isolates of Bradyrhizobium sp. (cajanus) were isolated from the nodules of pigeon pea plants grown in fields receiving petrochemical industrial wastewater for the past 12 years and characterized using standard methods. The heavy metal analysis of field soil and treated wastewater showed their presence in varying concentrations. All isolates showed resistance to one or more metals at concentrations >200 g/ml. Multiple metal resistance was a common phenomenon in these isolates. There was no correlation between extractable soil metal concentration and the ability of the isolates to tolerate metal salts in their growth medium as evidenced from their minimum inhibitory concentration (MIC). However, high incidence of metal resistance and the multiple nature of resistance might have been the result of continuous exposure of these strains to heavy metals in the treated wastewater of Mathura Oil Refinery. These strains were also found to be resistant to one or more of the 13 antibacterial drugs tested.     

Comparison of Salmonella enterica serovar distribution and antibiotic resistance patterns in wastewater at municipal water treatment plants in two California cities

AIM: To determine Salmonella enterica serovars and antibiotic resistance (ABR) in the human waste stream. METHODS AND RESULTS: Sampling of influent wastewater at municipal treatment plants in two California cities was performed by collecting composite samples, over a 24-h period, from the treatment plants on five to six occasions. Serial water quantities were filtered and cultured with a Salmonella selective method and an oxytetracycline-supplemented Salmonella selective method. Antibiotic susceptibilities to 12 antibiotics were determined and the isolates were grouped based on ABR patterns. From 983 S. enterica isolated, 102 represented unique sampling-serovar-ABR patterns. Thirty-five different serovars were identified to be distributed over 17 different ABR patterns. The serovar distribution differed between the sampling sites, whereas there was no significant trend in levels of multiple ABR. CONCLUSIONS: Salmonella enterica was recovered with ease from small sample volumes of wastewater received by municipal water treatment plants. A large variety of serovars and ABR profiles were represented in the recovered Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: The ease of sampling and recovery of Salmonella from municipal wastewater from treatment plants makes it a valuable sampling approach for monitoring the presence of Salmonella in the human population.     

增强型绿色荧光蛋白在集胞藻6803中的表达

利用聚球藻7942热休克基因groESL的启动子和报告基因egfp,构建了表达载体pUC-Tegfp并转化集胞藻6803,并通过所制备抗体对转基因藻进行蛋白免疫印迹检测.结果发现,在转基因藻株T-egfp的细胞粗提液中含有能与eGFP抗体特异结合的蛋白质,表明外源增强型绿色荧光蛋白基因(egfp)在集胞藻6803中成功表达.     

Prevalence and antibiotic resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in tropical seafood

Summary The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).     

Short Communication: Antibiotic resistance among faecal coliform bacteria isolated from wastewater before and after treatment by an experimental sand filter

During the functioning period of a sand filter, 254 and 234 faecal-coliform strains were isolated from raw wastewater and reclaimed effluent, respectively. When tested for their susceptibility to ampicillin, kanamycin, chloramphenicol, streptomycin and tetracycline, 53% of the strains from the raw sewage and 84% of those isolated from the effluent were resistant to at least one antibiotic. The high incidence of drug resistance in the effluent was connected with a high proportion of Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii.     

237例新生儿败血症的病原分布及耐药性分析

目的了解近年本地区新生儿败血症病原学及细菌耐药状况,用以指导临床治疗。方法对2006年5月至2010年4月收治的237例病例的血液进行培养,并对分离的菌株进行鉴定和药敏试验。结果革兰阳性菌为主要病原菌,排名前3位的病原菌分别是:表皮葡萄球菌(48.52%)、人葡萄球菌(16.03%)、凝固酶阴性葡萄球菌(10.13%)。对培养出的革兰阳性菌最敏感的抗生素是万古毒素、阿米卡星,对培养出的革兰阴性菌最敏感的抗生素是丁胺卡那霉素、亚胺培南。结论本地区新生儿血培养病原菌以革兰阳性菌为主,耐药菌株较多,临床医师应根据细菌鉴定及药敏试验选择敏感药物治疗。     

儿童细菌性腹泻病原菌分布及耐药性分析

目的 了解小儿细菌性腹泻的病原菌分布及耐药情况。方法 对773例临床上疑为细菌性腹泻病的患儿的粪便标本进行培养、鉴定及药物敏感试验、超广谱β-内酰胺酶（ESBLs）检测。结果 773例标本中分离出阳性菌株671株,总检出率为86．6％。其中埃希菌属、志贺菌属及耶尔森菌3种致病性细菌占阳性菌株的54．5％,肠杆菌属、非发酵菌群、酵母样真菌、肠球菌属、变形杆菌属、克雷伯菌属和枸橼酸杆菌属等条件致病菌占阳性菌株的45．5％。埃希菌属及志贺菌属对头孢他啶及氨曲南耐药率低（16．4％以下）,而肠杆菌属等条件致病菌对常用抗生素耐药率均较高。G^-杆菌中ESBLs的检出率为38．3％,其中大肠埃希菌占66．7％。结论 埃希菌属是儿童细菌性腹泻病的主要致病菌（50．2％）,且耐药率及ESBLs产生率高,但肠杆菌属等条件致病菌检出率也较高（45．5％）,且耐药率高,可能与抗生素广泛使用,肠杆菌群失调有关,应引起高度重视。     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.     

Effects of growth condition on the structure of glycogen produced in cyanobacterium Synechocystis sp. PCC6803

Growth and glycogen production were characterized for Synechocystis sp. strain PCC6803 grown under continuous fluorescent light in four variations of BG-11 medium: either with (G+) or without (G−) 5 mM glucose, and with a normal (N+, 1.5 g sodium nitrate/L) or a reduced (N−, 0.084 g sodium nitrate/L) nitrogen concentration. Glucose-supplemented BG-11 with a normal nitrogen concentration (N+G+) produced the highest growth rate and the greatest cell density. Although the maximum cell mass production was observed in the N+G+ medium, the highest glycogen yield (19.0 mg/g wet cell mass) was achieved under the glucose-supplemented, nitrogen-limiting condition (N−G+). The addition of glucose enhanced cell growth, while nitrogen limitation apparently directed carbon flux into glycogen accumulation rather than cell growth. Transmission electron microscopic analysis showed that, under nitrogen-limiting conditions (N−G+), glycogen particles accumulated in large amounts and filled the cytosol of the cells. Analysis by high-performance size-exclusion chromatography further revealed that the glycogen produced in N−G+ medium had the longest average branch chain-length (DP10.4) among the conditions tested. When the yield and structure of glycogen were examined in different growth phases, the greatest yield (36.6 mg/g wet cell mass) and the longest branch chain-length (DP10.7) were observed 2 days after the fully grown cells in the N+G+ medium were transferred to the growth restricting (N−G+) medium.     

幽门螺杆菌抗生素耐药机制研究进展

幽门螺杆菌(Helicobacter pylori,H.pylori)感染可引起消化性溃疡、胃粘膜相关淋巴组织淋巴瘤和胃癌。随着抗生素耐药性的问题越来越严重,耐药机制的研究也不断深入。分子检测方法,尤其是核酸检测技术,可高效、快速、准确地检测幽门螺杆菌抗生素耐药基因及突变,对幽门螺杆菌感染的临床治疗发挥重要的指导作用,同时也可对幽门螺杆菌抗生素耐药性进行大规模及时有效监控。本文讨论了关于幽门螺杆菌抗生素耐药机制并着重总结了相关耐药基因及突变。     

外科血培养标本中病原菌分布及耐药性分析

目的了解中山大学附属第一医院外科血标本中病原菌的菌种分布及常见菌株的耐药性。方法血标本用Bact/A lert-120全自动血培养仪进行血培养,阳性血培养转种后用VITEK-60 AMS细菌鉴定仪鉴定,用K-B法进行药敏试验。结果2002年1月至2005年12月血培养标本中共分离出病原菌256株,阳性率为10.6%。122株(47.7%)为革兰阴性杆菌,其中肠杆菌科细菌占72.1%(88/122),非发酵菌占27.9%(34/122);113株(44.1%)为革兰阳性球菌,其中葡萄球菌属占51.3%(58/113),肠球菌占38.1%(43/113);真菌21株(8.2%)。血培养中的革兰阴性杆菌对亚胺培南、美洛培南治疗敏感;革兰阳性球菌对万古霉素和替考拉宁敏感。结论肠杆菌科细菌和葡萄球菌是外科血培养中的主要病原菌,其耐药现象严重,宜根据药敏结果选用敏感抗菌药物治疗。     

水环境中耐药菌污染与危害研究进展

细菌在抗菌药选择性压力下产生耐药性并可传代,通过质粒和整合子等可移动基因元件将耐药基因在相同或不同种属中广泛传播,导致细菌多重耐药,并可通过多种途径进入水体,水环境日益成为庞大的耐药基因库,为致病菌及条件致病菌提供获得大量耐药基因的机会,若多重耐药菌再次侵入人体,可能引发严重的公共卫生问题。     

A high-throughput genetic system for assessing the inhibition of proteins: identification of antibiotic resistance and virulence targets and their cognate inhibitors in Salmonella

This study describes the development of a high-throughput genetic system for producing oligopeptides that can be used to identify molecular interactions leading to inhibition of specific proteins. Using a pathogenic bacteria model, we screened a library of clones expressing intracellular oligopeptides in order to identify inhibitors of proteins involved in antibiotic resistance and virulence. This method involved transforming the pathogen with an oligopeptide-encoding plasmid library, constructed using polymerase chain reaction and an oligonucleotide template designed to produce random oligopeptides composed of 2-16 amino acids, and high-throughput screening for phenotype alterations in the pathogen. A subsequent complementation phase enabled the identification of the full-length bacterial protein inhibited by the oligopeptide. Using this method we were able to identify oligopeptides that inhibit virulence and/or drug resistance in Salmonella, Shigella, and Escherichia coli; specific virulence and/or drug resistance proteins of Salmonella, Shigella, and E. coli that are sensitive to inhibition; and putative oligopeptide-binding sites on the inhibited proteins. This system is versatile and can be extended to other pathogens for analogous studies and it can be modified for used in eukaryotic models for identifying protein interactions that can be targeted for inhibition. Additionally, this system can be used for identifying protein domains involved in any biomolecular interaction.     

Role of mrgA in peroxide and light stress in the cyanobacterium Synechocystis sp. PCC 6803

In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the mrgA gene is part of the PerR regulon that is upregulated during peroxide stress. We determined that an Δ mrgA mutant was highly sensitive to low peroxide levels and that the mutant upregulated a gene cluster ( sll1722-26 ) that encoded enzymes involved with exopolymeric substance (EPS) production. We made mutants in this EPS cluster in both a wild type and Δ mrgA background and studied the responses to oxidative stress by measuring cell damage with LIVE/DEAD stain. We show that Synechocystis sp. PCC 6803 becomes highly sensitive to oxidative stress when either mrgA or the sll1722-26 EPS components are deleted. The results suggest that the deletion of the EPS cluster makes a cell highly susceptible to cell damage, under moderate oxidative stress conditions. Mutations in either mrgA or the EPS cluster also result in cells that are more light and peroxide sensitive, and produce significantly less EPS material than in wild type. In this study, we show that in the absence of MrgA, which is known to be involved in the storage or mobilization of iron, cells can be more easily damaged by exogenous oxidative and light stress.     

ggpS基因过表达对集胞藻PCC 6803甘油葡萄糖苷和甘油合成的影响

为了研究甘油葡萄糖苷磷酸合成酶(GgpS)在集胞藻PCC 803甘油葡萄糖苷和甘油合成中的作用,本研究在前期获得高产甘油葡萄糖苷藻株的基础上分别过量表达来自于集胞藻PCC 6803自身和聚球藻PCC7002的甘油葡萄糖苷磷酸合成酶基因ggpS,并测定了在不同浓度NaCl胁迫时突变藻株的甘油葡萄糖苷和甘油积累量。结果发现获得的突变株甘油葡萄糖苷合成没有提高,但是甘油合成显著增强。此外,当培养基NaCl浓度从600 mmol/L提高到900 mmol/L时,集胞藻PCC 6803自身ggpS过表达藻株的甘油合成进一步提高75%。这些结果显示了GgpS在将碳代谢流导入集胞藻甘油合成途径中的作用。研究成果也为进一步通过基因工程改造提高集胞藻甘油葡萄糖苷和甘油合成效率奠定了基础。