Usefulness of DNA Barcoding in Ecotoxicological Investigations: Resolving Taxonomic Uncertainties Using <Emphasis Type="Italic">Eisenia</Emphasis> Malm 1877 as an Example

Standard test species may differ in their response to toxicants. Accurate identification of test organisms is therefore of critical importance in correctly interpreting data generated from laboratory assays. This is not always possible when species are morphologically similar or where the taxonomy of the group has recently been revised. A case in hand concerns Eisenia sp. Based on recent genetic evidence two species, Eisenia andrei and Eisenia fetida, which were previously considered a single species, are currently recognized. In these instances, DNA barcoding, demonstrated and discussed herein, provides a method to accurately identify test organisms.     

In situ bioassay with Eisenia andrei to assess soil toxicity in an abandoned uranium mine

The goal of this study was to develop an in situ bioassay with Eisenia andrei, deploying it in several locations of an abandoned mining area. Our objectives were two-fold: (i) we intended to validate the in situ soil bioassay procedures, while (ii) providing ecologically relevant data to complement the ongoing risk evaluation based on laboratorial assays. To promote cost- and time-effectiveness, the in situ exposure was short (48 h) and the endpoints analysed included oxidative stress biomarkers and metal content in soil and organisms. The bioassay was carried out under different experimental conditions, simulating local (natural soil) vs. control conditions (LUFA soil), and irrigation with artificial rainwater vs. irrigation with diluted acidic effluent. Variation in the data was mostly due to soil type, rather than irrigation water, and substantial spatial heterogeneity was observed. Oxidative stress biomarkers did not fully work as sensitive parameters to environmental contamination. Earthworm metal burdens suggested a potential concern in terms of bioaccumulation of some metallic elements.     

The occurrence and sources of Campylobacter spp., Salmonella enterica and Escherichia coli O157:H7 in the Salmon River, British Columbia, Canada

In this study, we wished to assess the prevalence and determine the sources of three zoonotic bacterial pathogens (Salmonella, Campylobacter, and Escherichia coli O157:H7) in the Salmon River watershed in southwestern British Columbia. Surface water, sewage, and animal faecal samples were collected from the watershed. Selective bacterial culture and PCR techniques were used to isolate these three pathogens and indicator bacteria from these samples and characterize them. Campylobacter was the most prevalent pathogen in all samples, followed by Salmonella, and E. coli O157:H7. E. coli O157:H7 and Salmonella isolation rates from water, as well as faecal coliform densities correlated positively with precipitation, while Campylobacter isolation rates correlated negatively with precipitation. Analysis of DNA extracted from water samples for the presence of Bacteroides host-species markers, and comparisons of C. jejuni flaA-RFLP types and Salmonella serovars from faecal and water samples provided evidence that human sewage and specific domestic and wild animal species were sources of these pathogens; however, in most cases the source could not be determined or more than one source was possible. The frequent isolation of these zoonotic pathogens in the Salmon River highlights the risks to human health associated with intentional and unintentional consumption of untreated surface waters.     

肺炎克雷伯菌、类肺炎克雷伯菌和变栖克雷伯菌的研究进展

肺炎克雷伯菌临床分离株是一种有荚膜的革兰阴性粗短杆菌,是临床上重要的机会致病菌。肺炎克雷伯菌临床分离株基因间存在差异,依据系统发育树可分为肺炎克雷伯菌(Klebsiella pneumoniae, KpⅠ)、类肺炎克雷伯菌(Klebsiella quasipneumoniae, KpⅡ)和变栖克雷伯菌(Klebsiella variicola, KpⅢ),三者生态分布、基因型、耐药性、毒力特征以及致病性存在显著差异,为感染性疾病精准治疗带来新的挑战。本文对KpⅠ、KpⅡ、KpⅢ的鉴定、流行特点及致病特点的最新研究进展进行综述。     

Relationship between the Pfcrt T76 and the Pfmdr-1 Y86 mutations in Plasmodium falciparum and in vitro/in vivo chloroquine resistance in Burkina Faso, West Africa

The relationship between Pfcrt T76 and Pfmdr-1 Y86 mutations in Plasmodium falciparum was explored in samples from patients with uncomplicated malaria and tested in vitro and in vivo with chloroquine (CQ) in Burkina Faso. The two mutations were strongly related. The Pfcrt T76 mutation was found in 82% of the samples having the Pfmdr-1 Y86 mutation too (odds ratio (OR)=4.8 [95% CI: 1.7-13.3]; P=0.002). However, only half (16/34) of samples with Pfcrt T76 mutation had also the Pfmdr-1 Y86 mutation. The latter was apparently associated with in vitro resistance (OR=4.8 [95% CI: 1.4-16.5]; P=0.01) but such association disappeared (P=0.77) after adjusting for the presence of the Pfcrt T76 mutation. This suggests that the occurrence of the Pfmdr-1 Y86 mutation is dependent on that of Pfcrt T76 mutation and could explain previous reports linking the Pfmdr-1 Y86 mutation with CQ resistance (CQR). The isolates carrying both the Pfcrt K76 and Pfmdr-1 N86 alleles (wild/wild (WW)) and the single mutant Pfmdr-1 Y86 (WM) had the lowest IC50 geometric mean (GMIC50) values, while those carrying both Pfcrt T76/Pfmdr-1 Y86 alleles (mutant/mutant (MM)), and the single mutant Pfcrt T76 (MW) had the highest. Among pre-treatment samples there was a strong linkage disequilibrium with an excess of MM and WW and a deficit of single mutants (MW and WM), suggesting that parasite fitness is higher for the former and lower for the latter.     

Fluorescence analysis detects gp60 subtype diversity in Cryptosporidium infections

Ninety percent of human cryptosporidiosis infections are attributed to two species; the anthroponotic Cryptosporidium hominis and the zoonotic Cryptosporidium parvum. Sequence analysis of the hypervariable gp60 gene, which is used to classify Cryptosporidium to the subtype level, has highlighted extensive intra-species diversity within both C. hominis and C. parvum. The gp60 has also facilitated contamination source tracking and increased understanding of the epidemiology of cryptosporidiosis. Two surface glycoproteins, the gp40 and gp15 are encoded in the gp60 gene; both are exposed to the hosts’ immune system and play a pivotal role in the disease initiation process. The extent of genetic diversity observed within the gp60 would support the hypotheses of significant selection pressure placed on the gp40 and gp15. This study used a dual fluorescent terminal-restriction fragment length polymorphism (T-RFLP) analysis to investigate the genetic diversity of Cryptosporidium subtype populations in a single host infection. Terminal-RFLP showed subtype variation within one human Cryptosporidium sample and mouse samples from seven consecutive passages with C. parvum. Furthermore, this was the first study to show that differences in the ratio of subtype populations occur between infections. T-RFLP has provided a novel platform to study infection populations and to begin to investigate the impact of the hosts’ immune system on the gp60 gene.     

空肠弯曲菌、结肠弯曲菌检验方法团体标准解读

空肠弯曲菌和结肠弯曲菌是重要的食源性病原菌，是导致人类弯曲菌病的重要菌种。病原菌的培养、鉴定是食品污染以及人和动物感染诊断的“金标准”。中国CDC传染病预防控制所和国家食品安全风险评估中心等单位撰写了《空肠弯曲菌、结肠弯曲菌检验方法（T/CPMA 006－2019）》团体标准。标准以“科学性、规范性、适用性和可行性”为基本原则，提出从不同种类的标本、样品中空肠弯曲菌和结肠弯曲菌的分离培养以及鉴定的方法，用于指导和规范我国不同种类的标本以及样本中两种弯曲菌的检测过程、检测步骤和鉴定方法，提高空肠弯曲菌、结肠弯曲菌的检测水平。     

Changes in aromatic characteristics of Loureiro and Alvarinho wines during maturation

Changes in volatiles during maturation in bottles of monovarietal Vinhos Verdes wines from Loureiro and Alvarinho grape varieties, were followed by chemical and sensory analyses. Young wines and wines matured for 8 and 20 months were studied. The volatiles were determined by gas chromatography–mass spectrometry (GC-MS) after extraction on XAD-2 resin. Straight chain fatty acid ethyl esters and acetates of fusel alcohols decreased quicker for Loureiro wine, while the increase in ethyl esters of branched fatty acids was similar for both varieties. Linalool, Ho-trienol, α-terpineol and β-damascenone could be used to differentiate between each variety. However, linalool decreased to negligible values after 20 months of maturation. β-Damascenone decreased but remained high enough to be useful for differentiating each variety. Sensory analysis indicated a decrease of tropical fruit and tree fruit characters with conservation time for Alvarinho wine, and the opposite for Loureiro; moreover, citrus fruit character decreased in both varieties.     

Continuous Monitoring of Folsomia candida (Insecta: Collembola) in a Metal Exposure Test

Current recommended ecotoxicological tests with the parthenogenetic springtail Folsomia candida using standard OECD soil do not allow for continuous monitoring during the exposure period. Effects of chemicals cannot be determined until the end of the experiment (typically after 4 weeks), since the animals stay below the soil surface. In this study, F. candida were maintained on a plaster of Paris/graphite substrate for 7 weeks and were supplied with an aqueous suspension of yeast contaminated with Cd, Cu, Pb, and Zn as nitrate salts. Growth rate, time to first batch of eggs, quantity of food consumed, and the presence of graphite in the gut (a sign of avoidance of yeast) were all affected by metal contaminated diets. The relative toxicities of Cd:Cu:Pb:Zn in the yeast were 1.0:1.07:12.0:4.3, respectively (on a weight basis) with Cd being the most toxic. Internal body concentrations increased, and the concentration factor (metal concentration in F. candida/metal concentration in yeast) decreased with increasing metal exposure. In general, metals are much less toxic when added to the food of F. candida than when incorporated into soil in standard tests. It is suggested that Collembola have a greater tolerance of metals in the diet since they avoid contaminated food, and are able to excrete assimilated metals at moulting via exfoliation of the midgut epithelium where the elements are retained as part of a storage–detoxification system. The methodology described in this article allows effects on growth to be observed as early as 7 days after the beginning of the experiment.     

Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA

Legionella and Mycobacterium can proliferate within free-living amoebae (FLA) where they are protected from disinfectants at concentrations that can kill bacteria but not protozoa. Despite effective treatment of drinking water, microbes can enter water utility distribution systems (DS) and hence the plumbing within building premises. Additionally, biofilm formation may account for the persistence of microbes in the DS. In the present study a domestic water tap in north-central United States (USA) was sampled in March and September 2007 and analysed for FLA, Legionella and Mycobacterium. Identification of organisms was determined by growth on specific culture media, light and electron microscopy, and amplification of DNA probes specific for each organism. In both the spring and fall samples, amoebae, Legionella and Mycobacterium were detected. However, Acanthamoeba was prominent in the spring sample whereas Vahlkampfia and Naegleria were the amoebae detected in the autumn. Bacterial proliferation in laboratory cultures was noticeably enhanced in the presence of amoebae and biofilms rapidly formed in mixed amoebae and bacteria cultures. It is hypothesized that temperature affected the dynamics of FLA species population structure within the DS and that pathogenic bacteria that proliferate within FLA, which are themselves opportunistic pathogens, pose dual public health risks.     

Lack of pathogenicity and toxicity of the mycoinsecticide Metarhizium anisopliae var. acridum following acute gastric exposure in mice

Metarhizium anisopliae var. acridum, monospore culture EH-502/8 (CNRCB MaPL40), isolated in Mexico from Schistocerca piceifrons ssp. piceifrons (Orthoptera: Acrididae) was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated with one dose (10⁸ conidia/animal) of viable (72 mice), non-viable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies after the inoculation. No mice showed clinical symptoms of illness or died during the study. The fungus was able to persist in some organs until day 3, but did not cause any damage to the host. The gross pathology observed was splenomegaly in mice inoculated with viable and non-viable conidia. Non-germinated conidia, observed in several organs, suggest hematogenous spread, but without any histopathological tissue reaction. Results support the non-pathogenic and non-toxic status of this fungal strain when administered in a single intragastric dose to mice.     

Arsenic- and mercury-induced phytotoxicity in the Mediterranean shrubs Pistacia lentiscus and Tamarix gallica grown in hydroponic culture

Hg and As resistance and bioaccumulation were studied in hydroponically grown Pistacia lentiscus and Tamarix gallica plants. Both elements caused growth inhibition in roots and shoots, with mercury showing greater phytotoxicity than arsenic. Accumulation of both elements by plants increased in response to element supply, with the greatest uptake found in T. gallica. Both elements affected P and Mn status in plants, reduced chlorophyll a concentration and increased MDA and thiol levels. These stress indices showed good correlations with As and Hg concentration in plant tissues, especially in the roots. Toxic responses to mercury were more evident than for arsenic, especially in shoot tissues. T. gallica showed higher resistance to both Hg and As than P. lentiscus, as well accumulating more As and Hg.     

Coding and non-coding polymorphisms in VDR gene and susceptibility to pulmonary tuberculosis in tribes, castes and Muslims of Central India

Vitamin D receptor (VDR) plays an important role in activating immune response against various infectious agents. This study was aimed to investigate the association between VDR gene polymorphisms and different clinical forms of pulmonary tuberculosis (TB) in different population groups. Four common polymorphisms (TaqI, ApaI, BsmI and FokI) of VDR gene were studied in clinically diagnosed TB patients and healthy controls from Sahariya tribe (n = 377), Bhil tribe (n = 95), Chhattisgarh tribe (n = 33), general population from North-Central (NC) (n = 1021) and South-Eastern (SE) region (n = 646) and Muslims (n = 217). Genotyping was carried out using PCR-RFLP method and re-confirmed by direct sequencing. The haplotype analysis was performed on Haploview 4.1 and statistical analysis was done using SPSS 13.0 software. We found that bb genotype of BsmI polymorphism conferred significant risk to smear positive and multiple drug resistant (MDR) TB in tribes [OR (CI) = 3.7 (1.5–9.2), p = 0.002], SE population [OR (CI) = 2.1 (1.4–3.3), p = 0.0004] and Muslims [OR (CI) = 6.7 (1.1–39), p = 0.01]. The subjects with FF genotype of FokI polymorphism appeared less likely (p = 0.004) to develop MDR TB in NC population, whereas, those with Ff [OR (CI) = 2.5 (1.3–5.0), p = 0.004] and ff [OR (CI) = 3.4 (1.2–9.3), p = 0.01] genotypes were at high risk of MDR and smear positive disease, respectively. Similarly, tt genotype of TaqI polymorphism was found associated with high risk of smear positive TB in NC [OR (CI) = 3.6 (0.9–14.2), p = 0.05] as well as in SE [OR (CI) = 4.7 (1.8–12.3), p = 0.00003] population. Interestingly, tt genotype appeared strongly associated [OR (CI) = 8.9 (2.7–29), p = 0.00001] with high bacillary load outcome. In conclusion, genetic polymorphisms in VDR gene, alone or in combination (haplotypes) are associated with different clinical outcomes in pulmonary TB.     

Microsatellite markers from the Chagas disease vector, Rhodnius prolixus (Hemiptera, Reduviidae), and their applicability to Rhodnius species

Ten microsatellites were isolated and characterized from a partial genomic library of Rhodnius prolixus, the principal Chagas disease vector in Venezuela, Colombia and Central America. These polymorphic molecular markers could be particularly useful in Chagas disease control initiatives. A wider applicability of the primer-pairs isolated was shown, from 6 to 10 loci being amplifiable in five out of the ten Rhodnius species tested, namely R. domesticus, R. nasutus, R. neglectus, R. neivai and R. robustus. Interestingly, all the loci were amplified in the latter. These markers may be of interest to trace the colonization of human dwellings from triatomine sylvatic populations in order to better define epidemiological risk patterns.     

Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas

The protozoan parasites Giardia and Cryptosporidium have been described as important waterborne disease pathogens, and are associated with severe gastrointestinal illnesses. The objective of this paper was to investigate the presence of Giardia cysts and Cryptosporidium oocysts in samples from watershed catchments and treated water sources. A total of 25 water samples were collected and examined according to the US EPA—Method 1623, 2005, consisting of 12 from drinking water and 13 from raw water. Positive samples from raw water for Giardia cysts and Cryptosporidium oocysts were 46.1 and 7.6%, respectively. In finished water, positive samples were 41.7% for Giardia cysts and 25.0% for Cryptosporidium oocysts. Concentrations of Giardia cysts found in raw water samples ranged from “not detected” to 3.4 cysts/L, whereas concentrations of Cryptoporidium oocysts ranged from “not detected” to 0.1 oocysts/L. In finished water, Giardia concentrations ranged from “not detected” to 0.06 cysts/L, and Cryptosporidium, from “not detected” to 0.01 oocysts/L. Concentrations of Giardia cysts and Cryptosporidium oocysts were not high in the samples analyzed. Nevertheless, the results of this study highlight the need to monitor these organisms in both raw and drinking water.     

Rapid selection of dhfr mutant allele in Plasmodium falciparum isolates after the introduction of sulfadoxine/pyrimethamine in combination with 4-aminoquinolines in Papua New Guinea

To overcome the declining efficacy of the 4-aminoquinolines in Papua New Guinea, sulfadoxine/pyrimethamine (SP) was combined with the 4-aminoquinolines as the first line treatment for falciparum malaria since 2000. To assess how this change had affected SP resistant gene polymorphisms, we determined allele frequencies of dhfr and dhps in 113 Plasmodium falciparum isolates from Wewak, East Sepik of Papua New Guinea in 2002 and 2003. In dhfr, double mutant (ACNRNVI) was the predominant allele with a prevalence of 91%. We found a significant decrease of wild dhfr allele prevalence (7%) compared with that reported in the adjacent area of East Sepik called the Wosera region (57%), before the drug policy changed in 1990-1993. Between 2002 and 2003, the prevalence of this allele decreased from 15% to 3% (P=0.02). Two distinct microsatellite haplotypes flanking dhfr were found in isolates with dhfr double mutant, suggesting the selection of preexisting SP resistant parasites rather than a frequent occurrence of dhfr mutations. The dhfr/dhps quartet mutations (ACNRNVI in dhfr and SGEAA in dhps) were identified in six of the isolates (8%) from 2003. This genotype, which is associated with in vivo resistance to SP, has not been reported before in Papua New Guinea. These findings suggest that isolates resistant to SP were rapidly selected despite the use of the SP combination therapy, probably because of their preexisting high level of resistance to the 4-aminoquinoline partner drug.     

铜绿假单胞菌对碳青霉烯类抗生素异质性耐药的研究进展

 异质性耐药指同一克隆菌株中同时存在对某种抗菌药物耐药及敏感亚群的现象。碳青霉烯类抗生素是临床治疗铜绿假单胞菌感染的常用药物,随着其使用增加,碳青霉烯类异质性耐药铜绿假单胞菌（CHPA）的检出率日益增高。但是,至今没有标准、高效、低成本的CHPA检测方法应用于临床,常导致CHPA漏检和临床治疗失败。本文对异质性耐药的定义、检测方法、CHPA的耐药机制、临床及流行病学特征进行综述,为临床诊断和治疗CHPA感染提供参考。     

Pseudomonas entomophila and Pseudomonas mendocina: Potential models for studying the bacterial type VI secretion system

A diversity of molecular translocation mechanisms, including various secretion systems, has been elaborated in host–bacterial interactions. The newly described type VI secretion system (T6SS) appears to be involved in bacterial pathogenesis by acting as a nano-syringe, contributing in translocation of several effector-proteins into the eukaryotic host cell cytoplasm. Recent evidences revealed the involvement of T6SS machinery in inter-bacterial interactions.Several Pseudomonas species are found to harbour multiple and well organised T6SS loci, however, their genomic structural similarities as well as phylogenetic divergence suggest an independent evolution. Until now elementary evidence was provided for the presence of T6SS in the genomes of Pseudomonas entomophila (Pen), an aggressive insect pathogen as well as the human opportunistic pathogen Pseudomonas mendocina (Pme).In this report we evidenced by in silico genome mining along with bioinformatic analysis the presence of genes encoding for putative T6SS core components and secreted proteins in the sequenced Pen L48 and Pme ymp, strains and designated their putative promoters, sigma factors binding sites and various regulatory proteins. Moreover, we investigated the phylogenetic relatedness of four T6SS core proteins from these strains with their orthologues from various Pseudomonas species.Our analysis revealed two phylogenetically distinguishable T6SS loci in the genome of Pme that appeared to be highly homologous to Pseudomonas aeruginosa Hcp-Secretion Island-I (HSI-I) and -II. Our findings suggest that Pme could be excellent additional to P. aeruginosa model, for the elucidation of HSI-I and -II biological role(s), avoiding the overlapping activity HSI-III (Lesic et al., 2009), which is missing from Pme's genome.Likewise, our analysis revealed the presence of a unique entire T6SS in Pen genome, which appears to be phylogenetically close to Pme T6SS-II and P. aeruginosa HSI-II. Since Pen lacks the common secretion systems T3SS and T4SS, the single T6SS locus could have an enforced role in the insect–bacterial interactions, providing thus a promising model for studying its biological function.