牙菌斑培养菌群宏基因组文库构建及抗生素耐药基因筛选

[目的]构建牙菌斑培养菌群宏基因组文库,筛选牙菌斑生物膜中细菌的抗生素耐药基因.[方法]采集20例无龋健康人的集合牙菌斑并进行厌氧培养.提取牙菌斑培养菌群宏基因组构建Fosmid文库.用卡那霉素、四环素及氨苄西林对文库进行筛选,并对筛选到的抗性Fosmid克隆进行末端测序、亚克隆构建、亚克隆测序和序列分析.[结果]构建了牙菌斑培养菌群宏基因组Fosmid文库,插入片段长度在36-48 kb间约有15 120个克隆,插入片段长度小于36 kb的约有3 360个克隆.筛选获得一个氨基糖苷类双功能修饰酶AacA-AphD基因、一个核糖体保护蛋白型四环素耐药基因tet (M)及一个C家族β-内酰胺酶基因.[结论]证实了可以通过构建宏基因组文库的方法来筛选牙菌斑培养菌群中的抗生素耐药基因.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.     

The Ocean as a Global Reservoir of Antibiotic Resistance Genes

Recent studies of natural environments have revealed vast genetic reservoirs of antibiotic resistance (AR) genes. Soil bacteria and human pathogens share AR genes, and AR genes have been discovered in a variety of habitats. However, there is little knowledge about the presence and diversity of AR genes in marine environments and which organisms host AR genes. To address this, we identified the diversity of genes conferring resistance to ampicillin, tetracycline, nitrofurantoin, and sulfadimethoxine in diverse marine environments using functional metagenomics (the cloning and screening of random DNA fragments). Marine environments were host to a diversity of AR-conferring genes. Antibiotic-resistant clones were found at all sites, with 28% of the genes identified as known AR genes (encoding beta-lactamases, bicyclomycin resistance pumps, etc.). However, the majority of AR genes were not previously classified as such but had products similar to proteins such as transport pumps, oxidoreductases, and hydrolases. Furthermore, 44% of the genes conferring antibiotic resistance were found in abundant marine taxa (e.g., Pelagibacter, Prochlorococcus, and Vibrio). Therefore, we uncovered a previously unknown diversity of genes that conferred an AR phenotype among marine environments, which makes the ocean a global reservoir of both clinically relevant and potentially novel AR genes.     

Increased levels of sigJ mRNA in late stationary phase cultures of Mycobacterium tuberculosis detected by DNA array hybridisation

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.     

The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tc(r)) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tc(r) genes. Fecal fosmid libraries were functionally screened for Tc(r), and further PCR-screened for specific Tc(r) genes. Tc(r) fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tc(r), although encoded by different genes and organisms. Tc(r) organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tc(r) gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tc(r) was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tc(r) bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual.     

Genes homologous to glycopeptide resistance vanA are widespread in soil microbial communities

The occurrence of d-Ala : d-Lac ligase genes homologous to glycopeptide resistance vanA was studied in samples of agricultural (n=9) and garden (n=3) soil by culture-independent methods. Cloning and sequencing of nested degenerate PCR products obtained from soil DNA revealed the occurrence of d-Ala : d-Ala ligase genes unrelated to vanA. In order to enhance detection of vanA-homologous genes, a third PCR step was added using primers targeting vanA in soil Paenibacillus. Sequencing of 25 clones obtained by this method allowed recovery of 23 novel sequences having 86-100% identity with vanA in enterococci. Such sequences were recovered from all agricultural samples as well as from two garden samples with no history of organic fertilization. The results indicated that soil is a rich and assorted reservoir of genes closely related to those conferring glycopeptide resistance in clinical bacteria.     

Selected chitinase genes in cultured and uncultured marine bacteria in the alpha- and gamma-subclasses of the proteobacteria

PCR primers were patterned after chitinase genes in four gamma-proteobacteria in the families Alteromonadaceae and Enterobacteriaceae (group I chitinases) and used to explore the occurrence and diversity of these chitinase genes in cultured and uncultured marine bacteria. The PCR results from 104 bacterial strains indicated that this type of chitinase gene occurs in two major groups of marine bacteria, alpha- and gamma-proteobacteria, but not the Cytophaga-Flavobacter group. Group I chitinase genes also occur in some viruses infecting arthropods. Phylogenetic analysis indicated that similar group I chitinase genes occur in taxonomically related bacteria. However, the overall phylogeny of chitinase genes did not correspond to the phylogeny of 16S rRNA genes, possibly due to lateral transfer of chitinase genes between groups of bacteria, but other mechanisms, such as gene duplication, cannot be ruled out. Clone libraries of chitinase gene fragments amplified from coastal Pacific Ocean and estuarine Delaware Bay bacterioplankton revealed similarities and differences between cultured and uncultured bacteria. We had hypothesized that cultured and uncultured chitin-degrading bacteria would be very different, but in fact, clones having nucleotide sequences identical to those of chitinase genes of cultured alpha-proteobacteria dominated both libraries. The other clones were similar but not identical to genes in cultured gamma-proteobacteria, including vibrios and alteromonads. Our results suggest that a closer examination of chitin degradation by alpha-proteobacteria will lead to a better understanding of chitin degradation in the ocean.     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.     

Bacterial diversity and the antimicrobial resistome in the southwestern highlands of Saudi Arabia

Soil is a reservoir of microbial diversity and the most supportive habitat for acquiring and transmitting antimicrobial resistance. Resistance transfer usually occurs from animal to soil and vice versa, and it may ultimately appear in clinical pathogens. In this study, the southwestern highlands of Saudi Arabia were studied to assess the bacterial diversity and antimicrobial resistance that could be affected by the continuous development of tourism in the region. Such effects could have a long-lasting impact on the local environment and community. Culture-dependent, quantitative polymerase chain reaction (qPCR), and shotgun sequencing-based metagenomic approaches were used to evaluate the diversity, functional capabilities, and antimicrobial resistance of bacteria isolated from collected soil samples. Bacterial communities in the southwestern highlands were mainly composed of Proteobacteria, Bacteroidetes, and Actinobacteria. A total of 102 antimicrobial resistance genes (ARGs) and variants were identified in the soil microbiota and were mainly associated with multidrug resistance, followed by macrolide, tetracycline, glycopeptide, bacitracin, and beta-lactam antibiotic resistance. The mechanisms of resistance included efflux, antibiotic target alteration, and antibiotic inactivation. qPCR confirmed the detection of 18 clinically important ARGs. In addition, half of the 49 identified isolates were phenotypically resistant to at least one of the 15 antibiotics tested. Overall, ARGs and indicator genes of anthropogenic activities (human-mitochondrial [hmt] gene and integron-integrase [int1]) were found in relatively lower abundance. Along with a high diversity of bacterial communities, variation was observed in the relative abundance of bacterial taxa among sampling sites in the southwestern highlands of Saudi Arabia.     

Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc

Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.     

Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids

In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.     

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.     

Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

Influence of maize mucilage on the diversity and activity of the denitrifying community

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.     

口虾蛄肠道细菌种群多样性的分子分析

目的探讨口虾蛄肠道细菌种群的多样性。方法通过不依赖于分离培养的分子生物学分析方法,以直接提取虾蛄肠道细菌的总DNA为模板经过PCR扩增16S DNA,然后经与T载体连接建立质粒文库。用限制性内切酶(BsuRⅠ和Hin6Ⅰ)对阳性克隆的PCR扩增产物进行限制性酶切片段长度多态性(RFLP)分析,选取有代表性的克隆进行测序。结果16S DNA序列通过CLUSTALX进行多序列比对及NCBI数据库中的BLAST分析后发现口虾蛄肠道细菌主要分成4类:未培养细菌,未培养支原体科细菌,未培养δ变形菌和马特斯支原体。结论口虾蛄肠道细菌种类较为简单,且多为未培养的细菌。