Spraying of oxytetracycline and gentamicin onto field-grown coriander did not affect the abundance of resistant bacteria, resistance genes, and broad host range plasmids detected in tropical soil bacteria

Horticultural supplements containing oxytetracycline and gentamicin, two clinically relevant biocides, are widely marketed to prevent or control infections by bacterial phytopathogens. Despite their regular consumption in the world’s less developed countries, it is unknown whether exposure of tropical farmlands to these drugs results in an enrichment of resistant bacteria, resistance genes, and/or mobile genetic elements in the soil. These concerns were investigated under field conditions by repeatedly spraying recommended amounts of a commercial product containing oxytetracycline-HCl, and gentamicin- onto two coriander plots. Subsequent to five applications within 16 months, composite soil samples from control and treated sections were compared with respect to the abundance of resistant bacteria and the prevalence of conserved nucleotide sequences from tetracycline efflux proteins, tetracycline ribosomal protection proteins, four different families of gentamicin-modifying enzymes, and broad host range plasmids of the IncP-1 and IncQ incompatibility groups. The isolation frequency of oxytetracycline- and gentamicin-resistant bacteria and the detection rate of the aforementioned genes and elements were unrelated to application of the supplement. Despite the omnipresence of sequences from IncP-1 plasmids, conjugative plasmids conferring resistance to oxytetracycline or gentamicin were not captured in biparental matings. The widespread occurrence of resistant bacteria and resistance genes at the beginning of the trial emerges as a reasonable explanation for the lack of anticipated responses. Moreover, we assume that the biocides applied were inactivated by biotic and abiotic factors under tropical conditions.     

抗生菌肥对植烟土壤和烤烟生产的影响

烤烟移栽时施入抗生菌芽孢杆菌株 A03 菌肥,研究抗生菌肥对植烟土壤和烤烟生产的影响.小区实验结果表明:施用抗生菌芽孢杆菌 A03菌肥可明显提高烤烟根际细菌的数量,显著降低放线菌的数量,而对真菌没有影响.施用抗生菌肥能够提高烤烟对病毒病的抗性,圆顶期根际土壤中除 Ca 和 Mg 元素的有效性略有降低外,P、K、Cu、Zn、Fe和 Mn 6 种元素的有效性均有不同程度的提高,提高幅度在 16.57%～56.39%;烤烟杀青样和烤后烟中 N 和 P 的平均含量高于对照处理;烤烟亩产量比对照处理提高 10.45%,产值提高11.22%,上等烟率、均价也高于对照和商品菌肥处理.     

Plasmid transfer between introduced and indigenous bacteria in leaf litter, soil and vermicompost as affected by soil invertebrates

 Plasmid transfer between introduced strains of Pseudomonas stutzeri JM302 (pLV1013), Pseudomonas putida PAW340 (pLV1017), Pseudomonas aeruginosa PAO5 (RP4), and Enterobacter cloacae MF10 (RP4), all of them harbouring genetically modified or naturally occurring plasmids and bacteria indigenous to oak leaf litter, soddy-podzolic soil or vermicompost was monitored using non-sterile laboratory microcosms inhabited by the millipede Pachyiulus flavipes, the woodlouse Armadillidium vulgare, or the earthworms Aporrectodea caliginosa and Eisenia fetida, respectively. Plasmid transfer from genetically modified Streptococcus faecalis JH2-2 (pAMβ₁) to Bacillus thuringiensis var. israelensis 1-5 under similar conditions was also followed. A recombinant conjugative plasmid (pLV1017) encoding resistance to antibiotics and expressing catechol 2,3-dioxygenase was transferred into two facultatively anaerobic gram-negative bacteria, isolated from the excrement of A. vulgare. Presumed transconjugants of the facultatively anerobic gram-negative bacteria given above were isolated from the leaves and soil (four strains) and the excrement of the invertebrates (11 strains). They carried the same plasmids and exhibited the same enzymatic activity as the respective inoculant strains; for most of them, however, DNA fingerprints were slightly different. Transfer from S. faecalis JH2-2 to B. thuringiensis var. israelensis of a naturally occurring plasmid, pAMb₁, encoding resistance to erythromycin was observed in vermicompost. Received: 18 November 1997     

Persistence of plasmid RP4 in Pseudomonas putida and loss of its expression of antibiotic resistance in a groundwater microcosm

We examined the stability of plasmid RP4 and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RP4 was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80-90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics.     

Use of an instrumented disc coulter for mapping soil mechanical resistance

A soil mechanical resistance sensor with a large-diameter disc coulter was developed to delineate areas of differing soil strength across agricultural fields. The instrumented disc coulter consisted of a 76.2 cm disc with two depth-measuring sensors (rotary potentiometer and ultrasonic proximity sensor) along with a global positioning system (GPS) receiver to georeference operating depth measurements. The consistency and repeatability of the system response were evaluated by making six passes across long-term tillage comparison plots with different degrees of soil disturbance, including: 20 cm plowing, 15 cm disking, 30 cm chiseling, and no-till in several combinations. At the time of testing, standard soil cone penetrometer measurements were taken. The relationship between the average cone index in the 0–30 cm soil profile (CI_0–30 cm) and the disc operating depth was evaluated. In addition, the cumulative energy density of the given depth of penetration defined as specific cone penetration energy (J m⁻² or N cm⁻¹) for each tillage plot was calculated using the cone index profiles. The average measured depth in each tillage plot was compared to the average predicted depth (d_ci) of a fixed specific cone penetration energy (P_ci). Static calibration tests on the depth sensors showed excellent linearity with coefficients of determination (R²) greater than 0.99. The results showed that, on the average, the changes in the depth measured with the rotary potentiometer were 44 and 68% of the changes in the depth measured with the ultrasonic proximity sensor while the disc coulter was passing across, or along, the tillage plots. This difference was primarily due to the sinkage of the tractor wheels. The depth measured with the ultrasonic sensor had significant correlation with both CI_0–30 cm and d_ci. This was partially due to the fact that a significantly high correlation (R² = 0.97) between the CI_0–30 cm and d_ci was observed, which was not expected and originated from the type of soil profiles present. The instrumented disc coulter is a low soil disturbance system and could be used as an inexpensive and simple sensor to obtain information about the mechanical condition of the soil for spot tillage or other management decisions.     

Effects of soil invertebrates on the survival of some genetically engineered bacteria in leaf litter and soil

Seven bacterial strains, most of them bearing natural or recombinant plasmids, were introduced in oak leaf litter or soddy-podzolic soil. In these substrata, which contained litter-dwelling diplopods and isopods, or endogenic earthworms, bacteria survival was followed. In the absence of the animals, the numbers of introduced strains gradually decreased. In the presence of the animals, plasmid-bearing strains of Pseudomonas putida survived at 10⁵–10⁷ CFUs g^-1 up to 1.5 months in both leaves and soil. The total numbers of bacteria found in excrements from the soil macrofauna were 5–15 times higher than in the food. The numbers of P. putida in the excrements were equal to or higher than in the food. The numbers of Pseudomonas stutzeri JM302 (pLV1013) and Azospirillum brasiliense ATCC29710 (pFACII) in the excrements were always 2–10 times lower than in the food. The digestive fluid taken from the middle part of the gut of the diplopod Pachyiulus flavipes showed a strong antibacterial activity. Those bacteria with lower survival in the gut appeared to be more sensitive to digestion by the midgut fluid. In contrast, the hindgut fluid did not suppress the viability of P. stutzeri JM302 (pLV1013). We postulate that the introduced bacteria partially survive the midgut passage and then multiply with a high growth rate in the hindgut of the animals. The environmental consequences of the interactions between soil invertebrates and the released bacteria are discussed.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday     

Caution of intensified spread of antibiotic resistance genes by inadvertent introduction of beneficial bacteria into soil

The use of beneficial bacteria isolated especially from rhizosphere soils called plant growth-promoting rhizobacteria (PGPR) has been attracting particular interests. However, a potential source of antibiotic resistance genes (ARGs) carried by PGPRs and derived biocontrol agents and/or bio-fertilizers is widely forgotten and ignored. It is very urgent to raise the question if large-scale introduction of beneficial bacteria into soils can aggravate the situation in the spread of ARGs in environment. In this article, we conclude and analyze the possibility of a potential ARGs reservoir associated with introduction of PGPR strains into soils, and offer some advice on PGPR isolation, development and application.     

Enumeration and characterization of arsenate-resistant bacteria in arsenic free soils

The numbers of arsenate-resistant bacteria present in four different soils were determined. Counts were typically in the order of 10⁶ arsenate-resistant colony forming units g⁻¹ of soil, accounting from 7 to 50% of the total viable count. Arsenate-resistant bacteria obtained from woodland soil and leaf litter were further characterized. 16S rRNA sequence analysis revealed that these bacterial isolates represented six bacterial groups including the Proteobacteria, Bacteroidetes, and Firmicutes. Almost all of these isolates were bacteria that have not previously been shown to tolerate arsenic. Some of these bacteria were capable of growth when exposed to high levels of arsenate (over 250 mM), although arsenite tolerance was much lower. These results support the suggestion that arsenate-resistant bacteria are common in natural environments and may play an important role in arsenic cycling in soils.     

Growth rate of bacteria is affected by soil texture and extraction procedure

Effects of soil texture on the extraction efficiency of bacteria from soils and on biosynthetic activity of the extracted bacteria were studied. Bacterial extracts were prepared from three soils of different texture by homogenization (ultrasonication and mixing) or by homogenization-centrifugation at different speeds. Bacterial biosynthetic activity was estimated using thymidine and leucine incorporation techniques. In each step of the extraction procedure, a higher extractability of bacteria was obtained in finer soils than in coarse soil. Also cell-specific growth rates of bacteria were higher in the finer soils than in the coarse soil. However, in all soils, the extracted bacteria always had significantly lower cell-specific thymidine and leucine incorporation rates than the bacteria in soil slurries and thus did not represent so well the bacterial growth in the original soils. The total declines in cell-specific incorporation rates caused by the extraction were larger in fine soil (96-98%) than in coarse soil (90%), but bacteria in the coarse soil were more responsive to only minor intervention. The homogenization-centrifugation method eliminated the differences in bacterial biosynthesis found when working with soil slurries. Therefore, we recommend using of soil slurries or, optionally, soil suspensions to compare bacterial biosynthetic activity among soils of different textures.     

鱼菜共生系统中乳酸菌的筛选及其发酵矿化应用

针对鱼菜共生系统固体废弃物资源化利用效率低的问题,该研究旨在筛选出抗逆性好、矿化功能强的鱼源性乳酸菌,加强鱼粪残饵的发酵矿化性能。试验从鱼菜共生系统中的生物滤料和鱼体中分离乳酸菌,并通过抗逆性及发酵矿化性能检测,筛选出2株具有应用潜力的鱼粪残饵矿化菌株,经鉴定分别为乳酸乳球菌（Lactococcus lactis）L1和糊精乳杆菌（Lactobacillus dextrinicus）L2。耐温性、耐酸碱性和耐盐性检测结果显示,L1表现出较好的抗逆性。在 50 ℃时,L1存活率为96.60%,显著高于L2的存活率80.35%（P<0.05）;在pH值分别为5.0和9.0时,L1的存活率分别为65.43%和71.25%,高于L2的存活率31.10%和52.22%（P<0.05）;当盐浓度为60 g/L时,L1的存活率为37.33%,而L2无法存活。通过比较对照组（CK组,未添加乳酸菌）和乳酸乳球菌组（L组,添加乳酸乳球菌）发酵矿化过程中发酵液水质和矿物元素含量,结果显示 L组的有机物降解及矿化效果更好,除硫（S）元素以外,钾（K）、钙（Ca）、镁（Mg）、铁（Fe）、锰（Mn）和锌（Zn）元素的矿化率均在第3天达到最高（27.59%～94.67%）。综上所述,乳酸乳球菌（Lactococcus lactis）具有较强的抗逆性和显著的发酵矿化效果,且其最佳发酵矿化周期为3 d。该研究可为提高鱼菜共生系统固体废弃物资源化利用效率提供技术支持。     

Combination effects of heavy metals and fluoranthene on soil bacteria

The effects of (1) Cd, Cu, Zn, and fluoranthene (FLA), separately applied, and (2) combinations of one of these heavy metals with FLA on the growth of bacteria were studied in agar plate experiments. The bacteria were extracted from A horizons of a Eutric Regosol and a Calcic Chernozem. Significant reductions of bacterial counts were observed for both soils at concentrations > 1.0 mg Cd l^–1, 0.5 mg Cu l^–1, and 0.5 mg Zn l^–1, respectively. Additions of FLA up to 100 mg l^–1 did not result in increasing reductions of bacterial growth in the Regosol. Only 0.5, 2, and 100 mg FLA l^–1 caused significant reductions of 22–27%. Bacterial counts were not affected by 0.2 mg FLA l^–1. Low concentrations of heavy metals which were not affective when added separately were found to reduce bacterial growth when applied in combination with 0.2 mg FLA l^–1. At higher levels of heavy metals up to 2.5 mg l^–1, addition of FLA also increased the toxicity of the metals. It is assumed that the enhancement of toxicity by FLA is due to an alteration of the permeability of bacterial cell membranes. Received: 19 July 1996     

Edaphic factors as determinants for the distribution of intrinsic antibiotic resistance in a cowpea rhizobia population

 A large collection of cowpea rhizobia strains was obtained from soil samples collected from either a semi-arid or a tropical rain forest area located at about the same latitude in the north-eastern region of Brazil and evaluated for their intrinsic antibiotic resistance to eight commercial antibiotics. The aim of this study was to correlate antibiotic resistance of native rhizobia strains to edaphic-climatic factors as a way to establish suitable inoculants for specific areas. A large diversity regarding intrinsic antibiotic resistance was found, and 17 clusters were identified as varying from sensitive to gradually resistant up to 500 μg·ml^–1 of the antibiotics tested. Clustering analysis did not show any pattern related to the geographic region where isolates have been obtained. On the other hand, an increase in the antibiotic-resistant rhizobia population was associated with an increase in soil P and Al contents. lsolates which were sensitive to spectinomycin, ampicillin, streptomycin, chloramphenicol and tetracycline were present at higher rates in soils devoid of Al. Rhizobia bacteria producing mucus type I (fluid and capable of spreading over the solid media) were found preferentially in soil with Al concentrations up to 36 mg·kg^–1, diminishing quickly at higher levels. Received: 2 May 1997     

磺胺嘧啶对土壤氮矿化及相关微生物参数的影响

兽用抗生素进入土壤,已经带来了新的土壤污染生态学问题,并已引起广泛关注。本文研究了不同浓度磺胺嘧啶残留对土壤氮矿化量及相关微生物参数的影响。结果表明,磺胺嘧啶对土壤氮矿化产生抑制作用,且抑制作用在低浓度比高浓度更为敏感。通过主成分分析和多元回归分析可知,在各相关微生物参数中,土壤微生物群落功能多样性可作为预测磺胺嘧啶残留时土壤氮矿化强度变化的指标。     

几株产荚膜细菌在土壤水稳性团聚体形成中的作用

试验研究几株产荚膜细菌在土壤水稳定性团聚体形成中的作用结果表明,菌株次生代谢产物即发酵物在土壤水稳定性团聚体形成中起作用,而菌体本身不起作用。细菌能够显著促进>0.25mm土壤水稳定性团聚体的形成,其效果随加入C源物质而异,蔗糖效果优于小麦秸秆和玉米秸秆,风化煤几乎不起作用。加入不同C源物质时,菌株之间表现出明显差异。     

Modeling and correction of soil penetration resistance for varying soil water content

For this study penetration resistance (PR) was measured within the profiles of four Oxisols for a wide range of water contents (θ) and bulk densities. Obtained data were utilized to parameterize 23 previously applied regression models. The most promising models were selected to illustrate effects of soil texture on PR. Finally, a new correction method based on normalization of PR with θ corresponding to a matric potential of − 10 kPa was introduced. Evaluation of texture effects revealed that for very wet soils PR was lowest, but increased with clay content. PR at − 1500 kPa exhibited a maximum at clay content of 35% and at − 10 kPa PR was least affected by texture. From all regression models three- and two-parametric exponential and power functions yielded closest matches to measured data. The proposed correction significantly dampened the influence of θ on PR, which allows better comparison for a specific soil or among different soils.     

高产生物膜乳酸菌抗逆性及其抗氧化特性

为了揭示乳酸菌生物膜抵抗不良环境的作用机制,该研究以2株乳酸片球菌RJ2-1-4、TG1-1-10和2株植物乳杆菌RJ1-1-4、RM1-1-11(菌株均高产生物膜)为研究对象,探究浮游态、被膜态菌株对酸、碱、胆盐、模拟人工胃肠液的耐受能力以及抗氧化能力.结果表明:在极酸条件下,菌株生长受到抑制,但是pH值3.0时,被...     

Effects of long-term manure applications on the occurrence of antibiotics and antibiotic resistance genes (ARGs) in paddy soils: Evidence from four field experiments in south of China

Most studies of the effects of manure amendment on the occurrence of antibiotics and antibiotic resistance genes (ARGs) in soil employ the investigation of grab samples or short-term laboratory studies. However, the effects of long-term manure applications on antibiotics, ARGs and their vertical distribution in paddy soil in field experiments are lacking. We assessed the concentrations of antibiotics, ARGs and their vertical distribution in paddy soil receiving long-term manure applications in four field experiments. High concentrations of tetracyclines were detected in most manured soils, while sulfonamides were not detectable. Long-term manure amendments generally increased the antibiotic concentrations and ARGs abundances in the paddy soil over decades. However, in some sites such significant trends of ARGs could not be observed. The abundance of ARGs was statistically correlated with antibiotics and soil properties including pH and soil organic matter (SOM), indicating their importance in the selection of resistance genes. Tetracyclines could be detected in soil at different depths and the concentrations of tetracyclines and abundance of ARGs generally decreased with increasing soil depths.     

Co-selection for antibiotic resistance genes is induced in a soil amended with zinc

The likelihood of co-selection for antibiotic resistance induced by heavy metals is a potential threat to human health, however, direct evidence of heavy metal-induced co-selection of antibiotic resistance is lacking in soil. By using a metagenomic sequencing approach, zinc (Zn) driven co-selection of antibiotic resistance genes (ARGs) in soil was investigated through a microcosm experiment where Zn salts were added at different levels. The results showed that the abundance and diversity of ARGs had a tendency to increase along the gradient of increasing Zn contents, with the maximum values recorded in the soil amended with 800 mg Zn kg⁻¹. The abundance of mobile genetic elements (MGEs) such as integrons and insertion sequences was significantly increased by Zn exposure. Network analysis demonstrated significant associations between ARGs and MGEs, suggesting that Zn treatment might enhance the potential for horizontal transfer of ARGs. Furthermore, structural equation models revealed that the types of ARGs were primarily driven by variations in bacterial compositions and Zn exposure, followed by integrons. Thus, these results indicate that added Zn salts considerably changed the diversity, abundance and mobility potential of antibiotic resistance, and imply that application of amendments containing high levels of Zn to soil could be causing the emergence and release of ARGs to the environment, and should be carefully monitored.     

Tetracycline resistance genes persist in soil amended with cattle feces independently from chlortetracycline selection pressure

Antibiotic residues and antibiotic resistance genes originating from animal waste represent environmental pollutants with possible human health consequences. In this study, we addressed the question whether chlortetracycline (CTC) residues in soils can act as selective pressure enhancing the persistence of tetracycline (TC-r) resistance genes in grassland soils receiving cattle feces. We performed a soil microcosm experiment, using 3 grassland soils with different management history, which were incubated with feces from conventionally raised dairy cows. The microcosms included treatments with a low dose (0.2 mg kg⁻¹), a high dose (100 mg kg⁻¹) and no CTC. The presence and abundance of TC-r genes tet(O), tet(Q) and tet(W) and the intI1 gene coding for class 1 integrase were assessed with real-time PCR after 0, 14, 28, 56 and 86 d of incubation. The genes tet(Q) and intI1 persisted in all feces-containing treatments for at least 28 d, and tet(W) and tet(O) for at least 86 d, though they went close to limits of quantification after 14–28 d in most cases. The soil, but not the dose of CTC, significantly affected the gene persistence. Concluding, certain TC-r genes originating from cattle feces may persist in soil for several months independently from antibiotic selection pressure.     

红壤坡地利用方式对土壤细菌群落结构的影响

利用中国科学院桃源农业生态试验站坡地不同利用方式长期定位观测试验场农田、自然恢复和茶园土壤为研究对象,直接从土壤中抽提总DNA,应用T-RFLP和RT-PCR技术研究红壤坡地利用方式对土壤细菌群落结构的影响。结果表明,红壤坡地三种土地利用方式土壤细菌多样性指数农田>茶园>自然恢复(p<0.05),但土壤细菌数量茶园>自然恢复>农田(p<0.05),茶园土壤细菌数量是农田的8.76倍。基于T-RFLP图谱的样品间相似性指数和主要限制性片段(T-RFs)定性分析均表明,农田与茶园和自然恢复土壤均存在显著的差异(p<0.05),而茶园和自然恢复土壤细菌群落比较相似。不同土地利用方式土壤有机质、有效磷和速效钾均对土壤细菌群落结构产生显著影响(p<0.05)。综合考虑经济效益和保持红壤坡地的可持续利用,茶园将成为中国南方红壤丘陵坡地可持续利用的一种有效方式。