Genome analysis of probiotic bacteria for antibiotic resistance genes

To date, probiotic bacteria are used in the diet and have various clinical applications. There are reports of antibiotic resistance genes in these bacteria that can transfer to other commensal and pathogenic bacteria. The aim of this study was to use whole-genome sequence analysis to identify antibiotic resistance genes in a group of bacterial with probiotic properties. Also, this study followed existing issues about the importance and presence of antibiotic resistance genes in these bacteria and the dangers that may affect human health in the future. In the current study, a collection of 126 complete probiotic bacterial genomes was analyzed for antibiotic resistance genes. The results of the current study showed that there are various resistance genes in these bacteria that some of them are transferable to other bacteria. The tet(W) tetracycline resistance gene was more than other antibiotic resistance genes in these bacteria and this gene was found in Bifidobacterium and Lactobacillus. In our study, the most numbers of antibiotic resistance genes were transferred with mobile genetic elements. We propose that probiotic companies before the use of a micro-organism as a probiotic, perform an antibiotic susceptibility testing for a large number of antibiotics. Also, they perform analysis of complete genome sequence for prediction of antibiotic resistance genes.

     

Evolution and ecology of antibiotic resistance genes

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the 'antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria. Besides direct selection by antibiotics, there is a number of other factors that may contribute to dissemination and maintenance of antibiotic resistance genes in bacterial populations.     

ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

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.     

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.     

抗生素耐药基因在畜禽粪便-土壤系统中的分布、扩散及检测方法

抗生素耐药基因作为一种新型的环境污染物已引起研究者的高度关注。畜禽养殖业长期将抗生素添加到饲料中,在促进动物生长、预防和治疗动物疾病等方面起了重要作用。这些抗生素大多数不能被动物完全吸收,在动物肠道中诱导出耐抗生素细菌和抗生素耐药基因,并随着粪便排出体外。畜禽粪便作为重要的抗生素、耐抗生素细菌和抗生素耐药基因储存库,通过堆粪、施肥等农业活动进入土壤环境中,可刺激土壤中耐抗生素细菌和抗生素耐药基因的富集。耐药基因借助于基因水平转移等方式在土壤介质中进一步传播扩散,甚至进入植物中随食物链传播,对生态环境和人类健康造成极大的威胁。为了正确评估抗生素耐药基因的生态风险,本文结合国内外相关研究,系统阐述了畜禽粪便-土壤系统中抗生素耐药基因的来源、分布及扩散机制,同时探讨了细菌耐药性的主要研究方法,指出堆肥化处理仍是目前去除抗生素耐药基因的主要手段,并对今后的研究方向进行展望。     

Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer

Antimicrobial resistance (AMR) poses a worldwide threat to human health and biosecurity. The spread of antibiotic resistance genes (ARGs) via conjugative plasmid transfer is a major contributor to the evolution of this resistance. Although permitted as safe food additives, compounds such as saccharine, sucralose, aspartame, and acesulfame potassium that are commonly used as nonnutritive sweeteners have recently been associated with shifts in the gut microbiota similar to those caused by antibiotics. As antibiotics can promote the spread of antibiotic resistance genes (ARGs), we hypothesize that these nonnutritive sweeteners could have a similar effect. Here, we demonstrate for the first time that saccharine, sucralose, aspartame, and acesulfame potassium could promote plasmid-mediated conjugative transfer in three established conjugation models between the same and different phylogenetic strains. The real-time dynamic conjugation process was visualized at the single-cell level. Bacteria exposed to the tested compounds exhibited increased reactive oxygen species (ROS) production, the SOS response, and gene transfer. In addition, cell membrane permeability increased in both parental bacteria under exposure to the tested compounds. The expression of genes involved in ROS detoxification, the SOS response, and cell membrane permeability was significantly upregulated under sweetener treatment. In conclusion, exposure to nonnutritive sweeteners enhances conjugation in bacteria. Our findings provide insight into AMR spread and indicate the potential risk associated with the presence of nonnutritive sweeteners.Subject terms: Microbial ecology, Water microbiology     

Genetic determinants of antibiotic resistance in gram negative bacteria

Modern data on spreading, structural and functional organization and evolution of the genetic determinants of antibiotic resistance in the gram-negative bacteria are reviewed. Some mechanisms for resistance to trimethoprime, sulphonamides, tetracyclines, chloramphenicol aminoglycosides, beta-lactam antibiotics controlled by the plasmid and chromosomal genes are presented. The problem of using the molecular DNA-probes containing the genetical determinants for antibiotic resistance in the practical work of clinical laboratories is discussed.     

畜禽养殖废物中抗生素和重金属抗性基因的产生机制和控制方法研究进展

动物饲料中常混有抗生素和重金属,导致外排的动物粪便中携带有抗生素和重金属,引发细菌产生耐药性和重金属抗性,继而产生抗生素抗性基因和重金属抗性基因。抗生素和重金属抗性基因污染已成为威胁人类身体健康及破坏生态环境的重大问题。本文从细菌进化的角度,明确了细菌的抗生素和重金属长期进化试验对抗性机制研究的重要性;抗生素抗性基因与重金属抗性基因间存在复杂的协同选择抗性,两者间相互影响,共同决定着细菌环境行为;抗性基因的水平转移增加了细菌在环境中的可变性,可移动遗传元件在抗性基因水平转移中发挥着重要作用。在抗性基因污染控制方面,高级氧化技术具有很好的抗性基因去除效果,尤其是UV/TiO₂氧化技术,能使抗生素抗性基因丰度减少4.7～5.8 log,减少率大于99.99%。其他的控制策略,如抗生素替代品博落回提取物以及噬菌体与抗生素结合使用,对于抗性基因的控制也具有重要意义。     

Factors That Affect Transfer of the IncI1 β-Lactam Resistance Plasmid pESBL-283 between E. coli Strains

The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Impact of Manure Fertilization on the Abundance of Antibiotic-Resistant Bacteria and Frequency of Detection of Antibiotic Resistance Genes in Soil and on Vegetables at Harvest

Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.     

Uncultured soil bacteria are a reservoir of new antibiotic resistance genes

Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.     

Veterinary use and antibiotic resistance

Globally, an estimated 50% of all antimicrobials serve veterinary purposes. Bacteria that inevitably develop antibiotic resistance in animals comprise food-borne pathogens, opportunistic pathogens and commensal bacteria. The same antibiotic resistance genes and gene transfer mechanisms can be found in the microfloras of animals and humans. Direct contact, food and water link animal and human habitats. The accumulation of resistant bacteria by the use of antibiotics in agriculture and veterinary medicine and the spread of such bacteria via agriculture and direct contamination are documented.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.     

食物链中抗生素耐药性基因的转移

抗生素的使用,一方面起到预防和治疗疾病的作用,另一方面,饲料、食品和环境中抗生素残留增加,使抗生素耐药性菌产生并进化,从而导致将来治疗某些疾病时无有效抗生素可用,比如,结核病已经卷土重来,而且现在就有许多患者就不能用抗生素治愈。随着人们生活水平的提高,安全、健康问题受到人们广泛的关注和重视。本文初步对耐药性基因在食物链中怎样产生和转移进行综述。