Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non-Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain.     

The Culturable Soil Antibiotic Resistome: A Community of Multi-Drug Resistant Bacteria

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16–23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla _NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.     

水环境中耐药菌污染与危害研究进展

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

食品动物养殖环境中细菌耐药性研究进展

抗生素耐药性被世界卫生组织认为是21世纪人类面临的最大的公共卫生安全问题之一。近年来,抗生素耐药基因作为一种新型污染物而受到广泛关注。养殖场现已成为耐药基因的一个重要储库,耐药菌及耐药基因随着动物排泄物进入环境,从而加速了耐药基因在环境中的传播。畜禽养殖环境中耐药基因和耐药菌可能经食物链、空气等途径传至人类,给人类健康带来巨大威胁。文中结合最新文献,主要介绍了动物养殖场抗菌药物耐药菌和耐药基因的分布特点、耐药基因的持留和传播扩散、研究方法等方面的研究进展,为食品动物养殖环境的抗菌药物耐药性风险评估提供一定支持。     

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10¹⁹ bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.     

细菌中整合性接合元件的研究进展

整合性接合元件是近年来在细菌中发现的一种可移动的基因元件,它位于染色体上,可通过接合转移的方式介导细菌间基因的水平转移。这种基因的水平转移有助于细菌适应特定的环境条件,但许多整合性接合元件包含耐药基因,这些遗传元件的水平转移极大地加速了耐药基因在同种及不同种属之间的传播,造成细菌的耐药以至多重耐药问题日益严重,耐药机制日趋复杂;同时整合性接合元件与基因岛有着密切的联系,因此对其特征及转移机制进行研究很有必要。     

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.     

Bacterial evolution and the cost of antibiotic resistance.

Bacteria clearly benefit from the possession of an antibiotic resistance gene when the corresponding antibiotic is present. But do resistant bacteria suffer a cost of resistance (i.e., a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put resistance genes into naive bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments (in vitro and in vivo) show that the cost of antibiotic resistance can be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.     

Resistance to tetracycline, macrolide-lincosamide-streptogramin, trimethoprim, and sulfonamide drug classes

The discovery and use of antimicrobial agents in the last 50 yr has been one of medicine’s greatest achievements. These agents have reduced morbidity and mortality of humans and animals and have directly contributed to human’s increased life span. However, bacteria are becoming increasingly resistant to these agents by mutations, which alter existing bacterial proteins, and/or acquisition of new genes, which provide new proteins. The latter are often associated with mobile elements that can be exchanged quickly across bacterial populations and may carry multiple antibiotic genes fo resistance. In some case, virulence factors are also found on these same mobile elements. There is mounting evidence that antimicrobial use in agriculture, both plant and animal, and for environmental purposes does influence the antimicrobial resistant development in bacteria important in humans and in reverse. In this article, we will examine the genes which confer resistance to tetracycline, macrolide-lincosamide-streptogramin (MLS), trimethoprim, and sulfonamide.     

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。     

Genetics of Antimicrobial Resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

Acquired antibiotic resistance in lactic acid bacteria from food

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. -type replicating plasmids of the pAM1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29'871 bp resistance plasmid detected in Lactococcus lacti s subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabo lic traits to generate food-grade vectors.     

Genetics of antimicrobial resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

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.     

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.     

Combinatorial genetic evolution of multiresistance

The explosion in genetic information, whilst extending our knowledge, might not necessary increase our conceptual understanding on the complexities of bacterial genetics, or why some antibiotic resistant genotypes such as blaCTX-M-15 and blaVIM-2 appear to dominate. However, the information we have thus far suggests that clinical isolates have 'hijacked' plasmids, primarily built of backbone-DNA originating from environmental bacteria. Additionally, the combinatorial presence of other elements such as transposons, integrons, insertion sequence (IS) elements and the 'new' ISCR (IS common region) elements have also contributed to the increase in antibiotic resistance - an antibiotic resistant cluster composing four or five genes has become commonplace. In some instances, the presence of antibiotics themselves, such as fluoroquinolones, can mediate a bacterial SOS cell response, subsequently amplifying and/or augmenting the transfer of large genetic entities therefore, potentially promoting long-term detrimental effects.     

The food safety perspective of antibiotic resistance

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens. This issue has been the subject of heated debates for many years, however, there is still no complete consensus on the significance of antimicrobial use in animals, or resistance in bacterial isolates from animals, on the development and dissemination of antibiotic resistance among human bacterial pathogens. In fact, the debate regarding antimicrobial use in animals and subsequent human health implications has been going on for over 30 years, beginning with the release of the Swann report in the United Kingdom. The latest report released by the National Research Council (1998) confirmed that there were substantial information gaps that contribute to the difficulty of assessing potential detrimental effects of antimicrobials in food animals on human health. Regardless of the controversy, bacterial pathogens of animal and human origin are becoming increasingly resistant to most frontline antimicrobials, including expanded-spectrum cephalosporins, aminoglycosides, and even fluoroquinolones. The lion's share of these antimicrobial resistant phenotypes is gained from extra-chromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large, transferable, extra-chromosomal DNA elements, called plasmids, on which may be other DNA mobile elements, such as transposons and integrons. These DNA mobile elements have been shown to transmit genetic determinants for several different antimicrobial resistance mechanisms and may account for the rapid dissemination of resistance genes among different bacteria. The increasing incidence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many aspects of the development of antimicrobial resistance still remain uncertain. The emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms, and the surrounding environments. Although research has linked the use of antibiotics in agriculture to the emergence of antibiotic-resistant foodborne pathogens, debate still continues whether this role is significant enough to merit further regulation or restriction.     

多重耐药菌在人类、动物和环境的耐药和传播机制

抗生素等抗菌药物的滥用在全球范围内造成了多重耐药菌的传播。多重耐药菌(Multidrug resistant organisms,MDRO)以及耐药基因(Antibiotic resistance genes,ARGs)可在人类、动物和环境之间进行传播,尤其是ARGs可以通过水平转移的方式在同种属或者不同种属的菌群之间进行传播,使得细菌耐药问题日益严重,耐药机制趋于复杂,疾病治疗更加困难,对人类公众健康造成严重的威胁。因此抗生素等抗菌药物的使用应加以规范。