Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes

The rapid emergence of antibiotic-resistant (ART) pathogens is a major threat to public health. While the surfacing of ART food-borne pathogens is alarming, the magnitude of the antibiotic resistance (AR) gene pool in food-borne commensal microbes is yet to be revealed. Incidence of ART commensals in selected retail food products was examined in this study. The presence of 10(2)-10(7) CFU of ART bacteria per gram of foods in many samples, particularly in ready-to-eat, 'healthy' food items, indicates that the ART bacteria are abundant in the food chain. AR-encoding genes were detected in ART isolates, and Streptococcus thermophilus was found to be a major host for AR genes in cheese microbiota. Lactococcus lactis and Leuconostoc sp. isolates were also found carrying AR genes. The data indicate that food could be an important avenue for ART bacterial evolution and dissemination. AR-encoding plasmids from several food-borne commensals were transmitted to Streptococcus mutans via natural gene transformation under laboratory conditions, suggesting the possible transfer of AR genes from food commensals to human residential bacteria via horizontal gene transfer.     

Geographically widespread honeybee‐gut symbiont subgroups show locally distinct antibiotic‐resistant patterns

How long‐term antibiotic treatment affects host bacterial associations is still largely unknown. The honeybee‐gut microbiota has a simple composition, so we used this gut community to investigate how long‐term antibiotic treatment affects host‐associated microbiota. We investigated the phylogenetic relatedness, genomic content (GC percentage, genome size, number of genes and CRISPR) and antibiotic‐resistant genes (ARG) for strains from two abundant members of the honeybee core gut microbiota (Gilliamella apicola and Snodgrassella alvi). Domesticated honeybees are subjected to geographically different management policies, so we used two research apiaries, representing different antibiotic treatment regimens in their apiculture: low antibiotic usage (Norway) and high antibiotic usage (Arizona, USA). We applied whole‐genome shotgun sequencing on 48 G. apicola and 22 S. alvi. We identified three predominating subgroups of G. apicola in honeybees from both Norway and Arizona. For G. apicola, genetic content substantially varied between subgroups and distance similarity calculations showed similarity discrepancy between subgroups. Functional differences between subgroups, such as pectin‐degrading enzymes (G. apicola), were also identified. In addition, we identified horizontal gene transfer (HGT) of transposon (Tn10)‐associated tetracycline resistance (Tet B) across the G. apicola subgroups in the Arizonan honeybees, using interspace polymorphisms in the Tet B determinant. Our results support that honeybee‐gut symbiont subgroups can resist long‐term antibiotic treatment and maintain functionality through acquisition of geographically distinct antibiotic‐resistant genes by HGT.     

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.     

Antibiotics and anaerobes of gut origin

Hundreds of bacterial species make up human gut flora. Of these, 99% are anaerobic bacteria. Although anaerobes are part of the normal commensal flora, they can become opportunistic pathogens, causing serious, sometimes fatal infections if they escape from the colonic milieu. Most often, this escape occurs as a result of perforation, surgery, diverticulitis or cancer. Infections involving anaerobic bacteria are often difficult to treat because antibiotic resistance is increasing among the genera, mediated primarily through horizontal transfer of a plethora of mobile DNA transfer factors. Some of these transfer factors can also be transmitted to aerobic bacteria. It is becoming increasingly clear that antibiotic resistance trends have to be carefully monitored, and the transfer factors and mechanisms of transfer understood at a molecular level to avoid negative clinical outcomes when infections involve anaerobic bacteria.     

Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract,and impact on fish intestinal microbiota

The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid‐mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self‐transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4‐mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%–97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.     

The evolution of class 1 integrons and the rise of antibiotic resistance

Class 1 integrons are central players in the worldwide problem of antibiotic resistance, because they can capture and express diverse resistance genes. In addition, they are often embedded in promiscuous plasmids and transposons, facilitating their lateral transfer into a wide range of pathogens. Understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring how lateral gene transfer can seriously impact on, and be impacted by, human activities. We now show that class 1 integrons can be found on the chromosomes of nonpathogenic soil and freshwater Betaproteobacteria. Here they exhibit structural and sequence diversity, an absence of antibiotic resistance genes, and a phylogenetic signature of lateral transfer. Some examples are almost identical to the core of the class 1 integrons now found in pathogens, leading us to conclude that environmental Betaproteobacteria were the original source of these genetic elements. Because these elements appear to be readily mobilized, their lateral transfer into human commensals and pathogens was inevitable, especially given that Betaproteobacteria carrying class 1 integrons are common in natural environments that intersect with the human food chain. The strong selection pressure imposed by the human use of antimicrobial compounds then ensured their fixation and global spread into new species.     

双歧杆菌抗生素耐药性研究进展

目前,由于抗生素的广泛、大量使用,耐药菌株急剧增多,加剧了耐药菌对人类健康和生态环境的威胁。有研究表明,益生菌自身含有的耐药基因或耐药质粒等可通过基因水平转移传递给人体肠道中的致病菌,导致耐药菌感染。随着双歧杆菌相关微生态制剂的广泛应用,通常以活菌形式进入人体的双歧杆菌,与肠道内原籍菌群混合生长,致使其携带的耐药性基因片段在肠道菌群中水平转移,从而导致某些致病菌具有耐药性。因此,研究双歧杆菌等益生菌的耐药性基因转移有着十分迫切的意义。本文总结了双歧杆菌的耐药性,分析了双歧杆菌的耐药机理,为进一步筛选安全的双歧杆菌菌株提供理论依据。     

Monitoring horizontal antibiotic resistance gene transfer in a colonic fermentation model

The human microbiota is suggested to be a reservoir of antibiotic resistance (ABR) genes, which are exchangeable between transient colonizers and residing bacteria. In this study, the transfer of ABR genes from Enterococcus faecalis to Listeria monocytogenes and to commensal bacteria of the human gut microbiota was demonstrated in a colonic fermentation model. In the first fermentation, an E. faecalis donor harboring the marked 50-kb conjugative plasmid pRE25(*) and a chromosomal marker was co-immobilized with L. monocytogenes and infant feces. In this complex environment, the transfer of pRE25(*) to L. monocytogenes was observed. In a second fermentation, only the E. faecalis donor and feces were co-immobilized. Enumeration of pRE25(*) and the donor strain by quantitative PCR revealed an increasing ratio of pRE25(*) to the donor throughout the 16-day fermentation, indicating the transfer of pRE25(*) . An Enterococcus avium transconjugant was isolated, demonstrating that ABR gene transfer to gut commensals occurred. Moreover, pRE25(*) was still functional in both the E. avium and the L. monocytogenes transconjugant and transmittable to other genera in filter mating experiments. Our study reveals that the transfer of a multiresistance plasmid to commensal bacteria in the presence of competing fecal microbiota occurs in a colonic model, suggesting that commensal bacteria contribute to the increasing prevalence of antibiotic-resistant bacteria.     

Preventive antibiotic treatment of calves: emergence of dysbiosis causing propagation of obese state-associated and mobile multidrug resistance-carrying bacteria

In agriculture, antibiotics are used for the treatment and prevention of livestock disease. Antibiotics perturb the bacterial gut composition but the extent of these changes and potential consequences for animal and human health is still debated. Six calves were housed in a controlled environment. Three animals received an injection of the antibiotic florfenicol (Nuflor), and three received no treatment. Faecal samples were collected at 0, 3 and 7 days, and bacterial communities were profiled to assess the impact of a therapy on the gut microbiota. Phylogenetic analysis (16S-rDNA) established that at day 7, antibiotic-treated microbiota showed a 10-fold increase in facultative anaerobic Escherichia spp, a signature of imbalanced microbiota, dysbiosis. The antibiotic resistome showed a high background of antibiotic resistance genes, which did not significantly change in response to florfenicol. However, the maintenance of Escherichia coli plasmid-encoded quinolone, oqxB and propagation of mcr-2, and colistin resistance genes were observed and confirmed by Sanger sequencing. The microbiota of treated animals was enriched with energy harvesting bacteria, common to obese microbial communities. We propose that antibiotic treatment of healthy animals leads to unbalanced, disease- and obese-related microbiota that promotes growth of E. coli carrying resistance genes on mobile elements, potentially increasing the risk of transmission of antibiotic resistant bacteria to humans.     

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that shortterm antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ～3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.     

肠杆菌科细菌耐药基因表达的遗传和环境调控

细菌耐药性是21世纪国际关注的重要问题,也是全球面临的重大挑战.肠杆菌科细菌是医院感染的重要病原菌之一.近年来,随着抗生素的大量使用,多种肠杆菌科耐药菌,尤其是多重耐药肠杆菌开始大量出现,对人类健康形成了日益严重的威胁.细菌可以通过耐药基因突变或水平转移的方式获得耐药性,通常情况下,可以通过已知的耐药机制预测相应的耐药...     

The role of antibiotics and antibiotic resistance in nature

Investigations of antibiotic resistance from an environmental prospective shed new light on a problem that was traditionally confined to a subset of clinically relevant antibiotic‐resistant bacterial pathogens. It is clear that the environmental microbiota, even in apparently antibiotic‐free environments, possess an enormous number and diversity of antibiotic resistance genes, some of which are very similar to the genes circulating in pathogenic microbiota. It is difficult to explain the role of antibiotics and antibiotic resistance in natural environments from an anthropocentric point of view, which is focused on clinical aspects such as the efficiency of antibiotics in clearing infections and pathogens that are resistant to antibiotic treatment. A broader overview of the role of antibiotics and antibiotic resistance in nature from the evolutionary and ecological prospective suggests that antibiotics have evolved as another way of intra‐ and inter‐domain communication in various ecosystems. This signalling by non‐clinical concentrations of antibiotics in the environment results in adaptive phenotypic and genotypic responses of microbiota and other members of the community. Understanding the complex picture of evolution and ecology of antibiotics and antibiotic resistance may help to understand the processes leading to the emergence and dissemination of antibiotic resistance and also help to control it, at least in relation to the newer antibiotics now entering clinical practice.     

人体肠道4 644株代表菌次级代谢产物生物合成基因簇挖掘与耐药及毒力基因分析

为了更好地从肠道微生物组中挖掘新的次级代谢产物、了解肠道微生物组编码的抗生素耐药基因和毒力因子情况,本研究基于4 644株人体肠道微生物代表菌的基因组序列,对其编码的次级代谢产物基因簇、抗生素耐药基因和毒力因子进行了预测分析。经antiSMASH预测分析发现,超过60%的代表菌编码至少1个次级代谢产物基因簇,并从8个未可培养菌中发现了8个潜在的新颖次级代谢产物基因簇。人体肠道中的次级代谢产物主要由梭菌纲（Clostridia）、芽孢杆菌纲（Bacilli）、γ-变形菌纲（Gammaproteobacteria）、拟杆菌纲（Bacteroidia）、放线菌纲（Actinobacteria）和厚壁菌纲（Negativicutes）6类细菌编码的非核糖体多肽合成酶（nonribosomal peptide synthetase,NRPS）、细菌素、芳基多烯类化合物、萜烯、β-丙内酯、NRPS-样蛋白组成。经PathoFact预测分析发现,抗生素耐药基因和毒力因子在代表性菌株中分布广泛,但潜在病原菌编码频率更高。潜在病原菌中编码外膜蛋白、PapC N-端结构域、PapC C-端结构域、肽酶M16失活结构域等分泌型毒素和硝基还原酶家族、AcrB/AcrD/AcrF家族、PLD-样结构域、Cupin结构域、假定溶血素、S24-样肽酶、磷酸转移酶家族、内切核酸酶/外切核酸酶/磷酸酶家族、乙二醛酶/博莱霉素抗性等非分泌型毒素的频率较高。该研究将为进一步从肠道微生物组中挖掘新的微生物天然产物、了解肠道微生物的定殖与感染机制,为肠道微生物相关疾病提供靶向防治策略等奠定基础。     

Antibiotic resistance determinants in the interplay between food and gut microbiota

A complex and heterogeneous microflora performs sugar and lactic acid fermentations in food products. Depending on the fermentable food matrix (dairy, meat, vegetable etc.) as well as on the species composition of the microbiota, specific combinations of molecules are produced that confer unique flavor, texture, and taste to each product. Bacterial populations within such "fermented food microbiota" are often of environmental origin, they persist alive in foods ready for consumption, eventually reaching the gastro-intestinal tract where they can interact with the resident gut microbiota of the host. Although this interaction is mostly of transient nature, it can greatly contribute to human health, as several species within the food microbiota also display probiotic properties. Such an interplay between food and gut microbiota underlines the importance of the microbiological quality of fermented foods, as the crowded environment of the gut is also an ideal site for genetic exchanges among bacteria. Selection and spreading of antibiotic resistance genes in foodborne bacteria has gained increasing interest in the past decade, especially in light of the potential transferability of antibiotic resistance determinants to opportunistic pathogens, natural inhabitants of the human gut but capable of acquiring virulence in immunocompromised individuals. This review aims at describing major findings and future prospects in the field, especially after the use of antibiotics as growth promoters was totally banned in Europe, with special emphasis on the application of genomic technologies to improve quality and safety of fermented foods.     

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."     

Crossroads of Antibiotic Resistance and Biosynthesis

The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance.     

Characterization of Changes and Driver Microbes in Gut Microbiota During Healthy Aging Using A Captive Monkey Model

Recent population studies have significantly advanced our understanding of how age shapes the gut microbiota. However, the actual role of age could be inevitably confounded due to the complex and variable environmental factors in human populations. A well-controlled environment is thus necessary to reduce undesirable confounding effects, and recapitulate age-dependent changes in the gut microbiota of healthy primates. Herein we performed 16S rRNA gene sequencing, characterized the age-associated gut microbial profiles from infant to elderly crab-eating macaques reared in captivity, and systemically revealed the lifelong dynamic changes of the primate gut microbiota. While the most significant age-associated taxa were mainly found as commensals such as Faecalibacterium, the abundance of a group of suspicious pathogens such as Helicobacter was exclusively increased in infants, underlining their potential role in host development. Importantly, topology analysis indicated that the network connectivity of gut microbiota was even more age-dependent than taxonomic diversity, and its tremendous decline with age could probably be linked to healthy aging. Moreover, we identified key driver microbes responsible for such age-dependent network changes, which were further linked to altered metabolic functions of lipids, carbohydrates, and amino acids, as well as phenotypes in the microbial community. The current study thus demonstrates the lifelong age-dependent changes and their driver microbes in the primate gut microbiota, and provides new insights into their roles in the development and healthy aging of their hosts.     

Comparative genomics groups phages of Negativicutes and classical Firmicutes despite different Gram-staining properties

Negativicutes are gram-negative bacteria characterized by two cell membranes, but they are phylogenetically a side-branch of gram-positive Firmicutes that contain only a single membrane. We asked whether viruses (phages) infecting Negativicutes were horizontally acquired from gram-negative Proteobacteria, given the shared outer cell structure of their bacterial hosts, or if Negativicute phages co-evolved vertically with their hosts and thus resemble gram-positive Firmicute prophages. We predicted and characterized 485 prophages (mostly Caudovirales) from gram-negative Firmicute genomes plus 2977 prophages from other bacterial clades, and we used virome sequence data from 183 human stool samples to support our predictions. The majority of identified Negativicute prophages were lambdoids closer related to prophages from other Firmicutes than Proteobacteria by sequence relationship and genome organization (position of the lysis module). Only a single Mu-like candidate prophage and no clear P2-like prophages were identified in Negativicutes, both common in Proteobacteria. Given this collective evidence, it is unlikely that Negativicute phages were acquired from Proteobacteria. Sequence-related prophages, which occasionally harboured antibiotic resistance genes, were identified in two distinct Negativicute orders (Veillonellales and Acidaminococcales), possibly suggesting horizontal cross-order phage infection between human gut commensals. Our results reveal ancient genomic signatures of phage and bacteria co-evolution despite horizontal phage mobilization.     

Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes.

Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes.     

Exploring the bovine rumen bacterial community from birth to adulthood

The mammalian gut microbiota is essential in shaping many of its host''s functional attributes. One such microbiota resides in the bovine digestive tract in a compartment termed as the rumen. The rumen microbiota is necessary for the proper physiological development of the rumen and for the animal''s ability to digest and convert plant mass into food products, making it highly significant to humans. The establishment of this microbial population and the changes occurring with the host''s age are important for understanding this key microbial community. Despite its importance, little information about colonization of the microbial populations in newborn animals, and the gradual changes occurring thereafter, exists. Here, we characterized the overall bovine ruminal bacterial populations of five age groups, from 1-day-old calves to 2-year-old cows. We describe the changes occurring in the rumen ecosystem after birth, reflected by a decline in aerobic and facultative anaerobic taxa and an increase in anaerobic ones. Some rumen bacteria that are essential for mature rumen function could be detected as early as 1 day after birth, long before the rumen is active or even before ingestion of plant material occurs. The diversity and within-group similarity increased with age, suggesting a more diverse but homogeneous and specific mature community, compared with the more heterogeneous and less diverse primary community. In addition, a convergence toward a mature bacterial arrangement with age was observed. These findings have also been reported for human gut microbiota, suggesting that similar forces drive the establishment of gut microbiotas in these two distinct mammalian digestive systems.