Antibiotic Resistance In Environment Research Articles

A Major Mycobacterium tuberculosis outbreak caused by one specific genotype in a low-incidence country: Exploring gene profile virulence explanations

Denmark, a tuberculosis low burden country, still experiences significant active Mycobacterium tuberculosis (Mtb) transmission, especially with one specific genotype named Cluster 2/1112–15 (C2), the most prevalent lineage in Scandinavia. In addition to environmental factors, antibiotic resistance, and human genetics, there is increasing evidence that Mtb strain variation plays a role for the outcome of infection and disease. In this study, we explore the reasons for the success of the C2 genotype by analysing strain specific polymorphisms identified through whole genome sequencing of all C2 isolates identified in Denmark between 1992 and 2014 (n = 952), and the demographic distribution of C2. Of 234 non-synonymous (NS) monomorphic SNPs found in C2 in comparison with Mtb reference strain H37Rv, 23 were in genes previously reported to be involved in Mtb virulence. Of these 23 SNPs, three were specific for C2 including a NS mutation in a gene associated with hyper-virulence. We show that the genotype is readily transmitted to different ethnicities and is also found outside Denmark. Our data suggest that strain specific virulence factor variations are important for the success of the C2 genotype. These factors, likely in combination with poor TB control, seem to be the main drivers of C2 success.

Combined impact of fishmeal and tetracycline on resistomes in mariculture sediment

Mariculture sediment has been recognized as a major contributor of environmental antibiotic resistance genes (ARGs), which are challenging the treatment of infections worldwide. Both antibiotics and fishmeal are used in aquaculture, and each has the potential to facilitate ARG dissemination, however their combined impact on the sediment resistome and their relative contribution remain unclear. In this study, microcosms were exposed to varying concentrations of tetracycline with or without fishmeal (0.1% wt/wt) for 14 days. Sediment genomic DNA was analyzed using high throughput quantitative PCR and 16S rRNA gene amplicon sequencing to compare the contribution of fishmeal and tetracycline to antibiotic resistomes and bacterial communities in mariculture sediment. Sixty-seven ARGs were detected potentially correlating to resistance for several major antibiotics. Fishmeal, but not the dose of tetracycline, contributed to the significant increase of both ARG abundance and diversity in the sediment. Based on principle coordinate analysis and hierarchical clustering, ARGs were clustered into two groups depending on whether fishmeal was added. Aminoglycoside, macrolide–lincosamide–streptogramin b (MLSb) and tetracycline resistance genes were the most abundant when fishmeal was used, while a significant increase in mobile genetic element (MGE) abundance was also detected (P < 0.05). Meanwhile, bacterial community structures were detected with distinct patterns between the two groups (Adonis, P < 0.05). Using the Mantel test and partial least squares path modeling, we identified that sediment resistomes were significantly correlated with microbial community structures (P < 0.05) which were mainly driven by nutrients in fishmeal. Together our findings suggested that fishmeal plays a more important role than tetracycline in proliferation of ARGs in mariculture sediment. This study may provide new insights into the mitigation of ARG propagation in mariculture operations.

The diversity of uncharacterized antibiotic resistance genes can be predicted from known gene variants\u2014but not always

BackgroundAntibiotic resistance is considered one of the most urgent threats to modern healthcare, and the role of the environment in resistance development is increasingly recognized. It is often assumed that the abundance and diversity of known resistance genes are representative also for the non-characterized fraction of the resistome in a given environment, but this assumption has not been verified. In this study, this hypothesis is tested, using the resistance gene profiles of 1109 metagenomes from various environments.ResultsThis study shows that the diversity and abundance of known antibiotic resistance genes can generally predict the diversity and abundance of undescribed resistance genes. However, the extent of this predictability is dependent on the type of environment investigated. Furthermore, it is shown that carefully selected small sets of resistance genes can describe total resistance gene diversity remarkably well.ConclusionsThe results of this study suggest that knowledge gained from large-scale quantifications of known resistance genes can be utilized as a proxy for unknown resistance factors. This is important for current and proposed monitoring efforts for environmental antibiotic resistance and has implications for the design of risk ranking strategies and the choices of measures and methods for describing resistance gene abundance and diversity in the environment.

Diversity of herbaceous plants and bacterial communities regulates soil resistome across forest biomes.

Antibiotic resistance is ancient and prevalent in natural ecosystems and evolved long before the utilization of synthetic antibiotics started, but factors influencing the large-scale distribution patterns of natural antibiotic resistance genes (ARGs) remain largely unknown. Here, a large-scale investigation over 4000 km was performed to profile soil ARGs, plant communities and bacterial communities from 300 quadrats across five forest biomes with minimal human impact. We detected diverse and abundant ARGs in forests, including over 160 genes conferring resistance to eight major categories of antibiotics. The diversity of ARGs was strongly and positively correlated with the diversity of bacteria, herbaceous plants and mobile genetic elements (MGEs). The ARG composition was strongly correlated with the taxonomic structure of bacteria and herbs. Consistent with this strong correlation, structural equation modelling demonstrated that the positive effects of bacterial and herb communities on ARG patterns were maintained even when simultaneously accounting for multiple drivers (climate, spatial predictors and edaphic factors). These findings suggest a paradigm that the interactions between aboveground and belowground communities shape the large-scale distribution of soil resistomes, providing new knowledge for tackling the emerging environmental antibiotic resistance.

Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.11The recommendations from the workshop have also been communicated in a separate report published on the website of the JPIAMR (www.jpiamr.org).

Screening for ESBL producing bacterial isolates of agricultural soil and profiling for multidrug resistance

Abstract Objectives construction of agricultural environmental soil antibiotic resistance profile of the culturable bacteria in Kalyani, India, followed by the screening of ESBL producers harboring blatem, blashv, and blactx-M. Methods 158 Multi-Drug Resistant (MDR) bacteria were isolated from 18 agricultural sampling points in Kalyani, West Bengal, India followed by their Antibiotic resistance profiling against 15 test antibiotics. They were then screened for the presence of three Extended Spectrum Beta-Lactamase (ESBL) genes, namely blatem, blactx-M and blashv by Double Disk Diffusion test and Polymerase Chain Reaction. The positive candidates were further identified using Amplified Ribosomal DNA Restriction Analysis followed by 16S rDNA sequencing. Results lowest resistance was observed against Vancomycin (0%) whereas the highest against the β lactam Ampicillin (97.5%). Very high resistance was observed against the 3rd generation cephalosporin Cefotaxime (90%). 16/158 bacterial isolates were positive for one or more of the three ESBL genes under investigation. 11 out of those 16 isolates (68.75%) harbored at least 2 genes and the rest 31.25% either had the blashv or the blatem gene. Phylogenetic analysis of the prevalent ESBL carrying organisms in the region demonstrated that the genes are being horizontally transferred across different bacterial orders and classes. Conclusion Significant antimicrobial resistance noted against 12 out of 15 antibiotics tested. Stenotrophomonas, Myroides, Pseudomonas, Enterobacter, and Serratia are found as culturable MDR bacteria carrying ESBL genes among the isolates. It is the first report of environmental ESBL carriage by Myroides isolated from India.

Distinct effects of struvite and biochar amendment on the class 1 integron antibiotic resistance gene cassettes in phyllosphere and rhizosphere

Struvite recovered from wastewater is promising for recycling phosphorus into soil as fertilizers. However, struvite application may prompt the proliferation of antibiotic resistance in soil and plant. This study examined the impacts of struvite application and biochar amendment on integrons abundance and gene cassette contexts in rhizosphere soil and phyllosphere using quantitative PCR and clone library analysis. Microcosm experiments revealed that class 1 integron was the most prevalent in all samples, with higher concentration and higher relative abundance in rhizosphere than those in phyllosphere. The majority of resistance gene cassettes were associated with genes encoding resistance to aminoglycosides, beta-lactams and chloramphenicols. Struvite application significantly increased the genetic diversity of antibiotic resistance gene cassettes in both rhizosphere and phyllosphere. However, biochar amendment attenuated the increasing effect of struvite application exerting on the class 1 integron antibiotic resistance gene cassette pool in phyllosphere. These findings highlighted human activities to be the source of integron gene cassette pool and raised the possibility of using biochar amendment as an alternative mean for mitigating antibiotic resistance in environments.

Antibiotic Resistant Bacteria in Municipal Wastes: Is There Reason for Concern?

Recently, there has been increased concern about the presence of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG), in treated domestic wastewaters, animal manures and municipal biosolids. The concern is whether these additional sources of ARB contribute to antibiotic resistance levels in the environment, that is, "environmental antibiotic resistance." ARB and ARG occur naturally in soil and water, and it remains unclear whether the introduction of ARB in liquid and solid municipal and animal wastes via land application have any significant impact on the background levels of antibiotic resistance in the environment, and whether they affect human exposure to ARB. In this current review, we examine and re-evaluate the incidence of ARB and ARG resulting from land application activities, and offer a new perspective on the threat of antibiotic resistance to public health via exposure from nonclinical environmental sources. Based on inputs of ARBs and ARGs from land application, their fate in soil due to soil microbial ecology principles, and background indigenous levels of ARBs and ARGs already present in soil, we conclude that while antibiotic resistance levels in soil are increased temporally by land application of wastes, their persistence is not guaranteed and is in fact variable, and often contradictory based on application site. Furthermore, the application of wastes may not produce the most direct impact of ARGs and ARB on public health. Further investigation is still warranted in agriculture and public health, including continued scrutiny of antibiotic use in both sectors.

Chlorine disinfection increases both intracellular and extracellular antibiotic resistance genes in a full-scale wastewater treatment plant

The emergence and spread of antibiotic resistance has posed a major threat to both human health and environmental ecosystem. Although the disinfection has been proved to be efficient to control the occurrence of pathogens, little effort is dedicated to revealing potential impacts of disinfection on transmission of antibiotic resistance genes (ARGs), particularly for free-living ARGs in final disinfected effluent of urban wastewater treatment plants (UWWTP). Here, we investigated the effects of chlorine disinfection on the occurrence and concentration of both extracellular ARGs (eARGs) and intracellular ARGs (iARGs) in a full-scale UWWTP over a year. We reported that the concentrations of both eARGs and iARGs would be increased by the disinfection with chlorine dioxide (ClO2). Specifically, chlorination preferentially increased the abundances of eARGs against macrolide (ermB), tetracycline (tetA, tetB and tetC), sulfonamide (sul1, sul2 and sul3), β-lactam (ampC), aminoglycosides (aph(2’)-Id), rifampicin (katG) and vancomycin (vanA) up to 3.8 folds. Similarly, the abundances of iARGs were also increased up to 7.8 folds after chlorination. In terms of correlation analyses, the abundance of Escherichia coli before chlorination showed a strong positive correlation with the total eARG concentration, while lower temperature and higher ammonium concentration were assumed to be associated with the concentration of iARGs. This study suggests the chlorine disinfection could increase the abundances of both iARGs and eARGs, thereby posing risk of the dissemination of antibiotic resistance in environments.

Seasonal impact of multi-antibiotic resistant gram-negative pathogenic bacteria on the air environments and their associated health risks in a pharmaceutical wastewater treatment plant

The spread of antibiotic resistance in environments has been a serious public health threat. Many related studies have focused on waters, but bioaerosols, which can transmit pathogens to humans through air, have been largely ignored. Here, we evaluated health risks from airborne multi-antibiotic resistant bacteria by collecting bioaerosols in summer and winter in a full-scale plant treating antibiotics-producing wastewater. Gram-negative opportunistic pathogens were isolated, and antibiotic susceptibility tests against 18 commonly used antibiotics, including 11 β-lactam antibiotics, 3 aminoglycosides, 2 fluoroquinolones, 1 furan and 1 sulfonamide, were conducted. We found the percentages of strains resistant to β-lactam antibiotics were higher in summer than winter. However, the proportions resistant to other antibiotics did not significantly differ between both seasons. Quantitative microbial risk assessment (QMRA) showed the annual risks from isolated strains ranged from 10−4 to 10−3, all higher than the acceptable standard for microorganisms (less than 1 infection per 10 000 persons per year, 10−4) in both seasons. Multiple antibiotic resistance (MAR) indexes demonstrated that bioaerosols in and around the pharmaceutical wastewater treatment plant (WWTP) in summer were high exposure risks, whereas they possessed lower but still substantial exposure risks in winter. In addition, we determined the dominant genera of isolated strains in both seasons were Acinetobacter, Enterobacter, Pantoea, Pseudomonas and Sphingomonas. Together, we showed that the WWTP treating cephalosporin-producing wastewater imposed a potentially severe health risk for the staff and near residents, calling for measures to minimize the release of bacteria into the bioaerosols in pharmaceutical WWTPs.

ExtendedSpectrumBeta-Lactamase (ESBL)-producing bacteria isolated from hospital wastewaters, rivers and aquaculture sources in Nigeria.

Untreated wastewater is a risk factor for the spread of antibiotic resistance in the environment. However, little is known about the contribution of untreated wastewater to the burden of antibiotic resistance in the Nigerian environment. In this study, a total of 143 ceftazidime-/cefpodoxime-resistant bacteria isolated from untreated wastewater and untreated wastewater-contaminated surface and groundwater in Nigeria were screened for extended-spectrum β-lactamase (ESBL) genes, integrons and integron gene cassettes by PCR. The genetic environment of bla CTX-M-15 was mapped by PCR and potentially conjugative plasmids were detected among the isolates by degenerate primer MOB typing (DPMT). ESBL production was confirmed in 114 (79.7%) isolates and ESBL genes (bla SHV, bla CTX-M-15 and bla TEM) were detected in 85 (74.6%) ESBL-producing isolates. bla CTX-M-15 was associated with ISEcp1 and with orf477 in 12 isolates and with ISEcp1, IS26 and orf477 in six others. To the best of our knowledge, this is the first report of bla CTX-M-15 in hand-dug wells and borehole serving as sources of drinking water and a first report of the genetic environment of bla CTX-M-15 in environmental bacteria from Nigeria. The results of this study confirm untreated wastewater as an important medium for the spread of ESBL-producing bacteria within the Nigerian environment. Hence, the widespread practice of discharging untreated wastewater into the aquatic ecosystem in Nigeria is a serious risk to public health.

MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage.

Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.

Survival of Litopenaeus vannamei shrimp fed on diets supplemented with Dunaliella sp. is improved after challenges by Vibrio parahaemolyticus.

Survival of Litopenaeus vannamei was evaluated during a Vibrio parahaemolyticus infection. This bacteria has been causing significant economic losses in the shrimp industry due to the appearance of early mortality syndrome (EMS), also known as acute hepatopancreatic necrosis disease (AHPND). Control of bacteria in ponds is difficult to achieve with antibiotics due to environmental infections and antibiotic resistance. New methods have been proposed to control and prevent the impact of bacterial infections. The physiological response indicated by plasma biochemical parameters in shrimp can determine their health and stress status. Meanwhile, shrimp immunology is the key factor in establishing strategies to control diseases. Immunostimulants are the best alternative to antibiotics to prevent or minimize disease damage, and at the same time, these stimulants improve the immune system in shrimp. Four diets containing 1.5, 2, 2.5 and 3% of Dunaliella sp. with high β-carotene content were tested in the present study. After 20days of feeding, organisms were infected with V. parahaemolyticus. Protein, glucose, lactate, triglyceride and cholesterol levels, as well as activity of prophenoloxidase and phenoloxidase, were determined 48 h post-infection (hpi). Shrimp fed a diet with 3% Dunaliella sp. showed the highest survival. Glucose, cholesterol, and triglyceride levels, as well as prophenoloxidase and phenoloxidase activity, were not observed to be suitable indicators during this bacterial infection. The results indicated that the inclusion of Dunaliella sp. in diet increases survival in L. vannamei infected with V. parahaemolyticus.

Reclassification of the Specialized Metabolite Producer Pseudomonas mesoacidophila ATCC 31433 as a Member of the Burkholderia cepacia Complex

ABSTRACTPseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the β-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis. The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis. Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.

Contribution of bacteriophage and plasmid DNA to the mobilization of antibiotic resistance genes in a river receiving treated wastewater discharges

In this study, we quantified eleven antibiotic compounds and nine antibiotic resistance genes (ARGs) in water samples collected upstream and downstream of the discharge point from a municipal wastewater treatment plant (WWTP) into the Ter River. Antibiotics were analyzed by liquid chromatography coupled to mass spectrometry, whereas the concentration of ARGs in bacterial, phage and plasmid DNA fractions was determined by real-time PCR to explore their contribution to environmental antibiotic resistance. WWTP discharges resulted in higher concentrations of antibiotic residues as well as ARGs in water samples collected downstream the impact point. Specifically, genes conferring resistance to macrolides (ermB), fluoroquinolones (qnrS) and tetracyclines (tetW) showed significant differences (p<0.05) between upstream and downstream sites in the three DNA fractions (i.e. bacteria, plasmids and phages). Interestingly, genes conferring resistance to β-lactams (blaTEM, blaNDM and blaKPC) and glycopeptides (vanA) only showed significant differences (p<0.05) between upstream and downstream sites in phage and plasmid DNA but not in the bacterial DNA fraction. Our results show for the first time the extent to which phages and plasmids contribute to the mobilization of ARGs in an aquatic environment exposed to chronic antibiotic pollution via WWTP discharges. Accordingly, these mobile genetic elements should be included in further studies to get a global view of the spread of antibiotic resistance.

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii.

A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as Acinetobacter baumannii, a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of A. baumannii is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying A. baumannii pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTSNtr), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in A. baumannii, and coupling this pathway with the PTSNtr allows A. baumannii to integrate cellular metabolic status with external environmental cues.IMPORTANCE Opportunistic pathogens, including Acinetobacter baumannii, encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in A. baumannii has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we investigate two previously identified virulence determinants, GigA and GigB, and report that they are required for in vitro stress resistance, likely comprising upstream elements of a global stress response pathway. Additional experiments identify a connection between GigA/GigB and a widely conserved metabolic-sensing pathway, the nitrogen phosphotransferase system. We propose that coordination of these two pathways allows A. baumannii to respond appropriately to changing environmental conditions, including those encountered during infection.

Functional Metagenomics as a Tool for Identification of New Antibiotic Resistance Genes from Natural Environments.

Antibiotic resistance has become a major concern for human and animal health, as therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. The problem is compounded by low investment in antibiotic research and lack of new effective antimicrobial drugs on the market. Exploring environmental antibiotic resistance genes (ARGs) will help us to better understand bacterial resistance mechanisms, which may be the key to identifying new drug targets. Because most environment-associated microorganisms are not yet cultivable, culture-independent techniques are essential to determine which organisms are present in a given environmental sample and allow the assessment and utilization of the genetic wealth they represent. Metagenomics represents a powerful tool to achieve these goals using sequence-based and functional-based approaches. Functional metagenomic approaches are particularly well suited to the identification new ARGs from natural environments because, unlike sequence-based approaches, they do not require previous knowledge of these genes. This review discusses functional metagenomics-based ARG research and describes new possibilities for surveying the resistome in environmental samples.

Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291.

Background Clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. We examined the structure and function of a gene product with the locus tag CDR20291_0991 (cdPadR1) as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent C. difficile strain R20291. cdPadR1 is genomically positioned near genes that are involved in stress response and virulence. In addition, it was previously reported that cdPadR1 and a homologue from the historical C. difficile strain 630 (CD630_1154) were differentially expressed when exposed to stressors, including antibiotics.ResultsThe crystal structure of cdPadR1 was determined to 1.9 Å resolution, which revealed that it belongs to the PadR-s2 subfamily of PadR transcriptional regulators. cdPadR1 binds its own promoter and other promoter regions from within the C. difficile R20291 genome. DNA binding experiments demonstrated that cdPadR1 binds a region comprised of inverted repeats and an AT-rich core with the predicted specific binding motif, GTACTAT(N2)ATTATA(N)AGTA, within its own promoter that is also present in 200 other regions in the C. difficile R20291 genome. Mutation of the highly conserved W in α4 of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other PadR-s2 proteins, resulted in alterations of cdPadR1 binding to the predicted binding motif, potentially due to loss of higher order oligomerization.ConclusionsOur results indicate that cdPadR1 binds a region within its own promoter consisting of the binding motif GTACTAT(N2)ATTATA(N)AGTA and seems to associate non-specifically with longer DNA fragments in vitro, which may facilitate promoter and motif searching. This suggests that cdPadR1 acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0850-0) contains supplementary material, which is available to authorized users.

Field-based evidence for copper contamination induced changes of antibiotic resistance in agricultural soils.

Bacterial resistance to antibiotics and heavy metals are frequently linked, suggesting that exposure to heavy metals might select for bacterial assemblages conferring resistance to antibiotics. However, there is a lack of clear evidence for the heavy metal-induced changes of antibiotic resistance in a long-term basis. Here, we used high-capacity quantitative PCR array to investigate the responses of a broad spectrum of antibiotic resistance genes (ARGs) to 4-5 year copper contamination (0-800 mg kg-1 ) in two contrasting agricultural soils. In total, 157 and 149 unique ARGs were detected in the red and fluvo-aquic soil, respectively, with multidrug and β-lactam as the most dominant ARG types. The highest diversity and abundance of ARGs were observed in medium copper concentrations (100-200 mg kg-1 ) of the red soil and in high copper concentrations (400-800 mg kg-1 ) of the fluvo-aquic soil. The abundances of total ARGs and several ARG types had significantly positive correlations with mobile genetic elements (MGEs), suggesting mobility potential of ARGs in copper-contaminated soils. Network analysis revealed significant co-occurrence patterns between ARGs and microbial taxa, indicating strong associations between ARGs and bacterial communities. Structural equation models showed that the significant impacts of copper contamination on ARG patterns were mainly driven by changes in bacterial community compositions and MGEs. Our results provide field-based evidence that long-term Cu contamination significantly changed the diversity, abundance and mobility potential of environmental antibiotic resistance, and caution the un-perceived risk of the ARG dissemination in heavy metal polluted environments.

What is the evidence that point sources of anthropogenic effluent increase antibiotic resistance in the environment? Protocol for a systematic review.

Herein we describe a protocol for a systematic review of the evidence on whether point sources of anthropogenic effluent are associated with an increase in antibiotic resistance in the adjacent environment. The review question was based on the Population, Exposure, Comparator, Outcome, Study Design (PECOS) framework as follows: Is the prevalence or concentration of antibiotic resistant bacteria or resistance genes (O) in soil, water, air or free-living wildlife (P) higher in close proximity to, or downstream from, known or suspected sources of anthropogenic effluent (E) compared to areas more distant from or upstream from these sources (C)? A comprehensive search strategy was created to capture all relevant, published literature. Criteria for two stages of eligibility screening were developed to exclude publications that were not relevant to the question, and determine if the study used a design that permitted estimation of an association between a source and levels of resistance. A decision matrix was created for assessment of risk of bias to internal validity due to sample selection bias, information bias, and confounding. The goal of this protocol is to provide a method for determining the state of knowledge about the effect of point sources on antibiotic resistance in the environment.