The Ocean as a Global Reservoir of Antibiotic Resistance Genes

Recent studies of natural environments have revealed vast genetic reservoirs of antibiotic resistance (AR) genes. Soil bacteria and human pathogens share AR genes, and AR genes have been discovered in a variety of habitats. However, there is little knowledge about the presence and diversity of AR genes in marine environments and which organisms host AR genes. To address this, we identified the diversity of genes conferring resistance to ampicillin, tetracycline, nitrofurantoin, and sulfadimethoxine in diverse marine environments using functional metagenomics (the cloning and screening of random DNA fragments). Marine environments were host to a diversity of AR-conferring genes. Antibiotic-resistant clones were found at all sites, with 28% of the genes identified as known AR genes (encoding beta-lactamases, bicyclomycin resistance pumps, etc.). However, the majority of AR genes were not previously classified as such but had products similar to proteins such as transport pumps, oxidoreductases, and hydrolases. Furthermore, 44% of the genes conferring antibiotic resistance were found in abundant marine taxa (e.g., Pelagibacter, Prochlorococcus, and Vibrio). Therefore, we uncovered a previously unknown diversity of genes that conferred an AR phenotype among marine environments, which makes the ocean a global reservoir of both clinically relevant and potentially novel AR genes.     

Staphylococci Isolated from Carriage Sites and Infected Sites of Dogs as a Reservoir of Multidrug Resistance and Methicillin Resistance

The aim of this study was to compare the spread of multidrug-resistant (MDR) and methicillin-resistant (MR) staphylococci in healthy dogs and in dogs with evident symptoms of infection. The samples from 172 healthy and 197 infected dogs were examined. The staphylococci were identified with conventional methods and by means of the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method (MboI). Susceptibility to 15 antibiotics from 10 different antimicrobial classes was tested. Resistance to methicillin was confirmed by the presence of Staphylococcus aureus mecA and S. sciuri mecA genes. Multidrug resistance was defined as resistance to three or more antimicrobial classes. The oral mucosa to be the most frequent site of staphylococcal colonization (55.8 %), followed by nasal cavity (44.2 %), and anus (32.6 %). The prevalence of MDR staphylococci in infected dogs was significantly higher than in the healthy animals (74/137 vs. 34/95, P = 0.006). The MR strains of S. pseudintermedius (2.9 %) originated solely from infected dogs. In contrast, the MR coagulase-negative strains (7.4 %) were isolated solely from healthy dogs. S. aureus strains originated from nasal swabs, MRSA strains were not isolated. MDR staphylococci and MR S. pseudintermedius are more common among infected dogs, but coagulase-negative staphylococci (mostly S. sciuri) seem to be a reservoir of methicillin resistance in healthy dogs.     

Striatal Astrocytes Act as a Reservoir for L-DOPA

L-DOPA is therapeutically efficacious in patients with Parkinson’s disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.     

Commensal Bacteria Lipoteichoic Acid Increases Skin Mast Cell Antimicrobial Activity against Vaccinia Viruses

Mast cells (MCs) are considered sentinels in the skin and mucosa. Their ability to release antimicrobial peptides, such as cathelicidin, protects against bacterial infections when the epithelial barrier is breached. We recently described that MCs defend against bacterial and viral infections through the release of cathelicidin during degranulation. In this study, we hypothesize that cathelicidin expression is induced in MCs by the activation of TLR2 from bacterial products (lipoteichoic acid) produced by commensal bacteria at the epithelial surface. Our research shows that signaling through TLR2 increases the production and expression of cathelicidin in mast cells, thereby enhancing their capacity to fight vaccinia virus. MCs deficient in cathelicidin were less efficient in killing vaccinia virus after lipoteichoic acid stimulation than wild-type cells. Moreover, the activation of TLR2 increases the MC recruitment at the skin barrier interface. Taken together, our findings reveal that the expression and control of antimicrobial peptides and TLR signaling on MCs are key in fighting viral infection. Our findings also provide new insights into the pathogenesis of skin infections and suggest potential roles for MCs and TLR2 ligands in antiviral therapy.     

Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.     

Resistance Genes and Genetic Elements Associated with Antibiotic Resistance in Clinical and Commensal Isolates of Streptococcus salivarius

The diversity of clinical (n = 92) and oral and digestive commensal (n = 120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide-lincosamide-streptogramin B [MLS_B] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n = 13), followed by Tn6002 (n = 11) and Tn2009 (n = 4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut.     

Antibiotic Resistance of Commensal Staphylococcus aureus and Coagulase-Negative Staphylococci in an International Cohort of Surgeons: A Prospective Point-Prevalence Study

Nasal colonization with antibiotic resistant bacteria represents both a risk factor for the colonized individual and their immediate contacts. Despite the fact that healthcare workers such as orthopedic surgeons are at a critical interface between the healthcare environment and an at-risk patient population, the prevalence of antibiotic resistant bacteria within the surgical profession remains unclear. This study offers a snapshot of the rate of nasal colonization of orthopedic surgeons with multi-resistant staphylococci including methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS). We performed a prospective, observational study obtained at a single time point in late 2013. The participants were active orthopedic, spine and head & neck surgeons from 75 countries. The prevalence of nasal carriage of the different bacteria and the corresponding 95% confidence interval were calculated. From a cohort of 1,166 surgeons, we found an average S. aureus nasal colonization rate of 28.0% (CI 25.4;30.6) and MRSA rate of 2.0% (CI 1.3;2.9), although significant regional variations were observed. The highest rates of MRSA colonization were found in Asia (6.1%), Africa (5.1%) and Central America (4.8%). There was no MRSA carriage detected within our population of 79 surgeons working in North America, and a low (0.6%) MRSA rate in 657 surgeons working in Europe. High rates of MRCoNS nasal carriage were also observed (21.4% overall), with a similar geographic distribution. Recent use of systemic antibiotics was associated with higher rates of carriage of resistant staphylococci. In conclusion, orthopedic surgeons are colonized by S. aureus and MRSA at broadly equivalent rates to the general population. Crucially, geographic differences were observed, which may be partially accounted for by varying antimicrobial stewardship practices between the regions. The elevated rates of resistance within the coagulase-negative staphylococci are of concern, due to the increasing awareness of their importance in hospital acquired and device-associated infection.     

Drug Resistance of Staphylococci VI. Genetic Determinant for Chloramphenicol Resistance

Naturally occurring strains of staphylococci which are resistant to chloramphenicol (CM) inactivate this antibiotic. One of the inactivation products of CM showed the chromatographic behavior of 3-acetoxychloramphenicol. Induction of resistance occurred after prior exposure to subinhibitory concentrations of the antibiotic. The resistance of induced populations, as well as CM-inactivation ability, was decreased when they were grown in CM-free medium. The CM-inactivation property was transduced together with CM resistance. Transductional analysis and CM-resistance elimination experiments indicated that CM resistance in naturally occurring strains of staphylococci is mainly accounted for by inactivation of the drug.     

痢疾杆菌酸抗性系统相关基因缺失突变体的构建

依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础.     

Maternal Effects May Act as an Adaptation Mechanism for Copepods Facing pH and Temperature Changes

Acidification of the seas, caused by increased dissolution of CO₂ into surface water, and global warming challenge the adaptation mechanisms of marine organisms. In boreal coastal environments, temperature and pH vary greatly seasonally, but sometimes also rapidly within hours due to upwelling events. We studied if copepod zooplankton living in a fluctuating environment are tolerant to climate change effects predicted for 2100, i.e., a temperature increase of 3°C and a pH decrease of 0.4. Egg production of the copepod Acartia sp. was followed over five consecutive days at four temperature and pH conditions (17°C/ambient pH; 17°C/low pH; 20°C/ambient pH; 20°C/low pH). Egg production was higher in treatments with warmer temperature but the increase was smaller when copepods were simultaneously exposed to warmer temperature and lowered pH. To reveal if maternal effects are important in terms of adaptation to a changing environment, we conducted an egg transplantation experiment, where the produced eggs were moved to a different environment and egg hatching was monitored for three days. When pH changed between the egg production and hatching conditions, it resulted in lower hatching success, but the effect was diminished over the course of the experiment possibly due to improved maternal provisioning. Warmer egg production temperature induced a positive maternal effect and increased the egg hatching rate. Warmer hatching temperature resulted also in earlier hatching. However, the temperature effects appear to be dependent on the ambient sea temperature. Our preliminary results indicate that maternal effects are an important mechanism in the face of environmental change.     

Metagenomic Screening for Bleomycin Resistance Genes

A metagenomic library of activated sludge was screened for bleomycin resistance genes. Two genes were identified that differed greatly from each other, from the genes of bleomycin-producing actinomycetes, and from those of clinical isolates. Therefore, the nonclinical environment is a rich reservoir of new resistance elements, and metagenomics can be used to sample the resistome rapidly.     

Can Insulated Skin Temperature Act as a Substitute for Rectal Temperature when Studying Circadian Rhythms?

We measured rectal, lateral chest wall, and axillary temperature every half hour for at least 24 h while subjects were living normal life-styles and keeping a sleep/activity diary. We then used a purification method to estimate the decrease of temperature due to sleep and the increases due to sitting, standing, walking, or exercising, as well as the parameters of the cosine curve that described the “purified data.” Cosinor analysis of raw and purified data showed that the acrophases from both skin sites were much more variable and up to 8 h later than were those from the rectum (particularly if exercise had been taken), even though the acrophases from the two skin sites were similar to each other. For rectal temperature, there was an increase in the size of the masking effect as activity progressed through the sequence: sitting, standing or walking, exercising. In contrast, for both chest wall and axillary temperatures, although sitting produced masking effects similar to those for rectal temperature, masking effects due to standing or walking and exercising were much smaller, and sometimes they were even less than the masking effects due to sitting. These results indicate that our measurements of cutaneous temperature did not act as a substitute for rectal temperature, particularly when the subject was physically active rather than sedentary.     

A Collection of Popcorn as a Reservoir of Genes for the Generation of Lineages

In the present study, we analyze the genetic structure and diversity among accessions of popcorn obtained from the CIMMYT International Maize and Wheat Improvement Center that represent the diversity available for current use by breeding programs. The main objectives were to identify SSR loci that were the best indicators of genetic diversity, to measure the genetic diversity within popcorn genotypes, and to analyze the genetic structure of the promising populations destined for use in breeding programs. The mean gene diversity of all SSR loci was 0.6352. An extremely high population differentiation level was detected (F _st  = 0.3152) with F _st for each locus ranging from 0.1125 (Umc1229) to 0.4870 (Umc1755). Analyzing the genetic structure of eight popcorn accessions was especially important for identifying both SSR loci with high levels of heterozygosity and genotypes showing high heterozygosity (Boya462 and Arzm13 050). This analysis should be the medium and long-term selection goal for the generation of inbred lines and the future production of new cultivars. Plant accessions Arzm05 083, Arzm13 050, and Urug298 may also be useful varieties that exhibit important agronomic characteristics that can be used through crosses to broaden the genetic basis of popcorn.     

Enhanced Disease Susceptibility 1 and Salicylic Acid Act Redundantly to Regulate Resistance Gene-Mediated Signaling

Resistance (R) protein–associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non–race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA–synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.