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Antimicrobial resistance (AMR) is currently one of the most concerning threats in public health. The efforts to tackle the problem require a global One Health approach, using multidisciplinary approaches and a thorough understanding of the topic both by the general public and the experts. Currently, the lack of a shared mental model of the problem, the absence of a sense of responsibility amongst the different actors and a deficient education on the topic burden the efforts to slow down the emergency and spread of antimicrobial resistant infections. We here propose a multidisciplinary approach to tackle the AMR problem, taking into consideration not only the input from the biological and medical sciences but also the input from the social sciences. Specifically, we suggest strategies from education and psychology to increase awareness about antimicrobial resistance and to implement more effective interventions. Finally, we advocate for a comprehensive and a solidaristic model as the only solution for a problem which knows no borders. As such, political will and international cooperation will be key to achieve the desired change in antibiotic resistance trend.

Imaging through turbid media leads to a great loss of information decreasing the image quality. This loss of quality is due to the light interaction with the medium, since part of the light will pass through it, but another part will be scattered generating a blurred image pattern. In this work we try to decrease this problem by adding an absorbent, eliminating part of the scattered radiation responsible for the turbidity. With this aim, we use graphene nanoplates as an absorbent and we also compare the experimental results with black carbon powder.

Multidrug resistance (MDR) plasmids drive the spread of antibiotic resistance between bacterial lineages. The immediate impact of MDR plasmid acquisition on fitness and cellular processes varies among bacterial lineages, but how the evolutionary processes enabling the genomic integration of MDR plasmids vary is less well understood, particularly in clinical pathogens. Using diverse Escherichia coli lineages experimentally evolved for ∼700 generations, we show that the evolutionary response to gaining the MDR plasmid pLL35 was dominated by chromosomal mutations affecting metabolic and regulatory functions, with both strain-specific and shared mutational targets. The expression of several of these functions, such as anaerobic metabolism, is known to be altered upon acquisition of pLL35. Interactions with resident mobile genetic elements, notably several IS-elements, potentiated parallel mutations, including insertions upstream of hns that were associated with its upregulation and the downregulation of the plasmid-encoded extended-spectrum beta-lactamase gene. Plasmid parallel mutations targeted conjugation-related genes, whose expression was also commonly downregulated in evolved clones. Beyond their role in horizontal gene transfer, plasmids can be an important selective force shaping the evolution of bacterial chromosomes and core cellular functions.

By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multispecies communities are poorly understood. Here, we show, using experimental multispecies communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to ‘the dilution effect’; an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from coinfected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions reducing the rate of horizontal transmission.

Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversifica-tion. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plas-mid coexistence was unstable despite compensatory evolution. Positive selection dis-criminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids. IMPORTANCE Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful func-tion, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during peri-ods of strong selection for the useful plasmid-encoded function.

Plasmids play a key role in bacterial evolution by transferring adaptive functions between lineages that often enable invasion of new niches, including driving the spread of antibiotic resistance genes. Fitness costs of plasmid acquisition arising from the disruption of cellular processes could limit the spread of multidrug resistance plasmids., Multidrug-resistant (MDR) Escherichia coli strains are a major global threat to human health, wherein multidrug resistance is primarily spread by MDR plasmid acquisition. MDR plasmids are not widely distributed across the entire E. coli species, but instead are concentrated in a small number of clones. Here, we test if diverse E. coli strains vary in their ability to acquire and maintain MDR plasmids and if this relates to their transcriptional response following plasmid acquisition. We used strains from across the diversity of E. coli strains, including the common MDR lineage sequence type 131 (ST131) and the IncF plasmid pLL35, carrying multiple antibiotic resistance genes. Strains varied in their ability to acquire pLL35 by conjugation, but all were able to stably maintain the plasmid. The effects of pLL35 acquisition on cefotaxime resistance and growth also varied among strains, with growth responses ranging from a small decrease to a small increase in growth of the plasmid carrier relative to the parental strain. Transcriptional responses to pLL35 acquisition were limited in scale and highly strain specific. We observed transcriptional responses at the operon or regulon level—possibly due to stress responses or interactions with resident mobile genetic elements (MGEs). Subtle transcriptional responses consistent across all strains were observed affecting functions, such as anaerobic metabolism, previously shown to be under negative frequency-dependent selection in MDR E. coli. Overall, there was no correlation between the magnitudes of the transcriptional and growth responses across strains. Together, these data suggest that fitness costs arising from transcriptional disruption are unlikely to act as a barrier to dissemination of this MDR plasmid in E. coli. IMPORTANCE Plasmids play a key role in bacterial evolution by transferring adaptive functions between lineages that often enable invasion of new niches, including driving the spread of antibiotic resistance genes. Fitness costs of plasmid acquisition arising from the disruption of cellular processes could limit the spread of multidrug resistance plasmids. However, the impacts of plasmid acquisition are typically measured in lab-adapted strains rather than natural isolates, which act as reservoirs for the maintenance and transmission of plasmids to clinically relevant strains. Using a clinical multidrug resistance plasmid and a diverse collection of E. coli strains isolated from clinical infections and natural environments, we show that plasmid acquisition had only limited and highly strain-specific effects on bacterial growth and transcription under laboratory conditions. These findings suggest that fitness costs arising from transcriptional disruption are unlikely to act as a barrier to transmission of this plasmid in natural populations of E. coli.

Summary A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to the introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to higher titer as pseudotyped virions. In infected individuals G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, although not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus, and support continuing surveillance of Spike mutations to aid in the development of immunological interventions., Highlights: - A SARS-CoV-2 variant with Spike G614 has replaced D614 as the dominant pandemic form - The consistent increase of G614 at regional levels may indicate a fitness advantage - G614 is associated with lower RT PCR Ct’s, suggestive of higher viral loads in patients - The G614 variant grows to higher titers as pseudotyped virions

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed “sub-genomic RNAs”. sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5’ UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5’ end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/− cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

Bacteria exist in complex communities that include not only many other bacterial species but also diverse mobile genetic elements which accelerate bacterial evolution via horizontal gene transfer. How multiple mobile genetic elements interact in bacterial populations and how this affects plasmid dynamics remains poorly understood. We experimentally evolved populations of Pseudomonas fluorescens SBW25 either singly-infected or co-infected with two conjugative mercury resistance plasmids either with or without positive selection (i.e. addition of Hg(II)). We show that co-infection led to higher levels of mercury resistance in the bacterial population in the absence of positive selection. Consistent with this, in the absence of positive selection the plasmids could stably coexist within bacterial cells resulting in the maintenance co-infection. By contrast, with positive selection, plasmid coexistence was destabilised, leading to the dominance of a single plasmid in several replicate bacterial populations. Plasmid co-infection appears to alter the trajectory of compensatory evolution to ameliorate the cost of plasmid carriage and may have selected for alternative mechanisms compared to singly-infected populations. Stable plasmid co-infection without positive selection for plasmid-encoded traits suggests that environments where plasmids are useless may be hot-spots for genomic innovation via plasmid-plasmid recombination.

Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR , an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB , and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones.

Plasmid pB1000 is a mobilizable replicon bearing the bla ROB-1 β-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000′, in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae , giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 μg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae . Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae .

Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 β-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae .

Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli , corresponding to the genotypes rsmF + , Δ rsmF , rsmF + rmtC + , and Δ rsmF rmtC + . When analyzed for the antimicrobial resistance pattern, the Δ rsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli . Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.