Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.

The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, πVLC5 and πVLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage πVLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage πVLC6 is conferred by a second, divergent depolymerase. Phage πVLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications.

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The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae.

16 Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a 17 frequently used approach in microbial genomics. However, the choice of a reference may represent a 18 source of errors that may affect subsequent analyses such as the detection of single nucleotide 19 polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference 20 choice on short-read sequence data from five clinically and epidemiologically relevant bacteria 21 (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa 22 and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic 23 diversity of these species were selected as reference sequences, and read alignment statistics, SNP 24 calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the 25 mapping reference. The choice of different reference genomes proved to have an impact on almost all 26 the parameters considered in the five species. In addition, these biases had potential epidemiological 27 implications such as including/excluding isolates of particular clades and the estimation of genetic 28 distances. These findings suggest that the single reference approach might introduce systematic errors 29 during mapping that affect subsequent analyses, particularly for data sets with isolates from 30 genetically diverse backgrounds. In any case, exploring the effects of different references on the final 31 conclusions is highly recommended. 32 33 Author summary 34 Mapping consists in the alignment of reads (i.e., DNA fragments) obtained through high-throughput 35 genome sequencing to a previously assembled reference sequence. It is a common practice in genomic 36 studies to use a single reference for mapping, usually the 'reference genome' of a species -a high-37 quality assembly. However, the selection of an optimal reference is hindered by intrinsic intra-species 38 genetic variability, particularly in bacteria. Biases/errors due to reference choice for mapping in 39 bacteria have been identified. These are mainly originated in alignment errors due to genetic 40 differences between the reference genome and the read sequences. Eventually, they could lead to 41 misidentification of variants and biased reconstruction of phylogenetic trees (which reflect ancestry 42 between different bacterial lineages). However, a systematic work on the effects of reference choice 43 in different bacterial species is still missing, particularly regarding its impact on phylogenies. This 44 work intended to fill that gap. The impact of reference choice has proved to be pervasive in the five 45 bacterial species that we have studied and, in some cases, alterations in phylogenetic trees could lead 46 to incorrect epidemiological inferences. Hence, the use of different reference genomes may be 47 prescriptive to assess the potential biases of mapping. 48 49 Introduction 50 The development and increasing availability of high-throughput sequen...

The misuse of antibiotics is leading to the emergence of multidrug-resistant (MDR) bacteria, and in the absence of available treatments, this has become a major global threat. In the middle of the recent severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic, which has challenged the whole world, the emergence of MDR bacteria is increasing due to prophylactic administration of antibiotics to intensive care unit patients to prevent secondary bacterial infections. This is just an example underscoring the need to seek alternative treatments against MDR bacteria. To this end, phage therapy has been proposed as a promising tool. However, further research in the field is mandatory to assure safety protocols and to develop appropriate regulations for its use in clinics. This requires investing more in such non-conventional or alternative therapeutic approaches, to develop new treatment regimens capable of reducing the emergence of MDR and preventing future global public health concerns that could lead to incalculable human and economic losses.

Recombination is one of the main processes shaping the evolution of HIV-1, with relevant consequences for its epidemiology. In fact, Circulating and Unique Recombinant Forms (CRFs and URFs) cause 23% of current infections. The routine analyses of antiretroviral resistance yield partial pol gene sequences that can be exploited for molecular epidemiology surveillance but also to study viral diversity and to detect potential recombinant samples. Among the pol sequences derived from a large sample dataset from the Comunitat Valenciana (Spain), we identified nine putative recombinant samples. We aimed at fully characterizing these samples and performing a detailed analysis of the corresponding recombination events. We obtained nearly full-genome sequences and used jpHMM and RDP4 to detect and characterize recombinant fragments. We assessed the confidence of these inferences by likelihood mapping and phylogenetic placement with topology congruence tests. Next, we performed a phylogenetic analysis of each putative recombinant fragment to determine its relationships to previously described recombinant forms. We found that two samples related to CRF44_BF whereas the rest corresponded to new URFs (two URF_AD, one URF_BG that can constitute a new CRF resulting from subtype B and CRF24_BG, and two URF_cpx composed of A, G, K, H, and J subtypes). These URFs have a complex recombination pattern that cannot be determined accurately. They seem to have arisen by successive recombination events among lineages, including other CRFs. Our results highlight the usefulness of routine surveillance analysis for the detection of new HIV-1 recombination forms and, at the same time, the need for full-genome sequencing and recombination detection guidelines to properly characterize this complex process.

At a time when antimicrobial resistance has become one of the biggest concerns worldwide, the emergence of novel alleles and extremely drug-resistant plasmids is a threat to public health worldwide, especially when they produce carbapenem resistance in one of the most problematic pathogens, such as Klebsiella pneumoniae . We used genomic epidemiology to describe the emergence of a novel NDM-23 allele and identify it in a MDR plasmid that has disseminated through a K. pneumoniae ST437 clone in several hospitals in Spain.