Prophages play a significant role in bacterial evolution by shaping genomic diversity, virulence, and host adaptation. This study investigated the prophage composition of 109 clinical Staphylococcus aureus isolates obtained from four tertiary care hospitals in Mexico City and compared these results with data from 993 global genomes. Prophages were present in 97% of local isolates. Consistently, analysis of the global genome collection revealed a 99% prevalence, supporting the near ubiquity of prophages in S. aureus. Analysis identified 216 genomic regions corresponding to the predicted prophages within the Mexican S. aureus isolates. A substantial fraction (19%) of the predicted prophages was identified as phage-inducible chromosomal islands (PICIs), such as SaPI1, SaPI2, and SaPIpt1028-like elements. These PICIs encoded anti-phage defense systems (63%) and virulence genes (27%). Experimental treatment with mitomycin C induced 17 temperate phages, of which 12 demonstrated functional activity and the ability to undergo lysogenic-lytic switching and reinfection. No virulence or antibiotic resistance genes were identified in these temperate phages. Conversely, several uninduced prophages coincided with the virulence determinants. These findings highlight the complexity of the S. aureus mobilome, characterized by distinct functional profiles and heterogeneous mobilization capabilities, which may influence the dissemination of virulence factors.IMPORTANCEStaphylococcus aureus is a significant hospital-associated pathogen whose evolutionary processes are shaped by mobile genetic elements, including prophages and phage-inducible chromosomal islands (PICIs). While computational analyses suggest that nearly all S. aureus genomes contain prophages, our findings indicate that only a subset is inducible following mitomycin C treatment. These temperate phages do not possess virulence genes; however, other predicted prophages are associated with virulence factors. Additionally, we identified numerous predicted prophages as PICIs, which harbored anti-phage defense mechanisms and toxins. This study highlights the intricate mobilome of S. aureus and the various strategies that contribute to its horizontal gene transfer and pathogenic evolution.

Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus are major causes of hospital-acquired infections worldwide. However, their genomic features remain underexplored in many regions, particularly in low- and middle-income countries (LMICs). Here, we investigated the genomic diversity, antibiotic resistance, and virulence traits of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus isolates from three tertiary care hospitals in Mexico City. A total of 101 isolates collected between 2006 and 2019 from newborns and adults with diverse infections were analyzed using whole-genome sequencing. MRSA isolates were restricted to clonal complexes CC5 and CC8, whereas MSSA strains displayed a broader diversity across multiple lineages. A distinct clade of CC8-MRSA isolates identified in 2013 among newborns was closely related to the USA300 lineage but carried SCCmec IVc without the Panton-Valentine leukocidin genes and the COMER island. Similarly, a localized cluster of CC5-MSSA isolates from neonates in 2012 showed genomic variations largely driven by prophage-associated elements. MRSA strains carried a higher burden of resistance genes, including fluoroquinolone-associated mutations, whereas MSSA isolates exhibited greater heterogeneity in virulence genes and spa types. Both groups shared a conserved virulence gene repertoire; however, variations in the virulence genes highlighted lineage-specific pathogenic features.IMPORTANCEMRSA and MSSA S. aureus genomes are still largely unstudied in Mexico although they are frequently found in hospitals. This study provides a long-term genomic analysis of MRSA and MSSA isolates from three hospitals in Mexico. Our findings revealed the presence of S. aureus international clones of clonal complexes CC5 and CC8 over a decade, broader diversity in MSSA, and localized clonal transmission within hospitals. The contrasting resistance and virulence profiles of MRSA and MSSA clones underscore the need for genomic surveillance frameworks to inform infection control and antibiotic stewardship in healthcare settings.

Prophages are key drivers of bacterial evolution, genomic variability, virulence, and ecological adaptation. Here, we examined the prophage diversity of 109 Staphylococcus aureus clinical isolates from four tertiary care hospitals in Mexico City, integrating comparative analyses with 993 international genomes. Prophages were detected in 97% of the local strains, mirroring the near-ubiquitous presence (99%) observed globally. We identified 216 genomic regions encoding putative prophage functional elements. Using Mitomycin C induction, we recovered 17 temperate phages, 12 of which were functionally active and capable of lysogeny-lysis switching and reinfection. Induced phages lacked virulence or antibiotic resistance genes, in contrast to 55% of the predicted prophages in local S. aureus isolates that harbored known virulence factors. Moreover, 19% of the predicted prophages were phage-inducible chromosomal islands (PICIs) related to SaPI1, SaPI2, and SaPIpt1028. These PICIs encode anti-phage defense systems (63%) and virulence genes (27%), such as tsst-1 and hlb . Our findings reveal a complex prophage and PICI landscape in S. aureus circulating in Mexican hospitals, with implications for phage– host dynamics, horizontal gene transfer, and the evolution of pathogenic potential across geographically diverse populations. Importance Staphylococcus aureus is a major hospital pathogen whose evolution is driven by mobile genetic elements including prophages and phage-inducible chromosomal islands (PICIs). Although computational analyses predict prophages in nearly all S. aureus genomes, we demonstrate that only a small fraction is functionally active. Through experimental induction of clinical isolates from Mexican hospitals, we recovered active prophages that surprisingly lacked virulence genes, in contrast to the predicted prophages that frequently harbor virulence factors. Importantly, we discovered that many predicted “prophages” are PICIs carrying anti-phage defenses and toxins. These findings challenge the current prophage prediction methods and reveal the complex prophage-PICI landscape that shapes horizontal gene transfer and pathogenic evolution in clinical S. aureus populations.

Microarray data can be used to identify co-expressed genes that may play a role in the same biological process. An enormous amount of gene expression data is currently available online in repositories for several organisms. Therefore, the analysis and interpretation of this information could help us organize, make sense and notice the knowledge that it contains which represents a major challenge in the postgenomic era. Here, we grouped genes of Escherichia coli K-12 via expression data to infer meaningful transcriptional regulatory information, namely functionally relevant clusters, which were validated with curated transcriptional regulatory information in RegulonDB. Our method is based on the assumption that co-expressed genes reflect functional units provided by their genetic structure, i.e. the arrangement of the genes, their regulation, and their participation in defined biological processes. We showed that co-expressed genes are involved in the same metabolic pathways and type of regulation (through transcription factors, σ-factors, allosteric regulation or microRNA regulation) and are helpful for identifying novel transcriptional regulatory interactions.

An enormous amount of gene expression data is currently available online in repositories for several organisms. Microarray data can be used to identify co-expressed genes that may be involved in the same biological process. Therefore, the analysis and interpretation of this information could help organize and understand the knowledge it contains, representing a major challenge in the post-genomic era. Here, we grouped genes ofEscherichia coliK-12 using expression data to infer meaningful transcriptional regulatory information. Our method assumes that co-expressed genes reflect functional units, as evidenced by their genetic structure, including gene arrangement, regulation, and participation in defined biological processes. These functionally linked clusters were validated with curated transcriptional regulatory information from RegulonDB. From 907 growth conditions, 420 clusters were formed involving 1674 genes. Clusters contained from 2 to 64 genes. We found that co-expressed genes participate in related metabolic pathways and share similar types of regulation (through transcription factors,σ-factors, allosteric regulation, or micro-RNA regulation). This study is helpful for identifying novel transcriptional regulatory interactions.

Stenotrophomonas is a bacterial genus that can be found in various environments, such as water, soil, and clinical samples. Due to their high genetic and phenotypic diversity, it is difficult to properly identify and classify all isolates. The COVID-19 pandemic caused an increase in nosocomial infections, which played a major role in the high mortality rate among patients in intensive care. This is the first report of the identification of S. geniculata as a nosocomial opportunistic pathogen isolated from a patient with COVID-19. Their genome was isolated, sequenced, and assembled, and it consists of 4,488,090 bp in 24 contigs, 4,103 coding sequences, and a G+C content of 66.58%.

In this study, we examined the role of the lipopolysaccharide (LPS) core of Rhizobium etli in facilitating the adsorption and infection of phages with broad host range. When the plasmid-encoded LPS biosynthesis genes, wreU and wreV, were disrupted, distinct and contrasting effects on phage infection were observed. The wreU mutant strains exhibited wild-type adsorption and infection properties, whereas the wreV mutant demonstrated resistance to phage infection, but retained the capacity to adsorb phages. Complementation of the wreV mutant strains with a recombinant plasmid containing the wreU and wreV, restored the susceptibility to the phages. However, the presence of this recombinant plasmid in a strain devoid of the native lps-encoding plasmid was insufficient to restore phage susceptibility. These results suggest that the absence of wreV impedes the proper assembly of the complete LPS core, potentially affecting the formation of UDP-KdgNAg or KDO precursors for the O-antigen. In addition, a protein not yet identified, but residing in the native lps-encoding plasmid, may be necessary for complete phage infection.

The genus Campylobacter groups 32 Gram-negative bacteria species, several being zoonotic pathogens and a major cause of human gastroenteritis worldwide. Antibiotic resistant Campylobacter is considered by the World Health Organization as a high priority pathogen for research and development of new antibiotics. Genetic elements related to antibiotic resistance in the classical C. coli and C. jejuni species, which infect humans and livestock, have been analyzed in numerous studies, mainly focused on local geographical areas. However, the presence of these resistance determinants in other Campylobacter species, as well as in C. jejuni and C. coli strains distributed globally, remains poorly studied. In this work, we analyzed the occurrence and distribution of antibiotic resistance factors in 237 Campylobacter closed genomes available in NCBI, obtained from isolates collected worldwide, in different dates, from distinct hosts and comprising 22 Campylobacter species. Our data revealed 18 distinct genetic determinants, genes or point mutations in housekeeping genes, associated with resistance to antibiotics from aminoglycosides, β-lactams, fluoroquinolones, lincosamides, macrolides, phenicols or tetracyclines classes, which are differentially distributed among the Campylobacter species tested, on chromosomes or plasmids. Three resistance determinants, the blaOXA–493 and blaOXA–576 genes, putatively related to β-lactams resistance, as well as the lnu(AN2) gene, putatively related to lincosamides resistance, had not been reported in Campylobacter; thus, they represent novel determinants for antibiotic resistance in Campylobacter spp., which expands the insight on the Campylobacter resistome. Interestingly, we found that some of the genetic determinants associated with antibiotic resistance are Campylobacter species-specific; e.g., the blaOXA–493 gene and the T86V mutation in gyrA were found only in the C. lari group, whereas genes associated with aminoglycosides resistance were found only in C. jejuni and C. coli. Additional analyses revealed how are distributed the resistance and multidrug resistance Campylobacter genotypes assessed, with respect to hosts, geographical locations, and collection dates. Thus, our findings further expand the knowledge on the factors that can determine or favor the antibiotic resistance in Campylobacter species distributed globally, which can be useful to choose a suitable antibiotic treatment to control the zoonotic infections by these bacteria.

Staphylococcus epidermidis is a human commensal and opportunistic pathogen worldwide distributed. To ascertain which pathogenic S. epidermidis clones are circulating in a local tertiary hospital setting, we sequenced the complete genomes of 17 S. epidermidis isolates obtained from neonatal infections at a Hospital Care Unit in México City. Genomic comparisons between S. epidermidis isolates revealed high pairwise whole genome nucleotide identities of about 97% to 99% and essentially a clonal structure. We inferred eight Multilocus Sequence Types (MLST´s), six of them of worldwide distribution, and two showing allelic variants, not in MLST databases. The profile of virulence includes genes involved in biofilm and modulin formation; most of the strains are multi-resistant to methicillin and several other beta-lactams, fluoroquinolones, and macrolides. Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer. Rates of recombination between S. epidermidis strains were more frequent than the mutation rate and affected the whole genome. Therefore, recombination properties shape the population structure of local nosocomial S. epidermidis strains, formed by pathogenic and probably, non-pathogenic clones.

Staphylococcus epidermidis is a human commensal and pathogen worldwide distributed.In this work, we surveyed for multi-resistant S. epidermidis strains in eight years at a children's health-care unit in México City.Multidrug-resistant S. epidermidis were present in all years of the study, including resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides.To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17 S. epidermidis isolates.Whole-genome nucleotide identities between all the pairs of S. epidermidis strains were about 97% to 99%.We inferred a clonal structure and eight Multilocus Sequence Types (MLST´s) in the S. epidermidis sequenced collection.The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs).Half of the S. epidermidis analyzed lacked the ica operon for biofilm formation.Likely, they are commensal S. epidermidis strains but multi-antibiotic resistant.Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer.Rates of recombination between S. epidermidis strains were more prevalent than the mutation rate and affected the whole genome.Therefore, the multidrug resistance, independently of the pathogenic traits, might explain the persistence of specific highly adapted S. epidermidis clonal lineages in nosocomial settings.

The bacterial genus Rhizobium comprises diverse symbiotic nitrogen-fixing species associated with the roots of plants in the Leguminosae family. Multiple genomic clusters defined by whole genome comparisons occur within Rhizobium, but their equivalence to species is controversial. In this study we investigated such genomic clusters to ascertain their significance in a species phylogeny context. Phylogenomic inferences based on complete sets of ribosomal proteins and stringent core genome markers revealed the main lineages of Rhizobium. The clades corresponding to R. etli and R. leguminosarum species show several genomic clusters with average genomic nucleotide identities (ANI > 95%), and a continuum of divergent strains, respectively. They were found to be inversely correlated with the genetic distance estimated from concatenated ribosomal proteins. We uncovered evidence of a Rhizobium pangenome that was greatly expanded, both in its chromosomes and plasmids. Despite the variability of extra-chromosomal elements, our genomic comparisons revealed only a few chromid and plasmid families. The presence/absence profile of genes in the complete Rhizobium genomes agreed with the phylogenomic pattern of species divergence. Symbiotic genes were distributed according to the principal phylogenomic Rhizobium clades but did not resolve genome clusters within the clades. We distinguished some types of symbiotic plasmids within Rhizobium that displayed different rates of synonymous nucleotide substitutions in comparison to chromosomal genes. Symbiotic plasmids may have been repeatedly transferred horizontally between strains and species, in the process displacing and substituting pre-existing symbiotic plasmids. In summary, the results indicate that Rhizobium genomic clusters, as defined by whole genomic identities, might be part of a continuous process of evolutionary divergence that includes the core and the extrachromosomal elements leading to species formation.

Staphylococcus epidermidis is a human commensal and pathogen worldwide distributed. In this work, we surveyed for multi-resistant S. epidermidis strains in eight years at a children health-care unit in México City. Multidrug-resistant S. epidermidis were present in all years of the study. Resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides were included. To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17 S. epidermidis isolates. Whole-genome nucleotide identities between all the pairs of S. epidermidis strains were about 97% to 99%. We inferred a clonal structure and eight Multilocus Sequence Types (MLST´s) in the S. epidermidis sequenced collection. The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs). However, half of the S. epidermidis analyzed lacked the icaoperon for biofilm formation. Likely, they are commensal S. epidermidis strains but multi-antibiotic resistant. Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer. Rates of recombination between S. epidermidis strains were more prevalent than the mutation rate and affected the whole genome. Therefore, the multidrug-resistance, independently of the pathogenic traits, might explain the persistence of specific highly adapted S. epidermidis clonal lineages in nosocomial settings.

Staphylococcus epidermidis is a human commensal and pathogen worldwide distributed.In this work, we surveyed for multi-resistant S. epidermidis strains in eight years at a children's health-care unit in México City.Multidrug-resistant S. epidermidis were present in all years of the study, including resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides.To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17 S. epidermidis isolates.Whole-genome nucleotide identities between all the pairs of S. epidermidis strains were about 97% to 99%.We inferred a clonal structure and eight Multilocus Sequence Types (MLST´s) in the S. epidermidis sequenced collection.The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs).Half of the S. epidermidis analyzed lacked the ica operon for biofilm formation.Likely, they are commensal S. epidermidis strains but multi-antibiotic resistant.Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer.Rates of recombination between S. epidermidis strains were more prevalent than the mutation rate and affected the whole genome.Therefore, the multidrug resistance, independently of the pathogenic traits, might explain the persistence of specific highly adapted S. epidermidis clonal lineages in nosocomial settings.

Staphylococcus epidermidisis a human commensal and pathogen worldwide distributed. In this work, we surveyed for multi-resistantS. epidermidisstrains in eight years at a children’s health-care unit in México City. Multidrug-resistantS. epidermidiswere present in all years of the study, including resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides. To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17S. epidermidisisolates. Whole-genome nucleotide identities between all the pairs of S. epidermidis strains were about 97% to 99%. We inferred a clonal structure and eight Multilocus Sequence Types (MLSTs) in theS. epidermidissequenced collection. The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs). Half of theS. epidermidisanalyzed lacked the ica operon for biofilm formation. Likely, they are commensalS. epidermidisstrains but multi-antibiotic resistant. Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer. Rates of recombination betweenS. epidermidisstrains were more prevalent than the mutation rate and affected the whole genome. Therefore, the multidrug resistance, independently of the pathogenic traits, might explain the persistence of specific highly adaptedS. epidermidisclonal lineages in nosocomial settings.

Staphylococcus epidermidis is a human commensal and pathogen worldwide distributed. In this work, we surveyed for multi-resistant S. epidermidis strains in eight years at a children health-care unit in México City. Multidrug-resistant S. epidermidis were present in all years of the study. Resistance to methicillin, beta-lactams, fluoroquinolones, and macrolides were included. To understand the genetic basis of antibiotic resistance and its association with virulence and gene exchange, we sequenced the genomes of 17 S. epidermidis isolates. Whole-genome nucleotide identities between all the pairs of S. epidermidisstrains were about 97% to 99%. We inferred a clonal structure and eight Multilocus Sequence Types (MLST ́s) in the S. epidermidis sequenced collection. The profile of virulence includes genes involved in biofilm formation and phenol-soluble modulins (PSMs). However, half of the S. epidermidis analyzed lacked the icaoperon for biofilm formation. Likely, they are commensal S. epidermidis strains but multi-antibiotic resistant. Uneven distribution of insertion sequences, phages, and CRISPR-Cas immunity phage systems suggest frequent horizontal gene transfer. Rates of recombination between S. epidermidis strains were more prevalent than the mutation rate and affected the whole genome. Therefore, the multidrug resistance, independently of the pathogenic traits, might explain the persistence of specific highly adapted S. epidermidis clonal lineages in nosocomial settings.

In RegulonDB, for over 25 years, we have been gathering knowledge by manual curation from original scientific literature on the regulation of transcription initiation and genome organization in transcription units of the Escherichia coli K-12 genome. This unit describes six basic protocols that can serve as a guiding introduction to the main content of the current version (v9.4) of this electronic resource. These protocols include general navigation as well as searching for specific objects such as genes, gene products, transcription units, promoters, transcription factors, coexpression, and genetic sensory response units or GENSOR Units. In these protocols, the user will find an initial introduction to the concepts pertinent to the protocol, the content obtained when performing the given navigation, and the necessary resources for carrying out the protocol. This easy-to-follow presentation should help anyone interested in quickly seeing all that is currently offered in RegulonDB, including position weight matrices of transcription factors, coexpression values based on published microarrays, and the GENSOR Units unique to RegulonDB that offer regulatory mechanisms in the context of their signals and metabolic consequences. © 2018 by John Wiley & Sons, Inc.

The whole-genome sequences of three strains of Rhizobium gallicum reported here support the concept that the distinct nodulation host ranges displayed by the symbiovars gallicum and phaseoli can be largely explained by different symbiotic plasmids.

The evolution of bacterial pathogenicity, heavily influenced by horizontal gene transfer, provides new virulence factors and regulatory connections that alter bacterial phenotypes. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) are chromosomal regions that were acquired at different evolutionary times and are essential for Salmonella virulence. In the intestine of mammalian hosts, Salmonella expresses the SPI-1 genes that mediate its invasion to the gut epithelium. Once inside the cells, Salmonella down-regulates the SPI-1 genes and induces the expression of the SPI-2 genes, which favor its intracellular replication. The mechanism by which the invasion machinery is deactivated following successful invasion of host cells is not known. Here, we show that the SPI-2 encoded transcriptional regulator SsrB, which positively controls SPI-2, acts as a dual regulator that represses expression of SPI-1 during intracellular stages of infection. The mechanism of this SPI-1 repression by SsrB was direct and acts upon the hilD and hilA regulatory genes. The phenotypic effect of this molecular switch activity was a significant reduction in invasion ability of S. enterica serovar Typhimurium while promoting the expression of genes required for intracellular survival. During mouse infections, Salmonella mutants lacking SsrB had high levels of hilA (SPI-1) transcriptional activity whereas introducing a constitutively active SsrB led to significant hilA repression. Thus, our results reveal a novel SsrB-mediated mechanism of transcriptional crosstalk between SPI-1 and SPI-2 that helps Salmonella transition to the intracellular lifestyle.

A wide variety of Salmonella enterica serovars cause intestinal and systemic infections to humans and animals. Salmonella Patogenicity Island 1 (SPI-1) is a chromosomal region containing 39 genes that have crucial virulence roles. The AraC-like transcriptional regulator HilD, encoded in SPI-1, positively controls the expression of the SPI-1 genes, as well as of several other virulence genes located outside SPI-1. In this study, we applied a clustering method to the global gene expression data of S. enterica serovar Typhimurium from the COLOMBOS database; thus genes that show an expression pattern similar to that of SPI-1 genes were selected. This analysis revealed nine novel genes that are co-expressed with SPI-1, which are located in different chromosomal regions. Expression analyses and protein-DNA interaction assays showed regulation by HilD for six of these genes: gtgE, phoH, sinR, SL1263 (lpxR) and SL4247 were regulated directly, whereas SL1896 was regulated indirectly. Interestingly, phoH is an ancestral gene conserved in most of bacteria, whereas the other genes show characteristics of genes acquired by Salmonella. A role in virulence has been previously demonstrated for gtgE, lpxR and sinR. Our results further expand the regulon of HilD and thus identify novel possible Salmonella virulence genes.