Acinetobacter baumannii and Pseudomonas aeruginosa are the most relevant Gram-negative bacteria associated with hospital and opportunistic infections. This study aimed to evaluate the dynamics of drug-resistant A . baumannii and P . aeruginosa and biofilm formers from two public hospitals in northeastern Brazil. One hundred isolates (35 from A. baumannii and 65 from P. aeruginosa ) were identified using the automated Vitek ^® 2 Compact method (bioMérieux) and confirmed using the MALDI-TOF (MS) mass spectrometry technique. Molecular experiments were performed by polymerase chain reaction (PCR) to detect the frequency of bla _KPC , bla _IMP , bla _VIM , and bla _SHV genes. The biofilm formation potential was evaluated using crystal violet in Luria Bertani Miller and trypticase soy broth culture media under the following conditions: at standard concentration, one quarter (25%) of the standard concentration and supplemented with 1% glucose. In addition, the genetic diversity of the isolates was verified by the ERIC-PCR technique. Isolates presented distinct resistance profiles with a high level of beta-lactam resistance. The highest index of genes detected was bla _KPC (60%), followed by bla _SHV (39%), bla _VIM (8%), and bla _IMP (1%). All the isolates were sensitive to the polymyxins tested and formed biofilms at different intensities. Twelve clones of A . baumannii and eight of P . aeruginosa were identified, of which few were indicative of intra- and interhospital dissemination. This study reveals the dispersion dynamics of these isolates in the hospital environment. The results demonstrate the importance of monitoring programs to combat the spread of these pathogens.

The CRISPR/Cas system defends bacteria and archaea against invasive pathogens, such as phages, establishing an immunological memory from this interaction. Pseudomonas aeruginosa, an opportunistic pathogen, represents a significant public health concern due to its multidrug resistance. This study conducted a molecular epidemiological analysis of clinical isolates of Pseudomonas aeruginosa in Brazil using multilocus sequence typing (MLST) and characterization of CRISPR/Cas system. Most P. aeruginosa isolates harbored the type I-F CRISPR/Cas system (83%), with a subset also exhibiting the type I-E system. Additionally, some isolates presented incomplete CRISPR/Cas systems in their secondary loci. Notably, the isolate Pae93 exhibited a genetic composition rich in phage-related proteins proximal to the orphan CRISPR locus. The identification and characterization of spacer sequences, including previously undocumented ones, revealed a remarkable diversity of predatory mobile genetic elements (MGEs) among the P. aeruginosa isolates studied. The spacer sequences were incorporated into the MGE library. Additionally, the study identified the existence of prophages and anti-CRISPR genes. Two new sequence types (STs 3383 and 3384) were identified and added to the PubMLST database. No discernible correlation was established between the observed STs and the previously delineated CRISPR genotypes. However, the CRISPR system remains valuable for elucidating specific interactions between microorganisms and MGEs. The Brazilian population of clinical P. aeruginosa isolates was shown to be genetically heterogeneous with a non-clonal distribution, as revealed by MLST analysis. The presence of high-risk clones, such as ST 244 and ST 235, underscores the importance of robust epidemiological surveillance and infection control strategies for P. aeruginosa, especially in healthcare settings. This study significantly contributes to the understanding of the molecular epidemiology of these isolates in Brazil.

Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage–host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage–host relationship. The dual role of prophages—as vectors of antimicrobial resistance and as potential therapeutic agents—reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.

Staphylococcus aureus is a frequent cause of infections worldwide. Methicillin-resistant S. aureus (MRSA) is one of the main causes of Gram-positive infections, and methicillin-susceptible strains (MSSA) primarily colonize and infect community hosts. Multiple virulence factors are involved, with toxins playing a significant role in several diseases. In this study, we assess the prevalence of toxin genes in 89 S. aureus clinical isolates (31 MRSA and 58 MSSA). We evaluated the discriminatory power of the association of internal transcribed spacer-PCR (ITS-PCR) and 3’- end coa gene ( coa-PCR) when compared with other more commonly used and costly techniques. The isolates showed a high level of genetic diversity, and toxins were found in all the isolates. While most toxin classes displayed no statistically significant correlations and were equally distributed in isolates regardless of their resistance status, classic enterotoxins ( sea-see) showed a positive correlation with MSSA isolates. The combination of coa-PCR with ITS-PCR showed a discriminatory index of 0.84, discriminating 22 genotypes that agree with previously determined data by PFGE and MLST. This association between the two PCR-based methods suggests that they can be useful for an initial molecular epidemiological investigation of S. aureus in hospitals, providing significant information while requiring fewer resources.