Emergence and spread of resistant and biofilm-forming Acinetobacter baumannii in critically ill COVID-19 patients.

The COVID-19 pandemic has raised concerns beyond the viral infection itself. Bacterial co-infections, particularly those involving Acinetobacter baumannii, have become a significant worry in critically ill COVID-19 patients. A. baumannii is an opportunistic pathogen that can cause nosocomial infections, especially in patients with compromised immune systems. This study investigated 36 A. baumannii isolates obtained from COVID-19 patients during a concurrent outbreak. The isolates were collected over two years through routine medical requests sent to the Clinical Microbiology laboratory. Identification of the strains was confirmed through biochemical tests, the Phoenix BD® Automated Microbiology System, and MALDI-TOF Mass Spectrometry. The study assessed the antimicrobial sensitivity of the isolates, with a specific focus on resistance to the beta-lactam group as well as aminoglycosides. The presence of specific antibiotic resistance genes (blaOXA-23, -24, -51 and -58, blaKPC, blaSHV, blaIMP, blaVIM, aac(6')-Ib, ant(3″)-Ia, and aph(3')-Ia) was investigated using PCR and Sanger DNA sequencing. Biofilm-forming capabilities of the isolates were also evaluated. The findings revealed diverse resistance profiles, with a high prevalence of resistant strains, including resistance to carbapenems. Genetic analysis suggested potential clonal spread of certain strains within the hospital setting. Moreover, a significant proportion of the isolates demonstrated strong biofilm-forming abilities, which can enhance persistence and antibiotic resistance. In conclusion, this study highlights the need for vigilant monitoring and targeted interventions to address bacterial co-infections in COVID-19 patients. The diversity in resistance patterns, potential clonal spread, and robust biofilm-forming abilities among A. baumannii isolates underscore the importance of addressing this issue to better manage and treat critically ill COVID-19 patients.

Hidden ecotoxicological dangers: Investigating pathogen circulation and non-toxic risks hazards in a crucial brazilian watershed.

Numerous studies evaluate chemical contaminants released by human activities and their effects on biota and aquatic ecosystems. However, few of these studies address non-toxic agents and their potentially harmful effects, which, in a concealed manner, culminate in an increased ecotoxicological risk for aquatic life and public health. This study investigated the presence of toxic and non-toxic pollutants in one of the main watersheds in Northeast Brazil (Rio São Francisco) and proposed a model of dispersion and transfer of resistance among the analyzed bacteria, also assessing the health risks of individuals and aquatic organisms present there. The results are worrying because although most toxic parameters, including physical-chemical and chromatographic aspects, comply with Brazilian environmental standards, non-toxic (microbiological) parameters do not. This research reveals the circulation of pathogens in several points of this hydrographic basin, highlighting the hidden ecotoxicological potential of an aquatic environment considered unaffected by the usual patterns of toxic parameters.

Prevalence of cagA, cagM, vacA and oipA genes in isolates of Helicobacter pylori obtained from hospital patients in Northeast Brazil.

Helicobacter pylori is a major cause of gastrointestinal disorders such as chronic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. It is estimated that around half of the world's population is infected with this pathogen, with underdeveloped countries reporting the highest frequencies. The genes cagA, cagM, vacA, and oipA are some of the most important virulence factors of H. pylori; however, there are no recent studies from Recife-PE demonstrating their frequency, and their relationship with severe gastric modifications. This work aims to use qualitative PCR to detect the virulence genes cagA, cagM, vacA, and oipA in H. pylori isolates obtained from patients in a public hospital in Recife (PE). We collected samples from the stomach's body and antrum of 147 patients, from which 71 (48%) tested positive for H. pylori. Among positive samples, the most frequently infected gender was female (44/71, 62%), and the most frequently infected age group was those above the age of 46 (31/71, 44%). Histological examination of H. pylori-positive samples revealed alterations other than chronic gastritis, including metaplasia and atrophy. The frequency of cagA, cagM, and oipA genes were identified in 84%, 56%, and 69% of the samples tested, respectively, as well as the vacA-s1m1 allelic combination (77%). However, there was no statistically significant variation in the occurrence of these genes, therefore they cannot be considered unique markers of severity in our setting. New research with larger samples and investigations of other genetic markers can aid uncover local risk factors and lead to a better understanding of H. pylori's pathogenesis.

Comparative transcriptomics analysis of multidrug-resistant Acinetobacter baumannii in response to treatment with the terpenic compounds thymol and carvacrol.

Acinetobacter baumannii is a gram-negative opportunistic bacterium that has become a major public health concern and a substantial medical challenge due to its ability to acquire multidrug resistance (MDR), extended-drug resistance, or pan-drug resistance. In this study, we evaluated the antibacterial activity of thymol and carvacrol alone or in combination against clinical isolates of MDR A. baumannii. Additionally, we used RNA-sequency to perform a comparative transcriptomic analysis of the effects of carvacrol and thymol on the Acb35 strain under different treatment conditions. Our results demonstrated that thymol and carvacrol alone, effectively inhibited the bacterial growth of MDR A. baumannii isolates, with a minimum inhibitory concentration (MIC) lower than 500 µg/mL. Furthermore, the combination of thymol and carvacrol exhibited either synergistic (FICI ≤ 0.5) or additive effects (0.5 < FICI ≤ 4), enhancing their antibacterial activity. Importantly, these compounds were found to be non-cytotoxic to Vero cells and did not cause hemolysis in erythrocytes at concentrations that effectively inhibited bacterial growth. Transcriptomic analysis revealed the down-regulation of mRNA associated with ribosomal subunit assemblies under all experimental conditions tested. However, the up-regulation of specific genes encoding stress response proteins and transcriptional regulators varied depending on the experimental condition, particularly in response to the treatment with carvacrol and thymol in combination. Based on our findings, thymol and carvacrol demonstrate promising potential as chemotherapeutic agents for controlling MDR A. baumannii infections. These compounds exhibit strong antibacterial activity, particularly in combination and lower cytotoxicity towards mammalian cells. The observed effects on gene expression provide insights into the underlying mechanisms of action, highlighting the regulation of stress response pathways.

Comparison of genotyping methods and toxin gene profiles of Staphylococcus aureus isolates from clinical specimens.

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.

Genetic characteristics and phylogenetic analysis of Brazilian clinical strains of Pseudomonas aeruginosa harboring CRISPR/Cas systems.

The CRISPR-Cas are adaptive immune systems found in archaea and bacteria, responsible for providing sequence-specific resistance against foreign DNA. Strains of Pseudomonas aeruginosa may carry CRISPR/Cas system types I-F, I-E and/or I-C; however, several aspects related to the epidemiology and functionality of these systems have not yet been revealed. Here, we report 13 genomes of clinical strains of P. aeruginosa from Brazil that were positive for CRISPR/Cas system types I-F and I-E, a rare feature in this species. The phylogenetic tree, which was constructed with 161 other publicly available genomes, suggested no direct relationship between positive strains, and the various types of CRISPR/Cas systems were spread throughout the tree. Comparative analysis of the genetic locations of type I-F and a specific orphan CRISPR array (without cas genes), named the LES locus, showed sequence similarities between this orphan locus and type I-F, but these LES loci were inserted in a different genomic location. We also report the presence of prophages, the presence of anti-CRISPR genes, and possibly the presence of self-targeting spacers. Here, we conclude that CRISPR/Cas is highly associated with certain lineages and is spread throughout the phylogenetic tree, showing no clear pattern of evolutionary distribution. Moreover, the LES locus might be a CRISPR1 locus related to type I-F that may have been misplaced and maintained over time. Furthermore, strains carrying I-F and I-E are rare, and not all of them are closely related. Further functional work is needed to better comprehend if aspects reported in this study are functional, including the LES locus, self-targeting spacers, anti-CRISPR protection, and I-F/I-E-carrying lineages.

Occurrence and Diversity of Intra- and Interhospital Drug-Resistant and Biofilm-Forming Acinetobacter baumannii and Pseudomonas aeruginosa.

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 blaKPC, blaIMP, blaVIM, and blaSHV 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 blaKPC (60%), followed by blaSHV (39%), blaVIM (8%), and blaIMP (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.

Analysis of direct repeats and spacers of CRISPR/Cas systems type I-F in Brazilian clinical strains of Pseudomonas aeruginosa.

CRISPR/Cas is an adaptive immune system found in prokaryotes, with the main function of protecting these cells from invasion and possible death by mobile genetic elements. Pseudomonas aeruginosa is considered a model for type I-F CRISPR/Cas system studies. However, its CRISPR loci characteristics have not yet been thoroughly described, and its function has not yet been fully unraveled. The aims of this study were to find the frequency of the system in Brazilian clinical isolates; to identify the loci sequence, its spacer diversity and its origins; as well as to propose a unified spacer library to aid in future structural studies of the CRISPR loci of P. aeruginosa. We investigated types I-F and I-E gene markers to establish CRISPR/Cas typing, and observed two strains harboring both systems simultaneously, a very rare feature. Through amplification and sequencing of CRISPR loci related to type I-F system, we describe polymorphisms in DRs and 350 spacers, of which 97 are new. The spacers that were identified had their possible organisms or proteins of origin identified. Spacer arrays were grouped in five different CRISPR patterns and the plasticity was inferred by rearrangements in spacer arrays. Here, we perform the first detailed and focused description of CRISPR/Cas elements in Brazilian clinical strains of P. aeruginosa. Our findings reflect active and highly diverse CRISPR loci, and we suggest that CRISPR/Cas may also pose as a transcriptional regulatory mechanism. The structural and diversity features described here can provide insights into the function of CRISPR/Cas in this pathogen and help guide the development of new therapeutic strategies.