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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 (bla OXA-23 , -24 , -51 and -58 , bla KPC , bla SHV , bla IMP , bla VIM , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To assess the antibacterial effectiveness of Lippia macrophylla essential oil (LMEO) against multidrug-resistant Acinetobacter baumannii isolates, both as a standalone treatment and in combination with conventional antibiotics. LMEO demonstrated a significant inhibitory effect on the growth of A. baumannii, with a minimum inhibitory concentration (MIC) below 500 μg/mL. Notably, LMEO was capable of reversing the antibiotic resistance of clinical isolates or reducing their MIC values when used in combination with antibiotics, showing synergistic (FICI≤0.5) or additive effects. The combination of LMEO and imipenem was particularly effective, displaying synergistic interactions for most isolates. Ultrastructural analyses supported these findings, revealing that the combination of LMEO+ceftazidime compromised the membrane integrity of the Acb35 isolate, leading to cytoplasmic leakage and increased formation of Outer Membrane Vesicles (OMVs). Taken together our results point for the use of LMEO alone or in combination as an antibacterial agent against A. baumannii. These findings offer promising avenues for utilizing LMEO as a novel antibacterial strategy against drug-resistant infections in healthcare settings, underscoring the potential of essential oils in enhancing antibiotic efficacy., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

CRISPR/Cas is a molecular mechanism to prevent predatory viruses from invading bacteria via the insertion of small viral sequences (spacers) in its repetitive locus. The nature of spacer incorporation and the viral origins of spacers provide an overview of the genetic evolution of bacteria, their natural viral predators, and the mechanisms that prokaryotes may use to protect themselves, or to acquire mobile genetic elements such as plasmids. Here, we report on the CRISPR/Cas genetic structure, its spacer content, and strain epidemiology through MLST and CRISPR typing in Acinetobacter baumannii , an opportunistic pathogen intimately related to hospital infections and antimicrobial resistance. Results show distinct genetic characteristics, such as polymorphisms specific to ancestor direct repeats, a well-defined degenerate repeat, and a conserved leader sequence, as well as showing most spacers as targeting bacteriophages, and several self-targeting spacers, directed at prophages. There was a particular relationship between CRISPR/Cas and CC113 in the study of Brazilian isolates, and CRISPR-related typing techniques are interesting for subtyping strains with the same MLST profile. We want to emphasize the significance of descriptive genetic research on CRISPR loci, and we argue that spacer or CRISPR typing are helpful for small-scale investigations, preferably in conjunction with other molecular typing techniques such as MLST.

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.

Life-threatening bacterial infections are a major concern in health care services worldwide. This retrospective study aimed to demonstrate genetic and biochemical diversity in isolates of Acinetobacter baumannii and Pseudomonas aeruginosa from a public hospital in Brazil. A total of 63 isolates collected from different sites of infection and hospital sectors were characterized, and their susceptibility profile to antibiotics was assessed for 18 drugs belonging to 8 antimicrobial categories using the automated BACTEC system. Genetic diversity was assessed using the multiple locus variable number tandem repeat analysis. Among the isolates of A. baumannii , 83% were classified as extensively drug resistant (XDR), and 17 genotypic profiles were identified. About 67% of P. aeruginosa isolates were susceptible to antimicrobials and were distributed into 37 genotypic profiles, revealing genetic heterogeneity. This study has demonstrated the multicolonization of investigated pathogens and the high frequency (95.8%) of multidrug-resistant and XDR, as well as high genetic diversity, among the isolates supporting the continuous need to monitor these species in the hospital environment.

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.

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus , is synthesized by the expression of the norA gene. Menadione, also known as vitamin K 3 , is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

Acinetobacter baumannii is an opportunistic bacterial pathogen infecting immunocompromised patients and has gained attention worldwide due to its increased antimicrobial resistance. Here, we report a comparative whole-genome sequencing and analysis coupled with an assessment of antibiotic resistance of 46 Acinetobacter strains (45 A. baumannii plus one Acinetobacter nosocomialis ) originated from five hospitals from the city of Recife, Brazil, between 2010 and 2014. An average of 3,809 genes were identified per genome, although only 2,006 genes were single copy orthologs or core genes conserved across all sequenced strains, with an average of 42 new genes found per strain. We evaluated genetic distance through a phylogenetic analysis and MLST as well as the presence of antibiotic resistance genes, virulence markers and mobile genetic elements (MGE). The phylogenetic analysis recovered distinct monophyletic A. baumannii groups corresponding to five known (ST1, ST15, ST25, ST79, and ST113) and one novel ST (ST881, related to ST1). A large number of ST specific genes were found, with the ST79 strains having the largest number of genes in common that were missing from the other STs. Multiple genes associated with resistance to β-lactams, aminoglycosides and other antibiotics were found. Some of those were clearly mapped to defined MGEs and an analysis of those revealed known elements as well as a novel Tn 7 -Tn 3 transposon with a clear ST specific distribution. An association of selected resistance/virulence markers with specific STs was indeed observed, as well as the recent spread of the OXA-253 carbapenemase encoding gene. Virulence genes associated with the synthesis of the capsular antigens were noticeably more variable in the ST113 and ST79 strains. Indeed, several resistance and virulence genes were common to the ST79 and ST113 strains only, despite a greater genetic distance between them, suggesting common means of genetic exchange. Our comparative analysis reveals the spread of multiple STs and the genomic plasticity of A. baumannii from different hospitals in a single metropolitan area. It also highlights differences in the spread of resistance markers and other MGEs between the investigated STs, impacting on the monitoring and treatment of Acinetobacter in the ongoing and future outbreaks., (Copyright © 2020 Leal, Campos, Rezende, Docena, Mendes-Marques, de Sá Cavalcanti, Wallau, Rocha, Cavalcanti, Veras, Alves, Andrade-Figueiredo, de Barros, de Almeida, de Morais, Leal-Balbino, Xavier and de-Melo-Neto.)

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.

The widespread use of antibiotics created selective pressure for the emergence of strains that would persist despite antibiotic toxicity. The bacterial resistance mechanisms are several, with efflux pumps being one of the main ones. These pumps are membrane proteins with the function of removing antibiotics from the cell cytoplasm. Due to this importance, the aim of this work was to evaluate the inhibitory effect of tannic acid against efflux pumps expressed by the Staphylococcus aureus RN4220 and IS-58 strains. The efflux pump inhibition was assayed using a sub-inhibitory concentration of efflux pump standard inhibitors and tannic acid (MIC/8), observing their capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due the possible inhibitory effect of these substances. The MICs of EtBr and antibiotics were significantly different in the presence of tannic acid, indicating the inhibitory effect of this product against efflux pumps of both strains. These results indicate the possible usage of tannic acid asan inhibitor and an adjuvant in the antibiotic therapy against multidrug resistant bacteria (MDR)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Aeromonas spp. are gram-negative bacteria that can cause a variety of infections in both humans and animals and play a controversial role in diarrhea outbreaks. Our aim was to identify clinical and environmental Aeromonas isolates associated with a cholera outbreak in a northeast county of Brazil at the species level. We also aimed to determine the genetic structure of the bacterial population and the virulence potential of the Aeromonas isolates., Methods and Results: Analysis based on concatenated sequences of the 16S rRNA and gyrB genes suggested the classification of the 119 isolates studied into the following species: A. caviae (66.9%), A. veronii (15.3%), A. aquariorum (9.3%), A. trota (3.4%), A. hydrophila (3.4%) and A. jandaei (1.7%). One isolate did not fit any Aeromonas species assessed, which might indicate a new species. The haplotype network based on 16S rRNA gene sequences identified 59 groups among the 119 isolates and 26 reference strains, and it clustered almost all A. caviae isolates into the same group. The analysis of the frequency patterns of seven virulence-associated genes (alt, ast, hlyA, aerA, exu, lip, flaA/B) revealed 29 virulence patterns composed of one to seven genes. All the isolates harbored at least one gene, and three of them harbored all seven virulence genes., Conclusion: The results emphasize the need to improve local water supply and maintain close monitoring of possible bacterial contamination in the drinking water.

Background: Canine leishmaniasis caused by Leishmania infantum is a parasitic disease of great veterinary significance. Some dogs infected by L. infantum may mount a strong cellular immune response and clear the infection, while others may respond with exaggerated antibody production against the parasite and develop an overt disease, which may be fatal, if left untreated. The initial factors triggering the polarization of the immune response towards a predominantly T-helper 1 or T-helper 2 cytokines, as well as the markers of resistance and susceptibility to L. infantum infection and disease development in dogs, are not fully understood. Herein, we assessed the association between single nucleotide polymorphisms (SNPs) in the canine β-defensin-1 (CBD1) gene and the infection by L. infantum in two dog populations from Brazil (Sobral in Ceará State and São Raimundo Nonato in Piauí State) and one dog population from Italy., Results: A total of 387 dogs were assessed for L. infantum by real time PCR and 34.6% of them were positive. In CBD1 gene sequences from these positive dogs, nine polymorphic sites were detected, but only SNPs 3, 4, 7 and 8 were associated with L. infantum, in dogs from southern Italy. No association was found with dogs from Brazil., Conclusion: This study sets the basis for further studies on the usefulness of CBD1 as a marker of L. infantum infection susceptibility in dogs.

The aim of this study is to evaluate the effect of ergosterol on steroids and cholesterol efflux pumps in multidrug resistant strains of S. aureus. Were used RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein; and IS-58, which possesses the TetK tetracycline efflux protein; 1199B resists hydrophilic fluoroquinolones via a NorA-mediated mechanism and wild strain 1199B. The Minimal Inhibitory Concentration (MIC) was determined and the evaluation of possible inhibition of efflux pumps by reduction of MIC. Some of the detrimental effects on bacterial cells can be attributed to the detergent properties of cholesterol and ergosterol on account of their amphipathic structure. Besides the cholesterol did not affect directly the pump structure, a synergism was observed, maybe due the interaction with the cell membrane and interference in the lipid bilayer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Here, we report the isolation of 31 Acinetobacter baumannii strains producing OXA-253 in a single large Brazilian city. These strains belonged to five different sequence types (STs), including 4 STs not previously associated with bla OXA-253 In all strains, the bla OXA-253 gene was located in a plasmid within a genetic environment similar to what was found previously in Brazil and Italy. The reported data emphasize the successful transmission of the bla OXA-253 gene through a large area and the tendency for this resistance determinant to remain in the A. baumannii population., (Copyright © 2016 American Society for Microbiology.)

During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Staphylococcus aureus is the major cause of global and nosocomial infections with a significant impact in hospitals worldwide. Our objective was to investigate clinical and molecular characteristics of S. aureus isolates causing infections in patients admitted to hospitals from Recife city, Brazil, and investigate the prevalence of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in the region, as well as genetically characterize the isolates and compare with epidemic clones., Results: We characterized 89 isolates in total, 31 clinical methicillin-resistant S. aureus (MRSA) and 58 methicillin-sensitive (MSSA) isolates by PFGE, MLST, spa typing and SCCmec genotyping. Isolates belonging to international MRSA clones were present: Brazilian epidemic clone (BEC) (61 % of MRSA isolates), Paediatric (36 %), New York/Japan (3 %). Some MSSA isolates were related to MRSA clones: USA400-related (10 % of MSSA isolates), Berlin clone (2 %), Paediatric (14 %), New York/Japan (2 %) and Southwest Pacific clone (17 %). MLST revealed new sequence types (ST's): ST2381, ST2382, and ST2383 and new spa types: 10548 and 10550. Among isolates phenotypically identified as MSSA by antimicrobial susceptibility assays, we verified 30 oxacillin-susceptible isolates, which exhibited the mecA gene, without mec complex amplification and were thus classified as OS-MRSA. We observed clonal spread of MRSA and MSSA, including OS-MRSA, within several areas of the main hospital investigated and closely related isolates between hospitals analyzed., Conclusions: The results of this study suggest a possible spread of the strains in hospital environment that could be responsible for nosocomial infections. We documented the presence of several MRSA clones, as well as new MLST and spa types, that were responsible for severe infections in hospitalized patients. The finding of OS-MRSA isolates could have implications for therapy, because testing for mecA and PBP2a is not a routine procedure performed by clinical microbiology laboratories in Brazil and, as consequence, these isolates could be misclassified as MSSA. Our data alert to the necessity to develop more effective strategies for epidemiological control of S. aureus in order to avoid an increase of hospital infections provoked by this pathogen. We reinforce the use of genetic methods, in addition to phenotypic tests, for a precise identification of MRSA.

Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure.

In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and an isolated population has been identified in the state of Pernambuco, Northeastern region. This study assessed the phylogeographic structure of three allopatric Brazilian populations of L. umbratilis. Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the period gene. Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and one clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations revealed high indices of interpopulational divergence. Phylogenetic analysis indicated that geographical distance and environmental differences have not modified the ancestral relationship shared by the Recife and Rio Preto da Eva populations. Genetic similarities suggest that, in evolutionary terms, these populations are more closely related to each other than to the Manacapuru population. These results confirm the existence of an L. umbratilis species complex composed of at least two incipient species., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Background: Even one hundred years after being originally identified, aspects of the taxonomy of the sand fly Lutzomyia longipalpis, the principal vector of Leishmania infantum in the Americas, remain unresolved for Brazilian populations of this vector. The diversity of morphological, behavioral, biochemical, and ethological characters, as well as the genetic variability detected by molecular markers are indicative of the presence of a complex of species., Methods: In this study, a 525 bp fragment of the period gene was used to evaluate sympatric populations of L. longipalpis. A combination of probabilistic methods such as maximum likelihood and genetic assignment approach to investigate sympatric species of L. longipalpis were applied in three populations of Northeast Brazil., Results: Fixed polymorphisms in geographically isolated populations of L. longipalpis from two localities in the state of Ceará and one in the state of Pernambuco, Brazil, was identified in a 525 bp fragment of the gene period (per). Our results suggest a direct relationship between the number of spots found in males' tergites and the genetic variation in cryptic species of L. longipalpis. The fragment used in this study revealed the nature of the ancestral morphotype 1S., Conclusion: New polymorphisms were identified in the gene per which can be used as a genetic barcode to sympatric taxonomy of L. longipalpis. The per gene fragment confirmed the presence of two siblings species of L. longipalpis in Sobral and showed that these same species are present in two other localities, representing an expansion within the L. longipalpis species complex with regards to the states of Ceará and Pernambuco.

Aedes aegypti and A. albopictus represent the two most important species of mosquitoes in relation to dengue virus transmission both in the Americas and Asia. However, the study of theses species generally requires the establishment of a colony for the larvae to hatch, or waiting for the adult development to perform its taxonomic classification, which is time consuming. Thus, the establishment of new methods aimed at obtaining DNA directly from the mosquito eggs is relevant. Accordingly, we compared a new approach based on Chelex(®) 100 resin with the standard STE method to extract DNA from the eggs of Aedes spp to molecularly identify these vectors. The Chelex(®) 100 resin approach was very efficient, as satisfactory amounts of DNA were obtained, making it possible to amplify and sequence a mitochondrial DNA barcode region widely used to identify species. The STE protocol yielded substantial amounts of DNA, but the 260/280 optical density ratio indicated a low quality, precluding amplification. This new method proved quite effective in obtaining DNA from even a single mosquito egg, and it can thus be applied in population genetic studies of various vector insects to enhance monitoring programs.

The potential use of CRISPR loci genotyping to elucidate population dynamics and microevolution of 146 Yersinia pestis strains from different biovars and locations was investigated in this work. The majority of strains from the Orientalis biovar presented specific spacer arrays, allowing for the establishment of a CRISPR signature for their respective isolates. Twenty-one new spacers were found in the Y. pestis strains from plague foci in Brazil. Ninety-three (64%) strains were grouped in the G1 genotype, whereas the others were distributed in 35 genotypes. This study allowed observing a microevolutionary process in a group of Y. pestis isolated from Brazil. We also identified specific genotypes of Y. pestis that were important for the establishment of the bacteria in plague foci in Brazil. The data have provided supporting evidence for the diversity and dynamics of CRISPR loci present in the genome of Y. pestis strains from plague foci in Brazil.

This work aimed to assess the clonal distribution among 94 strains of Staphylococcus aureus isolated from cow's milk, raw cheese, and a milking machine in 12 dairy farms in northeast Brazil, by analyzing different typing methods and detecting resistance and toxigenic profiles. For the first time, isolates of this region were assessed simultaneously by the polymorphism of the 3'-end coa gene and 16S-23S rDNA, pulsed-field gel electrophoresis, antibiotic resistance phenotyping, and toxigenic arsenal. Although pulsed-field gel electrophoresis patterns showed a wider variation (discriminatory index 0.83) than the PCR-based methods, the internal transcribed spacer-PCR proved to be a useful and inexpensive procedure for conducting epidemiological surveys of S. aureus on a regional scale. Each dairy farm had its own resistance profile, and in two herds, 63% of the strains were multiresistant, probably due to the indiscriminate use of antibiotics in bovine mastitis treatment. No methicillin-resistant S. aureus strains were detected in this study; however, 93.6% of S. aureus strains harbored variable profiles of staphylococcal enterotoxin genes seg, seh, sei, and sej. Transcriptional analysis revealed that 53.3% of staphylococcal enterotoxin genes actually transcribed, pointing out the food poisoning risk of these dairy products to consumers in the region. Based on the detection of the most prevalent clones in a herd or region, appropriate antibiotic therapy and specific immunization can be used for the treatment and control of staphylococcal mastitis.

Two new examples of OXA-72-producing Acinetobacter baumannii isolate resistant to a broad spectrum of antimicrobials, but not polymyxin B, have been identified in Recife, Brazil. Molecular typing indicated a close genetic link with the OXA-72-producing A. baumannii previously isolated in São Paulo, suggesting the possibility of clonal dissemination within the country., (© 2013.)

We subtyped Brazilian Yersinia pestis strains by pulsed-field gel electrophoresis (PFGE). This was done with 22 Brazilian Y. pestis strains: 17 from an outbreak and 5 from endemic routine surveillance. The strains were divided into 2 groups (I and II), 8 subgroups (A-H) and 19 PFGE profiles or pulsotypes. PFGE did not separate outbreak from non-outbreak strains, as identical pulsotype patterns were found among outbreak strains and strains obtained from surveillance. However, it was able to detect intraspecific genetic diversity among Brazilian strains. This PFGE technique was able to differentiate a homogeneous group of Brazilian Y. pestis strains.

Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.

Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.

Aims: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates., Materials and Results: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found., Conclusion: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains., Significance and Impact of the Study: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.