Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative bacterium considered an emerging multi-drug-resistant pathogen. Furthermore, this bacterium can survive in extreme environmental conditions, which makes it a frequent cause of nosocomial infection outbreaks. Gene expression analyses by Reverse Transcription Quantitative real-time PCR (RT-qPCR) depend on a reference gene, also called an endogenous gene, which is used to normalize the generated data and thus ensure an accurate analysis with minimal errors. Currently, gene expression analyses in A. baumannii are compromised, as there are no reports in the literature describing the identification of validated reference genes for use in RT-qPCR analyses. For this reason, we selected twelve candidate reference genes of A. baumannii and assessed their expression profile under different experimental and culture conditions. The expression stability of the candidate genes was evaluated by using statistical algorithms such as BestKeeper, geNorm, NormFinder, Delta CT, and RefFinder, in order to identify the most suitable candidate reference genes for RT-qPCR analyses. The statistical analyses indicated rpoB, rpoD, and fabD genes as the most adequate to ensure accurate normalization of RT-qPCR data in A. baumannii. The accuracy of the proposed reference genes was validated by using them to normalize the expression of the ompA gene, encoding the outer membrane protein A, in A. baumannii sensible and resistant to the antibiotic polymyxin. The present work provides suitable reference genes for precise RT-qPCR data normalization on future gene expression studies with A. baumannii.

Co-carriage of Plasmid NDM and Chromosomal KPC in Klebsiella pneumoniae ST255 Human Wound Isolate in Brazil.

Multidrug-resistant K. pneumoniae is one of the main causes of hospital-acquired infections worldwide and frequently carries antimicrobial resistance genes in moving elements. In this study, we described a K. pneumoniae clinical isolate carrying simultaneous chromosomal blaKPC, and plasmid-mediated blaNDM and blaOXA-9. The isolate is multidrug-resistant and belongs to ST 225. While blaKPC were identified in the chromosome, the blaNDM was mediated by IncFII(K) plasmid and the blaOXA-9, in a IncFIB(K) plasmid. The blaKPC context was composed by Tn4401 transposon and two insertion sequences ISKpn6 and ISKpn7. The co-production of diverse ß-lactamases brings an alert about a new adaptive profile of K. pneumoniae strains and their dissemination in the hospital-acquired infectious.

Low performance of Policimbac® broth microdilution in determining polymyxin B MIC for Klebsiella pneumoniae.

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient's treatment.

Host-microbiome interactions regarding peri-implantitis and dental implant loss.

In the last decades, the ortho-aesthetic-functional rehabilitation had significant advances with the advent of implantology. Despite the success in implantology surgeries, there is a percentage of failures mainly due to in loco infections, through bacterial proliferation, presence of fungi and biofilm formation, originating peri-implantitis. In this sense, several studies have been conducted since then, seeking answers to numerous questions that remain unknown. Thus, the present work aims to discuss the interaction between host-oral microbiome and the development of peri-implantitis. Peri-implantitis was associated with a diversity of bacterial species, being Porphiromonas gingivalis, Treponema denticola and Tannerella forsythia described in higher proportion of peri-implantitis samples. In a parallel role, the injury of peri-implant tissue causes an inflammatory response mediated by activation of innate immune cells such as macrophages, dendritic cells, mast cells, and neutrophils. In summary, the host immune system activation may lead to imbalance of oral microbiota, and, in turn, the oral microbiota dysbiosis is reported leading to cytokines, chemokines, prostaglandins, and proteolytic enzymes production. These biological processes may be responsible for implant loss.

Genomic characterization of multidrug-resistant Salmonella Heidelberg E2 strain isolated from chicken carcass in southern Brazil.

Salmonella Heidelberg is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry products. Since 1962, Salmonella Heidelberg has been widely reported from poultry production systems in several countries, including Brazil. The emergence of multidrug-resistant (MDR) Salmonella Heidelberg strains in food animals underscores a significant food safety hazard. In our study, we performed antimicrobial susceptibility testing (AST) and Whole-genome sequencing (WGS) to identify the antimicrobial resistance (AMR) genes, pathogenicity mechanisms and virulence factors (VF) in Salmonella Heidelberg E2 strain recovered from a chicken carcass in Southern Brazil. Salmonella Heidelberg strain belonged to ST15 and showed to be susceptible to colistin (MIC ≤2 µg/mL) and multidrug-resistant to amoxicillin-clavulanic acid, gentamicin, ampicillin, cefaclor, cefazolin, ceftiofur, nalidixic acid, azithromycin, erythromycin, doxycycline, tetracycline and sulfonamide. We identified AMR genes mediating resistance to aminoglycosides (aac(6')-Iaa, aac(3)-VIa, aph(3')-Ia, aadA, 16S rrsD), ß-lactams (blaCTX-M-2), quinolones (parC), macrolides (acrB), tetracyclines (tet(A)), fosfomycin (fosA7) and sulfonamide (sul1). Interestingly, the mutation in parC T255S has never been reported among Salmonella Heidelberg strains. To our knowledge, this is the first report of a Salmonella enterica strain harbouring 16S rrsD 471G > A, acrB F28L and acrB L40P chromosomal point mutations. Three plasmid replicon types, ST2-IncHI2, ST2-IncHI2A and IncX1 were identified. Nine Salmonella Pathogenicity Islands and 98 virulence genes encoding virulence factors were identified associated with cell adhesion, invasion, intracellular survival and resistance to antimicrobial peptides. Although Salmonella Heidelberg E2 strain likely originated from poultry, cross-contamination during meat processing cannot be excluded. This study adds to our understanding of Salmonella Heidelberg transmission along the food-chain and informs ongoing regulatory discussions on Salmonella Heidelberg in poultry.

Biochemical and Toxinological Characterization of Venom from Macrorhynchia philippina (Cnidaria, Hydrozoa).

Macrorhynchia philippina is a colonial benthic hydroid from the Class Hydrozoa (Phylum Cnidaria) distributed in the tropical and subtropical marine waters from Atlantic Ocean, Indo-Pacific, and Mozambique. Its colonies somewhat resemble plants, causing confusion in the bathers who accidentally touch the animal. Acute burning/local pain, edema, erythema, and pruritus were symptoms already described, but its venom composition is unknown, as well as the participation of toxins for the symptom's development. Thus, herein, we show the biochemical composition and toxic effects of M. philippina venom. Colonies were collected and processed for histological analysis; alternatively, they were immersed into methanol containing 0.1% acetic acid for venom attainment, which was analyzed by mass spectrometry and submitted to edema and nociception evaluation in mice, hemolysis and antimicrobial assays in vitro. Before the molecule's extraction, it was possible to see the inoculation structures (hydrocladiums and hydrotheca) containing venom, which was released after the immersion of the animal in the solvents. The venom was composed mainly by low molecular mass compounds, able to cause significant reduction of the paw withdrawal latency from the hot plate test, 30 minutes after the injection. Moreover, significant edema was observed 10 and 30 minutes after the injection, indicating the activity of at least two inflammatory mediators. The venom caused no hemolytic activity but reduced the growth of A. baumannii and K. pneumoniae strains. This study is the first biochemical description of M. philippina venom, with molecules that cause fast inflammatory and painful effects, characteristic of the envenomation.

Influence of the Polysaccharide Capsule on the Bactericidal Activity of Indolicidin on Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide's bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Association between asthma and enamel defects in primary and young permanent teeth - A systematic review.

Childhood respiratory diseases, such as asthma, are important public health problems worldwide and could be associated with tooth enamel defects. This study aimed to verify the relationship between asthma and enamel defects in teeth, to answer the following question: "Could asthma in children be significantly associated with enamel defects in deciduous dentition and young permanent teeth?." PUBMED-MEDLINE, EMBASE, LILACS, and COCHRANE databases were systematically searched and assessed articles (2000-2021) were cautiously scored according to a predetermined criterion. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were considered. Twenty-two articles were critically appraised and used as a basis for conclusions. The relationship between asthma and enamel defects was confirmed in the majority of appraised papers, one with a high level of evidence, nine with a moderate level, and four with a low level. Out of the eight manuscripts investigating the influence of asthma medication on enamel defects, only three (one with high, one moderate, and another with a low level of evidence) suggested an association. It can be concluded that asthma is closely connected with enamel defects in young permanent teeth. However, as most of the papers appraised were of cross-sectional or case-control design, further well-designed clinical investigations with a prospective design are welcome to reinforce our findings.

Influence of the Polysaccharide capsule on the bactericidal activity of indolicidin on Streptococcus pneumoniae

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide’s bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Biochemical and toxinological characterization of venom from Macrorhynchia philippina (Cnidaria, Hydrozoa)

Macrorhynchia philippina is a colonial benthic hydroid from the Class Hydrozoa (Phylum Cnidaria) distributed in the tropical and subtropical marine waters from Atlantic Ocean, Indo-Pacific, and Mozambique. Its colonies somewhat resemble plants, causing confusion in the bathers who accidentally touch the animal. Acute burning/local pain, edema, erythema, and pruritus were symptoms already described, but its venom composition is unknown, as well as the participation of toxins for the symptom’s development. Thus, herein, we show the biochemical composition and toxic effects of M. philippina venom. Colonies were collected and processed for histological analysis; alternatively, they were immersed into methanol containing 0.1% acetic acid for venom attainment, which was analyzed by mass spectrometry and submitted to edema and nociception evaluation in mice, hemolysis and antimicrobial assays in vitro. Before the molecule’s extraction, it was possible to see the inoculation structures (hydrocladiums and hydrotheca) containing venom, which was released after the immersion of the animal in the solvents. The venom was composed mainly by low molecular mass compounds, able to cause significant reduction of the paw withdrawal latency from the hot plate test, 30 minutes after the injection. Moreover, significant edema was observed 10 and 30 minutes after the injection, indicating the activity of at least two inflammatory mediators. The venom caused no hemolytic activity but reduced the growth of A. baumannii and K. pneumoniae strains. This study is the first biochemical description of M. philippina venom, with molecules that cause fast inflammatory and painful effects, characteristic of the envenomation.

Firmicutes Dysbiosis After Chlorhexidine Prophylaxis in Healthy Patients Submitted to Impacted Lower Third Molar Extraction.

Prophylaxis with antiseptic and antibiotic therapy is common in impacted lower third molar surgeries, despite the lack of consensus among professionals and researchers in the indication for healthy patients. The aim of the present preliminary study was to verify the impact of prophylaxis therapy with antiseptic and antibiotic in healthy patients submitted to impacted lower third molar extraction, according to oral microorganism quantification. Eleven patients submitted to impacted lower third molar extraction, under prophylactic therapy with 0.12% chlorhexidine and amoxicillin in four experimental phases, were evaluated. Our results showed no significant reduction in total bacteria load, as well as in Bacteroidetes and C. albicans loads in the oral cavity, after prophylactic therapy with antiseptic and antibiotic. On the other hand, there was a significant difference between the Firmicutes levels across the follow-up, and this effect seems to be large (ηp²=0.94). Post-hoc test demonstrated that the levels of Firmicutes in T1 were higher than T0, T2, and T3, suggesting a microbiota dysbiosis, when 0.12% chlorhexidine use, which may be responsible for selection of antibiotic-resistant microorganisms. Our results alert for an overuse of antiseptic and antibiotics by dentists and for a better evaluation of the available protocols.

Current Stage in the Development of Klebsiella pneumoniae Vaccines.

Klebsiella pneumoniae is a bacterium capable of colonizing mucous membranes, causing serious infections. Widespread antibiotic resistance in K. pneumoniae-either through intrinsic mechanisms or via acquisition from different species, especially in hospital environments-limits the therapeutic options against this pathogen, further aggravating the disease burden. To date, there are no vaccines available against K. pneumoniae infection. Although formulations based on capsular polysaccharides have been proposed, the high variability in capsular serotypes limits vaccine coverage. Recombinant vaccines based on surface exposed bacterial antigens are a promising alternative owing to their conservation among different serotypes and accessibility to the immune system. Many vaccine candidates have been proposed, some of which have reached clinical trials. The present review summarizes the current status of K. pneumoniae vaccine development. Different strategies including whole cell vaccines, outer membrane vesicles (OMVs), ribosome, polysaccharide, lipopolysaccharide (LPS), and protein-based formulations are discussed. The contribution of antibody and cell-mediated responses is also presented. In summary, K. pneumoniae vaccines are feasible and a promising strategy to prevent infections and to reduce the antimicrobial resistance burden worldwide.

Genomic Characterization of mcr-1.1-Producing Escherichia coli Recovered From Human Infections in São Paulo, Brazil.

Polymyxins are one of most important antibiotics available for multidrug-resistant Gram-negative infections. Diverse chromosomal resistance mechanisms have been described, but the polymyxin resistance phenotype is not yet completely understood. The objective of this study was to characterize colistin resistant mcr-1-producing strains isolated from human infections over one year in a hospital setting (Hospital das Clínicas, São Paulo, Brazil). We isolated 490 colistin-resistant Gram-negative rods, of which eight were mcr-1.1-positive Escherichia coli, the only species with this result, indicating a low incidence of the mcr-1 production mechanism among colistin-resistant isolates. All mcr-1.1 positive isolates showed similarly low MICs for colistin and were susceptible to most antibiotics tested. The isolates showed diversity of MLST classification. The eight mcr-1.1-positive E. coli genomes were sequenced. In seven of eight isolates the mcr-1.1 gene is located in a contig that is presumed to be a part of an IncX4 plasmid; in one isolate, it is located in a contig that is presumed to be part of an IncHI2A plasmid. Three different genomic contexts for mcr-1.1 were observed, including a genomic cassette mcr-1.1-pap2 disrupting a DUF2806 domain-containing gene in six isolates. In addition, an IS1-family transposase was found inserted next to the mcr-1.1 cassette in one isolate. An mcr-1.1-pap2 genomic cassette not disrupting any gene was identified in another isolate. Our results suggest that plasmid dissemination of hospital-resident strains took place during the study period and highlight the need for continued genomic surveillance.

Dynamic of High-Risk Acinetobacter baumannii Major Clones in a Brazilian Tertiary Hospital During a Short Time Period.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 structure was found in the chromosome of five isolates, whereas blaOXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of ISAba1-blaADC-like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC50, > 512 mg/L) verified in all isolates. Only minocycline (MIC50, ≤ 0.5 µg/mL), polymyxin B (MIC50, 0.5 µg/mL), and tigecycline (MIC50, 0.5 µg/mL) were in vitro active against such isolates. A diversity of other antimicrobial resistance determinants (aph(3')-VIa, aadA1, aac(3')-IIa, strA, strB, sul2, drfA1, mph(E), msr(E), tetB, and floR) was also observed, which may confer resistance to at last six distinct antimicrobial classes. Four distinct pulsed-field gel electrophoresis (PFGE) profiles were observed during the study period, which belonged to ST79/ST258 (n = 2; IC5), ST25/ST229 (n = 2; IC7), ST1 (n = 1; IC1), and ST162/ST235 (n = 1; IC4). Although the ST1 isolate that carried blaOXA-23 and blaOXA-72 was introduced in this hospital setting by a transferred patient, two clonally related ST79/ST258 isolates carrying either one of these carbapenemase encoding genes were recovered from two patients who were hospitalized within the same period of time in the same hospital unit. Finally, a good correlation between PFGE/MLST, blaOXA-51 variant, and single nucleotide polymorphisms was also observed. Here we demonstrated that distinct extensively drug-resistant A. baumannii clones can circulate in the same hospital setting during a short time period, illustrating a very complex epidemiological scenario for this priority pathogen.

KPC-producing Enterobacterales with uncommon carbapenem susceptibility profile in Vitek 2 system.

KPC-producing K. pneumoniae is an endemic challenge. Seven KPC-producing Enterobacterales showed unusual carbapenems susceptibility profile. These strains were resistance at least one carbapenem and the ertapenem MIC was lower than imipenem and/or meropenem MICs using Vitek 2™ system (bioMerieux). When E-test™ and disk diffusion methods were performed the carbapenems showed susceptible results.

Temporal evolution of Acinetobacter baumannii ST107 clone: conversion of blaOXA-143 into blaOXA-231 coupled with mobilization of ISAba1 upstream occAB1.

Nine carbapenem-resistant Acinetobacter baumannii isolates carrying blaOXA-231 and an ISAba1 upstream occAB1 were evaluated. They were clonally related and belonged to ST107. An OXA-143-producing A. baumannii ST107 strain (Ac-148) that did not possess ISAba1 upstream occAB1 was included in the analysis. Reduction in the expression of occAB1 and a 4-fold increase of carbapenem MICs were observed for all isolates, except for the Ac-148 strain, probably due to the presence of ISAba1 upstream occAB1 but in the same transcriptional orientation. We reported an A. baumannii ST107 clone carrying blaOXA-143 that acquired a mutation resulting into blaOXA-231 and mobilized ISAba1 upstream occAB1.

Emergence of polymyxin B resistance in a polymyxin B-susceptible KPC-producing Klebsiella pneumoniae causing bloodstream infection in a neutropenic patient during polymyxin B therapy.

The emergence of resistance to polymyxins in KPC-producing Klebsiella pneumoniae isolates has been a major clinical problem. This study evaluated the molecular mechanisms associated with polymyxin B (PMB) resistance that emerged in a previously PMB-susceptible KPC-2-producing K. pneumoniae during PMB therapy for a bloodstream infection in a neutropenic patient. The first isolate (PMB-susceptible) was obtained while the patient was receiving meropenem and other isolates were recovered from 2 sets of blood cultures in different dates while the patient was receiving PMB therapy (4 of 6 blood cultures bottles yielded isolates with full PMB resistance). The population analysis profile of the first isolate revealed the growth of resistant subpopulations with PFGE profile distinct from the parental isolate but undistinguishable from those obtained in subsequent days under PMB exposure. Resistant subpopulations were obtained from all parental PMB-susceptible and in one PMB-resistant isolate recovered from the patient. The molecular mechanism observed in the hetero-resistant subpopulations (IS1-like in mgrB-promoter region, increased rstB transcription with no mutation and non-identified mechanism) differed from those found in the PMB-resistant isolates, in which no mutation or transcriptional alterations were detected. This study showed that the mechanism of resistance to PMB that emerged during PMB therapy was not related to those observed in subpopulations selected in vitro from PMB-susceptible isolates recovered from the patient. The absence of mutations in the former isolates may be due to adaptive resistance occurred because of sub-optimal PMB levels as well as amikacin and meropenem used in combination.

Plasmid-mediated mcr-1 in carbapenem-susceptible Escherichia coli ST156 causing a blood infection: an unnoticeable spread of colistin resistance in Brazil?

OBJECTIVE: We describe an IncX4 pHC891/16mcr plasmid carrying mcr-1 in a colistin-resistant and carbapenem-susceptible E. coli isolate (HC891/16), ST156, which caused a blood infection in a Brazilian patient with gallbladder adenocarcinoma. METHODS: Strain HC891/16 was subjected to whole genome sequencing using the MiSeq Platform (Illumina, Inc., USA). Assembly was performed using Mira and ABACAS. RESULTS: The isolates showed resistance only to ciprofloxacin, ampicillin and cefoxitin, and whole-genome sequencing revealed the presence of aac(6')Ib-cr and blaTEM1. CONCLUSION: Our findings warn of the possible silent dissemination of colistin resistance by carbapenem-susceptible mcr-1 producers, as colistin susceptibility is commonly tested only among carbapenem-resistant isolates.