Complexo Acinetobacter calcoaceticus - Acinetobacter baumannii (ACB): ocorrência e perfil de resistência aos carbapenêmicos e polimixina B durante pandemia de SARS-CoV-2 em Pelotas, RS

Bactérias do complexo Acinetobacter calcoaceticus - Acinetobacter baumannii (ACB) são causa frequente das chamadas Infecções Relacionadas à Assistência à Saúde (IRAS). Durante a pandemia de COVID-19, causada pelo vírus SARS-CoV-2, coinfecções e aumento da resistência bacteriana aos antibióticos têm sido observados. Assim, o presente estudo verificou a ocorrência e perfil de resistência aos carbapenêmicos (no ano de 2020) e a polimixina B (de outubro de 2020 a março de 2021) em bactérias do complexo ACB, durante a pandemia de SARS-CoV-2, no Hospital Escola (HE) da Universidade Federal de Pelotas (UFPel/EBSERH). Duas metodologias foram aplicadas, a de identificação e avaliação da suscetibilidade bacteriana por automação (BD Phoenix™), e avaliação da resistência a polimixina B utilizando painel de microdiluição (CIM POLIMIXINA B, Laborclin). Oitenta e um isolados pertencentes ao complexo ACB foram identificados, sendo 69,1% (56) da espécie A. baumannii e 30,9% (25) de outras espécies do complexo ACB. Foi observado um aumento da resistência aos carbapenêmicos de 75% em 2019, para 94,3% em 2020. Dentre as bactérias do complexo ACB resistentes aos carbapenêmicos, 4 delas foram resistentes também a polimixina B. As bactérias do complexo ACB resistentes aos carbapenêmicos foram mais frequentes na UTI COVID-19, representando 44,9% em relação as outras unidades. Esses isolados resistentes foram obtidos de amostras de aspirado traqueal e sangue. Os dados obtidos revelam um aumento da resistência aos carbapenêmicos, além de uma maior frequência de bactérias do complexo ACB obtidas de aspirado traqueal de pacientes diagnosticados com COVID-19, o que pode estar relacionado a quadros de Pneumonia Associada a Ventilação Mecânica (PAVM).

Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumanniicomplex

Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes isolated from their functional contexts. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and micro-synteny conservation analyses. This allowed to delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with, or are found preferentially in, the pathogenic ACB clade. They unveil, at an unprecedented resolution, the genetic makeup that coincides with the manifestation of the pathogenic phenotype in the last common ancestor of the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. Specifically, we could show that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. They disclose promising routes for future therapeutic strategies.

Synergism of imipenem with fosfomycin associated with the active cell wall recycling and heteroresistance in Acinetobacter calcoaceticus-baumannii complex

The carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complex has become an urgent threat worldwide. Here, we determined antibiotic combinations and the feasible synergistic mechanisms against three couples of ACB (A. baumannii (AB250 and A10), A. pittii (AP1 and AP23), and A. nosocomialis (AN4 and AN12)). Imipenem with fosfomycin, the most effective in the time-killing assay, exhibited synergism to all strains except AB250. MurA, a fosfomycin target encoding the first enzyme in the de novo cell wall synthesis, was observed with the wild-type form in all isolates. Fosfomycin did not upregulate murA, indicating the MurA-independent pathway (cell wall recycling) presenting in all strains. Fosfomycin more upregulated the recycling route in synergistic strain (A10) than non-synergistic strain (AB250). Imipenem in the combination dramatically downregulated the recycling route in A10 but not in AB250, demonstrating the additional effect of imipenem on the recycling route, possibly resulting in synergism by the agitation of cell wall metabolism. Moreover, heteroresistance to imipenem was observed in only AB250. Our results indicate that unexpected activity of imipenem on the active cell wall recycling concurrently with the presence of heteroresistance subpopulation to imipenem may lead to the synergism of imipenem and fosfomycin against the ACB isolates.

Identification and antimicrobial susceptibility of microbial agents of otitis externa in dogs

In this study, a total of 277 unmedicated dogs with otitis externa were used. Overall, 413 agents were isolated from 277 ear swab samples; 52.7% of the cases were mono-infections (146 cases), and 37.1% of the cases were poly-infections (103 cases). In 10.1% (28) of the cases, neither bacteria nor yeasts were isolated. Coagulase-positive Staphylococcus spp. were the most frequently isolated bacteria and were found in 90 (21.8%) of the samples. Fifty-eight samples, (14%) were positive for Staphylococcus aureus, 51 (12.3%) for Pseudomonas aeruginosa, 27 (6.5%) for Proteus mirabilis, 27 (6.5%) for Malassezia pachydermatis, 21 (5%) for Corynebacterium spp., 21 (5%) for β-haemolytic Streptococcus spp., 15 (3.6%) for Staphylococcus pseudointermedius, 12 (2.9%) for Proteus spp., 12 (2.9%) for Escherichia coli, 9 (2.1%) for Acinetobacter calcoaceticus, 7 (1.6%) for Trichophyton mentagrophytes, 5 (1.2%) for Staphylococcus auricularis, and 46 (11.1%) for different bacteria and yeasts. A total of 14 different bacteria and yeasts were isolated and identified. Kirby-Bauer antibiotic susceptibility testing was carried out for 10 different antibiotics. The bacterial isolates were found to be resistant to amoxicillin-clavulanic acid (45%), gentamycin (28%), ampicillin/cloxacillin (69%), tobramycin (28%), amikacin (23%), enrofloxacin (47%), chloramphenicol (58%), doxycycline (65%), lincomycin/spectinomycin (58%) and polymyxin B (62%). In conclusion, it is important to test the antimicrobial sensitivity of aetiological agents of otitis externa before treatment so as to prevent the development of antibiotic resistance in bacteria and yeasts.

A novel enzyme synthesized by Acinetobacter sp. SM04 is responsible for zearalenone biodegradation

Zearalenone (ZEA), a non-steroidal estrogenic mycotoxin produced by multiple Fusarium species, contaminates cereals and threatens the health of both humans and animals by inducing hepatotoxicity, immunotoxicity, and genotoxicity. A new alkali tolerant enzyme named Ase, capable of degrading ZEA without H2O2, was derived from Acinetobacter sp. SM04 in this study. The Ase gene shares 97% sequence identity with hypothetical proteins from Acinetobacter pittii strain WCHAP 100004 and YMC 2010/8/T346 and Acinetobacter calcoaceticus PHEA-2, respectively. Based on the Acinetobacter genus database, the gene encoding Ase was cloned and extracellularly expressed in E. coli BL21. After degrading 88.4% of ZEA (20 μg/mL), it was confirmed through MCF-7 cell proliferation assays that Ase can transform ZEA into a non-estrogenic toxic metabolite. Recombinant Ase (molecular weight: 28 kDa), produced by E. coli BL21/pET32a(+)-His-Ase, was identified as an oxygen-utilizing and cytochrome-related enzyme with optimal activity at 60 °C and pH 9.0.

Аcinetobacter baumannii bv Tryptophandestruens bv nov. isolated from clinical samples

The aim of the study was to determine the taxonomic status of a group consisting of atypical strains of Acinetobacter baumannii, outline relevant characteristics and methods necessary for their identification. There were examined 10 strains of A. baumannii (6 of them primary comprised) bearing similar profile of atypical features isolated from clinical samples (urine, sputum) in 2017–2019 at the Military Medical Academy. Сlinical strains of typical A. baumannii (n = 36), Acinetobacter nosocomialis (n = 14), Acinetobacter pittii (n = 9) and 1 strain of Acinetobacter calcoaceticus isolated from the external environment were used in comparative studies. Atypical strains had the characteristics of A. calcoaceticus — A. baumannii (ACB) complex bacteria and were identified as A. baumannii. The utilization of substrates as the only carbon source was studied on a dense synthetic medium added with 0.2 % substrate during incubation for 72 hours at 37°C. Carbohydrate oxidation coupled to acid formation was detected on the Hugh–Leifson medium by using a micromethod. Aromatic amino acid biotransformation was carried out in liquid and dense nutrient media assessed in chromogenic reaction. The rpoB gene was used for strain genetic characterization. Amplification of two 940 and 1210 base pair (bp)-long fragments from the rpoB gene was performed by the routine polymerase chain reaction using primers with previously described sequences. Amplification products were sequenced by Sanger using Big Dye Terminator v3.1 (Applied Biosystems, USA) and capillary electrophoresis on an automatic sequencer ABI PRISM 3130 (Applied Biosystems, USA), followed by using methods for determining the similarity levels of sequenced fragments with the rpoB gene sequences of the reference strain A. baumannii ATCC 17978 (GenBank accession no. CP053098.1). It was found that all strains belonging to atypical A. baumannii spp. had a specific set of features that distinguish them from typical strains of A. baumannii as well as other types of the ACB complex: detected biotransformation of L-tryptophan (via anthranilate pathway) and anthranilic acid under unambiguous lack of such signs in other bacteria; lack of utilized sodium hippurate and L-arabinose being unambiguously evident in other bacteria; lack of utilized L-tryptophan, putrescine, L-ornithine being utilized in the majority of strains of belonging to other bacterial species. Genetic analysis showed that the control strains of typical A. baumannii displayed 99.20–99.21% similarity within the sequenced fragments of the rpoB gene with those from the rpoB gene of the reference strain. All 10 strains of atypical A. baumannii had similar features (99.20–99.21%). At the same time, parameters of control strains from other bacterial species significantly differed: A. nosocomialis (95.10–95.97%), A. pittii (94.63–94.92%), A. calcoaceticus (93.00%). Hence, the strains of atypical and typical A. baumannii are genetically homogeneous and belong to the same species. The data presented allow us to consider this group of atypical A. baumannii strains as a new biovar. We propose the name for this new biovar — tryptophandestruens (tryptophan-destroying) stemming from the Latin word destruens — destroying. Identification of A. baumannii bv. tryptophandestruens bacteria can be carried out in laboratory of any level by using tests for L-tryptophan biotransformation as well as sodium hippurate utilization.

Bacteria Cell Hydrophobicity and Interfacial Properties Relationships: A New MEOR Approach

For microbial enhanced oil recovery (MEOR), different mechanisms have been introduced. In some of these papers, the phenomena and mechanisms related to biosurfactants produced by certain microorganisms were discussed, while others studied the direct impacts of the properties of microorganisms on the related mechanisms. However, there are only very few papers dealing with the direct impacts of microorganisms on interfacial properties. In the present work, the interfacial properties of three bacteria MJ02 (Bacillus Subtilis type), MJ03 (Pseudomonas Aeruginosa type), and RAG1 (Acinetobacter Calcoaceticus type) with the hydrophobicity factors 2, 34, and 79% were studied, along with their direct impact on the water/heptane interfacial tension (IFT), dilational interfacial visco-elasticity, and emulsion stability. A relationship between the adsorption dynamics and IFT reduction with the hydrophobicity of the bacteria cells is found. The cells with highest hydrophobicity (79%) exhibit a very fast dynamic of adsorption and lead to relatively large interfacial elasticity values at short adsorption time. The maximum elasticity values (at the studied frequencies) are observed for bacteria cells with the intermediate hydrophobicity factor (34%); however, at longer adsorption times. The emulsification studies show that among the three bacteria, just RAG1 provides a good capability to stabilize crude oil in brine emulsions, which correlates with the observed fast dynamics of adsorption and high elasticity values at short times. The salinity of the aqueous phase is also discussed as an important factor for the emulsion formation and stabilization.

MICROBIAL AGENTS AS TRIGGERS OF MULTIPLE SCLEROSIS DEVELOPMENT

Multiple sclerosis (MS) is currently an urgent medical and social problem. This is due to the high prevalence of this pathology among neurological diseases with the predominant lesion of young people and subsequent rapid disability. This disease still remains a mystery for medicine in view of its ambiguous etiology, polymorphism of clinical manifestations and unstable course. Despite the significant development of modern instrumental diagnostic methods, for multiple sclerosis, pathognomonic signs have not yet been identified that make it possible to diagnose the disease with high accuracy in the early stages. At the moment, we can only say with confidence that MS is a neurodegenerative disease accompanied by rapid demyelination and death of nerve cells. Complex and diverse pathogenetic mechanisms suggest a multifactorial nature of the disease, which develops with a combination of external factors and hereditary predisposition, which causes a violation of immune tolerance. The most substantiated is the polygenic theory of MS, which implies that the genotype of MS patients consists of many genes, each of which contributes to the development of the disease. More than 100 genes associated with MS have been identified, among which a special place is occupied by the HLA system (human leukocyte antigen), which controls the interaction of immunocompetent cells and carries out an immune response. In addition, new candidate genes have been identified that contribute to the development of MS: genes of interleukin 2 and 7 receptors (IL-2R, IL-7R), differentiation cluster 6 (CD6) and 58 (CD58), tumor necrosis factor α, interferon regulatory factor 8 (IRF8), interleukin 12А (IL12A) and others.However, to realize genetic predisposition, the influence of external trigger factors is necessary. The activation of the demyelinating process is quite often initiated by various infectious agents, among which the most studied viruses are Epstein-Barr, John-Cunningham, acute encephalomyelitis, and human endogenous retroviruses. Particular attention in the development of neurodegenerative disorders deserves a change in the intestinal microbiota, by counting microorganisms such as Candida albicans, Staphylococcus aureus, Acinetobacter calcoaceticus, Bacteroides, Proteobacteria and Firmicutes. This imbalance has a significant effect on the functioning of the immune and nervous systems, taking part in the processes of neurogenesis, myelination, activation of the cellular and humoral types of the immune response. This review presents and analyzes latest data from domestic and foreign literature on the study of the epidemiological features of MS, as well as microbiological risk factors for the development of the disease.

Anthracene and Pyrene Biodegradation Performance of Marine Sponge Symbiont Bacteria Consortium

Every petroleum-processing plant produces sewage sludge containing several types of polycyclic aromatic hydrocarbons (PAHs). The degradation of PAHs via physical, biological, and chemical methods is not yet efficient. Among biological methods, the use of marine sponge symbiont bacteria is considered an alternative and promising approach in the degradation of and reduction in PAHs. This study aimed to explore the potential performance of a consortium of sponge symbiont bacteria in degrading anthracene and pyrene. Three bacterial species (Bacillus pumilus strain GLB197, Pseudomonas stutzeri strain SLG510A3-8, and Acinetobacter calcoaceticus strain SLCDA 976) were mixed to form the consortium. The interaction between the bacterial consortium suspension and PAH components was measured at 5 day intervals for 25 days. The biodegradation performance of bacteria on PAH samples was determined on the basis of five biodegradation parameters. The analysis results showed a decrease in the concentration of anthracene (21.89%) and pyrene (7.71%), equivalent to a ratio of 3:1, followed by a decrease in the abundance of anthracene (60.30%) and pyrene (27.52%), equivalent to a ratio of 2:1. The level of pyrene degradation was lower than that of the anthracene due to fact that pyrene is more toxic and has a more stable molecular structure, which hinders its metabolism by bacterial cells. The products from the biodegradation of the two PAHs are alcohols, aldehydes, carboxylic acids, and a small proportion of aromatic hydrocarbon components.