Microbial traces and their role in forensic science.

Forensic microbiology, also known as the microbiology of death, is an emerging branch of science that is still underused in criminal investigations. Some of the cases might be difficult to solve with commonly used forensic methods, and then they become an operational field for microbiological and mycological analyses. The aim of our review is to present significant achievements of selected studies on the thanatomicrobiome (micro-organisms found in the body, organs and fluids after death) and epinecrotic community (micro-organisms found on decaying corpses) that can be used in forensic sciences. Research carried out as a part of the forensic microbiology deals with the thanatomicrobiome and the necrobiome-communities of micro-organisms that live inside and outside of a putrefying corpse. Change of species composition observed in each community is a valuable feature that gives a lot of information related to the crime. It is mainly used in the estimation of post-mortem interval (PMI). In some criminal investigations, such noticeable changes in the microbiome and mycobiome can determine the cause or the actual place of death. The microbial traces found at the crime scene can also provide clear evidence of guilt. Nowadays, identification of micro-organisms isolated from the body or environment is based on metagenome analysis and 16S rRNA gene amplicon-based sequencing for bacteria and ITS rRNA gene amplicon-based sequencing for fungi. Cultivation methods are still in use and seem to be more accurate; however, they require much more time to achieve a final result, which is an unwanted feature in any criminal investigation.

Comparison of Antibacterial Mode of Action of Silver Ions and Silver Nanoformulations With Different Physico-Chemical Properties: Experimental and Computational Studies.

The aim of this study was to compare the antibacterial mode of action of silver ions (Ag+) and selected silver nanoformulations against E. coli strains (E. coli J53, Escherichia coli BW25113 and its derivatives: Δ ompA, Δ ompC, Δ ompF, Δ ompR, ompRG596AcusSG1130A, cusSG1130A). In this research we used various experimental methods and techniques such as determination of the minimal inhibitory concentration, flow cytometry, scanning electron microscopy, circular dichroism as well as computational methods of theoretical chemistry. Thanks to the processing of bacteria and silver samples (ions and nanoformulations), we were able to determine the bacterial sensitivity to silver samples, detect reactive oxygen species (ROS) in the bacterial cells, visualize the interaction of silver samples with the bacterial cells, and identify their interactions with proteins. Differences between the mode of action of silver ions and nanoformulations and the action of nanoformulations themselves were revealed. Based on the results of computational methods, we proposed an explanation of the differences in silver-outer protein interaction between silver ions and metallic silver; in general, the Ag0 complexes exhibit weaker interaction than Ag+ ones. Moreover, we identified two gutter-like areas of the inner layer of the ion channel: one more effective, with oxygen-rich side chains; and another one less effective, with nitrogen-rich side chains.

How Bacteria Change after Exposure to Silver Nanoformulations: Analysis of the Genome and Outer Membrane Proteome.

OBJECTIVE: the main purpose of this work was to compare the genetic and phenotypic changes of E. coli treated with silver nanoformulations (E. coli BW25113 wt, E. coli BW25113 AgR, E. coli J53, E. coli ATCC 11229 wt, E. coli ATCC 11229 var. S2 and E. coli ATCC 11229 var. S7). Silver, as the metal with promising antibacterial properties, is currently widely used in medicine and the biomedical industry, in both ionic and nanoparticles forms. Silver nanoformulations are usually considered as one type of antibacterial agent, but their physical and chemical properties determine the way of interactions with the bacterial cell, the mode of action, and the bacterial cell response to silver. METHODS: the changes in the bacterial genome, resulting from the treatment of bacteria with various silver nanoformulations, were verified by analyzing of genes (selected with mutfunc) and their conservative and non-conservative mutations selected with BLOSUM62. The phenotype was verified using an outer membrane proteome analysis (OMP isolation, 2-DE electrophoresis, and MS protein identification). RESULTS: the variety of genetic and phenotypic changes in E. coli strains depends on the type of silver used for bacteria treatment. The most changes were identified in E. coli ATCC 11229 treated with silver nanoformulation signed as S2 (E. coli ATCC 11229 var. S2). We pinpointed 39 genes encoding proteins located in the outer membrane, 40 genes of their regulators, and 22 genes related to other outer membrane structures, such as flagellum, fimbria, lipopolysaccharide (LPS), or exopolysaccharide in this strain. Optical density of OmpC protein in E. coli electropherograms decreased after exposure to silver nanoformulation S7 (noticed in E. coli ATCC 11229 var. S7), and increased after treatment with the other silver nanoformulations (SNF) marked as S2 (noticed in E. coli ATCC 11229 var. S2). Increase of FliC protein optical density was identified in turn after Ag+ treatment (noticed in E.coli AgR). CONCLUSION: the results show that silver nanoformulations (SNF) exerts a selective pressure on bacteria causing both conservative and non-conservative mutations. The proteomic approach revealed that the levels of some proteins have changed after treatment with appropriate SNF.

First Speleomycological Study on the Occurrence of Psychrophilic and Psychrotolerant Aeromycota in the Brestovská Cave (Western Tatras Mts., Slovakia) and First Reports for Some Species at Underground Sites.

Most underground ecosystems are heterotrophic, fungi in these objects are dispersed in the air in the form of spores, and they may be potentially hazardous to mammals. Research in underground sites has focused on mesophilic airborne fungi and only a few concerned cold-adapted species. Therefore, the goal of our research was the first report of psychrophilic and psychrotolerant aeromycota in the Brestovská Cave using culture-based techniques with genetic and phenotypic identification. Plates with PDA medium containing sampled biological material were incubated at 8 ± 0.5 °C. The density of mycobiota inside the cave ranged from 37.4 to 71 CFU 1 m-3 of air and 63.3 CFU 1 m-3 of air outside the cave. Thus, the level of fungal spores did not exceed the standards for the mycological quality of the air. A total of 18 species were isolated during the study, and some species may be potentially dangerous to people with weakened immune system. All fungal species were present inside the cave and only seven of them were outside. Cladosporium cladosporioides dominated in the external air samples and Mortierella parvispora was cultured most frequently from internal air samples. To our knowledge, this is the first discovery of the fungal species such as Coniothyrium pyrinum, Cystobasidium laryngis, Filobasidium wieringae, Leucosporidium drummii, M. parvispora, Mrakia blollopis, Nakazawaea holstii, and Vishniacozyma victoriae in the air inside the underground sites. Moreover, C. pyrinum, C. laryngis, L. drummii, M. blollopis, and N. holstii have never been detected in any component of the underground ecosystems. There are possible reasons explaining the detection of those species, but global warming is the most likely.

Consequences Of Long-Term Bacteria's Exposure To Silver Nanoformulations With Different PhysicoChemical Properties.

PURPOSE: Resistance to antibiotics is a major problem of public health. One of the alternative therapies is silver - more and more popular because of nanotechnology development and new possibilities of usage. As a component of colloid, powder, cream, bandages, etc., nanosilver is often recommended to treat the multidrug-resistant pathogens and we can observe its overuse also outside of the clinic where different physicochemical forms of silver nanoformulations (e.g. size, shape, compounds, surface area) are introduced. In this research, we described the consequences of long-term bacteria exposure to silver nanoformulations with different physicochemical properties, including changes in genome and changes of bacterial sensitivity to silver nanoformulations and/or antibiotics. Moreover, the prevalence of exogenous resistance to silver among multidrug-resistant bacteria was determined. MATERIALS AND METHODS: Gram-negative and Gram-positive bacteria strains are described as sensitive and multidrug-resistant strains. The sensitivity of the tested bacterial strains to antibiotics was carried out with disc diffusion methods. The sensitivity of bacteria to silver nanoformulations and development of bacterial resistance to silver nanoformulations has been verified via determination of the minimal inhibitory concentrations. The presence of sil genes was verified via PCR reaction and DNA electrophoresis. The genomic and phenotypic changes have been verified via genome sequencing and bioinformatics analysis. RESULTS: Bacteria after long-term exposure to silver nanoformulations may change their sensitivity to silver forms and/or antibiotics, depending on the physicochemical properties of silver nanoformulations, resulting from phenotypic or genetic changes in the bacterial cell. Finally, adaptants and mutants may become more sensitive or resistant to some antibiotics than wild types. CONCLUSION: Application of silver nanoformulations in the case of multiple resistance or multidrug-resistant bacterial infection can enhance or decrease their resistance to antibiotics. The usage of nanosilver in a clinic and outside of the clinic should be determined and should be under strong control. Moreover, each silver nanomaterial should be considered as a separate agent with a potential different mode of antibacterial action.

Similarities and Differences between Silver Ions and Silver in Nanoforms as Antibacterial Agents.

Silver is considered as antibacterial agent with well-known mode of action and bacterial resistance against it is well described. The development of nanotechnology provided different methods for the modification of the chemical and physical structure of silver, which may increase its antibacterial potential. The physico-chemical properties of silver nanoparticles and their interaction with living cells differs substantially from those of silver ions. Moreover, the variety of the forms and characteristics of various silver nanoparticles are also responsible for differences in their antibacterial mode of action and probably bacterial mechanism of resistance. The paper discusses in details the aforementioned aspects of silver activity.