Such a strain would be called beta-hemolytic. Notice how the agar is completely transparent, showing objects behind the plate.
If the media around the bacteria changes to a dark green color but does not become transparent, the bacteria are displaying incomplete hemolysis, or alpha hemolysis. These bacteria are alpha-hemolytic. Notice how the media is discolored, but objects behind the blood agar plate cannot be seen. Knowing what type of hemolysis a bacterial strain is capable of can be helpful in identifying several types of bacteria, especially organisms isolated from human tissue such as Streptococcus and Staphylococcus species.
Staphylococcus aureus , Streptococcus pyogenes and Streptococcus agalactiae are b-hemolytic the picture on the right below shows the beta-hemolysis of pyogenes. Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic the picture in the middle below shows the a-hemolysis of S.
If no hemolysis occurs, this is termed gamma-hemolysis. There are no notable zones around the colonies. Staphylococcus epidermidis is gamma-hemolytic. Greenland and the Svalbard Archipelago are not drawn to scale. Table 1. Sample types and GPS coordinates of the sampling locations for this study. After the outer surface layer of subglacial ice was melted, it was discarded, the remaining ice was washed with sterile water and only the second round of meltwater was used for analyses.
Serial dilutions were used for soil and sediment samples. Water samples were filtered through Milli-pore membrane filters 0. Filters were placed onto R2A BioLife , an oligotrophic medium for heterotrophic microorganisms. Negative control plates were prepared with sterile water frozen prior to culturing and processed as the samples.
Two control agar plates were streaked for each testing temperature and incubated in the same conditions and for the same duration as the samples. Liquid and enrichment cultures were not used. Morphologically different colonies were selected and streaked onto fresh R2A plates in order to obtain pure cultures.
The use of human blood is generally discouraged Buxton, and thus, it was not used in our experiments. Only actively growing cultures were tested. Hemolytic activity of organic bacterial extracts was tested in real time using bovine and sheep erythrocyte suspensions rather than blood agar. Bacterial cultures environmental and 3 control strains described in Supplementary Table S2 were grown in sterile Falcon tubes containing 20 mL of Nutrient Broth BioLife.
When tested alone, ethanol did not induce any visible hemolysis during the 60 min. Results were observed as soon as the bacterial growth appeared between 1 and 7 days. A total of isolates were obtained from all the samples after incubation Supplementary Table S1.
Nutrient-rich samples, i. Based on 16S rDNA identification, abundance and morphological characteristics, unique strains were selected for further tests Supplementary Table S2. Bacillus sp. Two different experiments were performed to observe the hemolytic activity of the isolates.
The tested bacterial strains displayed all three phenotypes Figure 2. Our results showed that the hemolysis was influenced by the incubation temperature Table 2. Pseudomonas spp. Figure 2. Examples of hemolytic phenotypes expressed by the tested isolates.
Table 2. Bacillus spp. Of the Micromonospora spp. Isolates of Carnobacterium spp. Interestingly, isolates L Flavobacterium sp. In general, isolates that were non-hemolytic on bovine blood plates were also non-hemolytic on sheep blood plates Table 2 except for Pseudomonas spp. Species belonging to the genera Herminiimonas sp. The three control strains displayed their corresponding hemolytic phenotype on blood agar plates.
The second experiment involved the observation of hemolytic activity of the ethanolic extracts of bacterial lysates in a short assay within a min time frame using erythrocyte suspensions.
The objective of this experiment was to determine the potential presence of membrane-active secondary metabolites which could be present in the tested bacteria, and which could contribute to the pathogenicity of the producing organism. In the assays with bovine and sheep erythrocytes in suspension, only 5 extracts in total were hemolytic within 60 min of the assay: Pseudomonas sp. L, L, and L together with Micromonospora sp.
In general, for the Pseudomonas sp. Ethanolic extracts of the three control species displaying the hemolytic phenotype on blood agar plates were inactive on erythrocytes in suspension. Figure 3. Hemolytic activity of ethanolic extracts of Micromonospora sp.
A and Pseudomonas sp. The hemolytic activity was assayed against bovine full symbols and sheep open symbols erythrocytes. In total, isolates were tested for antimicrobial resistance.
The plates were read as soon as colonies were detected on them and the results are presented in Table 3 summary and Supplementary Table S3 all results , except for 14 isolates that did not grow on the used media or in the presence of any of the antimicrobials tested.
Resistant strains to higher concentrations of imipenem are mostly from the genera Flavobacterium , Enterococcus , Janthinobacterium , Raoultella , and, notably, Pseudomonas. Interestingly, not all Pseudomonas spp. For instance, strains L closely related to P. L , Janthinobacterium sp. L, Pseudomonas sp. L Supplementary Table S4. Table 3. The isolates mentioned above of which two were obtained from tap water , shared very similar antimicrobial resistance profiles, i.
All Pseudomonas sp. Generally, Bacillus spp. Micromonospora spp. Isolates belonging to the Enterobacteriaceae family i. L and Raoultella sp. Some isolates of the genera Cryobacterium L, L, L, and L , Massilia L and Undibacterium L and L , did not show growth on the control plates nutrient agar without added antimicrobials but generally showed better growth on the plates containing IPM.
Until recently, the Arctic represented a pristine environment, largely unaffected by anthropogenic influences. However, this geographic area is now experiencing the dramatic repercussions of climate change more than other regions on the planet Intergovernmental Panel on Climate Change [IPCC], Due to its low adaptability, these consequences are amplified Doney et al.
Moreover, bacterial species in the short term are expected to come in contact more frequently with humans, animals, and plants outside of their current environments Epstein, ; Cavicchioli et al. A considerable number of isolates in this study showed a hemolytic phenotype when cultured on blood agar plates, in accordance to recent reports Mogrovejo-Arias et al. The type of blood used bovine vs. While most of the bacterial isolates cultured on agar plates showed active growth, some genera Flavobacterium sp.
Several genera responded differently to the incubation temperature. For instance, Pseudomonas spp. Differences in the hemolysins structures and modes of action might explain that some of them have a lytic effect only above certain temperatures, at which the characteristics of erythrocyte membranes change due to increased membrane fluidity Carr et al. Alternatively, the expression of hemolytic genes might be temperature-regulated as described by Madrid et al.
Our study comprises a large and preliminary screening study of crude extracts where non-purified membrane-active compounds were present. To the best of our knowledge, it represents the first screening of this sort for hemolytic activity in Arctic environmental bacteria. The hemolytic activity of ethanolic extracts was tested in two types of erythrocyte suspensions: sheep and bovine. Sheep blood is commonly used as a standard for the determination of the hemolytic phenotype Yeh et al.
The hemolytic potential of human-associated, pathogenic or opportunistic bacteria is regularly assessed using animal blood Bevivino et al. Anuclear, mature red blood cells are often used as a model to assess the membrane damage, either by monitoring the hemoglobin release or by measuring the turbidity of erythrocyte suspension.
However, it should not be inferred that erythrocytes are the only target cells of the compounds observed in this study Tomita and Kamio, given that hemolytic compounds can often also be cytolytic for cells of the immune system, e.
The discrepancies in membrane composition among different mammalian erythrocytes influence the hemolytic activity of hemolysins, which act by recognizing specific membrane lipids or membrane receptors DuMont and Torres, ; Rojko and Anderluh, We observed that the lytic activity of the tested Pseudomonas sp.
Red blood cell membranes from different mammals display different composition and physical characteristics e. Cholesterol content, on the other hand, is higher in bovine red blood cells than in sheep ones Nelson, Thus, the faster hemolytic reaction on bovine erythrocytes observed might be because the hemolytic compound s produced by the isolates in this study have greater affinity for cholesterol-enriched membrane domains.
Based on our results, the use of more than one type of blood is recommended as it widens the spectrum of observed hemolytic activities. Future research could benefit from the inclusion of assays with human blood, the results of which could be predicted to some extend based on similarities with the types of blood used in the present study.
A greater number of isolates were found to have hemolytic activity on blood agar plates around 30 isolates vs. The culture conditions at which the isolates were grown in preparation for each test, i. Another possible explanation for such a remarkable difference between the tests is that each one assesses the presence of different compounds. Plates show the effects of a wider range of molecules produced by the bacteria in the course of their growth e. Crude extracts, moreover, might possess more than one hemolytically active and less polar compound, mainly acting as surfactants and whose function is independent of membrane lipid composition.
This might explain the observed absence of hemolytic activity of the ethanolic extract of S. In fact, the main hemolytic compounds characteristic of S. Finally, the growth phase of the culture at the moment of the test might vary, directly affecting the compounds produced by the bacteria. That is, compounds produced in actively growing cultures for instance, liquid cultures differ from those produced in cultures going through stationary phase for instance, solid cultures.
Studying antimicrobial resistance profiles in Arctic isolates is gaining in importance since climate change is exerting a stronger pressure on this environment and higher temperatures are associated with increased antimicrobial resistance in common pathogens Hutchins et al. Therefore, the definition of resistance for these bacteria is difficult to ascertain. In this study, we performed screening for phenotypic resistance on media supplemented with antimicrobials as an indicator for the presence of resistance genes.
On one hand, we observed that a majority of the Arctic environmental isolates analyzed were resistant to at least one of the antimicrobials tested, suggesting a strong competitiveness in the habitat. Some isolates were resistant even toward broad-spectrum antimicrobials, such as ciprofloxacin and chloramphenicol. Similar results were found in other natural environments such as Antarctica Tam et al.
On the other hand, almost all of the isolates were susceptible to tetracycline, contrary to what has been reported before for other cold environments Perron et al. Since tetracycline could be naturally occurring in soils, such widespread susceptibility is surprising, but could be due to a lack of tetracycline producers in the Arctic.
In accordance to our findings, Pal et al. The mutant phenotypes observed for ciprofloxacin are usually induced by low concentrations of this antimicrobial Cirz and Romesberg, Resistance to some antibiotics can occur quickly during selective enrichment, predominantly that which is triggered by point mutations in housekeeping genes, e. Accordingly, growth observed in plates containing ciprofloxacin, erythromycin, and kanamycin has been cautiously interpreted.
The constant appearance of a low number of individual, presumably mutant, colonies is indicated in Supplementary Table S3. Isolates belonging to the genus Pseudomonas were the ones with the strongest multidrug-resistant abilities, showing marked resistance tetracycline, and with frequent occurrence of individual mutant ciprofloxacin resistant colonies. Correspondingly, Pseudomonas spp. Pseudomonas sp. In addition, Pseudomonas sp.
Even though efflux pumps can be also associated with other functions in the cell, the responsible genes can be transferred to other environments and into other species where the resistance phenotype becomes of clinical relevance Graham et al. Finally, we observed that the concentration of imipenem used in this study favored, rather than inhibited, the growth of several isolates, notably Massilia sp.
Phenotypic resistance to imipenem is of high importance since carbapenemase-producing bacteria have become a major public health concern worldwide. In this study, we identified carbapenem resistant bacteria and predicted carbapenem resistance mechanisms CRM and the subsequent potential for horizontal spread.
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