Identification of Recovered Environmental Microbial Isolates

Thursday, October 21, 2021

Besides detecting and enumerating the number of microorganisms in an environmental test sample (i.e., air, compressed air, purified water and equipment surface monitoring), it is important to identify representative recovered environmental microbial isolates to detect changes in trends. A shift in the types of microorganisms being isolated in an environmental sample may indicate a deviation from the “norm.” There are several identification methodologies that can be used to identify recovered environmental microbial isolates.

Types of Identification Methods

To identify a recovered environmental microbial isolate, the following types of identification methodologies are commonly used:

  • Presumptive
  • Phenotypic
  • Phylogenetic

For conducting presumptive identification of an isolate, the following presumptive methods can be used such as color reactions on selective/ differential agars (i.e., Vogel-Johnson Agar, MacConkey Agar, Cetrimide Agar), Gram-stain results (e.g., positive or negative and morphology - cocci, bacilli, yeast) and diagnostic tests (i.e., Catalase, Coagulase, Lysostaphin, Bacitracin, Cytochrome Oxidase, Oxidation/Fermentation (O/F) tests).

To conduct a phenotypic identification of an isolate, the following methods are commonly used: biochemical identification kits and MALDI-TOF Mass Spectrometry. Carbon utilization and biochemical reactions are used in biochemical identification kits to identify an isolate to the genus/species level. MALDI-TOF Mass Spectrometry generates a proteomic fingerprint of an isolate to determine an identification of an isolate to the genus/species level.

For identification of bacteria, the most common phylogenetic method is 16S rRNA sequencing. For identification of fungi, the most common phylogenetic methods are Fungal Internal Transcribed Spacer (ITS2) and LSU-D2 rDNA sequencing.

Gram-Staining

The first identification step of recovered environmental bacterial isolates is to subculture representative isolates from the isolation plate based upon colony morphology characteristics such as color, form, elevation, surface, and margin onto a sterile Petri dish of Soybean-Casein Digest Agar Medium (SCDAM) for Gram-staining. However, it should be noted that water isolates that have been recovered by using R2A Agar or Plate Count Agar may instead need to be subcultured onto a low nutrient agar such as Plate Count Agar to obtain sufficient growth for Gram-staining and identification because they are nutritionally stressed and might not be able to grow on a high nutrient medium such as SCDAM.1,2 A Gram-stain of an 18-to-24-hour isolate is used to determine Gram reaction and colony morphology (e.g., bacilli, cocci, or yeast). Bacterial Gram-staining is not required if the identification of an isolate to the genus/species level is going to be conducted by using 16S rRNA sequencing or MALDI-TOF. If an incorrect Gram-stain for an isolate is obtained by either excessive heat fixation, over decolorization or using cultures older than 24-hours in age, it is very possible to use an incorrect biochemical identification kit resulting in an incorrect identification.

Environmental isolates such as Actinomyces, Corynebacterium and Cutibacterium species are hard to Gram-stain because they have cell walls sensitive to breakage during the cell division causing them to stain Gram-negative while being Gram-positive and the result is a Gram-variable reaction.3  For confirmation of Gram-stain results for a Gram-variable microorganism, a KOH string test is recommended to be performed.

Bacillus and Paenibacillus Species

Bacillus and Paenibacillus species are endospore forming Gram-positive bacilli and are common environmental isolates. In general, Bacillus species are not identified to genus/species level due to difficulty in identifying some members of the genus by using biochemical or phylogenetic identification methods. For example, it is difficult to biochemically separate out the individual members of the Bacillus cereus group.4,5 Furthermore, there are phylogenetic identification issues with the Bacillus cereus group. The 16S rRNA gene sequences of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis have high levels of sequence similarity (>99%) that support the close relationship shown by DNA hybridization studies.6,7 Some strains of Bacillus anthracis and Bacillus cereus differ by only a single nucleotide position.8 There also has been a report of Bacillus anthracis having a sequence that is identical to a Bacillus cereus sequence.9

It can be difficult to biochemically separate Bacillus and Paenibacillus species from one another.4  A combination of both genotypic and phenotypic identification methods may have to be used to identify isolate as a Paenibacillus species.10

Non-Spore Forming Gram-Positive Bacilli

Actinomyces, Corynebacterium, Erysipelothrix, Listeria, Cutibacterium and Lactobacillus species are non-spore forming Gram-positive bacilli that can be isolated in an environmental sample. Actinomyces species can be separated from the other non-spore forming Gram-positive bacilli because a Gram-stain will show a branched network of hyphae under a microscope. In addition, Actinomyces species can be distinguished from other bacterial colonies on an agar surface because they tend to be very dense and tough. To presumptively separate Corynebacterium, Cutibacterium, Erysipelothrix, Listeria and Lactobacillus, the following biochemical tests can be performed: Catalase, Hydrogen sulfide and Motility. Corynebacterium, Cutibacterium and Listeria species are Catalase-positive, while Lactobacillus and Erysipelothrix species are Catalase-negative. Erysipelothrix species are positive and Lactobacillus species are negative for Hydrogen sulfide production on TSI Agar. Corynebacterium and Cutibacterium species are non-motile while Listeria species are motile (Tumbling Motion) only at room temperature. It is exceedingly difficult to use presumptive biochemical reactions to separate Cutibacterium species from other non-spore forming Gram-positive bacilli. Gram-positive bacilli isolates can be identified to the species level by using biochemical identification kits, 16S rRNA sequencing or MALDI-TOF analysis.

Gram-Positive Cocci

It is common to isolate Gram-positive cocci in environmental samples. A presumptive Catalase test can be used to separate Gram-positive cocci into two groups: Catalase-positive (Staphylococcus, Micrococcus or Kocuria) and Catalase-negative (Enterococcus, Streptococcus, or Aerococcus). To presumptively separate Staphylococcus from Micrococcus and Kocuria, a Lysostaphin or a Bacitracin sensitivity test can be used. In a Lysostaphin sensitivity test, Staphylococcus species are sensitive while Micrococcus and Kocuria species are resistant. In a Bacitracin sensitivity test, Staphylococcus species are resistant while Micrococcus and Kocuria species are sensitive. 

It is difficult to differentiate Kocuria from Micrococcus with conventional biochemical methods. Therefore, these organisms are often reported together as Micrococcus/Kocuria species. Phenotypic identification methods may fail to recognize all the members of Kocuria species because the results of biochemical and carbon assimilation tests are shown to be heterogeneous in the different species of Kocuria. Most biochemical databases do not include all the classified Kocuria species. Identification of Kocuria species is now typically performed by using 16S rRNA sequencing and MALDI-TOF.11 

The Coagulase test is used as a presumptive test to determine whether a Staphylococcus isolate is Staphylococcus aureus. However, there are now seven recognized Coagulase-positive staphylococci: Staphylococcus aureus, S. intermedius, S. schleiferi subsp. coagulans, S. hyicus, S. lutrae, S. delphini, and S. pseudintermedius.(12,13,14,15,16,17) In addition, there are now Staphylococcus aureus isolates that are  Coagulase-negative.(18,19,20) It cannot be assumed that all Coagulase-positive Gram-positive cocci isolates are Staphylococcus aureus,  and all Coagulase-negative Gram-positive cocci isolates are not Staphylococcus aureus. Instead of using the Coagulase test, it is recommended that all recovered staphylococci isolates be identified to the species level by using biochemical identification kits, 16S rRNA sequencing or MALDI-TOF. 

Isolation of Catalase-negative Gram-positive cocci in environmental samples would be rare, but it can occur. Catalase-negative Gram-positive cocci that can be isolated is Aerococcus, Enterococcus and  Streptococcus species. Aerococcus is difficult to presumptively identify because it resembles alpha-hemolytic Streptococcus species on Blood Agar Plates and is also difficult to identify by just using biochemical tests.21 Presumptive biochemical tests such as Optochin, Bacitracin, Growth in 6.5% NaCl, Bile Esculin hydrolysis, and Hippurate hydrolysis can be used to separate Enterococcus and Streptococcus species. For identifying Aerococcus to the genus/species level, it is recommended that 16S rRNA sequencing or MALDI-TOF be used.21 For Enterococcus and Streptococcus, biochemical identification kits, 16S rRNA sequencing or MALDI-TOF are used.

Gram-Negative Bacilli

Gram-negative bacilli can be recovered in environmental samples. For presumptive biochemical testing, the Oxidase test is used to confirm the presence of Cytochrome C oxidase enzyme to differentiate Gram-negative bacilli that are Oxidase-positive (ox +) and Oxidase-negative  (ox -) groupings. The presumptive Oxidation- Fermentation (O/F) test can be used to determine whether Gram-negative bacilli can metabolize glucose by fermentation or aerobic respiration (oxidatively) to separate Oxidase-positive Gram-negative bacilli into Oxidative (i.e., Pseudomonas, Burkholderia, Sphingomonas species, Stentrophomonas maltophilia, and Flavimonas oryzihabitans), Fermentative (Vibrio, Aeromonas, Photobacterium, and Plesiomonas species) and Negative (Alcaligenes and Moraxella species)  groupings and Oxidase-negative Gram-negative bacilli into Oxidative (i.e., Acinetobacter species, Pseudomonas luteola, and Stentrophomonas maltophilia) and Fermentative (Enterobacteriaceae) groupings. Based upon the results of these two biochemical presumptive tests, appropriate biochemical identifications kits can be used to determine an identification of an isolate to the genus/species level. In addition, 16S rRNA sequencing or MALDI-TOF analysis can also be used to identify isolates to the genus/ species level.

Mold Isolates

It is very common to isolate mold in environmental samples. Phenotypic and phylogenetic identification methods can be used to identify a mold isolate to the species level. Phenotypic identification methods for mold are as follows: macroscopic and microscopic characteristics (Traditional Morphological Approach), Biolog FF MicroPlateTM, and MALDI-TOF. The Traditional Morphological Approach is based on macroscopic (colonial) features and microscopic characteristics of an isolate that is used in an identification key to determine a genus/species identification. Macroscopic features used in a key involves the usage of color, texture, colony reverse color and the presence of diffusible pigments. Microscopic characteristics used in a key includes the types of hyphae, type of spores and reproductive structures that are present for an isolate. Traditional morphological identification method will often lack specificity for determining a species identification of a recovered mold isolate and can be time-consuming. An identification of a mold isolate to the genus/species level is subjective based upon the presence of morphological structures. The Biolog FF MicroPlateTM utilizes both color development (reduction of tetrazolium) from the oxidation of substrates and turbidity from fungal growth to give a characteristic reaction pattern or fingerprint for an identification of a mold isolate to the species level. MALDI-TOF is generated to generate a proteomic fingerprint of a mold isolate that is used to determine an identification of a mold isolate to the species level. However, MALDI-TOF mold commercial databases needs to be improved because the library coverage is insufficient.22,23 Most people are using in-house databases as a supplement. 

For phylogenetic identification methods for mold isolates, there are two distinct approaches: rDNA sequencing of the D2 region of the large subunit ribosomal DNA (D2 LSU) and sequencing of either one or both of the internal transcribed spacer regions (ITS). The ITS2 has more variability than the D2 expansion segment DNA sequence, thus providing greater specificity.24 This yields a much higher resolution in fungi species identification than D2. In cases where species level identification cannot be obtained using D2 LSU sequences, it is often possible to obtain species level identification using ITS sequencing. Generally, there is a higher level of differentiation between ITS sequences. In some cases, D2 LSU sequencing alone cannot distinguish between different, but closely related genera or it was not possible to differentiate between an even larger group of closely related genera, ITS sequencing has been successful in providing a species level identification.

Yeast Isolates

Yeast isolates are commonly found in environmental samples and will Gram-stain as positive. Phenotypic methods such as biochemical identification kits and MALDI-TOF analysis can be used to identify yeast isolates to the genus/species level. The following presumptive biochemical tests can be used for an identification of a yeast isolate as Candida albicans: selective/differential agars such as Bismuth Glucose Glycine Yeast (BIGGY) Agar, various chromogenic agars, and a rapid biochemical test for detecting the presence of the following two enzymes: L-Proline aminopeptidase and ß-galactoaminidase. Phylogenetic rDNA sequencing of the D2 Region of the Large Subunit (LSU) and the ITS2 region can also be used to determine an identification of a yeast isolate to the genus/species level.

Conclusion

There are many different methods that can used for the identification of a recovered environmental microbial isolate to the genus/species level.

References

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