Enterobacteriaceae characteristics and identification

Melissa Cavallo, Vasili Giannoulis, Katie De Paoli and Jo Houlden

Aim:
To identify whether or not human enteric bacteria was present in a sample of river water in order to confirm or deny its contamination due to a leaking septic tank.

Discussion: Results:
The preliminary tests to identify the bacterium displayed a negative result for the Gram Stain, thus ruling out the Unknown B (Table 1). Positive and negative controls, using various bacteria, were applied for testing the efficiency in the conventional biochemical tests as illustrated in Tables 1 & 2. The key results highlighted in Table 1 identified the Enterobacteriaceae family for which was used in all subsequent tests.
The results of the Microbact, Enterotube and conventional biochemical tests led to three different identifications which were Enterobacter agglomerans, Klebsiella pneumoniae and Enterobacter gergoviae, respectively (Tables 2 & 3). The varying identifications were due to inconsistencies present within the test systems which pertained to the citrate, adonitol and motility tests (Tables 1, 2 & 3). The microbact system was the only test which illustrated a negative result for citrate utilisation (Tables 2 & 3). The microbact required the use of a fresh broth culture rather than the streak plate used for the subsequent tests as it was incubated for too long (J Markham [University of Western Sydney] 2010, 300321 practical, 1st September). The use of the different cultures can possibly explain the discrepancies observed (Table 3). Other explanations for the inconsistencies include contamination from other cultures or chemicals due to multiple groups using the same microbact plate, or misinterpretation of the colour indicators due to their subjective nature. The identifications made by the microbact test and the conventional biochemical tests illustrate a single distinguishing characteristic; the presence of ornithine decarboxylase. Tests for ornithine decarboxylase returned negative results (Table 3) which led to a possible identification of the unknown as E. agglomerans. However, as E. agglomerans is known to be lysine decarboxylase negative (Hosono & Iimura 1995), it was excluded as a possible identification as the unknown illustrated positive results in all three testing methods. Repetition of the ornithine decarboxylase tests would be ideal as it would provide conclusive data pertaining to the identity of the unknown bacterium. The lysine decarboxylase tests were therefore the most valuable tests performed as they were conducted using all three testing methods, and thus excluding E. Agglomerans. In contrast, the adonitol test was the least meaningful in forming an identification because its main purpose is to distinguish between species of Klebsiella (ed. OLeary 1989), and did not provide differences between K. pneumonia and E. gergoviae. Motility is the only distinguishing trait between K. pneumoniae and E. gergoviae (Staley et al 2007). The initial motility test performed illustrated a negative result, however in the indole production test the SIM agar demonstrated significant motility of the unknown culture as the organism had spread from the line of inoculation into the media. The initial negative result is likely to be due to the broth cultures remaining cold after refrigeration which could have slowed the organisms movement (J Markham [University of Western Sydney] 2010, 300321 practical, 25 August), thus becoming undetectable under the microscope. Similar results were observed in the duplicated indole test and also when conducted on E. coli which is known to be motile (Staley et al 2007). This suggests that the unknown was motile and consequently E. gergoviae. However due to the initial motility test being negative; repetition of the motility test using cultures at room temperature and control cultures of K. pneumoniae and E. gergoviae would be required to resolve the aforementioned discrepancies. T o determine if the isolated bacterium in the river sample is a result of contamination from the septic tank genetic analysis of the bacterium strain would need to be implemented. This is because Enterobacter is commonly found in the human gastrointestinal tract, as well as in soil and water samples (Murray, Rosenthal & Pfaller 2009). Subsequently, it is plausible for Enterobacter to exist within the environment of a septic tank. If the strain of Enterobacter is analysed and found to be consistent in both the septic tank and water supply it is then likely that there has been contamination of the water supply by the septic tank. The most efficient process of identifying this bacterium is through ribotyping as it is able to identify not only the genus and species of the bacterium, but also the strain by recognizing the ribosomal RNA. Ribotyping consists of the detection of genetic variation in the genomic sequence, rendering of this technique is the most versatile method for pathogen typing. As stated by Rapley & Walker (2005), the differences between strains of bacteria are small. In ribotyping, restriction enzymes break genes of rRNA into segments where it is then separated by electrophoresis into a gel. Automated ribotyping is then used to determine the length of DNA using specific enzymes (EcoRI) (Rapley & Walker 2005), which can distinguish between strains.

Introduction:
The Enterobacteriaceae are among the largest family of gram-negative rods of medical and ecological significance, with 44 genera and hundreds of species (Staley et al 2007). These facultative anaerobes are characterised by being oxidase positive and catalase negative, distinguishing them from other gram-negative rods (Staley et al 2007). They range from 0.3 6m in size, and as a family they share the Enterobacterial common antigen and have the ability to grow rapidly on both selective and non-selective media with simple nutrient requirements (Murray, Rosenthal & Pfaller 2009). These have made them the focus of intensive studies such as gene manipulation (ed. Krieg 1984) and intercellular communications (ed. Goldman & Green 2009). Enterobacteriaceae are ubiquitous and commonly found in soil, water, vegetation and animals - making up part of the commensal flora of both humans and insects (Murray, Rosenthal & Pfaller 2009). They are responsible for 30-35% of all bacteremias and 70% urinary tract infections (Staley et al. 2007). However, only a few significant species are accountable for all human infections (Murray, Rosenthal & Pfaller 2009). Bacteria of this family are considerably more potent than other families, such as Brucella, due to the endotoxins they possess and their developed resistance to antibiotics and other antimicrobial agents (Staley et al. 2007). As displayed in Table 2, a range of biochemical tests determining the identification of the unknown were used. Enterobacter gergoviae was identified as the unknown gram negative microorganism from the conventional biochemical tests conducted. Table 3 shows the results of the Enterotube and Microbact tests. The main differences observed were the results of the Adonitol and Citrate tests. The Enterotube does not cover the same range of tests as the Microbact. This is illustrated in table 3 by the different bacterial identifications of Klebsiella and Enterobacter.

Table 1: Preliminary identification of unknown bacteria including results of control bacteria.

Bacteria Gram Stain Acid Fast Stain NA Spore Stain Motility Diagnostic tests Catalase Oxidase Oxidati /Fermentati use of Glucose

Table 2: Identification of the Enterobacteriaceae using conventional biochemical tests

Diagnostic Tests Bacteria E. coli + + NA NA + + NA NA Enterobacter Salmonella Proteus aerogenes mirabilis + NA NA NA NA + NA NA NA NA NA NA NA NA NA NA NA NA NA NA + + NA NA NA NA + Enterobacter gergoviae Pseudomonas NA NA NA NA NA NA NA NA NA + NA Klebsiella NA NA NA NA + NA NA NA NA Unknown A + + + + + +

Methyl Red VogesProskauer Indole production

Staphylococcus

+, cocci

NA

NA

NA

NA

NA

Pseudomonas Mycobacterium Bacillus Streptococcus S. aureus Micrococcus Unknown A

NA NA NA NA NA -, rods

+ NA NA NA NA -

NA NA NA NA -

+ NA NA NA NA NA -

+ NA NA NA NA +

+ NA NA NA NA -

NA NA NA NA Oxidative Fermentative Fermentative

H2S production Lysine decarboxylase Phenylalanine deaminase Citrate Utilisation Lactose fermentation ONPG

Method:

Unknown B

+ , rods

Fermentative

RESULTS unknown A RESULTS unknown B

Enterobacteriaceae

Gelatin hydrolysis Urea hydrolysis RESULT Unknown A

Bacillus

NA = test not conducted

NA = test not conducted

Table 3: Identification of the Enterobacteriaceae using commercial rapid biochemical tests

Medium/Test Glucose Gas Production Lysine Decarboxylation Ornithine Decarboxylation H2S Production Indole formation Adonitol Lactose Arabinose Sorbitol Voges- Proskauer Dulcitol Phenylalanine Deaminase Urea Citrate ONPG Gelatin Hydrolysis Raffinose Mannitol Malonate Inhibition Rhamnose Salicin Xylose Tryptophan Deaminase Inositol Sucrose Arginine Dihydrolase
*only using unknown bacteria A RESULTS = Test not A NA unknown conducted.

Enterotube* + + + + + + + + + + NA NA NA NA NA NA NA NA NA NA NA NA Klebsiella pneumonia

Microbact* + NA + + + + + NA NA + + + + + + + + + Enterobacter agglomerans

Conclusion:
Considering the results of the biochemical tests and the aforementioned inconsistencies, the unknown microorganism was identified as Enterobacter gergoviae. This identification was based primarily on the results of the conventional biochemical tests as they were duplicated using two cultures of the isolate and illustrated consistent results. They also tested for motility which was important in distinguishing between Klebsiella and Enterobacter, whilst the lysine test differentiated between the Enterobacter species. It is possible that the septic tank could be responsible for contaminating the water supply, however additional ribotyping of the strains of E. gergoviae would be required to determine if the strains present in the septic tank and the river sample are identical.

References:
Goldman, E. Green, L. (ed) 2009, Practical Handbook of Microbiology, 2nd edn, CRC Press, New York. Hosono, A. Iimura, K. (1995). Biochemical characteristics of Enterobacter agglomerans and related strains found in buckwheat seeds, International Journal of Food Microbiology, vol. 30, issue 3, p.243253, viewed 1 October 2010, Science Direct database, DOI 10.1016/0168-1605(96)00949-X Krieg, N (ed.) 1984, Bergeys manual of Systematic Bacteriology: volume 1, Williams & Wilkins, USA. Murray, PR, Rosenthal, KS & Pfaller, MA 2009, Medical Microbiology, 6th edn, Elsevier, Philadelphia, viewed 7 September 2010, NetLibrary database.

Figure 1: Flow diagram illustrating experimental methodology. *For in depth explanations to each test procedure mentioned above, refer to the Microbiology 2 Practical Manual (University of Western Sydney 2010).

O'Leary, W (ed.) 1989, Practical handbook of Microbiology, CRC press, New York. Rapley, R & Walker, MJ 2005 Medical biomethods handbook, Humana Press, New Jersey. Staley, JT, Gunsalus, RP, Lory, S & Perry, JJ 2007, Microbial Life, 2nd edn, Sinaeur Associates, Inc., Massachusetts. University of Western Sydney 2010, Unit 300321 Microbiology 2: Laboratory Manual, Spring Session, University of Western Sydney, Hawkesbury.