Microbiology of brewing production-bacteria of the order Enterobacterales and culture methods for their detection

The growth of 7 strains belonging to the order of Enterobacterales, represented by the species of Citrobacter Freundii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Obesumbacterium proteus, Rahnella aquatilis, Raoultella terrigena, Serratia marcescens and Shimwellia pseudoproteus, was monitored on selected cultivation media. Three types of agars - Endo, MacConkey and Chromocult Coliform agar together with two incubation temperatures of 28 and 37 °C were tested under aerobic conditions. The aim of the study was to detect such essential enterobacteria harmful to beer that cannot be proven at 37 °C, which is the temperature usually used in operational laboratories in breweries. Our results showed that most of the tested strains of enterobacteria were able to grow at 28 °C on all selected types of agar. The exception was just the representatives detection of which is problematic at 37 °C. Nevertheless, a little or no growth was always observed on just one of the tested media.


Introduction
This article follows a review paper entitled Microbiology of brewery production -bacteria of the order of Enterobacterales (Matoulková et al., 2018). The Enterobacterales order contains 7 families with more than 40 bacterial genera that are isolated from diverse types of environments. Particularly members of Shimwellia, Obesumbacterium, Rahnella, Citrobacter, Klebsiella, Raoultella, Serratia and Enterobacter genera were found in breweries. On the other hand, pathogenic species, including Escherichia coli, have not been detected in breweries (Van Vuuren and Priest, 2003).
Mainly due to the sensitivity of enterobacteria to ethanol and acidic pH, their growth and ability to multiply in the finished beer is minimal and they occur mainly as a contamination of production yeast.
Contaminated water or unsatisfactory hygienic condition of surfaces and equipment (e.g. leakage of pipe connection) represent other usual sources of enterobacteria (Vaughan et al., 2005). These bacteria are harmful since they produce undesirable sensory substances (e.g. diacetyl and dimethyl sulphide) at the beginning of the main fermentation. Moreover, some species are involved in the formation of carcinogenic N-nitrosamines (Boulton and Quain, 2001).
Shimwellia pseudoproteus and Rahnella aquatilis present an increased risk as they can repeatedly penetrate into a new batch of wort as contaminants of pitching yeast and therefore they are able to damage several batches of beer in a row (Bokulich and Bamforth, 2013). The damaged beer then shows a sweet, honey, fruity to vegetable and even fecal character (Jespersen and Jakobsen, 1996;Briggs et al., 2004).
The detection of enterobacteria in operational brewing laboratories is a routine procedure that is carried out mostly on MacConkey agar or chromogenic media at 37 °C.
In this study, we compared the growth of selected species of enterobacteria on several types of cultivation agars -Endo, MacConkey and Chromocult Coliform agar at 28 °C (the temperature recommended for incubation of some Shimwellia, Obesumbacterium and Rahnella species) and 37 °C (the temperature recommended for incubation of some Citrobacter, Klebsiella, Raoultella and Serratia).

Microorganisms and cultivation conditions
The used bacterial strains came from the following col-lections: Czech Collection of Microorganisms (CCM), the Collection of Brewing Microorganisms (RIBM) and the German Collection of Microorganisms and Cell Cultures (DSMZ). The list of strains, their marks and origins is given in Table 1. The strains were incubated under aerobic conditions on PCA agar at 28 °C for 48 hours before inoculation on experimental media.

Preparation of bacterial suspension and cultivation conditions
Bacterial suspensions were prepared by stiring 1 colony obtained from agar plate into a sterile physiological solution. The concentration of resulting cell was approximately 3 × 10 8 cells/mL. The suspensions were diluted in order to grow a countable number of colonies on Petri dishes (i.e. 10-50). Their incubation was maintained under aerobic conditions at 28 and 37 °C for 48 hours.

Results and discussion
All bacterial strains grew on basic culture media (NA and PCA) and showed no differences in the appearance of their colonies. This is demonstrated in Figure 1., where the colonies of C. freundii CCM 7187 on NA are present. Regular, flat to slightly convex smooth colonies with a glossy surface and white to slightly cream colour were formed. A similar description of the colonies is reported by such authors as Holt et al. (1994), Back (2005)

The growth of selected enterobacteria on Endo agar
Because of the toxicity and low stability of fuchsin, Endo agar is used only to a limited extent for detection of coliform bacteria. The selectivity of Endo agar is determined by the content of basic fuchsin (decolorized with sulphite), with the carbon substrate being lactose. The colour of colonies makes it possible to distinguish between lactose-positive (pink or red colonies possibly with a greenish metallic lustre) and lactose-negative (colourless and cream colonies) bacteria. In our case, pink and red colonies with a metallic lustre or without any lustre were observed on Endo agar ( Figure 2).  Figure 2A) and E. coli RIBM CH1 ( Figure 2B).

The growth of selected enterobacteria on MacConkey agar
Endo agar was gradually replaced by MacConkey medium especially due to the toxicity of fuchsin and because of numerous aspects from the field of clinical microbiology. MacConkey agar contains lactose as a carbon source and a neutral red as a pH indicator. The medium enables detection of lactose-positive (coliform) bacteria, which grow on agar plates in the form of pink colonies without changing the colour of the culture medium. Lactose-negative bacteria (e.g. Obesumbacterium, Shimwellia) form cream-coloured colonies on MacConkey agar and they discolour the medium into light yellow to ochre. Gram-positive bacteria are inhibited by crystal violet and bile salts (Finney et al., 2003). Bacterial growth on MacConkey Agar is documented in Figure 3. Lactose-positive representatives of C. freundii CCM 4475, E. cloacae CCM 7931, E. coli CCM 7395 ( Figure  3A), K. oxycota CCM 3565 ( Figure 3B) and S. marcescens CCM 303 T ( Figure 3D) formed pink colonies without changing the colour of the medium at both incubation temperatures. An exception was the strain of R. aquatilis CCM 4086, which did not grow at 37 °C at all. This finding corresponds to the basic characteristics of the genus -the temperature optimum for most representatives is between 25-35 °C; at 37 °C they grow significantly slowly (Kämpfer, 2015).
Lactose-negative bacteria of O. proteus DSM 2777 and S. pseudoproteus DSM 3038 T , formed cream coloured colonies at 28 °C and the medium is discoloured into light yellow to ochre as a result of their metabolism (Figure 3C). They formed no or only very poorly observable colonies at 37 °C.
The temperature optimum for most representatives is between 25 and 32 °C. 37 °C is the temperature commonly used for incubation of enterobacteria in brewing laboratories. It should be noted that the bacteria grow substantially more slowly at this temperature (Sedláček, 2007). Thus, our results show a risk that detection of harmful bacteria belonging to the genera of Rahnella or Shimwellia in beer will fail during cultivation on MacConkey agar at the recommended temperature of 37 °C.

The growth of selected enterobacteria on Chromocult Coliform agar
Chromocult Coliform agar was developed so that it could detect the total number of coliform bacteria together with the number of E. coli in one sample simultaneously. The medium contains two chromogenic substrates: Salmon-GAL to detect β-D-galactosidase (an enzyme that breaks down lactose into galactose and glucose) and X-glucuronide to detect presence of β-D-glucuronidase enzyme. Coliform bacteria show β-D-galactosidase activity. Although E. coli belongs to coliform bacteria, it can be distinguished from other coliform bacteria in the same agar plate due to β-D-glucuronidase activity.
The growth of Gram-positive bacteria/organisms is inhibited by tergitol-7. Coliform bacteria grow on Chromocult Coliform agar in the form of pink to red colonies, while E. coli forms dark blue to purple colonies. The accompanying microflora grows in the form of colourless colonies (Finney et al., 2003). In our study the formation of pinkish or light pink colonies was observed in cases of Serratia, Enterobacter, Klebsiella, Citrobacter, Rahnella and Shimwellia. Detailed description for E. cloacae CCM 7931 is given in Figure 4A, for K. oxytoca CCM 3565 in Figure 4C. O. proteus strains DSM 2777 are elaborated in Figure 4D. S. pseudoproteus DSM 22121 grew as pink-purple colonies and both E. coli strains formed dark blue-violet colonies represented by E. coli strain RIBM CH1 shown in Figure 4B. Pink staining of E. cloacae and colonies of Klebsiella members, as well as blue-violet colour in E. coli have also been reported for example by Niemi et al. (2001).

The effect of incubation temperature on selected enterobacteria growth
The results of the growth of enterobacteria on various culture media at 28 and 37 °C are presented in Table 2  At 37 °C, these bacteria grow significantly more slowly (Sedláček, 2007). The cultivation of enterobacteria in operational brewing laboratories takes place as standard at the temperature of 37 °C (Analytica EBC, 2011). Thus, the most risky taxa, such as S. pseudoproteus ("O. proteus") and R. aquatilis, may not be detected during routine microbiological checks.

Conclusion
The presence of enterobacteria in samples that were taken in several breweries indicates deteriorating hygienic conditions and low levels of sanitation during the operation. Detection of enterobacteria is usually performed routinely in operational brewing laboratories. For diagnosis of en-terobacteria, usually MacConkey agar or chromogenic media are used and the cultivation is carried at 37 °C. In our study we compared cultivations on different media -Endo agar, MacConkey agar, and Chromocult Coliform agar at two incubation temperatures of 28 °C and the commonly used 37 °C in order to monitor the growth character of enterobacteria. We proved that the cultivation at the above-mentioned 37 °C led to a slower growth or no growth at all in the case of the strains Shimwellia pseudoproteus ("O. proteus")  and Rahnella aquatilis (i.e. the taxa with the greatest potential of harmfulness from the point of view of microbiological control in a brewery). Thus, in laboratories that use only the incubation temperature of 37 °C for the detection of enterobacteria, some harmful strains may not be detected.

Acknowledgments
Results were obtained under financial support from the Ministry of Agriculture CR (RO1918) and the Ministry of Education, Youth and Sports of the Czech Republic (LO1312).