Fast-growth bacteria

Environmental stresses such as reduced aeration or differential nutrient metabolism [1-3], the acquisition of virulence [4], and resistance to antibiotics [5, 6] are associated with fitness costs to bacteria. Growth rates of planktonic cells in the absence or presence of a variety of stressors can serve as one of the proxies for monitoring the fitness of bacterial populations under laboratory conditions [7]. Baseline culture history, quality of media used (rich [batch-to-batch variations in each recipe] vs defined media), oxygen supply, nature of the initial inoculum (at least a 1:10,000 dilution of an overnight culture) [8], and the type of assay employed [9] may introduce artifacts or variations in biomass. However, when identically grown cultures with the same exponential growth rates are compared, the assumption is that relative changes in bacterial biomass, viability, or macromolecular composition will be indicative of the influence of a stressor. In addition, growth of a reference (with an established doubling time range) and unknown strains under similar optimal conditions can provide insights into the growth rates of the unknown strains.

Bacterial numbers can be estimated by microscopy, flow cytometry or culture-based tests that provide insights about the growth and/or metabolic capabilities of populations. Of the seven quantification methods usually employed to assess absolute or viable cell numbers (microscopy, flow cytometry, methylene blue reduction test, luminescence-based growth curve assay, Start-of-Growth-Time method, colonies forming units [CFU] method, absorbance [6, 9]), CFU (range of detection: limited by nutrients and “viable-but-non-culturable” status; time-consuming [at least 1 to 3 days]) and absorbance measurements (range of detection: 108 to 1010 bacteria/mL) are inexpensive staples of most research laboratories. Depending on the proposed hypothesis under investigation, qualitative, high-throughput optical density comparisons using a micro-titer plate reader may suffice in estimating relative doubling time differences e.g, between control and treated populations or between reference and unknown strains.

As far as methodology goes, automated data generation is virtually indispensable in estimating key growth parameters in industrial settings, it is not possible to directly translate OD values to maximum specific growth rates due to the high detection limits of OD readers (approximately 107 viable cells [10]). These and other methodological aspects [8, 9, 11] are important considerations in assessing the effects of stressors on the growth rates of different strains, especially in light of tantalizing separate reports of fast-growing bacteria (defined here as bacteria with doubling times ≤15 minutes) [12-15].

The doubling time of one of the best-defined, batch-cultivated Gram-negative bacterial strains, Escherichia coli K-12 MG1655, has been reported to be as fast as 20 minutes in the widely-used Luria-Bertani rich media [16]. By comparison, three batch-cultivated, aerobic Gram-negative halophiles i.e.,  Vibrio natriegens strain DSMZ 759 [14], Vibrio natriegens ATCC 14048/Pseudomonas natriegens [15, 17], and Vibrio parahaemolyticus strain UM 4552 [12] have been reported to double at less than 7 [12, 14] and up to 9.8 minutes [13, 15] using dilute inocula in different types of rich media. Vibrio natriegens has also been reported to double as slow as 14 minutes with a large starting inoculum. Taken together with other studies, the doubling time range of less than 7 and up to 14 minutes for different strains of Vibrio natriegens [14, 15, 17] is still faster than the rapid doubling time of 20 minutes reported for a standard E. coli K-12 strain [16]. Another anaerobic, spore-forming Gram-positive foodborne pathogen, Clostridium perfringens, doubled at 7 to 8 minutes at relatively high temperatures (43 ◦C to 46 ◦C) in meat or thioglycollate broth [18]. The potential of different pathogens to exhibit bursts of fast growth in rich media opens up new avenues of research.

For instance, how is the growth physiology of fast-growers regulated relative to known mechanisms [19] in the well-characterized, Gram-negative E. coli K-12 strain? It has been known since the 1950s that, under conditions of balanced growth, higher growth rates correlated with increased cellular concentrations of DNA, RNA, and total protein [11]. The number of ribosomes per E. coli cell is also proportional to the growth rate in order to meet the demand for protein synthesis [19]. Aiyar et al.[13] estimated that V. natriegens produced 115,000 ribosomes per cell for a generation time of 4 doublings/hour compared with 70, 000 ribosomes per cell for E. coli doubling with a generation time of 25 minutes. In addition, V. natriegens has 14 rRNA-encoding genes [14] (compared with 7 rRNA operons for E. coli [19]) and its rRNA promoters are extremely strong [13]. The authors concluded that “V. natriegens rRNA transcription might be regulated by at least some of the same mechanisms used in E. coli, since V. natriegens rRNA promoters share sequence and kinetic features crucial for E. coli rRNA regulation” [13].

 References

1.            Monod, J., The Growth of Bacterial Cultures. Ann Rev Microb, 1949. 3: p. 371-394.

2.            McDaniel, L.E., E.G. Bailey, and A. Zimmerli, EFFECT OF OXYGEN SUPPLY RATES ON GROWTH OF ESCHERICHIA COLI. Appl Microbiol, 1965. 13: p. 109-14.

3.            Truong-Bolduc, Q.C., et al., Reduced aeration affects the expression of the NorB efflux pump of Staphylococcus aureus by posttranslational modification of MgrA. J Bacteriol, 2012. 194(7): p. 1823-34.

4.            Sturm, A., et al., The cost of virulence: retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1. PLoS Pathog, 2011. 7(7): p. e1002143.

5.            Schulz zur Wiesch, P., J. Engelstadter, and S. Bonhoeffer, Compensation of fitness costs and reversibility of antibiotic resistance mutations. Antimicrob Agents Chemother, 2010. 54(5): p. 2085-95.

6.            Kishony, R. and S. Leibler, Environmental stresses can alleviate the average deleterious effect of mutations. J Biol, 2003. 2(2): p. 14.

7.            Spoering, A.L. and K. Lewis, Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol, 2001. 183(23): p. 6746-51.

8.            Ryall, B., G. Eydallin, and T. Ferenci, Culture history and population heterogeneity as determinants of bacterial adaptation: the adaptomics of a single environmental transition. Microbiol Mol Biol Rev, 2012. 76(3): p. 597-625.

9.            Hazan, R., et al., A method for high throughput determination of viable bacteria cell counts in 96-well plates. BMC Microbiol, 2012. 12: p. 259.

10.          Biesta-Peters, E.G., et al., Comparison of two optical-density-based methods and a plate count method for estimation of growth parameters of Bacillus cereus. Appl Environ Microbiol, 2010. 76(5): p. 1399-405.

11.          Schaechter, M., From growth physiology to systems biology. Int Microbiol, 2006. 9(3): p. 157-61.

12.          Nackerdien, Z.E., et al., Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence. PLoS One, 2008. 3(2): p. e1671.

13.          Aiyar, S.E., T. Gaal, and R.L. Gourse, rRNA promoter activity in the fast-growing bacterium Vibrio natriegens. J Bacteriol, 2002. 184(5): p. 1349-58.

14.          Maida, I., et al., Draft Genome Sequence of the Fast-Growing Bacterium Vibrio natriegens Strain DSMZ 759. Genome Announc, 2013. 1(4).

15.          Eagon, R.G., Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J Bacteriol, 1962. 83: p. 736-7.

16.          Sezonov, G., D. Joseleau-Petit, and R. D'Ari, Escherichia coli physiology in Luria-Bertani broth. J Bacteriol, 2007. 189(23): p. 8746-9.

17.          Payne, W.J., Studies on bacterial utilization of uronic acids. III. Induction of oxidative enzymes in a marine isolate. J Bacteriol, 1958. 76(3): p. 301-7.

18.          Juneja, V.K., et al., Potential for growth of Clostridium perfringens from spores in pork scrapple during cooling. Foodborne Pathog Dis, 2010. 7(2): p. 153-7.

19.          Jin, D.J., C. Cagliero, and Y.N. Zhou, Growth rate regulation in Escherichia coli. FEMS Microbiol Rev, 2012. 36(2): p. 269-87.