Does bacterial growth refers to an increase in the numbers of cells in a bacterial culture?

Bacterial Growth

Bacterial replication is a coordinated process in which two equivalent daughter cells are produced. For growth to occur, there must be sufficient metabolites to support synthesis of the bacterial components and especially the nucleotides for DNA synthesis. A cascade of regulatory events (synthesis of key proteins and RNA), much like a countdown at the Kennedy Space Center, must occur on schedule to initiate a replication cycle.However, once it is initiated, DNA synthesis must run to completion, even if all nutrients have been removed from the medium.

Chromosome replication is initiated at the membrane, and each daughter chromosome is anchored to a different portion of membrane.Bacterial membrane, peptidoglycan synthesis, and cell division are linked together such that inhibition of peptidoglycan synthesis also will inhibit cell division. As the bacterial membrane grows, the daughter chromosomes are pulled apart. Commencement of chromosome replication also initiates the process of cell division, which can be visualized by the start of septum formation between the two daughter cells (Fig. 13.8; see alsoChapter 12). New initiation events may occur even before completion of chromosome replication and cell division.

Depletion of metabolites (starvation) or a buildup of toxic by-products (e.g., ethanol) triggers production of chemicalalarmones, which cause protein and other synthesis to stop, but degradative processes continue. DNA synthesis continues until all initiated chromosomes are completed, despite the detrimental effect on the cell. Ribosomes are cannibalized for deoxyribonucleotide precursors, peptidoglycan and proteins are degraded for metabolites, and the cell shrinks. Septum formation may be initiated, but cell division may not occur. Many cells die. Similar signals may initiatesporulation in species capable of this process (seeChapter 12). For some bacterial species, starvation promotes uptake of foreign DNA (transformation) that may encode the means to survive the challenge.

NOTES

1.

Campbell, 1957.

The term log arose because the data for bacterial growth give a straight line on semilogarithmic graph paper. The equation of growth is more properly described as exponential. For historical reasons, the term log phase has become synonymous with exponential growth.

The equation should be Fα = ln2·2(1 – α). The integral of this equation gives a unit amount of cells in the culture. This does not change the idea implicit in the equation, that there are twice as many young cells as old cells.

Buchanan, 1918.

Henrici, 1928.

Schaechter, Maaløe and Kjeldgaard, 1958.

The concentrations of DNA in the bacterial cell are referred to as nucleoids to distinguish them from the membrane enclosed genomes found in eukaryotic cells.

The original paper by Schaechter, Maaløe, and Kjeldgaard is notable for a number of other ideas. It may be the first explicit statement regarding the difficulty of deciding between a linear function and a logarithmic function when the data vary over a factor of two. They also used the chemostat to obtain very slow growth rates, and defined the difference between restricted and unrestricted growth.

Kjeldgaard, Maaløe, and Schaechter, 1958.

Longsworth, 1936.

Hershey, 1938, 1939, 1940; Hershey and Bronfenbrenner, 1937, 1938. This is the same Alfred Hershey who won a Nobel Prize for the analysis of T-even phage growth. It is forgotten that he also had an important place in defining the nature of bacterial growth.

The book by Maaløe and Kjeldgaard (1966) is a superb summary of the original ideas that form the core of the Copenhagen School.

Time to Detection of a Positive Blood Culture

Bacterial growth is evident in most cultures of blood from neonates within 48 hours [490–492]. With use of conventional culture techniques and subculture at 4 and 14 hours, only 4 of 105 cultures that had positive results (one GBS and three S. aureus) required more than 48 hours of incubation [491]. By use of a radiometric technique (BACTEC 460), 40 of 41 cultures that grew GBS and 15 of 16 cultures with E. coli were identified within 24 hours [492]. Controlled experiments suggest that delayed entry of the collected blood culture bottle into the automated blood culture machine can significantly prolong the time to positivity for common newborn pathogens [493].

Growth Requirements and Bacterial Products

Group B streptococci are quite homogeneous in their amino acid requirements during aerobic or anaerobic growth.62 A glucose-rich environment enhances the number of viable GBS during stationary phase and the amount of CPS elaborated.63 In a modified chemically defined medium, the expression of capsule during continuous growth is regulated by the growth rate.45 Invasiveness is enhanced by a fast growth rate and is optimal in the presence of at least 5% oxygen.64,65

GBS elaborate many products during their growth. Among these is the hemolysin that produces the β-hemolysis surrounding colonies on blood agar. Hemolysin is a surface-associated toxin active against the erythrocytes from several mammalian species. The GBS hemolysin recently has been characterized as the ornithine rhamnolipid pigment and shown to function as a virulence factor, promoting invasion of placental cells.66 GBS can hydrolyze hippuric acid to benzoic acid and glycine. The hippuricase of GBS is cell associated and is trypsin and heat labile.67 It is antigenic in rabbits, but its relationship to bacterial virulence, if any, has not been studied.

Most strains of GBS have an enzyme that inactivates complement component C5a by cleaving a peptide at the carboxyl terminus.68 GBS C5a-ase seems to be a serine esterase; it is distinct from the C5a-cleaving enzyme produced by group A streptococci,69 although the genes that encode these enzymes are similar.70 C5a-ase contributes to pathogenesis by rapidly inactivating the neutrophil agonist C5a, preventing the accumulation of neutrophils at the site of infection.71

Another group of enzymes elaborated by nearly all GBS are the extracellular nucleases.72 Three distinct nucleases have been physically and immunologically characterized. All are maximally activated by divalent cations of calcium plus manganese. These nucleases are immunogenic in animals, and neutralizing antibodies to them are detectable in sera from pregnant women known to be genital carriers of GBS. Their role in the pathogenesis of human infection is unknown.

An extracellular product that can contribute to virulence of GBS was originally defined as a neuraminidase and subsequently characterized as a hyaluronate lyase.73 Maximal levels are detected during late exponential growth in a chemically defined medium. Elaboration of large quantities can be a virulence factor for type III GBS. Musser and coworkers74 identified a high neuraminidase–producing subset of type III strains that were responsible for most serious GBS infections. Later studies indicated that these were from a single clonal complex, designated ST 17, that has been designated as “hypervirulent.” ST 17 is almost exclusively found in type III strains.

84 How long should one wait before a blood culture is designated negative?

Bacterial growth is evident in most cultures of infected blood within 48 hours or earlier. With the use of continuous monitoring techniques, a study at Children's Hospital of Philadelphia of 200 cultures from central venous catheters found that the median time for a positive blood culture was 14 hours. In addition, 99.2% of cultures with gram-negative bacteria were positive by 36 hours, and 97% of cultures with gram-positive bacteria were positive by 36 hours. A study from Australia of neonatal blood cultures found that the median time for positivity for group B streptococcus was 9 hours, that for Escherichia coli was 11 hours, and that for coagulase-negative staphylococci was 29 hours.

Although 36 to 48 hours is generally sufficient time to isolate common bacteria present in the bloodstream, fastidious organisms may take longer to grow. Therefore, when one suspects anaerobes, fungi, or other organisms with special growth requirements, a longer time should be allowed before concluding that a culture is negative.

Shah SS, Downes KJ, et al: How long does it take to “rule out” bacteremia in children with central venous catheters? Pediatrics 121:135–141, 2008.

Jardine L, Davies MW, Faoagali J: Incubation time required for neonatal blood cultures to become positive, J Paediatr Child Health 42:797–802, 2006.

Introduction

Bacterial growth and survival is dependent upon the ability of an organism to sense its environmental conditions and respond to external stimuli. Stimulus can come from a variety of sources including the nutrients available for growth, the presence of secondary metabolites, and the presence of other microorganisms. In the case of nutrients and secondary metabolites, sensor and regulatory proteins exist that affect a change in gene expression as these compounds change concentration. These changes in expression can cause bacteria to synthesize new proteins for catabolism in the case of diauxic growth, can lead to the production of extracellular enzymes to liberate nutrients from the environment in the case of starvation, or can cause changes in motility, chemotaxis, or metabolism that allow the bacteria to avoid or eliminate toxic concentrations of a secondary metabolite. In addition to these basic stimulus–response events, bacteria have evolved a mechanism to ‘count’ the local cell population. This process is known as quorum sensing and is mediated by the bacterium’s ability to produce and recognize soluble factors known as quorum signals.

The ability of bacteria to utilize extracellular signals to modify their behavior in a cell density-dependent manner was first described in the early 1970s by Kenneth Nealson, Terry Platt, Woodland Hastings, and Anatol Eberhard. These researchers discovered that the bioluminescent bacterium Vibrio fischeri only produced light when bacterial cell numbers were high (Figure 1). They also demonstrated that culture supernatants from high cell density cultures were able to stimulate light production in low cell density cultures. This phenomenon was deemed autoinduction and provided the first clues that bacteria were able to utilize a soluble extracellular signal to monitor population density.

Subsequent work characterized the autoinducer of V. fischeri and the genes responsible for its synthesis and detection by the cell. As autoinducer-related genes were studied, it was discovered that many other Gram-negative bacterium contained the genes necessary for autoinducer synthesis and detection. It was demonstrated that autoinducer sensing was dependent upon a critical number of cells in a defined volume. This threshold density of cells was first referred to as a quorum by Clay Fuqua, Stephen Winans, and Peter Greenberg in 1994, and they proposed the term quorum sensing to describe this event.

Quorum sensing has rapidly expanded as a field of study, and the discovery of new quorum signaling bacteria as well as new types of quorum signals is increasing at an ever growing rate. Quorum signals in Gram-negative species such as V. fischeri were the first to be studied at the genetic and chemical levels; however, a significant amount of work has subsequently been performed with Gram-positive species. It is notable that quorum signal-dependent behavior was observed in Gram-positive bacteria long before the discovery of autoinducers or quorum sensing.

Growth phases in broth culture

Bacterial growth in broth has been studied in great detail and has provided a framework within which the growth state or growth phase of any given pure culture of a single organism can be placed; these phases are summarized in the idealized growth curve shown in Figure 4.1. When growth is initiated by inoculation into appropriate broth conditions, the number of cells present appears to remain constant for the lag phase, during which cells are thought to be preparing for growth. Increase in cell number then becomes detectable, and its rate accelerates rapidly until it is established at the maximum achievable rate for the available conditions. This is known as the exponential phase, because the number of cells is increasing exponentially with time. To accommodate the astronomic changes in number, the growth curve is normally displayed on a logarithmic scale, which shows a linear increase in log cell number with time (hence the older term, log phase). This log-linear relationship is sufficiently constant for a given bacterial strain under one set of conditions that it can be defined mathematically, and is often quoted as the doubling time for that organism. Doubling times have been measured at anything between 13 min for Vibrio cholerae and 24 h for Mycobacterium tuberculosis. On this basis it is not surprising that cholera is a disease that can kill within 12 h, whereas tuberculosis takes months to develop. A further consequence is that, when specimens are submitted to diagnostic laboratories for the detection of these organisms by culture, a result is usually available for V. cholerae the next day, whereas several weeks are required for conventional culture of M. tuberculosis.

It is often difficult to grasp fully the scale of exponential microbial growth; the message may be strengthened by considering that the progeny of a lecture theatre containing 150 students would exceed the global population of humanity (6 × 109) within 8.5 h if they were able to breed like Escherichia coli!

Exponential growth cannot be sustained indefinitely in a closed (batch) system with limited available nutrients. Eventually growth slows down, and the total bacterial cell number reaches a maximum and stabilizes. This is known as the stationary or post-exponential phase. At this stage it becomes important to know what method has been used to determine the growth curve. If a direct method that assesses the total number of cells present is used then the count remains constant. Such methods include counting cells in a volumetric chamber observed by microscopy, electronic particle counters and measurement of turbidity. If, however, the growth potential of the individual cells present in the culture is assessed by taking regular samples, making tenfold dilutions of these and inoculating them on to agar, the number of colony-forming units (cfu) per unit volume can be determined at each sample time. Although such cfu counts closely parallel the results obtained by direct counting methods in the exponential and early stationary phases, a divergence begins to emerge towards the end of the latter; the total cell number remains constant whereas the colony count declines. This marks the beginning of the final, decline phase, in the sequence of growth states that can be observed in broth. The discrepancy between the total and cfu counts is conventionally held to represent the death of cells because of nutrient exhaustion and accumulation of detrimental metabolic end-products. However, there is some doubt concerning this interpretation (see below).

As noted above there has been increased interest in the properties of bacteria in non-growing states. While there are many different systems for studying non-replicating bacteria and their separate relevance to infection is argued, one phenomenon is of particular interest as it illustrates their capacity for adaptation based on mutation. The growth advantage in stationary phase (GASP) phenomenon is now well established and illustrates that while total cell numbers in a population may remain constant or decline, multiple genetic variants arise, some of which come to dominate the population. The genes and polymorphisms that lead to the growth advantage have been informative in improving our understanding of survival and competition under nutrient limited conditions.

The study of bacterial growth in broth provides a valuable point of reference to which practical, experimental and routine diagnostic procedures are often related. For example, the length of the lag phase and rates of exponential growth in different circumstances are used to make predictions and contribute to safety standards for storage in the food industry. An important feature to emerge is that cultures inoculated with cells prepared at different stages in the growth curve yield different results. The exponential phase is the most reproducible and readily identified, and is therefore used most frequently. It can be extended in an open system known as continuous culture using a chemostat in which cells of a growing culture are harvested continuously and nutrients replenished continuously. Chemostat studies have provided very detailed information on the chemistry of microbial growth and the way in which different organisms convert specific substrates into biomass. The extraordinary efficiency of this process has made natural and genetically manipulated microbes a powerful resource for the biotechnology industry.

In contrast to growth in broth, far less is known about the state of the bacteria in a mature macroscopic colony on an agar plate. Such a colony presents a wide range of environments, from an abundance of oxygen and nutrients at the edge to almost no oxygen or nutrients available to cells in the centre. It is likely that all phases of growth are represented in colonies, depending on the location of a particular cell and the age of the culture. Although in practice colonies can be used reliably to inoculate routine tests of antimicrobial susceptibility in clinical laboratories, they cannot be considered a defined starting point for experimental work because they comprise such a heterogeneous population of cells. In fact, colonies are complex and dynamic communities in which cells at different locations can show startlingly different phenotypes. In spite of its complexity, the capacity for and quality of colonial growth of specific organisms on specialized media is central to the laboratory description of medically important bacteria.

Corticosteroid Effects on Antimicrobial CSF Penetration and Bacterial Killing

Bacterial growth and death in the CSF both result in the release of proinflammatory bacterial products, such as peptidoglycan or lipopolysaccharide (LPS) that worsen CNS inflammation. For example, LPS activates Toll-like receptor 4 signaling in a wide variety of cells, ultimately leading to nuclear factor kappa B (NF-κB)-mediated transcription of proinflammatory genes like tumor necrosis factor-α (TNF-α) (Lu et al., 2008). Such inflammation mediates breakdown of the BBB and brain edema, with subsequent morbidity and/or mortality for the host. Both animal experiments and clinical trials have demonstrated a beneficial role for the early use of glucocorticoids to inhibit inflammation and brain edema during bacterial meningitis (Paris et al., 1994; de Gans and van de Beek, 2002; Nguyen et al., 2007). However, the reduced inflammation associated with glucocorticoids is also associated with reduced permeability at the BBB, thus potentially affecting the penetration of hydrophilic antimicrobials and immune cells into the CNS, leading to decreased clearance of bacteria from the CSF. Table 2.3 displays data on the effects of corticosteroids on CSF antimicrobial concentrations and bacterial clearance from CSF; this table includes data from both humans with bacterial meningitis and animals with experimental meningitis. The data in Table 2.3 for vancomycin and ceftriaxone are from in vivo studies of experimental meningitis in rabbits and are concerning in light of the increased incidence of invasive disease caused by both penicillin- and ceftriaxone-resistant pneumococci (Buke et al., 2003; Ricard et al., 2007). Current Infectious Diseases Society of America guidelines for the empiric treatment of suspected pneumococcal meningitis recommend a combination of vancomycin and ceftriaxone (or cefotaxime), along with early initiation of dexamethasone (Tunkel et al., 2004).

Table 2.3. The effects of corticosteroids on cerebrospinal fluid (CSF) antimicrobial concentrations and bacterial clearance

Modified from Lutsar et al. (1998).

PSSP, penicillin-sensitive Streptococcus pneumoniae; E. coli, Escherichia coli; PRSP, penicillin-resistant Streptococcus pneumoniae; CRSP, cephalosporin-resistant Streptococcus pneumoniae; MTb, Mycobacterium tuberculosis; iCRSP, intermediately cephalosporin-resistant Streptococcus pneumoniae; NE, not evaluated.

Fortunately, the clinical data available do not suggest that these observations for ceftriaxone and vancomycin in experimental meningitis translate into clinical failures or less favorable clinical outcomes in patients with pneumococcal meningitis treated with these antimicrobials (Buke et al., 2003; Ricard et al., 2007). A prospective, double-blind, randomized multicenter study in Europe of adjunctive dexamethasone therapy for adults with bacterial meningitis demonstrated a reduction in mortality among patients receiving the steroid (relative risk of death 0.48, 95% confidence interval 0.24–0.96, P = 0.04) (de Gans and van de Beek, 2002). A similar prospective study among adults in Malawi, 90% of whom were infected with human immunodeficiency virus (HIV), failed to demonstrate a benefit to adjunctive dexamethasone for bacterial meningitis, but did not find a difference in antimicrobial efficacy in those receiving steroids (Scarborough et al., 2007). Interestingly, a prospective, randomized, double-blind study of adjunctive dexamethasone for adults in Vietnam with bacterial meningitis found clinical benefits supporting dexamethasone use among patients with microbiologically proven bacterial meningitis (Mai et al., 2007). A cautionary note, however, is warranted because the vast majority of pneumococcal isolates in these three trials were cephalosporin- and vancomycin-susceptible.

Biosynthesis of UFAs

Bacterial growth requires an appreciable fraction of acyl chains of the membrane lipids to be in a disordered state (fluid). A disordered state is imparted by presence of either cis-unsaturated or terminally anteiso-branched-chain fatty acids both of which act to offset the closely packed ordered arrangement of the lipid bilayer acyl chains that is imparted by straight chain saturated acyl chain. In this section, we discuss the basic features of biosynthesis of UFA in organisms in which the proposed biosynthetic mechanisms have been well characterized. In E. coli, synthesis of the normal UFA content requires three enzymes, FabA, FabB, and FabF (Figure 2(a)). FabA, β-hydroxydecanoyldehydrase, is the key enzyme of the classic anaerobic pathway of UFA synthesis and introduces a cis double bond into a 10-carbon intermediate. FabA is a bifunctional enzyme that catalyzes both the removal of water to generate trans-2-decenoyl-ACP and the isomerization of this intermediate to cis-3-decenoyl-ACP. This cis-10 carbon intermediate is then elongated by FabB and later by FabF to form the two major UFA found in E. coli: C16:1Δ9 and C18:1Δ9. FabB and FabF have distinct and nonoverlaping roles in E. coli UFA synthesis. FabB is thought to elongate the product of the FabA enzyme to the C12 unsaturated intermediate, whereas FabF is required to convert C16 unsaturated species to the C18 species (Figure 2(a)).

Expression of the key UFA synthetic gene fabA from E. coli is positively regulated by FadR which was hitherto thought to function only as a repressor of the ß-oxidation pathway of fatty acids (see ‘Fatty Acid β-Oxidation’). Positive regulation of fabA expression is neutralized both in vivo and in vitro by long-chain acyl-CoAs that are small ligands which regulate the binding of FadR to the fabA promoter. FadR is also a positive regulator of fabB, the second UFA biosynthetic gene, although the changes in fabB expression in fadR mutants are not as great as with fabA. Thus, FadR acts as a repressor of ß-oxidation genes and as an activator of the two genes required for UFA synthesis. FabR is a second E. coli regulator of UFAs synthesis. It behaves as a repressor acting downstream of FadR operator in the regulation of fabA and fabB. The ligand regulating FabR activity is still unknown.

There is a variation of the FabA/FabB bacterial strategy for UFA synthesis, discovered in Enterococcus faecalis. This organism possesses a special FabZ enzyme (called FabN) that has a FabA-like activity and a unique FabF (called FabO) that replaces FabB. However, other organisms, such as S. pneumoniae, compensate FabA absence with an enzyme called FabM that is capable of isomerizing the trans-unsaturated bond at the key 10-carbon intermediate to its cis-isomer.

In the examples mentioned above, the proteins FabA, FabM, or FabN anaerobically introduce the double bond into a C10 intermediate and either FabB or FabF, in the organisms lacking FabB, channel this intermediate to the mainstream of the fatty acid synthetic pathway. In contrast, other bacteria like Bacillus and Cyanobacteria have completely separate systems for the synthesis of UFA and saturated fatty acids. These organisms use fatty acid desaturase enzymes, which require molecular oxygen and reducing equivalents obtained from an electron transport chain, to introduce the double bond into previously synthesized saturated fatty acids (Figure 2(b)).

The organisms mentioned above each have only a single pathway for the synthesis of the UFAs required to make functional membrane lipids. In marked contrast, UFA biosynthesis in Pseudomonas aeruginosa proceeds by three distinct pathways. In this organism, the FabA–FabB pathway does the bulk of UFA synthesis under all growth conditions. However, P. aeruginosa has two fatty acyl desaturases (DesA and DesB) that supplement the FabA–FabB pathway under aerobic conditions and thereby allows faster growth. DesA introduces the double bond into the acyl chains of intact phospholipids, whereas the substrates of DesB are acyl-CoAs that are derived from exogenous fatty acids. DesB is inducible and is regulated by DesT, a transcriptional regulator that has the property of being able to sense the fatty acid composition of the long chain acyl-CoA pools to adjust the expression of the desaturase.