Wednesday, August 31, 2011

Small colony variants- concluding remarks

Small colony variants (SCVs) can be isolated in vitro and in vivo under a variety of conditions, including antibiotic pressure. Various reports indicate that they are electron-deficient mutants. However, many SCVs may not revert in the presence of hemin, menadione, thiamine or thymidine and thus may not be auxotrophic mutants. It is also not known whether SCVs can revert to normal colony forms in vivo. Even though they can be isolated from many chronic infections, their role in causing these infections has not been proven conclusively. In most of the reported cases, SCVs were co-cultured with normal colony forms. Since there are no indications that the large colony forms are the revertants of SCVs, chronic infections could be due to the normal colony forms only that have persisted under those conditions. SCVs may not be responsible for fatal infections; reports associating SCVs with fatal infections are highly questionable. SCVs can be part of the normal life cycle of bacteria and may not have much clinical significance. However, the isolation of SCVs in vivo following antibiotic therapy may be indicative of the failure of antibiotics to reach throughout the site of infection at optimal concentrations, which may result in the survival of some normal bacteria and the selection of SCVs. The surviving normal bacteria may re-grow once the antibiotic pressure is removed, whereas SCVs may not have much role and may remain without causing infection since they are less virulent.

My blogs on SCVs are ending here. Next, I will start with senescent bacteria and bacterial aging model

Intracellular persistence of small colony variants

It has been suggested that small colony variants (SCVs) can persist intracellularly and are protected against antibiotics and host innate defense system which may contribute to chronic infections (von Eiff et al. 2001). They may produce low levels of alpha-toxins which may help in the intracellular persistence by preventing cell lysis or apoptosis.

Tuchscherr et al. (2010) investigated the infection of endothelial cells with highly virulent wild type isolates and isogenic SCVs of S. aureus. They found that wild type bacteria upregulated the expression of a number of endothelial genes and proteins whereas SCVs upregulated only a few of them. Similarly, the levels of chemokine release after 3 days of infection was much higher with wild type cells when compared to SCVs. The data from the above article indicates that SCVs are less virulent when compared to wild type. They may be better in avoiding the host innate defense system and hence may persist inside the cells. Since they divide slowly, they may produce fewer products that activate host cell responses.

However, whether SCVs are adapted phenotypes that can cause chronic infections is unproven. In fact, previous experiments with animal models have also shown that SCVs are less virulent than their wild type counterparts. But whether they can occasionally give rise to normal wild type bacteria through in vivo reversion has not been demonstrated even though such reversion has been shown in vitro.

von Eiff et al. (2001). Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease. Clin Infect Dis 32(11), 1643-7.
Tuchscherr et al. (2010). Staphylococcus aureus small colony variants are adapted phenotypes for intracellular persistence. Journal of Infectious Diseases 202(7): 1031-1040

Monday, August 29, 2011

Are small colony variants responsible for fatal infections?

There are a few reports suggesting that small colony variants (SCVs) may be responsible for fatal infections. Some are given below.

1. Haussler et al. (2003). Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex. Eur J Clin Microbiol Infect Dis 22(4), 249-53.
2. Seifert et al. (1999). Fatal case due to methicillin-resistant Staphylococcus aureus small colony variants in an AIDS patient. Emerg Infect Dis 5(3), 450-3.
3. Adler et al. (2003). Emergence of a teicoplanin-resistant small colony variant of Staphylococcus epidermidis during vancomycin therapy. Eur J Clin Microbiol Infect Dis 22(12), 746-8.

In all these cases, it can be noted that the patients had long and complicated clinical histories and had received antibiotic therapy for a long time. For example, in the first case, the patients had severe lung diseases and had undergone lung transplantation whereas in the second article, it was an AIDS patient who had a traffic accident. Similarly, in the third case, the patient was undergoing treatment for acute myeloid leukemia. Antibiotic therapy might have selected SCVs, and both large and small colony forms were cultured in all cases. However, there are no indications that SCVs are responsible for the fatal infections in any of the above cases. Just because SCVs were isolated from these patients, how can it be suggested that they are responsible for fatal infection especially considering their severe and complicated clinical histories?

Saturday, August 27, 2011

Are small colony variants responsible for chronic infections?

Researchers propose that during unfavorable conditions, especially antibiotic therapy, small colony variants (SCVs) are selected due to their ability to tolerate antibiotics. This is considered to be a survival strategy of bacteria to overcome adverse conditions. Upon the removal of unfavorable conditions, the SCVs revert to normal growth and cause re-infection. Moreover, they have enhanced capacity to persist intracellularly which may protect them from antibodies and complements. In many chronic infections, SCVs are isolated in large numbers. Isolation of SCVs from osteomyelitis and cystic fibrosis supports a pathogenic role for SCVs in such chronic diseases. In these cases, both large colonies and SCVs are isolated and researchers have shown that both colony types are clonal indicating a common origin. Thus, it is proposed that the normal large colonies are the revertants of SCVs.

Even though both the colony types might have originated from the common ancestor, however, there is no indication that the large colonies are truly the revertants of SCVs. In fact, there are no clear data that have proven the reversion of SCVs to large colony types in vivo. The isolation of both large and small colony types from such infections only indicates that, antibiotics are unable to kill all bacteria. Since some of the normal bacteria also survive, they may result in re-infection later. Thus, researchers have given undue importance to SCVs assuming that normal colonies are reverted from SCVs.

The fundamental flaw here- the assumption that antibiotics are capable of killing all normal bacteria under such chronic infections. (It is well established that antibiotics may not be able to kill all bacteria in biofilms or in other pathological conditions resulting in cystic fibrosis or chronic osteomyelitis). However, the reversion of SCVs to normal types in vitro made researchers to assume that the normal colonies found in vivo are also the revertants of SCVs.

Next- Are SCVs responsible for fatal infections?

Wednesday, August 24, 2011

The switching mechanism between the wild type and small colony variants as proposed by Massey et al. (2001) is not convincing

Massey et al. (2001) proposed that small colony variants (SCVs) could emerge by switching from the wild type and vice versa. They found that SCVs of S. aureus could be isolated after just 30 min of exposure to gentamicin. Their number increased as the exposure time increased and reached a maximum by 14 h, but subsequently declined due to the emergence and overgrowth of gentamicin-resistant wild type bacteria. The authors hypothesized that the increase in the frequency of SCVs after gentamicin treatment for the first 14 h was either due to the very short generation time of SCVs or due to the switching from wild type to SCVs. 

To test which of the above hypotheses was responsible for the increase in SCV numbers, the mean generation time of SCVs and the wild type population was compared. The former hypothesis was ruled out since the actual mean generation time was found to be much higher than the wild type. Since the first hypothesis was wrong, they studied whether the increase in the frequency of SCVs was due to their presence in the inoculum. For this purpose, they reduced the initial inoculum size of bacteria by 100-fold and calculated the percentage of SCVs. They found that the proportion of SCVs at 24 h increased when the initial inoculum size was reduced. They calculated that, if the emergence of SCVs actually depended on the initial inoculum, their number should have reduced at 24 h at low inoculum. Since they found an increased percentage of SCVs, it was concluded that the emergence of SCVs was not dependent on its initial numbers, but was due to the switching from the wild type bacteria.

However, the increase in the number of SCVs at a low inoculum size may not be due to the switching of wild type bacteria. Until 14 h (in the above experiment), the increase in the population of SCVs could be due to the selective killing of normal bacteria along with the gradual multiplication of SCVs. But, at a high inoculum size, antibiotics may not kill all normal bacteria. Some bacteria may escape killing and may remain dormant for a short period of time but later may undergo adaptation and re-grow and overcome the SCV population. On the other hand, at a low inoculum size, most of the normal bacteria get killed and thus the percentage of SCVs may increase. The switching mechanism proposed by Massey et al. (2001) could have resulted from the selective and complete killing of normal bacteria at a lower inoculum size, resulting in the selective multiplication and the increase in the number of SCVs.

The fundamental flaw with the above article is that they could find only two hypotheses for their results and assumed that if one of them is wrong, other should be correct. What, if there are more than two reasons or explanations for their results?

Massey, R. C., Buckling, A., and Peacock, S. J. (2001). Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus. Curr Biol 11(22), 1810-4.

Monday, August 22, 2011

Is SCV generation a survival strategy of bacteria?

Most of the SCVs are reported to be hemin, menadione, thymidine or thiamine auxotrophs. It is argued that SCV generation is a survival strategy of bacteria to resist adverse conditions, especially antibiotic therapy. Thus, in the presence of aminoglycoside antibiotics, only SCVs may survive whereas the normal wild type population gets killed by the antibiotic. However, once the antibiotic is removed, SCVs can revert to normal wild type and cause re-infection.

However, if the formation of SCV is a survival strategy, why do high frequencies of hemin-deficient mutants occur among the Enterobacteriacae family? It is documented that Enterobacteriacae lacks the ability to take up hemin (Sasarman et al. 1968). To revert to the normal wild type, it needs a second independent mutation that helps it take up hemin (Roggenkamp et al. 1998). However, the frequency of this second mutation is very low (Roggenkamp et al. 1998). This would mean that the hemin-deficient mutants of Enterobacteriacae will remain as SCVs even in the presence of hemin, thus offering them no growth advantages. If reversion is not possible, how it can be argued that SCVs are responsible for chronic infections?

Similarly, if the reversion to normal wild type is not possible, what is the fate of those Enterobacteriacae SCVs inside the body?

Next- A switching mechanism between the normal bacterial population and SCVs

Sasarman et al. (1968). Hemin-deficient mutants of Escherichia coli K-12. J Bacteriol 96(2), 570-2.
Roggenkamp et al. (1998). Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli. J Clin Microbiol 36(9), 2530-4.

Friday, August 19, 2011

Small colony variants- a survival strategy of bacteria: literature review

The formation of small colony variants (SCVs) is considered as a survival strategy of bacteria to evade antibiotic killing. In the presence of antibiotics, especially aminoglycosides, SCVs may survive due to reduced uptake of the antibiotic when most of the bacteria get killed. Thus, within a host, antibiotic pressure may select electron transport deficient mutants which may survive intracellularly due to low levels of free hemin and menadione within the host cell (McNamara and Proctor 2000). Since the intracellular milieu protects bacteria from antibodies, complements and many antibiotics, increased intracellular persistence of SCVs could be a survival strategy of S. aureus. However, once the antibiotic is removed, SCVs may revert to normal phenotype resulting in chronic infections.

In many clinical cases of chronic infections, both the SCVs and large colony types have been isolated. Researchers have shown that both colony types are clonal indicating a common origin. Isolation of SCVs from osteomyelitis and cystic fibrosis supports a pathogenic role for SCVs in such diseases (Proctor et al. 2006). SCVs have also been isolated from device-related infections, persistent wound infections and persistent bovine mastitis.

SCVs are implicated not only in chronic infections, but also in fatal infections. For example, a fatal infection due to SCVs of methicillin-resistant S. aureus in a patient with AIDS has been reported (Seifert et al. 1999). Similarly, SCVs of the Burkholderia cepacia complex has been reported to be responsible for the fatal outcome of lung transplantation in CF patients (Haussler et al. 2003).

Next- Is SCV generation a survival strategy of bacteria?

Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.
McNamara, P. J., and Proctor, R. A. (2000). Staphylococcus aureus small colony variants, electron transport and persistent infections. Int J Antimicrob Agents 14(2), 117-22.
Seifert et al. (1999). Fatal case due to methicillin-resistant Staphylococcus aureus small colony variants in an AIDS patient. Emerg Infect Dis 5(3), 450-3.
Haussler et al. (2003). Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex. Eur J Clin Microbiol Infect Dis 22(4), 249-53.

Thursday, August 18, 2011

Biochemical aspects of SCV formation- literature review

The phenotypic characteristics of SCVs can be related to defects in electron transport pathways. Defects in electron transport may affect the capacity of bacteria to produce ATP resulting in slow growth. Genetic mutations in hemB, menD, thyA and ctaA produce the SCV phenotype (Proctor et al. 2006). A mutation in hemB and ctaA blocks the biosynthesis of hemin, which is used in the cytochrome synthesis, whereas a mutation in menD blocks the synthesis of menadione, used in menaquinone synthesis. Since both menaquinone and cytochromes are components of the electron transport system, these mutations result in defective electron transport (Proctor et al. 2006). Similarly, a mutation in thyA, which encodes thymidylate synthase, results in impaired thymidine metabolism leading to SCV formation. Thus, the defects in electron transport are mainly responsible for the generation of SCVs.

The physiological characteristics of SCVs are mainly studied using hemB, menD  or thyA  mutants of S. aureus. All of these mutants show typical characteristics of SCVs such as slow growth, small colonies, decreased hemolytic and coagulase activity, reduced pigment formation and resistance to aminoglycosides. Complementing these mutants with hemB, menD or thyA respectively could restore the normal phenotype.

Defects in electron transport result in reduced carotenoid biosynthesis, which leads to reduced pigment formation by SCVs. Similarly, defects in electron transport decrease the amount of ATP used for cell-wall biosynthesis, leading to a slower growth rate and reduced membrane potential.  Reduction in the membrane potential results in the decreased uptake of cationic compounds including the aminoglycoside antibiotic (Proctor et al. 2006). The uptake of aminoglycosides depends on the membrane potential. Initiation of aminoglycoside uptake by bacteria requires a threshold level of membrane potential; above this level, the drug uptake is directly dependent on the magnitude of the membrane potential. Since SCVs show reduced membrane potential, uptake of aminoglycosides will be low, resulting in reduced killing by the antibiotic. Thus, SCVs can be selected by using aminoglycosides where the normal bacteria will be killed by the antibiotic wheras SCVs survive due to reduced uptake of the antibiotic. SCVs may be resistant to killing by other antibiotics such as penicillin, probably due to their slow growth rate.

Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.

Wednesday, August 10, 2011

Small colony variants- a short introduction

I will discuss SCVs and their role in chronic infections in the next few blogposts. Below are the first few introductory paragraphs from the chapter on SCVs in my book.

Small colony variants (SCVs) constitute a naturally occurring, slow-growing subpopulation of bacteria that form small colonies (less than one-tenth of the size of parent colonies) on solid media (Proctor et al. 2006). SCVs were first described around 100 years ago and since then have been reported in a wide range of bacterial genera and species. However, they have been most extensively studied for Staphylococcus aureus (Proctor et al. 2006). The major characteristics of SCVs include slow growth rate, lack of pigmentation, reduced hemolytic activity, reduced coagulase activity, increased antibiotic resistance especially to aminoglycosides, reduced carbohydrate utilization and low virulence with reduced production of virulence factors.  Since the growth rate of SCVs is approximately nine times lower than the parent strains, they require a longer incubation time (48-72 h) to form pinpoint colonies on agar. The slow growth rate and formation of small colonies is often due to the inability of the bacteria to synthesize certain substances required for their growth; thus, supplementation of these substances in the growth medium can result in a normal growth rate (Proctor et al. 2006). Most of the SCVs reported are auxotrophic for hemin, menadione or thiamine, even though other compounds such as thymidine, unsaturated fatty acids  and CO2 may also stimulate the growth of SCVs (Proctor et al. 2006).

They are implicated in a number of chronic infections, especially cystic fibrosis (CF) and osteomyelitis. SCVs are difficult to recognize using routine laboratory biochemical tests because of their atypical morphological and physiological features, thus presenting a challenge to clinical microbiologists.

Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.