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J Bacteriol. Feb 2007; 189(3): 807–817.
Published online Nov 17, 2006. doi:  10.1128/JB.01258-06
PMCID: PMC1797308

Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei[down-pointing small open triangle]

Abstract

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Burkholderia pseudomallei is a biothreat agent and the cause of melioidosis (29). This gram-negative motile bacillus is present in soil and water over a wide area of the Far East, where infection is acquired by inoculation or inhalation (29). B. pseudomallei causes 20% of community-acquired septicemias in northeast Thailand (7) and is the most common cause of fatal community-acquired pneumonia in Darwin, Australia (10, 14). Overall, mortality is around 50% in northeast Thailand (35% in children) and 20% in Australia (10, 11, 29).

A major feature of melioidosis is that bacterial eradication is difficult to achieve. The clinical response to intravenous antibiotics is slow (median fever clearance time, 8 days), and recurrent disease is common (6% in the first year in Thailand), despite appropriate antibiotic therapy for 12 to 20 weeks (6, 9). A prolonged period of dormancy may also occur between exposure to B. pseudomallei and clinical manifestations of infection, with the maximum recorded time being 62 years (8, 20, 21). It is clear that B. pseudomallei can become adapted for survival in vivo, but the mechanisms by which this occurs in humans have not been demonstrated.

In the 1930s, it was observed that colony morphology could change in vitro between rough and smooth colonies (22). We have observed over a period of 20 years of diagnostic culture of patients with melioidosis that B. pseudomallei cultures typically appear as dry, rough colonies on Ashdown's (selective) agar but that colony morphology often demonstrates considerable variability both within and between clinical samples. We hypothesize that B. pseudomallei undergoes a process of adaptation involving altered expression of surface determinants and associated colony morphology that facilitates bacterial survival in vivo. Here we describe a robust typing tool for the classification of B. pseudomallei colony morphology. We provide evidence for switching of B. pseudomallei colony morphotypes in response to stress in vitro and during both experimental infection and human melioidosis. We demonstrate that switches in colony morphology are associated with complex shifts in phenotype and that this is associated with altered interactions with epithelial cells and macrophages in vitro and persistence in an animal model. This process has major relevance to clinical disease, therapeutic response, and vaccine development.

MATERIALS AND METHODS

Bacterial isolates.

Three libraries of clinical B. pseudomallei isolates were examined. The first was obtained from 212 positive clinical specimens identified from 204 patients presenting with melioidosis to Sappasithiprasong Hospital, northeast Thailand, between 2002 and 2003 (strain set 1). Specimen types were blood (n = 100), pus (n = 59), sputum/respiratory secretions (n = 26), throat swabs (n = 9), urine (n = 13), and surface wound swabs (n = 5). This strain set was used to define morphotypes. The second independent library was obtained from 600 positive clinical specimens identified from 568 patients presenting with melioidosis between 1989 and 2002 (strain set 2). Specimen types were blood (n = 97), pus (n = 116), sputum/respiratory secretions (n = 109), throat swabs (n = 91), urine (n = 98), and surface wound swabs (n = 89). This set was used to define the relationship between clinical specimen type and the frequency of mixed morphotypes. The third independent library was obtained from 214 positive clinical specimens identified from 62 patients presenting between 1994 and 2002. This was used to examine variation in colony morphotypes in multiple samples from a given patient. Active observation for the presence of variable colony morphology in a given sample was undertaken in our diagnostic laboratory throughout the period of strain collection. All suspected B. pseudomallei colonies were confirmed using standard techniques (12, 31), and a final freezer vial was prepared to contain representatives of all colony morphology types from a given specimen. Vials were stored in Trypticase soy broth (TSB) with 15% glycerol at −80°C.

In addition, colony morphology was defined during a prospective study conducted at Sappasithiprasong Hospital between July and September 2006 in which clinical specimens taken from patients with suspected melioidosis were serially diluted with sterile normal saline and spread plated onto Ashdown's agar. Plates were examined after incubation for 4 days at 37°C in air. Each colony type present was examined using a specific latex agglutination reaction for B. pseudomallei (3), and the presence of more than one B. pseudomallei morphotype in the sample was determined. Ethical approval for collection of these isolates and associated clinical data has been obtained from the Ministry of Public Health, Royal Government of Thailand.

Observation of bacterial colony morphology.

A single investigator defined colony morphotypes throughout the study. Spread plates were prepared directly from freezer vials. A frozen bacterial colony was scraped from the top of the vial, suspended in sterile saline, serially diluted, and spread plated onto Ashdown's agar to give approximately 100 single colonies per plate. The first 50 isolates were examined to determine the optimum day of colony observation. Plates were incubated at 37°C in air and observed every day for 7 days, using a combination of a hand-held magnifier and colony photography. The following features of colony morphology were recorded: colony size (measured in mm by ruler), color (red, purple, or pink), translucency (opaque or translucent), degree of color (pale, bright, or dark), wetness (wet or dry), colony circumference (smooth or irregular), surface texture of center of colony, presence of roughness in outer half of colony, and overall surface shape (convex, crater, lobulated, radial, radio-umbilicated, radio-umbonated, peaked, rugose, segmented-rugose, segmented-umbilicated, umbilicated, umbilicated with irregular edge, umbilicated with heaped-up irregular edge, or umbonated). From these observations, we determined that day 4 gave the maximum observable colony variation between strains. The remaining samples were then examined. A morphotyping algorithm was subsequently developed to include discriminatory features, using Clementine 7.2 software. This divided colony morphotypes into seven types (I to VII).

Isolation of isogenic strains with variable colony morphology.

B. pseudomallei strains 153 and 164 were randomly selected from clinical isolates identified in 2002-2003 from those observed to have more than one morphotype present in a single sample. A single colony of morphotype I was harvested from Ashdown's agar for each strain after incubation at 37°C in air for 4 days and was placed into 3 ml of TSB. The colonies were maintained statically at 37°C in air for 28 days, after which spread plates were prepared on Ashdown's agar. Single colonies with morphology type I, II, or III were spread plated onto Ashdown's agar, incubated at 37°C in air for 4 days, harvested in purity into freezer vials, and maintained at −80°C. These were used to directly inoculate agar or broth for each assay comparing characteristics of isogenic colony morphotypes.

Switching of colony morphology during starvation stress.

Single type I colonies of B. pseudomallei strains 153 and 164 were inoculated into TSB and maintained statically at 37°C in air for 28 days. Aliquots were removed at 0, 7, 21, and 28 days, serially diluted, spread plated onto Ashdown's agar, and incubated at 37°C in air for 4 days. Plates with 100 to 300 colonies were examined, and the entire contents of the plates were defined with respect to colony morphology.

Rate of colony switching during starvation stress.

Starvation cultures of B. pseudomallei strains 153 and 164 (see above) were each plated in duplicate at 0, 7, 14, and 21 days onto Ashdown's agar and incubated at 37°C in air for 4 days. Eight single isolated colonies of type I, II, or III were identified from duplicate plates (giving a total of 16 colonies per type for each strain); this was termed the recovery morphotype. Each isolated colony was placed into a separate 500-μl aliquot of sterile normal saline and mixed well, and then 100-μl aliquots of 100-, 1,000-, 10,000-, and 100,000-fold dilutions were spread plated onto Ashdown's agar (secondary plating). The total number of bacteria and the proportion of each morphotype were determined for each primary colony after incubation of the secondary plates at 37°C in air for 4 days. Colonies that had different morphologies from that of the original colony were termed emergent morphotypes. The frequency of emergent morphotypes was determined and expressed as the mean for the 16 colonies.

Laboratory conditions as triggers for colony morphology switching.

Different laboratory culture conditions were examined to define conditions under which 24 randomly chosen clinical B. pseudomallei strains starting with the type I morphotype were triggered to change to one or more alternative morphotypes. Conditions were growth in 1 ml TSB for 7 days statically in air at 4°C, 30°C, or 42°C or at 37°C in the presence of Chelex (10 μg/ml), ceftazidime (20 μg/ml), or ciprofloxacin (125 μg/ml). Aliquots were removed, serially diluted, spread plated onto Ashdown's agar, and incubated at 37°C in air for 4 days. Plates with 100 to 300 colonies were examined, and the entire contents of the plates were defined with respect to colony morphology.

Phenotypic assays.

Bacterial aggregation was defined when a colony picked from Ashdown's agar following 4 days of incubation in air at 37°C did not disperse in phosphate-buffered saline (PBS). Positive colonies broke into visible fragments only. Attachment of a bacterial colony to the surface of Ashdown's agar was defined as a colony that did not transfer to a nitrocellulose membrane during colony blotting of the agar plate after 2 days of incubation in air at 37°C. Quantification of B. pseudomallei biofilm formation was performed using a microtiter plate assay. Biofilm formation was induced in Trypticase soy broth for 24 h, and the quantity was defined as previously described (19, 25). Each B. pseudomallei isolate was assayed in duplicate, using eight wells per experiment. The absorbance (optical density [OD]) was measured at 630 nm using a microplate reader (Bio-Rad).

The presence of elastase in the culture supernatant following bacterial incubation of B. pseudomallei for 24 h in LB at 37°C statically in air was detected using elastin Congo red (Sigma) as a substrate (5). The activity of each isolate was represented by the average OD at 490 nm for duplicate assays. Production of protease by B. pseudomallei colonies was detected using dialyzed brain heart infusion-milk medium (24). Isolates were incubated in TSB at 37°C statically in air for 20 h and spread onto dialyzed brain heart infusion medium to give single colonies, and the zone of clearing was measured around the colony after 48 h of incubation at 37°C in air. The size of the zone was measured and averaged for 10 colonies per isolate. Isolates were screened for lipase activity using an agar plate assay (30). Lipase-positive colonies were identified by a zone of precipitate extending from the edge of the colony. This was measured to the nearest mm after 72 h of incubation at 37°C in air for 10 colonies. Pseudomonas aeruginosa was used as a positive control for elastase and protease assays. Bacillus subtilis and Bacillus firmus were used as positive controls for the lipase assay. Escherichia coli was used as a negative control for protease and lipase assays.

Lipopolysaccharide (LPS) was extracted by proteinase K digestion and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the LPS bands were visualized by silver staining (2). The LPS pattern for B. pseudomallei in this technique has been described as either smooth LPS serotype A (typical ladder), smooth LPS serotype B (atypical ladder), or rough LPS (without ladder); this relates to variability in the O antigen. The presence of exopolysaccharide was examined by colony immunoblotting of colonies on Ashdown's agar. A single representative colony from each strain was transferred to a nitrocellulose membrane and blocked with 5% skim milk in PBS. This was probed with monoclonal antibody 4B11, specific for B. pseudomallei exopolysaccharide (4), followed after washing by a secondary horseradish peroxidase-conjugated rabbit anti-mouse whole immunoglobulin (Dako A/S) and detection using DAB (3′,3′-diaminobenzidine tetrahydrochloride) and hydrogen peroxide. A positive result was taken as the presence of any detectable color. Controls were B. pseudomallei strain 844 (positive) and a clinical E. coli isolate grown on Columbia agar (negative). Isolates that failed to transfer were grown manually on the membrane, which was placed on top of the agar medium; these were then incubated with monoclonal antibody as described above.

Swimming and swarming motilities were assessed using agar plate assays as previously described (13). Bacterial cells were point inoculated with a sterile toothpick onto swim or swarm agar. Plates were observed after incubation at 37°C in air for 72 h. Evidence of bacterial motility was taken as the presence of bacterial growth around the point of inoculation. The widest diameter of the zone was measured using a vernier micrometer. Burkholderia mallei strain EY 100 was used as a negative control, and Pseudomonas aeruginosa ATCC 27853 was used as a positive control in all motility assays. The presence of flagella was assessed by transmission electron microscopy. Bacteria were taken directly from freezer vials and incubated in TSB for 24 h at 37°C statically in air. A drop of suspension was applied to Formvar-coated carbon grids, stained with 1% uranyl acetate, and observed using an electron microscope. The presence and number of flagella were recorded for 100 bacteria per strain.

Bacterial genotyping.

Pulsed-field gel electrophoresis (PFGE) was performed as follows. A single bacterial colony was streaked onto Columbia agar and incubated for 48 h at 37°C in air. Colonies were harvested and suspended to an OD at 600 nm of 1.2 in suspension buffer (75 mM sodium chloride, 25 mM EDTA, pH 7.5). This was mixed (1:1) with molten 2% low-melting-point agarose (Gibco Ultrapure) and pipetted into PFGE plug molds (Bio-Rad Laboratories, Hercules, CA). Plugs were lysed overnight at 56°C in lysis buffer (0.1% sodium dodecyl sulfate, 25 mM EDTA, pH 8.0) containing 500 μg/ml proteinase K (Invitrogen) and then rinsed three times with TE buffer (10 mM Tris and 10 mM EDTA). Prior to PFGE, plugs were digested overnight at 37°C with 10 U SpeI (New England Biolabs) before being loaded into a 1% agarose gel (Gibco) in 0.5× Tris-borate-EDTA buffer. Each well was overlaid with 0.8% low-melting-point agarose. PFGE was performed on a CHEF-DRIII system (Bio-Rad Laboratories, Richmond, CA) for 44 h at 14°C and 6 V/cm, using the following parameters: initial switch time (block I), 18 h at 10 to 60 V; final switch time (block II), 18 h at 30 to 40 V; and run time (block III), 8 h at 50 to 90 V. Lambda concatemers were run as the standard (Promega). Gels were stained with ethidium bromide, washed in water, and photographed under UV light using a Gel Doc 1000 system (Bio-Rad). Analysis of PFGE banding patterns was performed using BioNumerics (version 2.5) software (Applied Maths, Belgium). Multilocus sequence typing (MLST) was performed as previously described (28). Alleles at each of the seven loci were assigned, and the allelic profile (a string of seven integers) was used to define the sequence type, using the B. pseudomallei MLST website (http://bpseudomallei.mlst.net/).

Cell lines.

The murine macrophage cell line J774A.1 (ATCC TIB-67) and the human respiratory epithelial cell line A549 (ATCC CCL-185) were used in this study. Cells were cultured and maintained in appropriate cell culture media supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 2 mM l-glutamine (Invitrogen). Dulbecco's modified Eagle medium (Invitrogen) was used for J774A.1 cells, and F-12K culture medium (HyClone, Logan, UT) was used for the A549 cells. Throughout the study, cells were incubated at 37°C in 5% CO2.

Bacterial adherence and invasion of macrophage and epithelial cell lines.

Adherence and invasion assays using B. pseudomallei were performed as previously described (1, 17). In brief, macrophages or epithelial cells were seeded at 1 × 105 cells per well into 24-well plates. These were incubated overnight at 37°C with 5% CO2. On the day of infection, overnight cultures of 153 isogenic type I, II, and III colonies were harvested from Ashdown's agar, washed, and resuspended in PBS to a final concentration of 5 × 107 CFU/ml. Bacteria were then added to each well to give a multiplicity of infection (MOI) of approximately 25 or 50 bacteria per cell. The MOI was confirmed by plating cells onto Ashdown's agar. To determine initial adherence and invasion, assay plates were incubated for 2 h at 37°C in 5% CO2, after which the cell monolayers were washed three times with PBS and lysed with 0.1% Triton X-100. Bacteria were enumerated by plating serial dilutions of lysate onto Ashdown's agar. Colony morphology was recorded for all experiments. To determine the ability of the three isogenic morphotypes to remain viable and replicate after internalization into cells, experiments were repeated as described above, except that after 2 h of incubation, cell lines were washed with PBS, and external bacteria were removed. This was achieved by adding fresh culture medium containing 250 μg of kanamycin/ml (Invitrogen) and incubating the culture for 2 h at 37°C in 5% CO2. The medium was then replaced with culture medium containing 20 μg of kanamycin/ml to prevent the outgrowth of the bacteria that might be released during prolonged incubation. Intracellular bacteria were enumerated as described above 4, 6, and 8 h after initial inoculation. All experiments were performed in duplicate on two independent occasions.

Animal model.

Approval was obtained from the Animal Ethics Committee of The Faculty of Tropical Medicine, Mahidol University. Mice were maintained in standard cages at 25°C with food, water, and light. Groups of eight 6- to 8-week-old BALB/c mice received a target inoculum of 100 CFU of isogenic B. pseudomallei strain 153 type I, II, or III by intraperitoneal injection. The inoculum was prepared by harvesting a single defined colony from Ashdown's agar into sterile PBS and adjusting the OD. Type II is difficult to suspend and was subjected to repeated passages through a sterile needle under appropriate biological containment. In view of this, the size of each inoculum was defined by serial dilution and plating onto Ashdown's agar; this was defined as 79 CFU for type I, 79 CFU for type II, and 49 CFU for type III. Sterile PBS was used for the control group. Death was determined over 28 days of observation, after which survivors underwent euthanasia. Immediately after death, organs were removed aseptically, and half of each organ was homogenized in 1.5 ml of sterile PBS, using a sterile multidirectional stopcock (B Braun). One-hundred-microliter aliquots of 10-fold serial dilutions of either homogenized tissue or blood were plated onto Ashdown's agar in duplicate and incubated at 37°C for 4 days. The colony morphotype was recorded, and the bacterial load was expressed as CFU per gram of organ or per milliliter of sample, as appropriate.

Statistical analysis.

Statistical analysis was performed using the statistical program Intercooled STATA, version 8.0 (College Station, TX). Fisher's exact test was used to test proportions. Data for phenotypic assays for the population of 241 strains were not normally distributed; data are presented using the median and interquartile range (IQR) and were analyzed using the Kruskal-Wallis test. Data for biofilms produced by isogenic strains were right skewed; these were log transformed to present geometric means and analyzed using analysis of variance.

RESULTS

Identification of seven unique B. pseudomallei colony morphotypes and development of morphotyping scheme.

Colony morphology on Ashdown's agar was observed for the 212 samples of strain set 1. Seven major morphotypes were defined after 4 days at 37°C in air, and they are referred to hereafter as types I to VII. A striking feature was that 25 cultures (12%) demonstrated more than one morphotype on the same plate; 22 contained two morphotypes, 2 contained three morphotypes, and 1 contained four morphotypes (giving a total of 241 “strains”). One hundred eighty-one (75.1%) of these strains were type I, and 60 (24.9%) belonged to the remaining six types (II to VII). Photographic representations of the seven morphotypes are shown in Fig. Fig.1A.1A. Type I is the classical morphotype described in the literature for B. pseudomallei. The presence of smooth colony morphotypes was confirmed, being observed for two morphotypes (types III and VI) that were differentiated on the basis of colony diameter and color. Type VI colonies were always pale purple or pale pink with a diameter of <2 mm. Type III colonies varied from pale purple to dark purple and were ≥2 mm in diameter. Discriminatory features in colony morphology were identified, and Clementine 7.2 software was used to develop the morphotyping algorithm shown in Fig. Fig.1B1B.

FIG. 1.
Seven unique B. pseudomallei colony morphotypes on Ashdown's agar. Colony morphology was observed for 212 samples of B. pseudomallei identified from 204 consecutive unselected patients presenting to a hospital in northeast Thailand with melioidosis. (A) ...

Genotyping of B. pseudomallei strains with mixed colony morphologies in the same clinical sample.

Mixed morphotypes in the same clinical specimen could be due to coinfection with two or more strains of B. pseudomallei. We examined the 25 samples with mixed morphotypes in strain set 1 by using PFGE, comparing the banding patterns of colonies with different morphologies from the same sample. The banding patterns were identical for 23 samples (data not shown), indicating morphotypic variability of a single infecting clone. One sample with two morphotypes demonstrated a PFGE pattern difference of one band; MLST of this discrepant pair demonstrated an identical sequence type, indicative of clonality. One sample with three morphotypes demonstrated a PFGE pattern difference of two bands for one of the morphotypes; MLST indicated a mixed infection with two different strains in this one case. Thus, mixed colony morphologies in a given sample were associated with a single B. pseudomallei clone in 24/25 (96%) cases.

Rates of mixed B. pseudomallei colony morphologies in samples from different human body compartments.

An independent collection of 600 clinical B. pseudomallei isolates cultured from blood, respiratory secretions, pus, urine, or throat or surface wound swabs (strain set 2) was studied to determine whether the rate of mixed morphotypes in a given sample varied between different body sites. Samples were examined in random order by a single investigator who was blinded to sample type.

The overall frequency of samples containing two or more morphotypes was 11.3%, consistent with the rate found for strain set 1. However, the rates of mixed morphotypes varied significantly between different sample types (P < 0.001) (Table (Table1).1). Almost one-quarter of respiratory isolates and 15% of pus and blood isolates demonstrated mixed colony morphology, compared with only 1% and 4% for wound swab and urine samples, respectively. Type I represented the majority population in all specimen types but was least common in respiratory samples and most common in wound swab samples (60.4% versus 87.8%; P < 0.001).

TABLE 1.
Frequency of mixed B. pseudomallei colony morphologies in a given sample according to clinical specimen type

Morphotypic switching is readily observed in vitro for a population of B. pseudomallei isolates associated with invasive disease.

We next considered the relationships between morphotypes and whether clinical isolates of B. pseudomallei could change from one morphotype to another. Responses to starvation in vitro were defined for 100 B. pseudomallei isolates randomly selected from strain set 1. A single colony with clearly defined morphology for each isolate was subcultured from Ashdown's agar into TSB and incubated at 37°C statically in air for 3 weeks, after which initial and final colony morphologies were compared. Summary results are shown in Table Table2.2. Sixty-seven of 100 cultures contained two or more morphotypes at 3 weeks. Type I cultures preferentially changed to type III (30 of 79 strains [38.0%]), type II (30.4%), or type V (25.3%). Types IV, VI, and VII were each observed in <5% of morphotype I starting cultures. These data indicate that the majority of the B. pseudomallei population associated with human disease can undergo morphotypic change in vitro; type I cells can switch to a range of other morphotypes, but types II and III are the preferential types under these conditions.

TABLE 2.
Change in colony morphotypes of a population of invasive B. pseudomallei isolates in response to starvation stress in vitro

Indirect evidence for switching of colony morphotype during human melioidosis.

Sixty-two patients who had more than one sample positive for B. pseudomallei during a single hospital admission were randomly identified using our existing databases. These patients had a total of 214 positive samples (range, 2 to 7 samples per patient; median, 4 samples per patient) (strain set 3). Isolates were examined, and the morphotypes were classified using the typing algorithm. Twelve patients had different morphotypes identified in at least two independent samples taken on the same day (n = 5), consecutive days (n = 5), or 2 days apart (n = 2). Two of these patients had three samples that each contained a different morphotype. The PFGE banding patterns were identical for within-patient strains in nine cases. Three patients had isolates with differing patterns (6, 12, and 14 bands different between the respective pairs); MLST of discrepant pairs gave different sequence types, indicating mixed infections for these three patients. These data demonstrate that most patients with variable colony morphotypes in different samples were infected with a single clone of B. pseudomallei; we propose that this represents indirect evidence for switching of colony morphology during human disease.

Parental phenotype in human isolates.

The relationship between morphotypes in a given clinical specimen was defined; for this purpose, we used 68 of the 600 cultures (set 2) that contained mixed morphologies in single samples. Type I cells were present in the majority of samples (n = 60 [88%]). The most frequent combinations of morphotypes were types I and III (22%), types I and VI (21%), types I and II (15%), and types I and IV (12%). Uncommon morphotypes rarely occurred in isolation; types II, III, IV, V, and VII were each present in purity in 0.7% to 1.7% of samples, and type VI was present in purity in 7% of samples. From these observations, we hypothesized on the basis of parsimony that type I is the parental type in vivo from which others arise.

Prospective validation of colony morphology variability in primary clinical cultures.

A potential criticism of using bacterial libraries from freezer stocks is that the process of freezing and/or thawing represents an environmental stress that could lead to changes in colony morphology. A prospective study was performed to address whether colony morphology variability occurs in primary cultures. This was examined using 218 unselected clinical samples taken from 128 patients presenting with melioidosis that were culture positive for B. pseudomallei. Specimen types were blood (n = 75), pus (n = 16), sputum or tracheal secretions (n = 53), throat swabs (n = 27), urine (n = 22), and wound swabs (n = 25). Samples were plated directly onto Ashdown's agar, and the colony morphology was determined after 4 days of incubation at 37°C in air. The presence of mixed morphotypes was observed in 18 specimens (8.3%), among which 7 samples had two morphotypes, 8 samples had three morphotypes, and 3 samples had four morphotypes. Type I was present in 202 of 218 specimens (93%), including 13 of the 18 samples (72%) that contained more than one morphotype. No previously unrecognized colony morphotypes were observed. We concluded from this prospective evaluation of primary cultures that variability in B. pseudomallei colony morphology is common in primary clinical samples.

Morphotypic switching over time during increasing environmental stress in vitro.

Having shown that the majority of clinical B. pseudomallei isolates demonstrated colony morphology switching at a fixed time point of 21 days in response to starvation stress in vitro, we next explored the sequence of events over time. This was examined for two type I isolates (strains 153 and 164) which were randomly selected from samples that contained mixed morphologies. Strains 153 and 164 were initially isolated from a blood culture. A single colony of each was subcultured into aliquots of TSB that were plated onto Ashdown's agar after 7, 21, and 28 days of static incubation at 37°C in air. The proportions of type I, II, and III colonies at each time point are shown in Fig. Fig.2.2. The behaviors of the two strains were comparable. The proportions of type I and III colonies were inversely related, with type III increasing over time, so that by 28 days of starvation it represented almost the entire population. Type II colonies demonstrated nonsustained increases at 7 and 21 days for strains 153 and 164, respectively. These data indicate that B. pseudomallei may switch to more than one morphotype over time in response to changing environmental cues.

FIG. 2.
Colony morphology of B. pseudomallei during starvation stress over time. Two type I isolates (strains 153 and 164) were randomly selected from clinical samples that contained mixed morphologies. A single colony of each was subcultured into aliquots of ...

Morphotypic switching over time during increasing environmental stress in vitro.

Having observed that type I switched preferentially to types II and III in response to starvation in vitro, we next determined whether this process was reversible. Day 7, 14, and 21 starvation cultures were used to define the dominant direction and rate of morphotypic change for strains 153 and 164 once starvation stress was removed. The results are summarized in Table Table3.3. Type I colonies remained true to type upon subculture after removal from stress. The overall trend for type II and type III colonies was to revert to type I upon subculture after removal from stress. These data represent evidence that colony morphology can switch in a reversible manner in response to environmental conditions.

TABLE 3.
Emergent B. pseudomallei morphotypes after removal of starvation stressa

Laboratory conditions as triggers for switching.

A range of laboratory conditions was used to define conditions that favor colony morphology switching. Incubation of 24 clinical B. pseudomallei isolates at temperatures lower than normal body temperature (4°C and 30°C) for 7 days had no effect on colony morphology, while incubation at 42°C was a strong stimulus for change, with 16/24 strains demonstrating one or more different morphologies. Iron limitation resulting from the presence of Chelex resulted in colony morphology changes in 19/24 strains, and the presence of subinhibitory concentrations of ceftazidime and ciprofloxacin led to changes in morphology in 10/24 and 16/24 strains, respectively. A spectrum of morphology types was observed within the strain collection, but a switch to type II predominated overall (2/16 strains with a switch of colony morphology at 42°C, 16/19 switchers in the presence of Chelex, 9/10 switchers in the presence of ceftazidime, and 12/16 switchers in the presence of ciprofloxacin).

Morphotype is related to phenotype for a population of invasive B. pseudomallei cells.

Bacterial switching between morphotypes is likely to reflect a change in expression of one or more phenotypic determinants. We examined the relationship between bacterial phenotype and morphotype for the 241 strains used during morphotypic characterization. Major putative virulence determinants were evaluated. LPS was extracted, and the LPS pattern was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and Western blotting. A smooth LPS serotype A (typical LPS) pattern was demonstrated for 97% of the strains, and a smooth LPS serotype B (atypical LPS) pattern was observed in 3% of strains; there was no association between LPS type and morphotype (P = 0.12). Qualitative expression of exopolysaccharide was ubiquitous, as defined by colony blotting and reaction against a monoclonal antibody to exopolysaccharide. Colony adherence to Ashdown's agar and the ability to disperse in PBS were expressed as binary characteristics. Eight strains demonstrated aggregation in PBS, among which 7 were type II (P < 0.0001), and 11 strains adhered to the agar surface, among which 7 were type II (P < 0.0001). Quantitative assays were used to assess biofilm formation and the production of protease, lipase, and elastase. Swimming and swarming motilities were assessed by quantitative plate assays. Patterns of phenotypic expression were clearly evident for this bacterial population (Table (Table4).4). Using type I as the reference against which other types were compared, expression of three or more of the six factors tested was significantly different for types II, III, and VI.

TABLE 4.
Association between B. pseudomallei morphotypes and phenotypes for population of 241 invasive isolates

Phenotypic switching was explored further by using isogenic type I, II, and III colonies of strains 153 and 164 obtained by starvation stress. A single freezer vial was created for each and used as a primary stock culture for all experiments. Specific phenotypic assays were performed to investigate putative factors associated with either concealment or motility. Only isogenic type II colonies (strains 153 and 164) adhered to agar and aggregated in PBS. Type II colonies produced significantly more biofilm than did parental type I colonies in a microtiter plate assay (geometric mean [95% confidence interval] OD630 values for strain 153 were 0.051 [0.047 to 0.056] for type I versus 2.27 [1.73 to 2.86] for type II [P < 0.0001], and those for strain 164 were 0.058 [0.042 to 0.078] for type I versus 0.637 [0.353 to 1.15] for type II [P < 0.0001]). In contrast, type III was associated with increased flagellum expression compared with type I, as defined by transmission electron microscopy. The proportions of flagellate bacteria of type I were 9.6% and 19% for strains 153 and 164, respectively, compared with 85% and 53% for type III colonies of strains 153 and 164, respectively (P < 0.0001 for both type I and type III comparisons).

Morphotype influences B. pseudomallei adherence to and intracellular replication in macrophages and epithelial cells in vitro.

B. pseudomallei is known to be taken up by epithelial cells and macrophages in vitro. We predicted that morphotypic switching would have a strong bearing on interactions with the host, in particular with host cells. We compared B. pseudomallei strain 153 isogenic types I, II, and III during interactions with the human respiratory epithelial cell line A549 and the mouse macrophage cell line J774A.1 in vitro. Three variations of the assay were performed. The initial interaction (adherence and invasion) was assessed after coculture for 2 h in the absence of antibiotics. Efficiency of invasion was assessed following removal of extracellular bacteria at 2 h and incubation for a further 2 h. Persistence and multiplication of intracellular bacteria were assessed at 6 h and 8 h postinoculation; extracellular bacteria were removed after 2 h, followed by extended culture. The integrity of epithelial monolayers was confirmed by inverted light microscopy for all assays. Colony morphology was examined at the end of all assays and expressed as a proportion of the final population.

The initial interaction at 2 h between epithelial cells and type II colonies was more efficient than that for parental type I colonies at MOIs of 25:1 and 50:1 (P = 0.01 and 0.04, respectively) (Fig. 3A and B), while the interaction of type III colonies was not different from that for type I colonies. There was a marked increase in replication fitness at MOIs of 1:25 and 1:50 for type II colonies compared with that for parental type I colonies after 8 h of incubation (P = 0.0001 and P = 0.0002, respectively) (Fig. 3C and D); the interaction of type III colonies was not different from that for type I colonies. Assessment of colony morphology switching indicated that type I and type II were stable throughout all assays, with no other morphotypes observed at any time point. In contrast, the assay inoculated with bacterial cells derived from a single type III colony contained an increasing proportion of type II colonies over time (Fig. 3E and F). Parallel assays were performed in the absence of antibiotics; the switching observed could not be accounted for by the presence of kanamycin (data not shown). We propose that the stable type II isogenic variant of parental type I strain 153 is associated with an adaptive phenotype associated with both increased efficiency of the initial interaction and enhanced fitness in epithelial cells in vitro. However, this was not observed during the time course of the experiment for type II variants that arose in the intracellular milieu from type III variants.

FIG. 3.
Interaction in vitro between the human respiratory epithelial cell line A549 and B. pseudomallei strain 153 type I (parental) or isogenic type II or III derived from type I strain 153 during experimental starvation stress. The initial interaction (adherence ...

The initial interaction at 2 h between macrophages and isogenic type III strain 153 was more efficient than that for parental type I colonies at MOIs of 25:1 and 50:1 (Fig. 4A and B) (P = 0.08 and 0.005, respectively), while the interaction of type II colonies was not different from that for type I colonies. There was a higher replication fitness at MOIs of 1:25 and 1:50 for type III colonies than that for parental type I colonies after 8 h of incubation (P = 0.03 and 0.01, respectively) (Fig. 4C and D); the interaction of type II colonies was not different from that for type I colonies. As described above, colony morphotypes I and II were stable throughout, while the assay inoculated with bacterial cells derived from a single type III colony demonstrated an increasing proportion of type II colonies over time (Fig. 4E and F). We propose that either the isogenic type III variant of parental type I strain 153 or the emergent type II morphotype that arose from type III during the course of the experiment is associated with an adaptive phenotype associated with enhanced interaction and fitness in macrophages in vitro. Based on numerical probability, it is possible that the emergent type II morphotype represents the adaptive phenotype associated with a fitness advantage. However, this is inconsistent with the lack of fitness advantage in cells directly inoculated with a type II colony.

FIG. 4.
Interaction in vitro between the mouse macrophage cell line J774A.1 and B. pseudomallei strain 153 type I (parental) or isogenic type II or III derived from type I strain 153 during experimental starvation stress. The initial interaction (adherence and ...

Morphotype is related to lethality and persistence in an animal model.

Groups of BALB/c mice (eight in each group) were inoculated with strain 153 type I (parental) or an isogenic type II or type III strain derived from type I strain 153. A single colony with a clearly defined morphotype was selected from Ashdown's agar and suspended in PBS to achieve a target concentration of 100 CFU. Bacteria were not used from broth culture to control for phenotypic switching during bacterial preparation. A control group (none of whom died during the experimental period) was inoculated with PBS. Survival of mice to 28 days is shown in Fig. Fig.5.5. Overall survival rates were comparable between animals infected with parental type I and type III strains (P = 1.0), but there was a trend towards increased survival of type II-inoculated animals compared with that of type I-inoculated animals, with 50% of animals inoculated with type II still alive at 28 days (P = 0.08). The time to death was also different for each group; the time taken for half of the population in each group to die was 4, 9, and 13 days for mice inoculated with type II, III, and I strains, respectively. The outcome difference between mice inoculated with type I and type II strains was reproducible. Using identical experimental conditions with a second independent test population, 4/8 mice survived to 28 days in the type II group, in contrast to 1/8 mice in the type I group; the type I survivor died on day 30.

FIG. 5.
Time to death of BALB/c mice inoculated with isogenic types of B. pseudomallei strain 153. Groups of BALB/c mice (eight in each group) were inoculated with strain 153 type I (parental) or isogenic type II or III derived from type I strain 153 during experimental ...

Cultures of spleens, livers, kidneys, lungs, hearts, blood, bladders, and brains were performed on all animals at the time of death or euthanasia. There was widespread bacterial dissemination to all organs examined, and this did not differ between the isogenic types. Colony morphology of B. pseudomallei was observed for all postmortem cultures. Types I and II were stable in vivo, with no detectable change of colony morphology in any of the postmortem samples. In contrast, all samples from all animals inoculated with type III colonies were positive only for type II B. pseudomallei at postmortem. The PFGE banding patterns of the type III starting inoculum and at least one representative bacterial type II colony from every animal obtained at postmortem were identical for this group (data not shown).

Five mice survived to 28 days. One mouse was culture negative at time of death; this was the 28-day survivor in the type III group. One explanation for bacterial clearance is that the founding inoculum did not contain a bacterial cell capable of switching from type III to an alternative type and that this was associated with a fitness disadvantage and more effective clearance by the host. In contrast, all four type II survivors were culture positive at 28 days. Quantitative postmortem cultures, as expressed in CFU per gram of tissue, were compared between groups. The spleen had the greatest bioburden in all three groups and was used for the purpose of comparative analysis. The median (IQR) CFU per gram of spleen at time of death or euthanasia was 1.15 × 109 (5.29 × 108 to 1.88 × 109) for type I-inoculated animals, 4.35 × 108 (2.57 × 108 to 9.76 × 108) for type III-inoculated animals, and 1.31 × 108 (1.44 × 108 to 2.69 × 108) for type II-inoculated animals (for type I versus III, P = 0.12; for type I versus II, P = 0.006). There was no significant difference in the splenic loads for type II animals who died within the first 4 days compared with those who survived to 28 days (P = 0.39). Thus, a proportion of mice appeared to tolerate a high bacterial bioburden of type II cells without clinical symptoms. There was a clear difference in the numbers of bacteria in the lungs of mice infected with type I (median, 1.35 × 108; IQR, 3.25 × 107 to 2.16 × 108) and type II (median, 1.44 × 106; IQR, 2.48 × 104 to 6.24 × 106 [P = 0.0008]) strains. There was a less striking reduction in bacterial numbers in the lung for type III versus type I infection (for type III, the median was 6.74 × 106, and the IQR was 5.66 × 104 to 7.66 × 107 [P = 0.02]).

DISCUSSION

Eradication of B. pseudomallei from patients with melioidosis who survive the early stages of clinical illness is the most significant challenge faced by those caring for this patient population. Recurrence is common and results in death of around one-quarter of these initial survivors. An important reason for treatment failure is that prolonged therapy with two or three oral drugs is associated with side effects and poor compliance. However, bacterial factors, such as dissemination within the host and the development of a protected niche, are likely to play a major role.

In this study, we have demonstrated the presence of colony morphology variability in a large population of clinical B. pseudomallei isolates. Using a morphotyping scheme, we classified the common, textbook colony morphotype as type I. Mixed morphotypes in a given specimen were common and were not due to infection with more than one strain in most cases. This led us to explore whether morphotypes were fixed or could change in response to stress in vitro. Our data indicated that the majority of invasive B. pseudomallei strains were capable of morphotypic change; more detailed evaluation of two strains indicated that this process was reversible. We suggest that type I is the parental morphotype during clinical infection, from which other morphotypes arise in vivo, and that this is associated with changes in expression of major surface-expressed bacterial determinants and altered host-pathogen interactions. Morphotypic and phenotypic plasticity on this scale could explain the persistent nature of human melioidosis. An alternative explanation is that bacteria in the founder infecting inoculum contain colony variants that are either stably maintained or selected for during human infection. Arguing against this is our unpublished observation that morphotypes other than type I are very rarely observed during culture of B. pseudomallei from soil.

Our finding that mixed morphotypes were more common in certain specimen types suggests that colony variants more readily arise in specific body compartments. The efficacy of the host response to infection is likely to be higher in blood, solid organs, and the lungs than in urine and surface wounds, and it is possible that initial host-pathogen interactions provide an environmental stimulus for colony morphology switching. The reason for the low rate of mixed morphotypes from throat swabs is unclear. B. pseudomallei cells are taken up by epithelial cells in vitro (17), but the possibility that this organism may exist within epithelial cells during human infection has not been examined. In our experience, a throat swab positive for B. pseudomallei may be associated with respiratory secretions that are negative for B. pseudomallei but blood cultures or pus that are positive. This indicates that a positive throat swab may not be a simple reflection of contamination of the pharynx by sputum containing B. pseudomallei. A possible criticism of the methodology used to define mixed or single morphotypes in relation to sample type is that cultures from sterile sites, such as blood and deep-site pus, usually contain a single pathogen, and detection of mixed B. pseudomallei morphotypes will be high compared with that for samples obtained from colonized sites, such as urine, throat swabs, and respiratory secretions, that will inevitably contain a range of bacterial species. However, the finding that respiratory secretions had the highest rate of mixed B. pseudomallei morphologies argues against this. A further possible criticism is that the large isolate sets examined were derived from freezer stocks, and morphology changes could have arisen during laboratory processing. We suggest that instability of colony morphology in nutritionally rich laboratory culture broth would, based on our in vitro data, favor overreporting of type I and an associated underreporting of other morphotypes, since non-type I variants revert to type I variants under favorable conditions. Furthermore, a prospective study of colony morphology variability on primary culture plates from patients presenting with melioidosis confirmed that morphology is variable.

The mouse model of infection provided clear evidence of morphotypic switching in vivo. Isogenic types II and III did not revert to parental type I, but rather favored continued persistence as type II or a switch from type III to type II. This indicates that type II had a fitness advantage in this model. In contrast, type III appeared to have a fitness disadvantage, since this type was not cultured at postmortem, and one mouse in this group was the only animal to clear the infection. The in vivo data also give an indication that bacterial adaptation may lead to host survival. Animals inoculated with type II cells demonstrated a biphasic response in that half of them died rapidly, but the remainder survived to 28 days. The lower virulence potential of bacteria in the type II survivors may relate to modification of gene expression over time, together with the development of biofilms. The observation that type II survivors had bacterial counts in the spleen that were equivalent to those seen in lethal type II infection is consistent with bacterial persistence in a low-virulence state.

The in vitro epithelial cell and macrophage assays provided further support for the idea that different colony morphotypes reflect adaptive phenotypes associated with enhanced fitness in a given environment. B. pseudomallei is an intracellular organism and is taken up by epithelial cells and macrophages in vitro (1, 15-18, 23). Uptake by macrophages is followed by bacterial escape from endocytic vacuoles into the cytoplasm (15, 16, 23). Induction of actin polymerization at one pole leads to the formation of membrane protrusions and cell-to-cell spread (18). B. pseudomallei mutants lacking components of a type III secretion system (TTSS) that shares homology with the inv/spa/prg TTSS of Salmonella enterica serovar Typhimurium and the ipa/mxi/spa TTSS of Shigella flexneri have reduced replication in murine macrophage-like cells, have an inability to escape from endocytic vacuoles, and fail to form membrane protrusions and actin tails (26, 27). It is possible that phenotypic switching has a direct effect on this secretion system or leads to altered expression or function via changes in one or more global regulators. Further studies are required to define the reproducibility of these findings, using a range of clinical strains, and to determine the mechanisms of such altered interactions. Expression microarray analysis should provide insights into differential expression levels, including those that may be involved in cell-cell interactions.

The emergence of type III in prolonged starvation cultures in vitro following a nonsustained emergence of type II appears to be somewhat at odds with the in vivo findings. One explanation is that type III is favored under harsh environmental conditions in the absence of challenge from human host factors. The increased expression of flagella may be associated with an enhanced ability to move towards more favorable conditions. It is highly likely that the evolution of switching mechanisms has been driven in response to survival within the environment, and it is possible that both types II and III arise in response to specific environmental stimuli, such as drying, heat, and limited nutrients.

In summary, our data suggest that B. pseudomallei has a high degree of phenotypic plasticity and that complex changes in phenotype are associated with altered interactions with the host. Experimentally, types II and III emerge under conditions of stress. Type II is morphotypically stable after host cell uptake in vitro and during animal infection and is associated with biological characteristics that could favor enhanced survival and persistence. However, we sound a note of caution about automatically linking a given colony morphotype with a specific phenotype and biological behavior. The isogenic type II and III strains used during this study were derived from two strains of B. pseudomallei; we are currently assessing whether other strains demonstrate identical patterns of switching and biological behavior. Additional studies are also being conducted to determine the relationships of other types defined during examination of clinical isolates (IV, V, VI, and VII) to types II and III. The phenotypic plasticity of B. pseudomallei has important implications for treatment and vaccine development. Understanding the full repertoire of morphotypic and phenotypic switching, together with the signals and mechanisms for this process, represents key areas for further study.

Acknowledgments

We are grateful for the support of the staff at Ubon Ratchathani Hospital, in particular Wipada Chaowagul. We thank the staff of the Wellcome Trust-Mahidol University-Oxford University Tropical Medicine Research Program, including Sirinuch Rajchaiboon for technical support and Ekawat Pasomsub, Faculty of Medicine (Ramthibodi Hospital), for his assistance in using Clementine software for the development of the morphotype algorithm. We thank Stitaya Sirisinha, Faculty of Science, Mahidol Unversity, for his assistance.

S. Peacock holds a Wellcome Trust career development award in clinical tropical medicine. This work was supported by The Wellcome Trust and the Thailand Research Fund.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 November 2006.

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