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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2020 Oct 21;58(11):e01218-20. doi: 10.1128/JCM.01218-20

Enhancement of Culture of Legionella longbeachae from Respiratory Samples by Use of Immunomagnetic Separation and Antimicrobial Decontamination

Ali Mohammadi a, Stephen T Chambers a,b,✉, Amy Scott-Thomas a, John G Lewis c, Trevor Anderson d, Ros Podmore d, Jonathan Williman e, David Murdoch a,c, Sandy Slow a
Editor: Geoffrey A Landf
PMCID: PMC7587110  PMID: 32817229

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae.

KEYWORDS: Legionella, clinical methods, immunomagnetic separation

ABSTRACT

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae. A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from −80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.

INTRODUCTION

Legionella longbeachae is a major cause of community-acquired pneumonia in New Zealand and Australia, and it has been identified in several European countries, the United States, and Japan (1–6). Quantitative PCR (qPCR) testing of respiratory samples is substantially more sensitive than current culture methods, but culture of L. longbeachae from sputum is often regarded as the gold standard diagnostic test (7). Culture also offers the opportunity to isolate disease-causing organisms for future study, such as conducting detailed genetics analyses and identifying factors that are important for its virulence/pathogenesis and molecular epidemiology.

Screening of respiratory samples by qPCR testing, followed by acid wash for bacterial decontamination and culture, has been adopted by our clinical laboratory as the preferred strategy for identifying Legionella species and investigating potential cases of Legionnaires’ disease. Despite the high index of suspicion provided by a positive qPCR test result, positive culture rates remain low, at about 40% (8). Previous studies have indicated that confirmation of the qPCR results by culture is uncommon at threshold cycle (CT) values greater than about 30 cycles (9). Possible reasons for the low recovery of viable organisms from respiratory samples include inhibition of growth by the acid wash protocol that is currently used to inhibit contaminating organisms from the respiratory tract and low numbers of viable organisms in samples.

Immunomagnetic separation has been successfully adopted for identifying Legionella pneumophila serogroup 1 from tap water and other environmental samples as well as other microorganisms from clinical samples and foods (10–15). We thought that isolation rates may be improved by eliminating the acid wash step and using decontamination with glycine, vancomycin hydrochloride, polymyxin B sulfate, and cycloheximide (GVPC) together with immunomagnetic separation. It was believed that this method could concentrate L. longbeachae to remove contaminating organisms and potential inhibitors from the sample prior to culture.

(This study was presented in part as a poster at the ESGLI Conference, Lyon, France, 28–30 August 2018.)

MATERIALS AND METHODS

Preparation of Legionella longbeachae antigen.

Wild-type strains of Legionella longbeachae serogroups 1 and 2 (Sg1 and Sg2) from human clinical specimens were acquired (Canterbury Health Laboratories [CHL], Christchurch, New Zealand), and the identity of the organisms was confirmed by culture on BYCE medium and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT system (Bruker Daltonics, Bremen, Germany) after culture on modified Wadowsky and Yee agar (MWY) agar with added BYCE. The MALDI Biotyper version 3.1 and 6903 MSP database were used for species identification.

The organisms were cultured on BCYE agar (Fort Richard Laboratories, Auckland, New Zealand), harvested, suspended in sterile PBS buffer, pH 7.2, and incubated at 60°C for 30 min to kill the bacteria. The final concentration of the bacterial suspension was adjusted to 200 μg/ml in sterile PBS. The purity and identity of the organisms were tested by culture before the killing step and sterility confirmed after killing.

Antibody production.

All animal work was performed at the Christchurch Animal Research Area (CARA) facility, University of Otago Christchurch. This work was approved by the University of Otago Animal Ethics Committee (AEC), approval C8/15C.

Rabbit antiserum against L. longbeachae Sg1 and Sg2 was raised using the method of Vaitukaitis et al. (16). In brief, 6-month-old female New Zealand White rabbits (Oryctolagus cuniculus) housed under specific pathogen-free (SPF) conditions and maintained on a standard commercial diet were immunized. The prime inoculum was 500 μl of the bacterial suspension (200 μg/ml) emulsified with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Four 6-month-old female New Zealand White rabbits (two rabbits per serogroup) were injected subcutaneously with the antigenic emulsion. Booster injections were administered on days 20 and 35, respectively, with incomplete Freund’s adjuvant, with a final injection on day 40 using sterile antigen in PBS buffer without any adjuvants (16). The rabbits were anesthetized and bled by cardiac puncture before being euthanized. The blood was stored at 4°C for 2 h to allow it to clot. The coagulated blood was separated from the serum by centrifugation at 1,500 × g for 5 min.

Purification of rabbit polyclonal antibody.

The antibody was purified by ion-exchange chromatography (DEAE-Sephadex A-50 [Sigma]) with 10 mM phosphate buffer, pH 6.5 (17). The fractions containing the peak anti-L. longbeachae Sg1 and Sg2 fractions were identified using a semiquantitative enzyme-linked immunosorbent assay (ELISA), using a commercially available conjugated goat anti-rabbit IgG horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, PA, USA). Light absorbance (optical density) of the samples was measured on a FLUOstar reader (Hidex, Finland).

Activity and specificity testing of antibody.

The activity and cross-reactivity with other Legionella species and non-Legionella organisms were tested by ELISA (17). The organisms tested were L. pneumophila serogroup 1 (ATCC 33152) and serogroup 4 (NZESR 3001), Legionella micdadei (NZESR 2609), Aspergillus fumigatus (clinical isolate, CHL), and Pseudomonas aeruginosa (ATCC 27853). A. fumigatus was chosen, as this species often overgrows selective medium plates on prolonged incubation required to isolate Legionella species and may survive acid wash. P. aeruginosa was chosen as a Gram-negative species that may contaminate respiratory samples from patients pretreated with antimicrobial agents. The identity and purity of the organisms were confirmed by culture. The organisms were prepared by culture on MWY agar or blood agar plates as appropriate, harvested, diluted in normal saline, and heat killed to make antigenic suspensions. The concentration of antigenic suspensions was adjusted to 10 μg/100 μl. Aliquots of 100 μl of the suspensions were stored at –80°C.

The ELISA of the pooled purified fractions showed that the antibody raised in rabbit A, which had been inoculated with L. longbeachae Sg1 antigens only, reacted with anti-rabbit HRP antibody. Samples from the same fractions were responsive to antigens of both L. longbeachae Sg1 and Sg2. The level of this response was higher against the L. longbeachae serogroup 1 antigens. On ELISA testing, the peak absorbance of Ig fractions was 2.0 against L. longbeachae Sg1 antigens and 1.5 against L. longbeachae Sg2.

There was high specificity for both L. longbeachae Sg1 and L. longbeachae Sg2 and no cross-reactivity between the antibody and any of the non-L. longbeachae organisms tested.

Coupling antibody with magnetic beads.

M-280 tosyl-activated Dynabeads (Invitrogen, Norway) were coupled to the harvested antibody. Coupling buffers were made according to the manufacturers’ instructions, and the antibody was coupled to the beads at a final concentration of 100 μg antibody/5 mg beads. The bound antibodies were separated from unbound antibodies with a magnet and washed with PBS buffer. The mean concentration of unbound antibodies was 0.46 μg/ml, meaning that 66% of the anti-L. longbeachae antibodies were coupled to the beads.

The ability of the antibody-coupled beads to bind L. longbeachae Sg1 cells was demonstrated by incubating them with a bacterial suspension (105 CFU/ml) in PBS and culturing bound cells on selective media. The efficacy of IMS capture of L. longbeachae was assessed by qPCR. The magnetic bead-bound bacteria were washed and suspended in PBS buffer, and DNA on washed beads (200 μl) was extracted from the bound bacteria using the GenElute bacterial genomic DNA kit protocol (Sigma). The binding was assessed by qPCR using Legionella primers and an L. longbeachae-specific probe (18, 19).

The qPCR amplification plot showed a quantitative signal from bacterial suspensions of 106, 104, and 102 CFU/ml with CT values of 20, 25, and 30, respectively.

qPCR testing for Legionella longbeachae.

DNA was extracted from the respiratory samples using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA). The presence of L. longbeachae DNA was detected by qPCR that targeted the ITS region using primers (Integrated DNA Technologies, Inc., IA, USA) and a specific probe (minor groove binder [MGB] 5′-FAM-ACGTGGGTTGCAA-MGB-NFQ-3′ [Applied Biosystems, CA, USA]) as described elsewhere (20). qPCR inhibition controls were used to validate negative results. All qPCR assays were carried out in 96-well plates using the LightCycler 480 real-time platform (Roche Diagnostics GmbH, Mannheim, Germany) using standard protocols (18).

Stored respiratory samples.

Residual portions of 62 respiratory specimens that were processed at CHL and Legionella positive by the genus-specific ssrA qPCR were identified (8). These samples, collected throughout 2017, were stored frozen as 2-ml aliquots at –80°C by CHL without added cryopreservative. Of these, 53 L. longbeachae-positive samples 17 (32%) were culture positive when originally tested. The CT values of the ITS region qPCR testing performed by CHL for species identification were available (8). The organisms identified in these samples were 53 L. longbeachae, 8 L. pneumophila, and 1 L. micdadei organism.

Culture of stored respiratory samples.

The stored sputa were thawed at room temperature, and equal volumes of HCl-KCl acidic buffer and specimen were mixed in 2-ml microtubes. The buffer was made fresh by mixing 39 ml of 0.2 M HCl with 250 ml of 0.2 M KCl (pH 2.2). After 4 min, the sample (100 μl) was streaked onto MWY plates (FortRichard, Auckland, New Zealand) and incubated at 36°C for up to 6 days. Plates were examined visually, and the identity of presumptive Legionella colonies was confirmed by MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany).

GVPC-IMS culture for the isolation of L. longbeachae from respiratory samples.

To reduce the contamination rate of respiratory samples, an equal volume of GVPC (Legionella selective supplement; Sigma-Aldrich) supplement dissolved in sterile ultrapure water was added to the samples and incubated at room temperature for 4 min. Dynabeads (50 μl) coated with anti-L. longbeachae antibody were added to 1 ml of each GVPC-treated sample in a microcentrifuge tube. The sample was incubated at room temperature with gentle agitation for 2 h and placed on a magnetic stand for 2 min to allow the magnetic beads to be pulled from the solution. The bead-bacterium complexes were washed twice with 1 ml of sterile ultrapure water (pH 7.0). The final sample was resuspended in 1 ml GVPC suspension and 100 μl inoculated onto an MWY plate, and the sample was incubated at 36°C aerobically for 3 to 7 days. Legionella-like colonies were examined under a stereomicroscope and identity confirmed by MALDI-TOF MS. Contaminant species were not identified further.

Pneumonia severity scores.

The clinical severity of community-acquired pneumonia was classified by CURB-65 scores (21). The severity of pneumonia was defined as a CURB-65 score: 0 to 1 was mild (mortality, 1.5%), 2 was moderate (mortality, 9.2%), and 3 to 5 was severe (mortality, 22%).

Statistical analysis.

Statistical analyses were carried out using R statistical software (R Core Team, 2019, Vienna). Culture positivity following acid wash versus IMS was compared using the McNemar's chi-squared test with continuity correction with and without stratification for CURB-score. Mean ITS qPCR CT values were compared using t tests. Linear regression was used to test the relationship between the CT values and CURB-65 scores of patients.

RESULTS

Culture results after acid wash of stored sputum specimens.

After acid wash, 10/53 (19%) samples were L. longbeachae culture positive. After decontamination and GVPC-IMS, 26/53 (49%) were culture positive, including all those that were positive after acid wash. There was strong evidence that GVPC-IMS decontamination produced a higher proportion of culture-positive samples than acid wash (McNemar’s chi-squared test with continuity correction, P < 0.001). There was no significant difference between the number of samples that were culture positive on the fresh samples and after GVPC-IMS treatment (P = 0.08 by Mid P exact test). No formal plate counts were done. Photographs of one sample processed after direct plating, pretreatment with acid wash, and IMS-GVPC on MWY selective agar are shown as an example in Fig. 1.

FIG 1.

FIG 1

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Representative culture recovery comparison of an untreated (left), acid-washed (middle), and IMS-GVPC (right)-treated cultures using different methods. All were plated on MWY selective agar.

qPCR and culture.

The mean qPCR CT values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7, 95% CI ±2.48; P = 0.16). After acid wash, the proportion of culture-positive samples reduced as the CT value increased (Fig. 2). There was a similar but less marked trend in the proportion that was culture positive following the GVPC-IMS procedure. In the band with a qPCR CT value of 38+, all 10 were culture negative with acid wash and 5/10 (50%) were positive after GVPC-IMS.

FIG 2.

FIG 2

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Plot of proportion of stored sputum samples that were culture positive after acid wash and IMS at various bands of CT values for the 53 qPCR-positive samples. AW, acid wash; IMS, immunomagnetic separation.

All 9 stored samples that were originally found to have L. pneumophila or L. micdadei DNA on ssrA qPCR were culture and ITS qPCR negative after GVPC-IMS treatment.

Severity of community-acquired pneumonia in patients and CT values of qPCR of L. longbeachae.

The CURB-65 severity scores for community-acquired pneumonia were available for 45 of the 62 patients from whom sputum samples had been stored. Of these, 8 (18%) had positive cultures after acid wash and 24 (53%) after GVPC-IMS. The proportion of positive cultures after acid wash and IMS, respectively, for mild pneumonia were 3/21 (14%) and 12/21 (57%) (acid wash versus GVPC-IMS, P = 0.008), for moderate pneumonia were 2/15 (13%) and 6/15 (40%) (P = 0.14), and for severe pneumonia were 3/10 (30%) and 6/10 (60%) (P = 0.25).

There was evidence of an inverse relationship between patients’ CURB-65 scores and the CT value of their samples (P = 0.001 by analysis of variance [ANOVA]). Mean CT values decreased as CURB scores increased, although evidence of a difference between consecutive CURB scores was only present for a score of 4 versus 3 (mean difference, −7.8; 95% CI, −15.2 to −0.4; P = 0.034) (Fig. 3).

FIG 3.

FIG 3

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Relationship between the CT values of ITS qPCR and the severity of the disease. Score 0 indicates the mildest level of pneumonia, while a score of 4 indicates the most severe in the cohort of patients that provided the samples for this assessment. Dots present individual CT values, and horizontal lines represent the mean and shaded boxes show the 95% confidence interval of each group.

DISCUSSION

In this study, we found strong evidence that L. longbeachae was more frequently cultured from stored sputum samples following preparation with antimicrobial decontamination (GVPC) and IMS separation than after acid wash. The culture plates with both methods were essentially free from contaminating organisms, making identification of Legionella colonies easy. No formal counts of contaminating bacteria were done, but there were some organisms of different colony types on the plates after 3 days of incubation, including occasional fungal species. These organisms were rarely seen after GVPC-IMS preparation.

There was some evidence that IMS improved the sensitivity of culture in samples with a high CT value, and a lower load of organisms, than after acid wash, as the median CT values were lower in the culture-negative samples than culture-positive samples but not after GVPC-IMS. There was also a trend for the proportion of culture-positive specimens to reduce as the CT values increased after acid wash, but this was less marked after GVPC-IMS separation. The most likely explanation is that viable organisms were concentrated by the IMS procedure and were missed after acid wash, as only a small portion of the acid-washed samples was cultured. The improved recovery after GVPC-IMS is unlikely to represent contamination, as the samples were run as a batch, the negative controls remained culture negative, and there was no evidence of carryover from culture-positive samples.

The data from the CURB-65 severity scores are consistent with this finding and suggest that GVPC-IMS is of particular value in culturing organisms in milder disease when there are likely to be lower numbers of organisms present in the samples (8). These results have important implications for defining the range of clinical severity of Legionnaires’ disease (LD), which is often considered primarily as a severe form of pneumonia with a mortality of 8 to 10% (7, 22). Improved sensitivity of culture would help define the epidemiology of LD and potentially improve the public health response to this infection. The recovery of organisms from the extremes of disease severity may also facilitate investigation of the pathogenic factors of organisms in these disease phenotypes.

The tolerance of L. longbeachae to the inhibitory effects of acid as a pretreatment has not been well studied. The acid wash protocol used here follows that used by others and developed in our local clinical laboratory for the isolation of all Legionella species (23). It is possible the short-term exposure to acid used in this study (4 min) reduces the viability of L. longbeachae at low inocula. We have not been able to compare the susceptibility of L. longbeachae in respiratory samples to acid pretreatment, but others have found that the recovery rate of other Legionella bacteria (L. pneumophila serogroups 1 to 14 and L. micdadei) from seeded sterile water samples was reduced after acid treatment compared with that after filtration and heat treatment (24–26). Further studies on fresh clinical isolates are needed to determine precisely how sensitive L. longbeachae is to acid conditions and the critical timing of acid exposure.

The GVPC-IMS method may offer other advantages over acid wash treatment. The GVPC-IMS procedure may remove more commensal organisms from the sputum matrix, making it easier to identify sparse colonies of L. longbeachae, although we did not quantitate this in our study. The GVPC-IMS procedure may also remove inhibitory compounds released by contaminating organisms in the sample. While there is little information on the inhibitory effects of common upper respiratory organisms on the growth of Legionella species, organisms such as Staphylococcus warneri may release bacteriocins that inhibit L. longbeachae ATCC 33484 (27). This raises the possibility that biologically active compounds produced by organisms found in the upper respiratory tract that contaminate respiratory samples have an inhibitory effect on the growth of Legionella species.

We are unaware of any possible unfavorable effects of capture of L. longbeachae by paramagnetic beads. Previous work has shown that the attachment and capture of other bacteria, such as Escherichia coli, Staphylococcus aureus, and Yersinia enterocolitica, to the paramagnetic beads has no unfavorable effect on their growth (14, 28–30). It is possible that the bacterial load is underestimated by colony counts. Steric hindrance and spatial limitations during the attachment of the antibodies on the surface of the beads to the surface of bacterial cells are supposedly random, but one bead may get attached to several organisms. This will produce only one colony when cultured on a plate and may lead to an underestimate of the number of viable organisms present in a given sample.

There was an apparent decline in the proportion of culture-positive results after acid wash in the stored respiratory samples (–80°C without cryopreservative) compared with the initial cultures when fresh. These results should not be compared directly, as they were done in different laboratories, with different preparations of reagents, and by different laboratory staff. Nevertheless, it is possible that there was a loss of viability of some, as has been observed in other species, and the viability of Legionella species with various methods of cryopreservation should be studied further (31).

There are significant limitations in this study. First, the process only detects L. longbeachae as currently configured. It is unclear whether L. longbeachae sg2 would be detected with the current antibody, as this organism was not present in our stored samples. This is not critical, as this organism is currently identified in about 1% of cases and our laboratory-prescreened respiratory samples with a genus-specific qPCR test. Detection of other species would require other species-specific antibodies. Second, the number of samples was limited and had been stored rather than run on fresh samples. Finally, we could not compare recovery using GVPC and IMS alone because of the small aliquots of samples that were available. Some preliminary studies indicated that fungal elements tended to separate from the matrix with bacteria IMS complex and contaminate the plates. This was not a problem after GVPC-IMS separation of these clinical samples but may be relevant in samples from patients with chronic obstructive airway disease who are at risk from LD (22). A prospective study on fresh samples is needed to determine the value of this technique in the clinical setting. It would be useful to include a control group that was cultured on Legionella selective media without pretreatment with acid wash or GVPC-IMS.

In conclusion, the recovery of viable L. longbeachae bacteria on culture from the stored respiratory specimens was improved by the GVPC-IMS procedure, including samples with a higher CT result and lower load of organisms.

The GVPC-IMS technique needs to be tested prospectively in a clinical laboratory with fresh specimens but could eliminate the use of acid wash. The GVPC-IMS method is amenable to automation in diagnostic laboratories with high-volume workloads if monoclonal antibodies were available and could be applied to other Legionella species for isolation of organisms from both clinical and environmental samples.

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