Bacteremia in an Immunocompromised Patient Caused by a Commensal Neisseria meningitidis Strain Harboring the Capsule Null Locus (cnl)

We recently described the capsule null locus (cnl) of constitutivelyunencapsulated Neisseria meningitidis clonal lineages. cnl meningococciwere recovered from healthy carriers at high frequency. We herereport on the first case of invasive disease caused by cnl meningococciin a severely immunosuppressed patient with chronic graft-versus-hostdisease after allogeneic peripheral blood stem cell transplantation.The sequence type 845 strain was extensively typed and, furthermore,shown to be sensitive to serum bactericidal activity.

INTRODUCTION

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Introduction

Neisseria meningitidis is a leading cause of sepsis and meningitisin toddlers and adolescents (16). The organism also is partof the human nasopharyngeal flora. Population biology studiesrevealed that clonal lineages responsible for most cases ofinvasive disease are rarely encountered in healthy carriers(1, 8). The capsular polysaccharide is the dominant virulencefactor (24). Invasive disease is usually caused by the serogroupsA, B, C, W-135, and Y. Other serogroups, e.g., 29E, X, and Z,are mostly found among healthy carriers. We recently demonstratedthat a significant proportion (i.e., 16.4%) of strains in healthycarriers in Bavaria, Germany, lack the genes for capsule synthesis,modification, and transport (2). Instead, the organization ofthe chromosomal locus harboring those genes resembled that ofconstitutively unencapsulated neisserial species such as N.gonorrhoeae and N. lactamica. Meningococci harboring the capsulenull locus (cnl) were found to be highly specific to few clonallineages. Those lineages might therefore be considered truecommensals.

In most cases of meningococcal disease, there is no obviousunderlying immunological disorder. Nevertheless, immunodeficiencymay render the host susceptible to disease. Deficiencies inthe terminal complex and the factor properdin of the complementsystem (3, 5), as well as functional or anatomic asplenia, havebeen described as a risk factors for meningococcal disease (13).There are case reports of patients with Waldenstrom's disease(18), AIDS (11), and hypogammaglobinemia (17) suffering frommeningococcal infection. In a 5-year population-based analysisof sporadic meningococcal disease in adults in Atlanta, Ga.,an area with low incidence during the study period, Stephenset al. found that two-thirds of those aged 24 years or oldershowed one of a variety of disorders, including human immunodeficiencyvirus infection, corticosteroid treatment, diabetes, chronicrenal failure, and bone marrow transplantation (19). It is unclearwhether these findings also hold true for regions with a highincidence. Nevertheless, it was interesting that meningococcalisolates from 6 of the 44 adult patients did not belong to serogroupB, C, W-135, or Y. In line with this observation, serogroup29E meningococci have once been reported to cause meningococcaldisease in a patient with multiple myeloma, suggesting thatotherwise purely commensal meningococci can be responsible foropportunistic infection in the immunocompromised host (26).Until now, cnl meningococci to our knowledge have not been describedas a cause of disease. We here report the first case of cnlmeningococcal bacteremia in a severely immunocompromised patient.This is another example of unusual organisms causing invasiveinfections in this cohort of patients (4, 20).

CASE REPORT

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Case report

In January 2001, a 42-year-old patient at the University HospitalUlm was diagnosed with Philadelphia chromosome-positive (bcr-abl⁺)common acute lymphatic leukemia with a delayed response to chemotherapy.A peripheral blood stem cell transplantation from an HLA-identicalsibling was performed (in April 2001) after a myeloablativeconditioning regimen. A second HLA-identical sibling peripheralblood stem cell transplantation without T-cell depletion wasperformed in February 2003 due to a relapse. The clinical coursewas complicated by chronic graft-versus-host disease.

In September 2003, the patient was readmitted to the hospitaldue to chronic progressive diarrhea. The immunosuppression wasintensified. Mucositis was not observed. No infectious causefor enteritis could be identified. During hospitalization, clinicalsigns of sepsis developed, with acute fever (39°C) and chills,which were associated neither with a rash nor with petechiae.The white blood cell count was 6.7/nl. C-reactive protein (34.8mg/liter) and interleukin-8 (3,279 U/liter) levels were increased.Blood cultures were taken, and an empirical antibacterial therapywas initiated (3 g of piperacillin-combactam three times a dayintravenously). The clinical symptoms of sepsis disappeareda few hours after initiation of the antibacterial therapy. Aftermicrobiological diagnosis of a penicillin-sensitive meningococcalbacteremia, the antibacterial therapy was continued with penicillinfor the following 4 weeks.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains Meningococci were isolated from the patient by the Instituteof Microbiology and Immunology, Ulm, Germany, and were referredto the National Reference Center for Meningococci, Würzburg,Germany. The strain grew on Martin-Lewis agar, was oxidase positive,and was identified as N. meningitidis by the API NH system (BioMerieux,Marcy-Etoile, France). The serogroup B strain MC58 was isolated1985 in the United Kingdom and belongs to the sequence type32 (ST-32) complex. The unencapsulated derivative of strainMC58 was described recently (14).

Meningococcal typing Multilocus sequence typing was performed as described previously(http://pubmlst.org/neisseria/) (9). Polysialyltransferase geneswere analyzed by PCR as described recently (22). The capsulenull locus (cnl) was identified by PCR and sequencing as describedpreviously (2). PorA genotyping was performed by using the PorAdatabase (http://neisseria.org/nm/typing/pora/). The FetA databasewas used for FetA typing (http://neisseria.org/nm/typing/feta/)(21). The cnl allele was determined as described recently (2).The presence of the lipopolysaccharide (LPS) immunotype L3,7,9was confirmed by enzyme-linked immunosorbent assay (ELISA) withthe monoclonal antibody 9-2-L379 (supplied by W. D. Zollinger).The ability of meningococci to exogenously sialylate the LPSwas confirmed by ELISA as described previously, using monoclonalantibody 3F11, whose binding is blocked by terminal sialylationof the lacto-N-neotetraose structure of the LPS (10). ExogenousLPS sialylation was also confirmed by Tricine gel electrophoresisas described previously (23).

Serum killing assay The serum killing assay was performed with normal human serum(NHS). NHS was obtained from a healthy volunteer with no historyof meningococcal infection, gonorrhea, or meningococcal vaccination.The donor was selected because the serum did not kill the encapsulatedstrain MC58 (ST-32 complex) but did kill the unencapsulatedvariant thereof and therefore matched the criteria previouslyapplied to pooled sera (23). The killing assay was performedas described previously for strain MC58, yielding results comparableto the published data (23). Meningococci were grown in the presenceor absence of CMP-N-acetylneuraminic acid (CMP-NANA) (Sigma,Taufkirchen, Germany) as described previously (25). CMP-NANAis essential for exogenous sialylation of neisserial LPS, whichcontributes to gonococcal serum resistance (6, 15). NHS wasapplied for 30 min in various concentrations. The bactericidalassays were performed two (in the case of MC58) or three (inthe case of DE9536) times, yielding comparable results. Extensiveclumping of strain DE9536 was observed, giving rise to slightlylower colony counts in untreated bacteria than for MC58 derivativesdespite of the use of identical optical densities of bacterialsuspensions.

RESULTS

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Results

A meningococcal isolate was recovered from two different bloodcultures from the patient. The meningococcal strain (isolateDE9536) was sent to the National Reference Laboratory for Meningococcifor fine typing. The isolate did not react with monoclonal antibodiesdirected against the serogroup A, B, C, W-135, and Y capsulesin ELISA and slide agglutination. PCR analysis demonstratedthat there was no evidence for polysialyltransferase genes (siaD)specific to serogroups B, C, W-135, and Y. However, geneticanalysis performed as described recently (2) revealed a lackof genes needed for capsule synthesis and transport. Furthermore,the isolate was shown by multilocus sequence typing to be ST-845,which has been shown to comprise exclusively cnl meningococci(2). The cnl allele (cnl-2) of the patient's isolate correspondedto the one found in the ST-845 complex. Furthermore, the outermembrane protein porin A of the isolate was typed as P1.18,25.This PorA type was predominantly found in ST-845 strains fromthe Bavarian meningococcal carriage strain collection (our unpublishedobservations). The FetA allele was 5-2, and the isolate expressedan LPS that was reactive with an L379 antibody. The isolatewas sensitive to the killing activity of a human serum obtainedfrom a healthy donor with no history of meningococcal or gonococcaldisease or meningococcal vaccination (Fig. 1). In contrast,a virulent meningococcal serogroup B isolate was serum resistant.Addition of CMP-NANA to the growth medium did not significantlyenhance the serum resistance of the patient's isolate, althoughthe isolate's LPS could be sialylated by the addition of exogenousCMP-NANA as evidenced by 3F11 ELISA and Tricine gel electrophoresis.This finding indicated that exogenous sialylation of LPS didnot render the strain serum resistant, in contrast to the casefor gonococcal strains (15). In order to test whether othercnl isolates were as serum sensitive as DE9536 even after growthin the presence of CMP-NANA, we also tested an ST-53 isolateand an ST-845 isolate from healthy carriers. Both isolates yieldeda rate of survival in the bactericidal assay which was comparableto that of DE9536 (data not shown).

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FIG. 1. Serum killing assay with NHS. NHS was applied to meningococci for 30 min in various concentrations. The serogroup B strain MC58 and its unencapsulated derivative MC58 siaD mut. were used as controls in addition to the test strain DE9536. Strains were tested after growth without or with an exogenous supply of CMP-NANA. The bactericidal assays were performed two (in the case of MC58) or three (in the case of DE9536) times. Mean values of log₁₀ CFU per milliliter are shown. Vertical bars represent the standard errors of the means, which were calculated for values derived from DE9536. The limit of detection was 1.

DISCUSSION

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Discussion

We report here the case of a severely immunocompromised patientwith septicemia due to a constitutively unencapsulated meningococcalisolate. cnl meningococci frequently colonize the nasopharynxof the human host. Disturbance of the nasopharyngeal barriermay provide access to the bloodstream for oral and nasopharyngealbacteria. The patient did not exhibit nasopharyngeal mucositisbut did show signs of a sicca syndrome caused by chronic graft-versus-hostdisease, which may have weakened the barrier functions of themucosa. Unencapsulated meningococci lack resistance to opsonophagocytosis,for which the capsule is a prerequisite (24). We show here thatthe patient's isolate (DE9536) was even more sensitive to thekilling activity of NHS than genetically engineered unencapsulatedderivatives of a pathogenic serogroup B strain. In severelyimmunocompromised hosts, however, a systemic proliferation ofunencapsulated cnl meningococcal strains may occur, as was shownin this patient. Bacteremia with the cnl meningococcal strainwas associated with a benign clinical course without signs ofmeningitis and a rapid clinical response after initiation ofantimicrobial therapy, which may support the less virulent natureof this strain.

Vaccination of bone marrow transplant recipients against meningococcaldisease has been considered (12). It should be noted that thecase reported here could not have been prevented by prophylacticimmunization with polyvalent polysaccharide vaccines due tothe lack of a capsule.

Meningococcal disease may give rise to concerns about transmissionto health care workers or other patients (7). However, in severelyimmunocompromised patients meningococcal disease may be causedby apathogenic isolates, as demonstrated here and by others(19, 26). Rapid detection of the cnl locus and of serogroupsother than A, B, C, W-135, and Y by specialized laboratoriescould reduce anxiety among health care workers and avoid unnecessaryantibiotic prophylaxis among persons in close contact with thepatient.

In conclusion, we report the first case of invasive diseasedue to cnl meningococci. This report provides further evidencethat meningococcal disease in severely immunocompromised patientsmay well be considered opportunistic in some cases.

ACKNOWLEDGMENTS

We gratefully acknowledge the expert technical assistance ofChristine Meinhardt, Gabi Heinze, and Carmen Roldan. This publicationmade use of the Neisseria Multi Locus Sequence Typing website(http://pubmlst.org/neisseria/) developed by Man-Suen Chan andKeith Jolley and sited at the University of Oxford (curator,Keith Jolley). We furthermore made use of the N. meningitidisporA and fetA typing websites (http://neisseria.org/nm/typing/),which were developed by Keith Jolley (curators, Janet Sukerand Ian Feavers, respectively).

The National Reference Laboratory for Meningococci is supportedby the Robert Koch-Institute (Berlin, Germany). The developmentof the Neisseria Multi Locus Sequence Typing website site hasbeen funded by the Wellcome Trust and European Commission.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany. Phone: 49(931)20146802. Fax: 49(931)20146445. E-mail: uvogel{at}hygiene.uni-wuerzburg.de.

REFERENCES

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