Rifampin-resistant meningococcal disease.

Rifampin-resistant meningococcal disease occurred in a child who had completed rifampin chemoprophylaxis for exposure to a sibling with meningococcemia. Susceptibility testing of 331 case isolates found only 1 other case of rifampin-resistant disease in Minnesota, USA, during 11 years of statewide surveillance. Point mutations in the RNA polymerase [beta] subunit (rpoB) gene were found in isolates from each rifampin-resistant case-patient.

**********

Chemoprophylaxis is recommended for close contacts of persons with invasive meningococcal disease to prevent secondary cases. In the 1960s, rifampin replaced sulfonamides as the recommended agent for chemoprophylaxis of household members and other close contacts of persons with invasive meningococcal disease when sulfonamide-resistant meningococci became common (1). In recent years, ciprofloxacin and ceftriaxone have been established as acceptable alternatives to rifampin for prophylaxis of meningococcal disease. However, rifampin remains a popular choice due to its low cost, ease of administration, and well-established record among infants and children.

Pharyngeal colonization with rifampin-resistant meningococci following chemoprophylaxis with rifampin of persons exposed to meningococcal disease was documented soon after treatment was initiated (2) and has continued to be observed over time (3). However, although rifampin has been used routinely worldwide for more than 30 years, few cases of rifampin-resistant meningococcal isolates in cases of invasive disease have been reported (4-7), and reports of only 3 instances in the United States could be found (8-10).

Rifampin targets the [beta] subunit of DNA-directed RNA polymerase by inhibiting extension of the RNA strand. The [beta] subunit is encoded by the rpoB gene. Previous studies have demonstrated that one of the mechanisms of rifampin resistance in Neisseria meningitidis is associated with single point mutations of the rpoB gene that result in amino acid substitutions (11-13). The data presented in this study confirm the rapid development of rifampin resistance upon exposure of meningococci to rifampin as a result of point mutations in the rpoB gene.

The Study

Cases of invasive meningococcal disease in Minnesota residents are required to be reported to the Minnesota Department of Health (MDH). Laboratories throughout the state routinely submit isolates from patients with this disease to the MDH Public Health Laboratory, where they are serogrouped by slide agglutination (Difco, Detroit, MI, USA). In 1995, the MDH began routinely testing antimicrobial susceptibilities on meningococcal isolates and retrospectively conducted susceptibility testing on all available meningococcal isolates that had been submitted since 1993.

Antimicrobial susceptibilities were determined by using broth microdilution. Panels contained cation-adjusted Mueller-Hinton broth with 2%-5% lysed horse blood (PML Microbiologicals, Wilsonville, OR, USA) and were incubated at 35[degrees]C in C[O.sub.2] for 20-24 h. An Etest (AB Biodisk, Solna, Sweden) was also used for isolates that demonstrated resistance to further quantify degree of resistance. MIC breakpoints have recently been established by the Clinical and Laboratory Standards institute for N. meningitidis (14). An MIC [greater than or equal to] 2 [micro]g/mL is considered resistant to rifampin.

Molecular subtyping of the sibling isolates was done by pulsed-field gel electrophoresis (PFGE) as described previously (15). The rpoB genes from rifampin-resistant and rifampin-sensitive isolates (Table 1) were amplified by polymerase chain reaction and sequenced by using primers described previously (13). DNA and peptide sequences were analyzed with BioNumerics (Applied Maths, Austin, TX, USA) and Vector NTI Suite (InforMax, North Bethesda, MD, USA).

The first known case of rifampin-resistant invasive meningococcal disease in Minnesota occurred in 1996. A 5-month-old infant had a clinical syndrome consistent with meningococcemia. He was hospitalized for 10 days, received antimicrobial drug therapy, and survived. By Etest, his serogroup B N. meningitidis isolate had a rifampin MIC [greater than or equal to] 32 [micro]g/mL. This was a sporadic case with no apparent links to any other previous or subsequent cases.

In 2002, fever, vomiting, and irritability developed in a 2-month-old infant, followed 12 hours later by labored breathing and a generalized rash. She was taken to a clinic where she experienced cardiac arrest and underwent cardiopulmonary resuscitation. She was transferred to a nearby emergency room where she died [approximately equal to] 1 hour later. Meningococcemia was suspected and household members were given prescriptions for rifampin. Waterhouse-Friderichsen syndrome was noted on autopsy, and N. meningitidis was isolated from a swab of brain tissue. Three days after the death of the case-patient and 1 day after completing a 2-day course of rifampin, a fever and lethargy developed in the case-patient's 6-year-old sister. Blood cultures were obtained and she was hospitalized, given antimicrobial drug treatment (ceftriaxone), and observed. No cerebrospinal fluid was collected. Blood cultures were subsequently positive for N. meningitidis. She responded to ceftriaxone and continued treatment as an outpatient after a short hospitalization. Household contacts, along with other close contacts of the 6-year-old girl, again received chemoprophylaxis. It was recommended that adults be treated with ciprofloxacin and children be treated with ceftriaxone because of concerns that 1 or both siblings could have had rifampin-resistant meningococcal infections. No additional related cases were identified over the following weeks.

Isolates from both siblings were identified as serogroup C. The PFGE patterns were indistinguishable and had, in fact, the most common PFGE pattern seen for that serogroup in Minnesota. Antimicrobial susceptibility testing showed that the isolate from the case-patient was susceptible to ceftriaxone, penicillin, chloramphenicol, ciprofloxacin, and rifampin. The MIC for rifampin was 0.008 [micro]g/mL. The isolate from the 6-year-old patient was susceptible to the same drugs, except for rifampin, which had an MIC >1 [micro]g/mL by broth microdilution and an MIC >32 [micro]g/mL by Etest.

A comparison of the nucleotide sequence of the rpoB gene of both sibling isolates showed they were identical except for a single nucleotide change. This change resulted in a substitution of serine for phenylalanine at amino acid position 548. This substitution has previously been associated with rifampin resistance in N. meningitidis (12).

The PFGE subtype of the isolate from the rifampinresistant case in 1996 differed from that of the siblings' isolates. Sequencing of the rpoB gene from this isolate showed an amino acid substitution of histidine for tyrosine at position 552. This substitution has also been previously associated with rifampin resistance in JV. meningitidis (Table 1; MDH97-498) (11,13).

Susceptibility results on meningococcal isolates from 1993 to 2003 for other antimicrobial agents are shown in Table 2. Using the newly established breakpoints, we observed that 92% (303/331) of the isolates were susceptible to penicillin, 100% (205/205) were susceptible to ceftriaxone, 100% (331/331) were susceptible to meropenem, 100% (205/205) were susceptible to ciprofloxacin, 100% (331/331) were susceptible to chloramphenicol, and 48% (158/331) were susceptible to trimethoprim-sulfamethoxazole.

Conclusions

Primary cases of rifampin-resistant meningococcal disease are rare. While more common, secondary cases with rifampin resistance can develop following chemoprophylaxis with rifampin. All N. meningitidis isolates tested at MDH were susceptible to ceftriaxone and ciprofloxacin. Ceftriaxone must be given parenterally but is the recommended prophylactic agent for infected pregnant women. According to the 2003 American Academy of Pediatrics Report of the Committee on Infectious Diseases, ciprofloxacin may be used by persons >15 years of age. While few instances of ciprofloxacin resistance have been reported, its widespread use may result in greater resistance in N. meningitidis (as has occurred in related pathogens such as Neisseria gonorrhoeae) (16,17). Persons receiving chemoprophylaxis should be advised about the potential of meningococcal disease developing, even though they have taken antimicrobial agents as prescribed. If a close contact who has been treated with rifampin becomes ill with meningococcal disease, alternative antimicrobial agents should be used for prophylaxis until rifampin sensitivity of the secondary infection can be established. Although rifampin-resistant meningococcal disease is still rare after 30 years of using rifampin for chemoprophylaxis and ciprofloxacin resistance has rarely been observed, susceptibilities to chemoprophylactic agents should be monitored to ensure that recommendations are sufficiently effective to minimize the occurrence of secondary cases.

Table 1. Rifampin phenotype and genotype
of Neisseria meningitidis isolates

Strain                   Description

MDH02-2342   Sporadic rifampin-susceptible
               serogroup C case isolate
MDH02-2271   Sporadic rifampin-susceptible
               serogroup B case isolate
MDH97-498    Isolate from sporadic rifampin-
               resistant serogroup B case in 1996
MDH02-2398   First sibling's isolate: rifampin
               susceptible, serogroup C
MDH02-2408   Second sibling's isolate: rifampin
               resistant, serogroup C

                  Rifampin MIC
Strain            ([micro]g/mL)        Amino acid change *

MDH02-2342      0.004 ([dagger])            None (WT)
MDH02-2271      <0.002 ([dagger])           None (WT)
MDH97-498        >4, ([dagger])         [His.sub.552]Tyr
              >32 ([double dagger])        ([section])
MDH02-2398      0.008 ([dagger])            None (WT)
MDH02-2408       >1, ([dagger])         [Ser.sub.548]Phe
              >32 ([double dagger])        ([section])

* WT, wildtype.

([dagger]) Determined by broth microdilution.

([double dagger]) Determined by Etest.

([section]) Numbering based on the entire N.
meningitidis rpoB gene (GenBank accession no.
Z54353). Accession numbers of isolate sequences
submitted to GenBank: MDH02-2342 (AY746965),
MDH02-2271 (AY746964), MDH97-498 (AY746963),
MDH02-2398 (AY746966), MDH02-2408 (AY746967).

Table 2. Antimicrobial drug susceptibilities for
meningococcal invasive disease Neisseria meningitidis
isolates, Minnesota, USA, 1993-2003 *

Antimicrobial drug
susceptibility
([section])               1996      1997       1998      1999

Rifampin                    97       100       100        100
Ceftriaxone                 NA        NA       100        100
Ciprofloxacin               NA        NA       100        100
Chloramphenicol            100       100       100        100
Meropenem                  100       100       100        100
Penicillin                  89        92        83         96
Trimethoprim-               62        32        42         29
sulfa methoxazole
No. of cases                40        40        36         56
No. of isolates tested   37 (93)   38 (95)   36 (100)   55 (98)
(%)

Antimicrobial drug
susceptibility
([section])               1997       1998      1999       2000

Rifampin                   100       100        100       100
Ceftriaxone                 NA       100        100       100
Ciprofloxacin               NA       100        100       100
Chloramphenicol            100       100        100       100
Meropenem                  100       100        100       100
Penicillin                  92        83        96         95
Trimethoprim-               32        42        29         36
sulfa methoxazole
No. of cases                40        36        56         22
No. of isolates tested   38 (95)   36 (100)   55 (98)   22 (100)
(%)

Antimicrobial drug
susceptibility
([section])                2001       2002       2003

Rifampin                   100         97        100
Ceftriaxone                100        100        100
Ciprofloxacin              100        100        100
Chloramphenicol            100        100        100
Meropenem                  100        100        100
Penicillin                  96         86         93
Trimethoprim-               52         58         66
sulfa methoxazole
No. of cases                27         36         29
No. of isolates tested   27 (100)   36 (100)   29 (100)
(%)

* NA, not available.

([dagger]) Values for antimicrobial drugs are % of
isolates susceptible by broth microdilution.

Acknowledgments

We thank the hospital infection control practitioners and local public health agencies of Minnesota for their cooperation in reporting cases; the microbiology laboratory personnel for submitting isolates to the Minnesota Department of Health; Richard Danila and Harry Hull for thoughtfully reviewing this manuscript; Nancy Rosenstein for guidance; and James Jorgenson for input on recommended breakpoints for N. meningitidis susceptibility.

This work was supported by the US Centers for Disease Control and Prevention Emerging Infections Program Cooperative Agreement U50/CCU511190-09.

References

(1.) Deal WB, Sanders E. Efficacy of rifampin in treatment of meningococcal carriers. N Engl J Med. 1969;281:641-5.

(2.) Weidmer CE, Dunkel TB, Pettyjohn FS, Smith CD, Leibovitz A. Effectiveness of rifampin in eradicating the meningococcal carrier state in a relatively closed population: emergence of resistant strains. J Infect Dis. 1971;124:172-8.

(3.) Jackson LA, Alexander ER, Debolt CA, Swenson PD, Boase J, McDowell MG, et al. Evaluation of the use of mass chemoprophylaxis during a school outbreak of enzyme type 5 serogroup B meningococcal disease. Pediatr Infect Dis J. 1996;15:992-8.

(4.) Cooke RPD, Riordan T, Jones DM, Painter MJ. Secondary cases of meningococcal infection among close family and household contacts in England and Wales, 1984-7. BMJ. 1989;298:555-8.

(5.) Yagupsky P, Ashkenazi S, Block C. Rifampicin-resistant meningococci causing invasive disease and failure of chemoprophylaxis. Lancet. 1993;341:1152-3.

(6.) Almog R, Block C, Gdalevich M, Lev B, Wiener M, Ashkenazi S. First recorded outbreaks of meningococcal disease in the Israel Defence Force: three clusters due to serogroup C and the emergence of resistance to rifampicin. Infection. 1994;22:69-71.

(7.) Dawson SJ, Fey RE, McNulty CA. Meningococcal disease in siblings caused by rifampicin sensitive and rifampicin resistant strains. Commun Dis Public Health. 1999;2:215-6.

(8.) Cooper ER, Ellison RT, Smith GS, Blaser MJ, Reller LB, Paisley JW. Rifampin-resistant meningococcal disease in a contact patient given prophylactic ritampin. J Pediatr. 1986;108:93-6.

(9.) Berkey P, Rolston K, Zukiwski A, Gooch G, Bodey GE Rifampin-resistant meningococcal infection in a patient given rifampin chemoprophylaxis. Am J Infect Control. 1988;16:250-2.

(10.) Levy DI, del Rio C, Stephens DS. Meningococcemia in identical twins: changes in serum susceptibility after rifampin chemoprophylaxis. J Infect Dis. 1988;157:1064-8.

(11.) Carter PE, Abadi FJR, Yakubu DE, Pennington TH. Molecular characterization of rifampin-resistant Neisseria meningitidis. Antimicrob Agents Chemother. 1994;38:1256-61.

(12.) Nolte O. Rifampicin resistance in Neisseria meningitidis: evidence from a study of sibling strains, description of new mutations and notes on population genetics. J Antimicrob Chemother. 1997;39:747-55.

(13.) Stefanelli P, Fazio C, La Rosa G, Marianelli C, Muscillo M, Mastrantonio P. Rifampicin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of the strains. J Antimicrob Chemother. 2001; 47:219-22.

(14.) National Committee for Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 15th informational supplement. CLSI/NCCLS document M100-S15. Wayne (PA): CLSI; 2005.

(15.) Popovic T, Schmink S, Rosenstein NA, Ajello GW, Reeves MW, Plikaytis B, et al. Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C. J Clin Microbiol. 2001;39:75-85.

(16.) Schultz TR, Tapsall JW, White PA, Newton PJ. An invasive isolate of Neisseria meningitidis showing decreased susceptibility to quinolones. Antimicrob Agents Chemother. 2000;44:1116.

(17.) Alcala B, Salcedo C, de la Fuente L, Arreaza L, Uria MJ, Abad R, et al. Neisseria meningitidis showing decreased susceptibility to ciprofloxacin: first report in Spain. J Antimicrob Chemother. 2004;53:409.

Jean Rainbow, * Elizabeth Cebelinski, * Joanne Bartkus, * Anita Glennen, * Dave Boxrud, * and Ruth Lynfield *

* Minnesota Department of Health, Minneapolis, Minnesota, USA

Ms. Rainbow is a surveillance officer for the Centers for Disease Control and Prevention Emerging Infections Program Active Bacterial Core Surveillance at the Minnesota Department of Health. Her research interests include the epidemiology of invasive bacterial diseases and surveillance for unexplained deaths that may have infectious causes.

Address for correspondence: Jean Rainbow, Minnesota Department of Health, 717 Delaware St SE, Minneapolis, MN 55414, USA; fax: [phone]; email: [e-mail]