[Emerging Infectious Diseases]
[Volume 4 No. 2 / April - June 1998]

Dispatches

Emergence of the M Phenotype of Erythromycin-Resistant Pneumococci in South
Africa

Carol A. Widdowson and Keith P. Klugman
South African Institute for Medical Research, Johannesburg, South Africa
-------------------------------------------------------------------------
      Erythromycin-resistant pneumococci have been isolated in South
      Africa since 1978; however, from 1987 to 1996, resistance to
      macrolides was only detected in 270 (2.7%) of 9,868 blood or
      cerebrospinal fluid (CSF) pneumococcal isolates, most of which
      were obtained from the public sector. In South Africa,
      macrolide use in the public sector is estimated at 56% of that
      in the private sector. Most erythromycin-resistant strains
      (89%) exhibited resistance to erythromycin and clindamycin
      (macrolide-lincosamide-streptogramin B phenotype). In the
      United States, most erythromycin-resistant pneumococci exhibit
      the newly described M phenotype (resistance to erythromycin
      alone), associated with the mefE gene. The M phenotype in South
      Africa increased significantly in the last 10 years, from 1 of
      5,115 to 28 of 4,735 of blood and CSF isolates received from
      1987 to 1991 compared with 1992 to 1996 [p = 5x10(sup -7)]. These
      data suggest that, although macrolide resistance in pneumococci
      remains low in the public sector, the mefE gene is rapidly
      emerging in South Africa.

Resistance to erythromycin in pneumococci has been observed since 1967 (1)
and was first reported in South African multiresistant pneumococcal strains
in 1978 (2). Until recently, the only mechanism described for resistance to
erythromycin in the pneumococcus was the N(sup 6)-methylation of a specific
adenine residue (A2058) in 23S rRNA, which resulted in reduced affinity
between the antibiotic and the ribosome (3,4). This resistance is
associated with the gene ermAM (5), first described in Streptococcus
sanguis (6). Since then, other mechanisms of erythromycin resistance in the
pneumococcus have been reported. In fact, most resistance in the United
States appears to be due to efflux of the antibiotic from the cell,
associated with the gene mefE (7,8). While ermAM confers coresistance to
most macrolides, lincosamides, and streptogramin B antibiotics (resulting
in the so-called MLS phenotype) (3,9), mefE confers resistance only to the
14- and 15-membered macrolides (resulting in the M phenotype) (7,8). We
report the emergence of M-phenotype erythromycin resistance in South
African blood and cerebrospinal fluid (CSF) pneumococcal isolates from 1987
to 1996.

The South African Institute for Medical Research (SAIMR), Johannesburg,
South Africa, regularly receives all pneumococcal isolates from
participating laboratories in eight of the nine provinces of South Africa.
We examined all erythromycin-resistant blood and CSF isolates received by
SAIMR from 1987 to 1996.

Erythromycin-, clindamycin-, and penicillin-resistance phenotypes were
determined by using disk diffusion assays (erythromycin, 15µg/disk,
clindamycin, 2 µg/disk, oxacillin, 1 µg/disk) on 5% horse blood agar plates
(Mueller-Hinton base) after overnight growth at 37°C under aerobic
conditions. Strains showing resistance (zone diameters </= 20mm for
erythromycin, </= 18mm for clindamycin, and </= 20mm for oxacillin) on the disk
diffusion plates were tested by the agar dilution method to obtain MICs
according to the National Committee for Clinical Laboratory Standards
guidelines (10). We evaluated (by the chi-square test) increases in the
prevalence of erythromycin resistance and the incidence of resistance to
erythromycin and susceptibility to clindamycin, which represents the M
phenotype.

The M phenotype increased significantly from 1987 to 1991 compared with
1992 to 1996 in blood and CSF isolates-from 1 of 5,115 isolates to 28 of
4,753 isolates (p = 5x10(sup -7), odds ratio [OR] 30.13, 95% confidence interval
[CI] 4.1-221) (Table 1; Figure).

Data on oral macrolides in the public sector (from the Division of Medical
Schemes Supplies and Pharmaceutical Services of the Department of Health)
show that 16.4 million defined daily doses (ddd) of macrolides were
purchased for the estimated 30.3 million persons who obtain health care
from the public sector (0.54 ddd per capita). Private sector use for the
year ending December 1997 (Intercontinental Medical Statistics South
Africa, Pty, Ltd., unpub. data) show that 7.3 million ddd of macrolide were
purchased in an estimated population of 7.57 million (0.96 ddd per capita).

Table 1. Prevalence of South African erythromycin-resistant pneumococcal
isolates, 1987–1996(sup a)
------------------------------------------------------------------
                   Total        No. of E-R      No. of M strains
Years       isolates(sup b) strains (%)(sup c) (% of E-R strains)
------------------------------------------------------------------
1987-1991      5,115          128 (2.5)            1 (0.8)
1992-1996      4,753         142 (3.0)            28 (19.7)
Total          9,868         270 (2.7)            29 (10.7)
  ----------------------------------------------------------------
(sup a)Of 9,868 blood and cerebrospinal fluid (CSF) isolates received by the
SAIMR from 1987 to 1996, 270 were fully resistant to erythromycin. While
the number of erythromycin-resistant blood and CSF isolates received
increased from 1987 to 1991 compared with 1992 to 1996 (2.5% to 3.0%), the
increase was not significant. There was no significant relationship
between erythromycin resistance and the M phenotype within any given
province throughout the 10 years.
(sup b)Blood and cerebrospinal fluid isolates.
(sup b)E-R= erythromycin-resistant.



                 [fig]
  Figure. Number of erythromycin-resistant blood and cerebro-
  spinal fluid isolates of pneumococci.

  --------------------------------------------------

All 78 MLS isolates hybridized with the ermAM probe or produced a
616-bp-amplification product during polymerase chain reaction (PCR)
amplification using the ermAM-specific primers. The 12 M isolates tested
contained the mefE gene as shown by a 348bp-amplification product when
amplified using primers specific for mefE. There were no
erythromycin-resistant isolates that contained neither the ermAM nor the
mefE gene.

(ft)
DNA was extracted from pneumococcal isolates by using a lysis solution
consisting of 0.1% sodium deoxycholate as described in (11), except that we
used plate rather than broth cultures.
        Seventy-eight MLS strains were probed for the ermAM gene by using
dot blots. The probe (supplied by P. Courvalin, Pasteur Institute, Paris,
France) (Escherichia coli JM83/pUC19 560bp Ssp1 intragenic fragment of
ermB) was labeled with digoxygenin by using random primed labeling (DIG DNA
Labeling and Detection Kit; Boeringer, Mannheim, Germany). Hybridization
and detection were performed following manufacturer's instructions (DIG DNA
Labeling and Detection Kit; Boeringer, Mannheim, Germany). PCR was also
used to detect ermAM in 30 strains according to standard conditions, with
an annealing temperature of 58°C. We used the following primers: forward
primer, 5'-CGAGTGAAAAAGTACTCAACC, reverse primer,
5'-GGCGTGTTTCATTGCTTGATG).
        Published primers for the mefE gene (5'-AGTATCATTAATCACTAGTGC, and
5'-TTCTTCTGGTACTAAAAGTGG) (12) were used to detect mefE through PCR
amplification in 13 M strains. Amplification was performed in a Perkin
Elmer Cetus DNA Thermal Cycler under standard reaction conditions, with an
annealing temperature of 56oC.


Erythromycin-resistant strains were serotyped by using the quellung
reaction and antisera from the Staten Seruminstitut, Copenhagen, Denmark.
Over the 10 years, the five most common serotypes and groups among the
erythromycin-resistant isolates in decreasing order of frequency were
serotype 14, serogroups 6, 19, 23, and serotype 1 (Table 2). Serotype 1
erythromycin-resistant pneumococci appeared only after 1992; serotype 14
was the most common in MLS isolates; serogroup 23 was the most common
serogroup in M isolates (Table 2). Serotypes 14 and serogroups 6, 23, and
19 are the most common serotypes and groups isolated from children with
serious infections (13,14). Of the 157 isolates from patients whose age was
supplied, 98 (62%) were obtained from children (</= 12 years). There was a
trend that was not significant toward more macrolide resistance in children
than adults (OR 1.17 [95% CI 0.98-1.39]). This trend may have been
significant if age data had been supplied with all the isolates received.
The proportion of pediatric isolates did not change significantly over the
10-year period (63% during 1987 to 1991 and 62% the 1992 to 1996 period).

Table 2. Serotype distribution among erythromycin-resistant pneumococci
-----------------------------------------------------------------------
Serotype/           No. (%) of       No. (%) of          Total no.
group           MLS(sup a)isolates   M isolates       (%) of isolates
  ---------------------------------------------------------------------
14                  96 (39.8)         9 (31.0)          105 (38.9)
 6                  71 (29.5)         3 (10.3)           74 (27.4)
19                  36 (14.9)         3 (10.3)           39 (14.4)
23                  26 (10.8)        10 (34.5)           36 (13.3)
 1                   7 (2.9)          1 (3.5)             8 (3.0)
Other                5 (2.1)          3 (10.3)            8 (3.0)
Total              241               29                 270
-----------------------------------------------------------------------
(sup a)MLS, macrolides-lincosamides-streptogramin B.

Approximately half of all the macrolide-resistant isolates were also either
intermediate or fully resistant to penicillin (60 of the 128 isolates from
the 1987 to 1991 period, and 72 of the 142 isolates from the 1992 to 1996
period). There was a trend (not significant) toward greater resistance to
penicillin in MLS strains. Of the 78 strains with MICs available, 38 (49%)
were fully resistant (MIC(sup 3) 2µg/ml) to penicillin, while the rest showed
intermediate resistance (1 µg </= MIC </= 0.12 µg). Previous data have
indicated that penicillin-intermediate resistance is far more common in
South Africa than full resistance to penicillin (15). In 1992, Friedland
and Klugman (15) reported that only 3 of 35 penicillin-resistant strains
showed high-level resistance.

Coresistance to penicillin limits the use of most macrolides as treatment
of penicillin-resistant pneumococcal infections. New semisynthetic
macrolides such as the ketolide RU 64004 (16) are being developed, however,
that do not show cross-resistance to penicillin or to erythromycin.

Compared with total erythromycin resistance (middle ear fluid, blood, and
CSF) in Europe (17-19), the United Kingdom (18), and the United States
(19,21,22), the overall prevalence of macrolide resistance is low. In
Europe erythromycin resistance varies by country. In Slovakia, almost all
pneumococcal isolates are resistant (17), whereas in Portugal only 0.6% of
isolates are resistant and the proportion appears to be declining (23). In
the United Kingdom, erythromycin resistance increased from 3.3% to 8.6%
between 1989 and 1992 in England and Wales (20). In the United States, 10%
of pneumococcal isolates appear to be erythromycin resistant (21). Most
isolates received by SAIMR are from the public sector, where macrolides are
not normally prescribed for pneumococcal infections. Only 4 of the 128
erythromycin-resistant isolates from 1987 to 1991 and 14 of the 142
erythromycin-resistant isolates from 1992 to 1996 were from the private
sector. Resistance data from the private sector may show much higher levels
of macrolide resistance, a contention supported by previous South African
resistance data (1986), where the carriage rates of multiresistant
pneumococci were 17.7% in children from more affluent communities and 0% in
children from less affluent areas (24). MIC data were available for 15 of
the 20 multiresistant isolates, and all 15 were fully resistant to both
erythromycin and clindamycin (24).

Before the M phenotype was observed, erythromycin resistance was assumed to
indicate cross-resistance to lincosamides and streptogramin B antibiotics
in the pneumococcus. The increase in the incidence of M phenotype may
warrant investigation into the use of these antibiotics for the treatment
of pneumococcal infections. Sutcliffe et al. (7) suggested that clindamycin
be considered for the treatment of bacteremia and middle ear and sinus
infections caused by Streptococcus pneumoniae. Treatment with clindamycin
is feasible only if infection with gram-negative pathogens has been
excluded and if the S. pneumoniae phenotype is known because the strain may
show MLS resistance and studies indicate that many penicillin-resistant
strains are also clindamycin-resistant (16,25). Visalli and colleagues (25)
found that clindamycin concentrations of only 0.06 µg/ml were required to
inhibit 90% of penicillin-susceptible strains when grown in air, while
clindamycin concentrations of > 64 µg/ml were required to inhibit 90% of
penicillin–intermediate and –resistant strains.

Studies of streptogramin use against pneumococci show some promise. The
streptogramin RP 59500, a mixture of type A streptogramin, dalfopristin,
and type B streptogramin quinupristin, is active against pneumococci
regardless of their susceptibilities to penicillin or erythromycin (26,27).
In contrast to erythromycin, RP 59500 is rapidly bactericidal (26).
Clinical and bacteriologic failure has, however, already been reported
using pristinamycin (28), an oral streptogramin combination from which RP
59500 was derived.

The M phenotype is thus relatively new in South African pneumococci but is
emerging as an important factor in erythromycin-resistant pneumococci.
Although the low overall rate of resistance makes the use of streptogramins
and lincosamides potentially more feasible for the treatment of
pneumococcal infections, coresistance to penicillin and the present high
rate of MLS resistance necessitate antibiotic susceptibility testing before
these antibiotics are administered.

Acknowledgments

The authors thank Patrice Courvalin for the probe used in this study, Thora
Capper for assistance with MICs, Avril Wasas for performing serotyping, and
Robyn Hubner for assistance with data analysis.

This work was supported by the Medical Research Council, the South African
Institute for Medical Research, and the University of the Witwatersrand.

Carol Widdowson is completing her Ph.D. at the South African Institute for
Medical Research, through the University of the Witwatersrand. Her research
focuses mainly on resistance to the nonbetalactam antibiotics such as
erythromycin, tetracycline, chloramphenicol, and streptomycin, in the
pneumococcus.

Keith Klugman is the director of the South African Institute for Medical
Research. He also heads the Pneumococcal Research Unit of the Medical
Research Council, the South African Institute for Medical Research, and the
University of the Witwatersrand. He has an interest in all aspects of
pneumococcal research.

Address for correspondence: Carol A. Widdowson, Department of Clinical
Microbiology and Infectious Diseases, SAIMR, P.O. Box 1038, Johannesburg
2000, South Africa; fax: 27-11-489-9332; e-mail:
174carol@chiron.wits.ac.za.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: http://www.cdc.gov/ncidod/EID/vol4no2/widdowsn.htm

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