# Antibiotic Resistance Patterns in Invasive Group B Streptococcal Isolates

Authors: Mei L. Castor; Cynthia G. Whitney; Kathryn Como-Sabetti; Richard R. Facklam; Patricia Ferrieri; Joanne M. Bartkus; Billie A. Juni; Paul R. Cieslak; Monica M. Farley; Nellie B. Dumas; Stephanie J. Schrag; Ruth Lynfield
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2008)

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## Abstract

Antibiotics are used for both group B streptococcal (GBS) prevention and treatment. Active population-based surveillance for invasive GBS disease was conducted in four states during 1996–2003. Of 3813 case-isolates, 91.0% (3471) were serotyped, 77.1% (2937) had susceptibility testing, and 46.6% (3471) had both. All were sensitive to penicillin, ampicillin, cefazolin, cefotaxime, and vancomycin.  Clindamycin and erythromycin resistance was 12.7% and 25.6%, respectively, and associated with serotype V (P<.001). Clindamycin resistance increased from 10.5% to 15.0% (X2 for trend 12.70; P<.001); inducible clindamycin resistance was associated with the erm genotype. Erythromycin resistance increased from 15.8% to 32.8% (X2 for trend 55.46; P<.001).  While GBS remains susceptible to beta-lactams, resistance to alternative agents such as erythromycin and clindamycin is an increasing concern.


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## Body

## 1. Introduction

Early-onset group BStreptococcal (GBS)
infection, occurring in the first 7 days of life, is a leading cause of
invasive bacterial infection among newborns and generally results from vertical
transmission of GBS from colonized mothers to their infants. Neonatal infection
is commonly characterized by bacteremia, pneumonia, and meningitis; mortality
from early-onset disease is estimated at 5%. GBS disease among pregnant women
manifests as urinary tract infections, bacteremia, chorioamnionitis, endometritis,
or septic abortions [1]. Among non-pregnant adults, GBS disease typically occurs
among older or immunocompromised persons.In the 1990s, the incidence of
early-onset neonatal GBS disease declined dramatically after widespread
implementation of intrapartum antibiotic prophylaxis (IAP). Revisions to IAP
guidelines in 2002 included universal GBS screening of pregnant women with
vaginal and rectal cultures at 35–37 weeks'
gestation, with IAP given to those with positive cultures. Susceptibility testing
for erythromycin and clindamycin of GBS isolates is recommended for colonized
women who are thought to be at high risk for anaphylaxis to penicillin or
ampicillin. Cefazolin is recommended for those who cannot take penicillin but
are at low risk for anaphylaxis [2].Publications from
the United States and Canada report clindamycin resistance (3%–21%) and
erythromycin resistance (5%–29%) in GBS
isolates [3–8]. Alteration of
the erythromycin-binding site on the 50S ribosomal RNA subunit, conferred by the erm gene, and active efflux of the
erythromycin, conferred by the mef gene, are the most common macrolide resistance mechanisms in beta-hemolytic
streptococci [9]. The erm gene
confers resistance to macrolides, lincosamides, and streptogramin B-type antibiotics
(MLSB) and may be expressed constitutively or it may be inducible. The mef gene confers resistance only to
macrolide antibiotics, the M phenotype. The inducible MLSB phenotype
may be detected in the laboratory by a disk approximation method (D-test),
which involves placement of an erythromycin and a clindamycin disk in close
proximity on the surface of an agar plate. Clinical and Laboratory Standards
Institute (CLSI) guidelines for antimicrobial susceptibility testing now include a method for
performing the D-test on beta-hemolytic streptococci [10].Serotype
distribution in invasive GBS disease is of interest in determining potential
vaccine composition. Serotype III has a high association with late-onset
neonatal disease, specifically meningitis. Serotype V has emerged as a more
frequent serotype in recent years [11]. Other common serotypes include Ia and
II.We analyzed GBS surveillance data from four
distinct US sites to understand serotype and antimicrobial susceptibility patterns in
invasive isolates from perinatal (pregnant and neonatal cases) and non-pregnant
adult populations.


## 2. Methods

Invasive GBS
isolate data regarding antimicrobial susceptibility, serotype, and
epidemiologic data were collected from selected counties in four states
(Georgia, Minnesota, New York, and Oregon); all participated in the Centers for
Disease Control and Prevention's (CDC) Active Bacterial Core surveillance (ABCs)
system of the Emerging Infections Program network for 1996–2003. Georgia's initial surveillance area of eight
counties in the Atlanta area expanded in 1997 to 20 counties. New York's
initial surveillance area of seven counties in 1997 expanded to 15 counties in
2000 (seven-county Rochester area and eight-county Albany area). Oregon's three-county and Minnesota's statewide
surveillance areas remained unchanged throughout the study period. Total
surveillance populations ranged from 8.7 million in 1996 to 13.2 million in
2003. By state, 2003 surveillance populations were 4.5 million in Georgia, 5
million in Minnesota, 2.1 million in New York, and 1.5 million in Oregon.A case of invasive
GBS was defined by bacterial isolation from a normally sterile site in a
surveillance area resident. GBS isolation from amniotic fluid or placentas was
included when fetal demise occurred. Early-onset neonatal cases were classified
by a first positive culture on days 0–6 of life and late-onset
at 7–89 days of life.
Maternal cases were those identified during the antepartum or postpartum
periods (30 days after a delivery or miscarriage). The case-finding methodology
used for the ABCs system has been previously described [12]. Laboratory audits
are performed every 6 months to capture any missed cases. Antimicrobial
susceptibility testing was performed at the Minnesota Department of Health
(MDH) laboratory or CDC laboratories. Serotyping of Minnesota isolates was
performed at the University of Minnesota and CDC, and isolates from other geographic sites
were tested at CDC. Lancefield immunoprecipitation techniques were used for
serotyping. Antimicrobial susceptibility testing was performed using broth
microdilution in microtiter plates containing cation-adjusted Mueller Hinton
broth with 3% lysed horse blood (PML Microbiologicals, Wilsonville, Ore)
with CLSI susceptibility definitions used when available.The constitutive (c) MLSB phenotype was inferred from broth microdilution MIC data.
Isolates found to be erythromycin-resistant and clindamycin-susceptible were
evaluated for inducible (i) clindamycin resistance using the double disk diffusion
(D-test) method with clindamycin (2 μg) and erythromycin (15 μg) disks (Becton
Dickinson Diagnostic Systems, Sparks, Md) placed 15 mm apart. Strains that
remained clindamycin-susceptible (those that did not have flattening of the
susceptibility zone adjacent to the erythromycin disk) were determined to be of
the M phenotype.PCR was used to
detect the presence of the most common macrolide resistance determinants in all
isolates with an M or iMLSB phenotype. Testing was also done on a
small subset of isolates with a cMLSB resistance phenotype. PCR for
detection of ermA, ermB, and ermC was performed as described by Sutcliffe et al. [13]. Detection
of ermTR and mef (the primers amplify both mefA
and mefE) was performed using primers
designed at MDH. The PCR reactions for ermTR
and mefA/E contained 10 mM Tris HCl,
50 mM KCl, 1.5 mM MgCl2, 1 μM forward and reverse primers, and 2 μl
of template DNA in a total volume of 50 μL. Primers used for the amplification
of ermTR, a subclass of ermA [14], were TR-322U,
5′-GGGTCAGGAAAAGGACAT-3′, and TR619L, 5′-CCTAAAGCTCGTTGGGTATT-3′. Primers used
for the amplification of mefA/E were
mef-3301U, 5′-AGGGCAAGCAGTATCATTAATCA-3′, and mef-3673L, 5′-CTGCAAAGACTGACTATAGCCT-3′. Optimum
annealing temperatures for ermTR and mefPCR were 56°C and 55°C, respectively.
PCR products were resolved on a 2% agarose gel. The expected fragment size for
ermTR was 317 bp and for mef was 394 bp.SAS (version 9.1)
was used for frequency and univariate analysis. Trend analyses were performed
with Epi Info (CDC, Atlanta, Ga) statistical software (version
6.04c). Chi-square calculations were used to compare continuous variables, with
Yates continuity-corrected chi-square used to compare categorical variables.
The Mantel-Haenszel chi-square test for trend was used to examine trends across
the study period; the Mantel-Haenszel extended test adjusted for possible
confounders. An alpha ≤0.05-significance level was used.


## 3. Results

Of the 5 373
patients, 71% had isolates submitted to a public health laboratory and the antimicrobial
susceptibility testing was performed on 77.1% (2 937).
The plurality of isolates were from Minnesota (46.8% or 1 375),
followed by Georgia (26.1% or 768), New York (16.9% or 496), and
Oregon (10.2% or 300). The perinatal category comprised 24.4% (1 311)
of the total sample with 12.5% (671) early-onset, 7.9% (424) late-onset, and
4.0% (216) maternal patients. The non-pregnant adult category accounted for
73.5% (3 951) of the total.All 2 937
isolates were susceptible to penicillin, ampicillin, cefotaxime, and
vancomycin. Although no CLSI
breakpoints existed for the first-generation cephalosporins (cephalothin and
cefazolin), all the isolates tested had minimum inhibitory concentrations
(MICs) ≤ 0.5 mcg/mL and were, therefore, considered susceptible.
Similarly, no breakpoints existed for cefuroxime and cefoxitin. All 1 201
Minnesota isolates tested for cefuroxime susceptibility had MICs ≤ 0.12 mcg/mL
and were, therefore, considered susceptible. Of 2 937
isolates tested for cefoxitin susceptibility, 36.7% (1 078) had MICs ≤ 2 mcg/mL; 62.5%
(1 835) had MICs = 4 mcg/mL; 0.7% (22) had
MICs = 8 mcg/mL; and 0.1% (4) had MICs ≥ 16 mcg/mL.Clindamycin
resistance was identified in 12.7% (374), and erythromycin resistance was
identified in 25.6% (752) of isolates tested. During the study period, a
significant increase was observed in the proportion of isolates resistant to
each of these antibiotic
(for clindamycin 10.5% in 1996 versus 15.0% in 2003,X2 for trend = 12.70, P<.001, for erythromycin
15.8% in 1996 versus 32.8% in 2003, X2 for trend = 55.46, P<.001) (see Figure 1). Concurrent resistance to both clindamycin and
erythromycin was identified in 12.6% (370) of isolates tested.Figure 1
Clindamycin and erythromycin resistance among invasive Group B streptococci isolates by year (n= 2 937 isolates).Significant
differences were identified between Minnesota and New York
isolates in clindamycin resistance (13.2% versus 9.3%,P=.03) and
erythromycin resistance (24.5% and 31.4%, P<.01). Georgia isolates were more likely to be
resistant to clindamycin than New York isolates (15.1% and 9.3%,
P=.02), but Georgia isolates were less likely to be resistant
to erythromycin than to New York isolates (26.3% and 31.4%, P=.04) (see Figure 2).Figure 2
Clindamycin and erythromycin resistance among invasive Group B streptococci isolates by state (n= 2 937 isolates).The proportion of
isolates resistant to clindamycin and erythromycin did not differ significantly
between the perinatal and non-pregnant adult patient groups (see Figure3).
Within the perinatal group, the overall clindamycin resistance was 13.1%, with
no significant changes between the early-onset (14.9%), late-onset (10.3%), and
maternal populations (13.7%). Likewise, no significant differences in
erythromycin resistance levels were identified between the different perinatal
groups (21.9% overall; 21.2% for early-onset, 22.1% for late-onset, and 23.9%
maternal).Figure 3
Resistance invasive Group B streptococci isolates among pregnant women and newborns (perinatal) and non-pregnant adults (n=2887  isolates).Serotyping was performed on 64.6% (3 471) of isolates. The largest proportion was
from Georgia (34.7%), followed by Minnesota (28.5%), New York (23.9%), and
Oregon (12.9%). Table1 illustrates the serotypes identified during the study period,
with predominant serotype groups being V, Ia, and III. The proportion of
isolates with serotype III was observed to have a significant downward trend (X2 trend = 38.8; P<.001) during the study period from 25% in 1996 to 17%
in 2003; no such trend was observed with the other predominant serotypes.Table 1
Distribution of serotypes and resistance to erythromycin and clindamycin among
invasive group B streptococcus isolates.
Serotyped isolates with susceptibility resultsSerotypeSerotyped isolatesTotalErythromycin-resistantClindamycin-resistantno. (%)*no. (%)no. (%)(n=3471)(n=2551)(n=669)(n=301)Ia, Ia/c835 (24.1)573112 (17)13 (4)Ib, Ib/c296 (8.5)23137 (6)10 (3)II, II/c357 (10.3)25568 (10)46 (15)III, III/c680 (19.6)49678 (12)26 (9)V, V/c951 (27.4)695293 (44)166 (55)NT, NTc247 (7.1)17565 (10)30 (10)Other**105 (3.0)12016 (2)10 (3)*of isolates serotyped.
**IV, IV/c, VI, VI/c, and VIII.The most common serotype in the perinatal
population was serotype III (37.0%; 351/948). This was significantly different
than for non-pregnant adults in which serotype V was most common (31.5%;P<.001).Both susceptibility and serotyping testing were
performed on 2 551 isolates. The largest proportion of these isolates were
from Minnesota (38.9%), followed by Georgia (30.1%), New York (19.4%), and
Oregon (11.8%). Clindamycin resistance was 3.9 times (P<.001) more likely to occur in
serotype V isolates (25.8%) than in other serotypes (8.2%). In addition,
erythromycin resistance was 2.9 times (P<.001) more likely to occur
in serotype V isolates (42.2%) than in other serotypes (20.2%). Subanalysis
within the different disease categories indicated a significant association
among clindamycin or erythromycin resistance and early-onset (P<.001),
late-onset (P<.001), and non-pregnant adult populations (P<.001).
No such association was observed among the maternal population.Three
hundred fifty-five erythromycin-resistant (47%) isolates were further
characterized. Seventy-eight isolates (22%) had an M phenotype and all of these
isolates containedmef only. Fifty-two percent (184/355) were
constitutively resistant to clindamycin (cMLS). Seventeen were further tested. Ten were positive for ermB and
seven contained ermTR; one isolate
had both mef and ermTR and one isolate had both mef and ermB. Twenty-five percent of isolates had inducible
clindamycin resistance (iMLS). All of the iMLS isolates were tested for
resistance genes; 83 (92%) contained ermTR,
1 isolate contained ermB, and 6 isolates
were negative for ermTR and ermB.Univariate analysis was conducted comparing
resistance genes to serotypes. Serotype V was associated with a lower
proportion ofmef positive isolates
compared to all other serotypes (P<.01). In contrast, serotypes Ia,
Ia/c, and III were associated with a higher proportion of mef-positive isolates compared to all other serotypes (all P values <.01). Serotype V had a higher proportion of isolates with ermTR, 73% of 37 tested
compared with 29% among all other serotypes (P<.01).


## 4. Conclusions

This study demonstrated the prevalence of
resistance to antibiotics commonly used for prophylaxis or treatment of GBS
infections among a large, multisite, collection of isolates. We found that all invasive GBS isolates tested were susceptible to
penicillin and ampicillin, the first-line agents recommended for IAP and to
cefazolin and vancomycin, second-line agents recommended for use among IAP
candidates who report penicillin allergies. In contrast, GBS resistance
to clindamycin (12.7%) and erythromycin (25.6%) was common. Subanalysis of early-onset
patient isolates indicated clindamycin resistance at 14.9% and erythromycin
resistance at 21.2%. These findings are consistent with previous reports in the
literature [3–8] and support the
2002 guidelines requiring susceptibility testing of isolates from IAP
candidates with penicillin allergy. Of note, because of reports of
subtherapeutic concentrations in amniotic fluid and fetal serum, certain healthcare specialists do
not recommend using erythromycin for IAP [15]. Clinicians should be aware of
the potential for MLS resistance when determining empiric regimens that target
GBS disease.We found an overall
concordance between the MLS phenotype and resistance genotype. D-testing
revealed inducible clindamycin resistance inerm-containing GBS isolates and also detected resistance in
isolates with determinants not identifiable by existing PCR methods. Our findings support the 2002 CDC guideline
to test both erythromycin and clindamycin susceptibilities if one of these
agents is being considered. Further D-testing should be done if erythromycin
resistance and clindamycin susceptibility are found. Rarely, clindamycin
resistance without concurrent erythromycin resistance can be observed; this has
been associated with the linB gene [9]. Minimal differences were identified in
clindamycin and erythromycin resistance levels across the four geographic areas
as well as between the perinatal and non-pregnant adult groups. This indicates
that GBS-resistance patterns might reflect more of a national than a regional
phenomenon.We noted high cefoxitin MICs to GBS among our
GBS isolates. This is not surprising because cefoxitin is a cefomycin, a class
of antibiotics which typically provides optimal coverage for anaerobes but lacks adequate activity
against gram-positive bacteria. Cefoxitin is likely to provide suboptimal GBS
coverage for chorioamnionitis and postpartum endometritis.The predominant GBS serotypes were Ia, III,
and V, which together comprised approximately 70% of all isolates serotyped.
The predominance of these serotypes among the perinatal population is
consistent with reports in the literature [4, 11] as is the association between
serotype V and erythromycin or clindamycin resistance [4, 7]. We found an association of serotype V with the erm genotype which confers resistance to
both erythromycin and clindamycin.This study has several
limitations, including variability in surveillance periods for isolates
collected, a varying proportion of cases with isolate collection and
susceptibility and serotyping results, and differences in surveillance
populations. However, the ABCs surveillance system is an active, multiregional, and
population-based system,
and the methods to determine susceptibilities and serotyping were
standardized. Continued surveillance of invasive GBS disease including
susceptibility and serotype determinations will impact plans for prevention
including IAP agents and vaccine design.



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