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Antimicrobial Agents and Chemotherapy, February 2000, p. 444-446, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Characterization of the 
-Lactamases
from Clinical Isolates of Moraxella (Branhamella)
catarrhalis Obtained from 24 U.S. Medical Centers during
1994-1995 and 1997-1998
Sandra S.
Richter,
Patricia
L.
Winokur,
Angela B.
Brueggemann,
Holly K.
Huynh,
Paul R.
Rhomberg,
Elizabeth M.
Wingert, and
Gary V.
Doern*
Medical Microbiology Division, Department of
Pathology, University of Iowa College of Medicine, Iowa City, Iowa
Received 21 June 1999/Returned for modification 4 October
1999/Accepted 25 October 1999
 |
ABSTRACT |
The 
-lactamases from 403 Moraxella
(Branhamella) catarrhalis clinical isolates
obtained during 1994-1995 and 1997-1998 U.S. multicenter surveillance
studies were characterized by isoelectric focusing. The overall
prevalences of the BRO-1 and BRO-2 enzymes among -lactamase-positive
isolates were estimated to be 97.5 and 2.5%, respectively. The minimum
inhibitory concentrations (MICs) of ampicillin for all BRO-2-producing
isolates were 
1 µg/ml; however, numerous -lactamase-positive
isolates for which the ampicillin MICs were 1 µg/ml produced the
BRO-1 enzyme (88.1%).
| |
TEXT |
Isoelectric focusing (IEF) of
-lactamases extracted from Moraxella
(Branhamella) catarrhalis typically reveals one
of two different banding patterns; these have been referred to as
Ravasio (BRO-1) and 1908 (BRO-2) (7, 15). The BRO-1 pattern
has been the most prevalent, accounting for 87 to 95% of M. catarrhalis -lactamases characterized in the United States,
France, Denmark, Sweden, England, and Scotland, with the remaining 5 to
13% identified as BRO-2 (2, 3, 5, 6, 8, 11, 13).
Although substrate hydrolysis rates of BRO-1 and BRO-2 enzymes are
similar, BRO-1-producing isolates are more resistant to penicillins
than BRO-2 strains (8, 10, 15). The higher -lactam
minimum inhibitory concentrations (MICs) associated with BRO-1 strains
have been attributed to larger amounts of enzyme produced
(15).
The purpose of this study was to characterize the -lactamases of
M. catarrhalis isolates collected during 1994-1995 and
1997-1998 U.S. multicenter surveillance studies. Enzyme type was
determined by IEF, and the relationship to ampicillin susceptibility
was examined.
Collection and identification of isolates.
Between 1 November
1994 and 30 April 1995, 606 clinical isolates of M. catarrhalis were obtained from 24 U.S. health care institutions.
Between 1 November 1997 and 30 April 1998, 513 additional clinical
isolates of M. catarrhalis were obtained from the same 24 centers. Stock cultures of all isolates were prepared by using an
absorbent bead system (Prolab Diagnostics, Austin, Tex.), and organisms
were stored at 
70°C until further use. The identity of the
organisms was confirmed using conventional criteria (9). The
details of these two studies and antimicrobial susceptibility patterns
of the isolates collected have been described previously (4,
14).
-Lactamase production and susceptibility testing.
-Lactamase production was assessed with a nitrocefin disk assay
(Cefinase; Becton-Dickinson Microbiology Systems, Cockeysville, Md.).
MICs of ampicillin were determined by a broth microdilution procedure
(total volume per well, 100 µl; final inoculum concentration, ca.
5 × 105 CFU/ml) in cation-adjusted Mueller-Hinton
broth (Difco Laboratories, Detroit, Mich.) according to the National
Committee for Clinical Laboratory Standards (NCCLS) guidelines
(12). Microdilution trays were incubated at 35°C in
ambient air for 22 to 24 h prior to the determination of the
results. Ampicillin was tested at 12 different concentrations (from
0.015 to 32 µg/ml) to limit the number of off-scale results.
Staphylococcus aureus ATCC 29213 and Escherichia
coli ATCC 25922 were used as controls. The lowest concentration of
antimicrobial tested that resulted in no visible growth was defined as
the MIC.
Selection of strains for IEF.
A total of 403 isolates were
selected for enzyme extraction and IEF on the basis of -lactamase
production and ampicillin MIC (202 from the 1994-1995 collection and
201 from the 1997-1998 collection). All of the -lactamase-positive
isolates in these collections for which ampicillin MICs were 1
µg/ml and 
32 µg/ml were focused. Among isolates for which MICs
were 2 µg/ml, 48 of 113 isolates from the 1994-1995 study and 53 of
83 strains from the 1997-1998 study were analyzed by IEF. For each of
the remaining MIC categories (4 µg/ml, 8 µg/ml, and 16 µg/ml), 10 or 11 strains from each collection were analyzed.
-Lactamase extraction.
-Lactamase extraction was
accomplished by a modification of the acetone method as described by
Nash and colleagues (11). The isolates were subcultured
twice on 5% sheep blood agar, suspended in 0.05 M phosphate-buffered
saline, and centrifuged for 15 min at 2,000 × g. The
pellets were washed three times in 10 ml of cold acetone and dried for
24 h at 4°C. The pellets were resuspended in 0.5 ml of 1%
glycine with 0.025% sodium azide and held for 4 to 5 days at 4°C.
Samples were centrifuged for 20 min, and the supernatant was
immediately focused or stored at 70°C for up to 1 week prior to IEF analysis.
IEF.
IEF of the enzymes was performed using commercially
prepared polyacrylamide gels with a pH range of 5.5 to 8.5 according to the manufacturer's instructions (Ampholine
PAGplate; Pharmacia Biotech, Uppsala, Sweden). -Lactamase
staining was accomplished by flooding the gels with nitrocefin (500 µg/ml in 0.05 M phosphate-buffered saline). Control -lactamases
(TEM-4, SHV-1, and SHV-3) with known isoelectric points (pIs) and
extracts from control strains of M. catarrhalis known to
produce the BRO-1 and BRO-2 -lactamases were included on each gel.
Among the 606 M. catarrhalis isolates collected during the
1994-1995 surveillance study, 576 (95%) were -lactamase positive (4). The same prevalence of -lactamase production was
observed with isolates from the 1997-1998 survey (i.e., 487 of 513 isolates were -lactamase positive) (14). A total of 202 -lactamase-positive isolates from the 1994-1995 study and 201 -lactamase-positive isolates from the 1997-1998 study were chosen
for characterization by IEF (Table 1).
These included all isolates from both collections for which the MICs
were 1 µg/ml, 42 to 64% of isolates for which the MICs was 2 µg/ml, 10 or 11 isolates each from organisms for which the MIC was 4, 8, or 16 µg/ml, and all isolates for which the MICs were 32
µg/ml.
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TABLE 1.
Characterization of -lactamases of M. catarrhalis isolates obtained during multicenter U.S. national
surveillance studies conducted from 1994 to 1995 and from 1997 to 1998
|
|
The banding patterns observed are illustrated in Fig.
1. The BRO-1
-lactamase was identified
by unique major bands with pIs
of 6.4 and 6.7. The BRO-2 enzyme
demonstrated unique bands at
pIs of 6.5 (major) and 7.5 (minor). Many
common bands at pIs of
5.8 and less were seen with both BRO-1- and
BRO-2-producing organisms,
as has been previously reported for
reference strains (
5,
11).
All of the
-lactamase-positive
isolates focused revealed either
the BRO-1 or BRO-2 banding pattern. No
bands were seen in the
lanes of the three
-lactamase-negative
isolates focused.
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FIG. 1.
Lane 1, TEM-4 (pI = 5.9) and SHV-1 (pI = 7.6)
-lactamases; lane 2, SHV-3 (pI = 7.0); lanes 3 and 4, Ravasio
(BRO-1) and 1908 (BRO-2) control strains, respectively; lanes 5 and 7 and 9 to 13, clinical isolates showing BRO-1 banding patterns; lanes 6 and 8, clinical strains showing BRO-2 banding patterns; lane 14, a
-lactamase-negative isolate.
|
|
As can be seen in Table
1, the ampicillin MICs for large numbers of
-lactamase-positive isolates of
M. catarrhalis from
both
1994-1995 and 1997-1998 were very low. Indeed, the ampicillin
MICs
for 116 of 576 (20.1%) 1994-1995 isolates and 110 of 487
(22.6%)
isolates from the 1997-1998 study were
1 µg/ml; these
isolates
would thus have been classified as being susceptible
to ampicillin
according to MIC interpretive criteria presently
advocated for use by
the NCCLS with
Haemophilus influenzae (nota
bene: presently,
there are no NCCLS MIC interpretive criteria
for tests with
M. catarrhalis).
The relative frequency of production of the BRO-1 and BRO-2 enzymes
together with the relationship between ampicillin MICs
and the
production of a specific enzyme is depicted in Table
1.
It is apparent
from Table
1 that all tested isolates from both
collections for which
ampicillin MICs were
2 µg/ml produced the
BRO-1
-lactamase. The
ampicillin MICs for BRO-2-producing strains
were always
1 µg/ml;
however, most isolates for which the MICs
were this low (i.e., 104 of
116 [89.7%] isolates from the 1994-1995
study and 95 of 110 [86.4%] isolates from the 1997-1998 study)
produced the BRO-1
enzyme. Among the isolates for which ampicillin
MICs were low, the
likelihood of an organism producing the BRO-2
enzyme was greatest as
ampicillin MICs dropped to 0.12 and 0.06
µg/ml.
The only prior U.S. study of this type identified 8% of 146
M. catarrhalis -lactamases from 1982-1983 as BRO-2
(
11). Based
on the results of this study, we estimate that
2.1 and 3.1% of
-lactamase-producing isolates of
M. catarrhalis from the United
States in 1994-1995 and 1997-1998,
respectively, produced the
BRO-2
-lactamase. Despite the fact that
not all
-lactamase-positive
isolates were characterized in this
study, it is likely that the
remaining strains produced the BRO-1
enzyme.
Our study showed the same general trend of higher ampicillin resistance
among BRO-1-producing strains reported previously
(
8,
10,
15); however, the wide range of ampicillin MICs
observed for
BRO-1 strains was unexpected and suggests that factors
other than
enzyme type determine antibiotic
susceptibility.
Bootsma et al. reported a 21-bp deletion in the promoter region of the
M. catarrhalis -lactamase gene (
bla) from a
BRO-2-producing
strain as a possible cause of the lower level of enzyme
production
(
1). Sequencing the
bla gene and
regulatory sequences from
a greater number of BRO-1- and
BRO-2-producing isolates showing
a range of ampicillin resistance may
reveal additional mutations
or deletions to explain the variation in
antibiotic susceptibility
observed in our
study.
| |
ACKNOWLEDGMENTS |
We are indebted to the following individuals for provision of
clinical isolates: Carla Clausen, Children's Hospital (Seattle, Wash.); Susan Rossmann, Texas Children's Hospital (Houston); Paul Southern, University of Texas Southwestern Medical Center (Dallas); Michael Wilson, Denver General Hospital (Denver, Colo.); Michael Saubolle, Good Samaritan Medical Center (Phoenix, Ariz.); John Washington, Cleveland Clinic (Cleveland, Ohio); Michael Dunne, Henry
Ford Hospital (Detroit, Mich.); Gerald Denys, Methodist Hospital
(Indianapolis, Ind.); Melodie Beard, Rush-Presbyterian St. Luke's
Medical Center (Chicago, Ill.); Richard Thompson, Evanston Hospital
(Evanston, Ill.); Susan Kehl, Children's Hospital (Milwaukee, Wis.);
Franklin Cockerill, Mayo Clinic (Rochester, Minn.); Patrick Murray,
Barnes-Jewish Hospital (St. Louis, Mo.); Ann Robinson, Hartford
Hospital (Hartford, Conn.); Betty Forbes, SUNY Health Sciences Center
(Syracuse, N.Y.); Dwight Hardy, University of Rochester Medical Center
(Rochester, N.Y.); Phyllis Della-Latta, Presbyterian Hospital (New
York, N.Y.); Allan Truant, Temple University Hospital (Philadelphia,
Pa.); Joseph Campos, National Children's Hospital (Washington, D.C.);
Paul Bourbeau, Geisenger Medical Center (Danville, Pa.); Peter
Gilligan, University of North Carolina Hospital (Chapel Hill); Robert
Jerris, Dekalb General Hospital (Decatur, Ga.); Kim Chapin-Robertson,
University of South Alabama Medical Center (Mobile); and Susan Sharp,
Mount Sinai Medical Center (Miami Beach, Fla.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, Clinical Microbiology, University of Iowa College of
Medicine, Iowa City, IA 52242. Phone: (319) 356-8616. Fax: (319)
356-4916. E-mail: gary-doern{at}uiowa.edu.
| |
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Antimicrobial Agents and Chemotherapy, February 2000, p. 444-446, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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