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Journal of Clinical Microbiology, April 2000, p. 1575-1580, Vol. 38, No. 4
Department of Pathobiology, University of
Washington, Seattle,1 and Infections
Limited, Tacoma,3 Washington; National
Center for Infectious Diseases, Centers for Disease Control and
Prevention, Atlanta, Georgia2; and
Departments of Pediatrics and Microbiology and Infectious
Diseases, University of Calgary, Calgary, Alberta,
Canada4
Received 16 September 1999/Returned for modification 24 December
1999/Accepted 31 January 2000
In 1997, a cluster of multiresistant invasive serogroup 19 pneumococcus infections, including two fatalities, was reported in
Washington State. Further investigation identified other cases. Fourteen Washington Streptococcus pneumoniae isolates, four
from Alaska, and eight isolates from eastern Canada with reduced
penicillin susceptibility (MIC of Streptococcus pneumoniae
is the leading bacterial cause of community-acquired pneumonia, otitis
media, bacteremia, and meningitis in the United States (14).
In the past 20 years, a worldwide increase in the incidence of
antibiotic-resistant S. pneumoniae has been observed
(3, 13, 30). Although more than 90 serotypes of S. pneumoniae exist, resistance to two or more different classes of
antibiotics (i.e., multiresistance) is currently limited to a few major
serotypes (6B, 9V, 14, 19F, and 23F) (11, 12, 30). The first
non-penicillin-susceptible multiresistant S. pneumoniae
strain, described in the 1970s, contained a conjugative transposon,
Tn1545, which carried four resistance genes:
erm(B) (macrolides, lincosamides, and streptogramin B),
tet(M) (tetracycline), aphA-3 (aminoglycosides),
and cat (chloramphenicol). This family of transposons has
since disseminated through the pneumococcal population (5,
8). Since the first description of an S. pneumoniae
clone, Spain-23F-1, other clones have been identified (18,
30). A Pneumococcal Molecular Epidemiology Network has been newly
established to collect, study, and assign systematic number
designations to S. pneumoniae clones that meet the
Network's criteria (K. Klugman, Letter, ASM News 64:371, 1998).
In February 1997, the Washington State Health Department was notified
of three cases of pneumonia due to ceftriaxone-resistant S. pneumoniae, two of which were fatal. Further investigation found
that all three invasive isolates were from patients in the same
community. The isolates were serogroup 19, were nonsusceptible to
penicillin, and were resistant to ceftriaxone, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Although serogroup 19 represents approximately 10% of pneumococci tested in Washington, ceftriaxone-resistant S. pneumoniae had only rarely been
identified in Washington (2, 10; Centers for Disease
Control and Prevention [CDC], unpublished data). To determine the
magnitude of the problem and to further characterize the isolates, we
selected 14 serogroup 19 multiresistant S. pneumoniae
isolates collected from the hospitals in the community where the
original cluster occurred and from other hospitals throughout
Washington State. We also included four randomly chosen Alaskan
isolates from a pool of multiresistant serotype 19F isolates. We also
chose eight Canadian isolates, serogroup 19, which had antibiograms,
including resistance to erythromycin, cephalosporins, and penicillin,
similar to those of the Washington isolates for comparison. These 26 isolates were compared with previously characterized multiresistant
S. pneumoniae clones using pulsed-field gel electrophoresis
(PFGE) and insertion sequence (IS) restriction fragment length
polymorphism (RFLP) pattern typing.
(This study was presented in part as abstract 10638 at the Eighth
International Congress of Infectious Diseases in Boston, Mass., May
1998, where it won the North American Pasteur-Merieux Connaught Award
in Epidemiology.)
Bacteria.
We examined 26 S. pneumoniae serogroup
19 isolates with diminished susceptibility to penicillin (MIC of >1
µg/ml) and resistance to at least three other antibiotics (Table
1). Seven isolates, including the initial
outbreak cluster, were from three hospitals around Tacoma, Wash., and
were collected during February and March 1997 (WA1 to WA7). Seven
isolates were from other hospitals in Washington and were collected
between December 1995 and April 1996 in a prior survey (WA8 to WA14).
The Washington isolates were from adults; most were from hospitals in
the Puget Sound region, which includes Tacoma and represents the major
portion of the state's population. The Arctic Investigations Program
(AIP), National Center for Infectious Disease, CDC, provided four
serogroup 19 isolates from adults treated at hospitals in Alaska (AK15
to AK18). The Alaskan isolates were randomly chosen from a pool of multiresistant serotype 19F isolates. Eight isolates (CN19 to CN26)
from children in metropolitan Toronto and the neighboring urban Peel
region of Canada were chosen because they were serogroup 19 and had
antibiograms similar to those of the Washington isolates, being
resistant to erythromycin, cephalosporins, and penicillin. Some
clinical and antimicrobial susceptibility data from the Canadian isolates have been previously reported (15). The isolates
outside of Washington allowed us to determine if genetically related
S. pneumoniae strains were present in regions outside
Washington State. In addition, six isolates representing six known,
characterized clones (267-Spain-23F-1, 681-Spain-6B-2, 665-France-9V-3,
17219-South Africa-19A-7, 50803-South Africa-6B-8, and 51702-South
Africa-19A-unnumbered) were provided by the Pneumococcal Diseases
Research Unit at the South African Institute for Medical Research,
University of the Witwatersrand, Johannesburg, South Africa (SP27 to
SP32).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Novel Multiresistant Streptococcus
pneumoniae Serogroup 19 Clone from Washington State Identified by
Pulsed-Field Gel Electrophoresis and Restriction Fragment Length
Patterns
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 µg/ml) were included in the
study. Pulsed-field gel electrophoresis (PFGE) with ApaI,
SacII, and SmaI restriction enzymes and
IS1167 and mef restriction fragment length
polymorphism (RFLP) pattern analysis were performed. Twenty of the 26 isolates had identical or related PFGE patterns, with two or all three enzymes, and identical or related IS1167 RFLP patterns,
indicating that they were genetically related. These 20 isolates
contained the mef gene conferring erythromycin resistance
and had identical mef RFLP patterns. The PFGE and RFLP
patterns were distinct from those of six multiresistant clones
previously described and suggest that a new multiresistant clone has
appeared in Washington, Alaska, and eastern Canada. This newly
characterized clone should be included in the Pneumococcal Molecular
Epidemiology Network.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Characteristics of S. pneumoniae isolates
Serology. Serogroups were determined by the Quellung reaction (25) by our laboratory and confirmed by the University of Washington Medical Center and/or the AIP. Subtyping was performed by counterimmunodiffusion electrophoresis at the AIP.
Antibiograms.
The MIC was determined using agar dilution
following the National Committee for Clinical Laboratory Standards
guidelines (9, 19). The Washington and Canadian isolates
were tested with the following antibiotics: penicillin, cefotaxime,
ceftriaxone, cefprozil, ceftazidime, loracarbef, erythromycin,
azithromycin, clindamycin, ciprofloxacin, grepafloxacin, levofloxacin,
sparfloxacin, trovafloxacin, linezolid, and HMR3647 (a new ketolide)
(Table 2). Isolates from Alaska were not
included in the full antibiogram determination.
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Media and growth conditions.
Bacteria were grown on brucella
blood agar (Difco) supplemented with 5% sheep red blood cells and
incubated for 18 to 24 h with 5% CO2 at 36.5°C.
Mueller-Hinton agar (Difco) supplemented with 5% sheep red blood cells
and appropriate concentrations of antibiotics was used for the agar
dilutions (19). Bacterial stocks were maintained at 
70°C
in sterile skim milk. Aliquots were subcultured onto appropriate media
from the frozen stocks as needed. Purity was maintained by stringent
aseptic techniques and confirmed by biochemical methods as described
previously (25).
PFGE analysis. Bacteria were grown for 18 to 24 h on brucella blood agar plates at 36.5°C in CO2, harvested, and made into blocks with 1% low-melting-point agarose (Bio-Rad Laboratories, Richmond, Calif.) in a PFGE mold provided by the manufacturer (Bio-Rad) as previously described (16, 23). The blocks were digested with proteinase K (Sigma) (100 µg/ml), washed, and stored at 4°C as described previously (16, 23). Gel plugs (approximately 3 by 6 mm) were cut from the blocks and digested for 20 to 24 h with 35 U of ApaI (Promega, Madison, Wis.) or SacII (Promega) at 37°C or SmaI (Promega) at 25°C as described previously (16, 23).
The digested gel blocks were embedded in a 1% agarose gel (SeaKem; FMC Corporation, Rockland, Maine) prepared with 0.5× Tris-borate-EDTA (TBE) (pH 8.0) and run using a CHEF-DRII (contour-clamped horizontal electrophoresis) apparatus (Bio-Rad) at 175 V for 20 h for SmaI-digested DNA gel plugs and 22 h for SacII- and ApaI-digested gel plugs. DNA bands were visualized by ethidium bromide and UV light and photographed as previously described (23, 31). Analysis of the PFGE patterns was performed by visual inspection of the photographs. ApaI and SacII produced PFGE patterns of 10 to 15 DNA bands between 45.5 and 291.5 kb, while SmaI digests produced PFGE patterns with 8 to 12 DNA bands between 45.5 and 291.5 kb. The most common PFGE patterns were designated with the first letter of the enzyme and an assigned number (A37 to A50 for ApaI and S26 to S37 for SmaI). We did not start with A1 because these PFGE patterns have previously been assigned to S. pneumoniae 6B isolates from other parts of the United States (23). To distinguish SacII from SmaI patterns, C was used for the designated name for SacII patterns (C8 to C21). Isolates with DNA patterns that differed by three or fewer bands from the main pattern were considered to be related and given a subscripted number starting with 1 (A371, A382, etc.), as the band difference could be explained by one genetic event (23, 31). Isolates which had a difference of more than three DNA bands were considered unrelated and were given consecutive numbers as they appeared (A38, A39, A40, etc.). Isolates that were identical or highly related (three or fewer bands) by two or three restriction enzyme PFGE patterns were considered to be genetically related. We have found this criterion valuable in other studies with S. pneumoniae as well as for other pathogens (23, 31). This classification is more stringent than the five-band difference previously suggested by Tenover et al. (29).IS1167 RFLP typing. Whole-cell DNA extracts were prepared from isolates grown in 100 ml of brain heart infusion broth supplemented with 0.6% D-glucose plus 0.03% DL-threonine and incubated at 36.5°C in 5% CO2, as previously described (1, 16, 17). DNA was digested with 80 U of HindIII restriction enzyme and run on a 0.7% agarose gel in 0.5× TBE buffer at 100 V for 3.0 h (26). Southern blots were prepared from the agarose gels and hybridized with a 32P-labeled oligonucleotide probe, DAMO13 (5'-TGG ATA TTA TGG AGC CT-3') (22, 32). This probe is specific for an upstream region of insertion sequence IS1167 (22, 32). The bands were counted, and patterns that differed in band number or size of band by more than one band were considered unrelated. RFLP typing and analysis were performed multiple times.
mef RFLP typing. Whole-cell DNA extracts were prepared from isolates grown in 100 ml of brain heart infusion as described for the IS1167 RFLP typing. DNA was digested with 80 U of HindIII restriction enzyme and run on a 0.7% agarose gel in 0.5× TBE buffer at 100 V for 3.0 h (26). Southern blots were prepared from the agarose gels and hybridized with a 32P-labeled oligonucleotide probe, MF5 (5'-GGT GCT GTG ATT GCA TCT ATT AC-3'), that is specific for the mef gene (17, 28). The bands were counted and analyzed as described for the IS1167 RFLP analysis. RFLP typing and analysis were performed multiple times. This is the first time that the mef gene has been used for RFLP typing.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antibiogram analysis.
Nonsusceptibility to penicillin
was a criterion for inclusion in the study. The 22 Washington and
Canadian isolates were also generally resistant to the cephalosporins.
Cefprozil, ceftazidime, and loracarbef showed the highest MICs (MIC
ranges, 8 to 16, 16 to 32, and 64 to 128 µg/ml, respectively) (Table
2). Of the 26 isolates, 20 were resistant to erythromycin and
azithromycin (MIC, 1 µg/ml). One isolate (WA9) was inducibly
resistant to the macrolides and clindamycin (MIC, 128 µg/ml for
both) and carried both the ermB and mef genes
(17, 27). The other 19 isolates carried the mef
gene and were resistant to macrolides (MIC range, 1 to 8 µg/ml) but
susceptible to clindamycin (MIC, 
0.25 µg/ml). The quinolones,
except for ciprofloxacin, had very low MICs. The MICs of linezolid were
comparable to those of the quinolones. The new compound HMR3647 had the
lowest MICs of any of the antibiotics tested (MIC range, 0.002 to 0.016 µg/ml).
PFGE analysis of the 26 clinical isolates.
Among the 26 S. pneumoniae isolates, 20 were considered to be genetically
related because they had identical or highly related PFGE patterns with
two or three enzymes (Table 3). Of these
20 isolates, 6 Washington isolates (WA7, WA8, WA9, WA11, WA12, and WA14) and 2 Alaska isolates (AK15 and AK18) of S. pneumoniae
had identical PFGE patterns with all three enzymes. Three Washington isolates (WA1, WA3, and WA5) had identical patterns with two enzymes and had highly related PFGE patterns with one enzyme (ApaI).
Two Washington isolates (WA2 and WA4) had highly related PFGE patterns with SacII and identical patterns with the other two
enzymes. Two Washington isolates (WA10 and WA13) and one Alaska isolate (AK17) had identical PFGE patterns with one enzyme (SacII)
and highly related patterns with the other two enzymes. One Alaskan (AK16) and three Canadian (CN19, CN20, and CN24) isolates had related
PFGE patterns for each of the three enzymes.
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PFGE comparison of related isolates with multiresistant
clones.
Isolate WA8, with the most common PFGE pattern for all
three enzymes (A37, C8, and S26), was selected as a representative of
the related isolates and was compared to the six previously known,
multiresistant clones (Fig. 1). Five of
the six clone isolates had unique PFGE patterns with all three enzymes
from the PFGE patterns of isolate WA8. Isolate SP27 (267-Spain-23F-1)
had distinct PFGE patterns with ApaI and SacII
enzymes (Fig. 1) and a three-band difference for the SmaI
PFGE pattern compared to the SmaI PFGE pattern of isolate
WA8.
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IS1167 RFLP analysis. Of the 20 isolates with the same or related PFGE patterns, restriction fragment length patterns were either identical or had a one-band difference in the IS1167 RFLP patterns. The six clone isolates (SP27 to SP32) had unique IS1167 RFLP patterns distinctly different from that of the Washington clone (data not shown). The remaining Washington isolate (WA6) and the Canadian isolates (CN21, CN22, CN23, CN25, and CN26) had six- to nine-band differences in patterns from the Washington clone (data not shown).
mef RFLP analysis.
All 20 isolates with the same
or related PFGE patterns had identical mef RFLP patterns and
were distinct from unrelated S. pneumoniae isolates that
carried the mef gene (Fig. 2).
The unrelated Washington isolate (WA6) and the unrelated Canadian
isolates (CN21, CN22, CN23, CN25, and CN26) had distinct RFLP patterns
having one to five bands with more than one band different from the
Washington clone (data not shown). In contrast, none of the clone
isolates (SP27 to SP32) carried the mef gene, and RFLP
analysis could not be performed.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PFGE analysis using three different enzymes and IS1167 and mef RFLP typing identified a unique, multiresistant, serogroup 19 S. pneumoniae group of 20 isolates. This clone was characterized by reduced susceptibility to penicillin and resistance to extended-spectrum cephalosporins, erythromycin, and ciprofloxacin. Recent reports have indicated increases in ceftriaxone-resistant pneumococcus-caused illness in Washington (D. B. Jernigan, I. Kargacin, A. Poole, and J. Kobayashi, Program Abstr. 36th Annu. Meet. Infect. Dis. Soc. Am., abstr. 565-Sa, 1998). This newly identified clone most likely contributed to these increases. The Washington isolates were genetically related to the multiresistant isolates obtained from Alaska and eastern Canada but unrelated to five previously characterized, multiresistant clones (30). The Spanish isolate (SP27) representing the Spanish clone (Spain-23F-1) differed from the Washington clone in PFGE patterns with two enzymes, ApaI (Fig. 1) and SacII, and in IS1167 RFLP typing, indicating that it was completely different from the Washington clone. In previous work with S. pneumoniae and Neisseria gonorrhoeae, if any two isolates had an identical or related PFGE pattern with only one of three enzymes used, we did not consider the isolates to be related (23, 31).
The initial three isolates that prompted our investigation were fully resistant to penicillin, cefotaxime, erythromycin, and other antibiotics but were susceptible to vancomycin, the newer quinolones (grepafloxacin, levofloxacin, and trovafloxacin), linezolid, and the investigational antibiotic HMR3647 (Table 2). Reports of these multiresistant isolates in Tacoma, with their associated mortality, presented a challenge to clinicians choosing empiric treatment for severe community-acquired pneumonia. In regions where multiresistant isolates have been identified, clinicians may choose to request that vancomycin, newer quinolones, or linezolid be added to routine laboratory susceptibility testing of invasive S. pneumoniae isolates. In addition, the new compound HMR3647 may offer another therapeutic choice in the future.
The Washington clone was identified in both serotype 19F and serotype 19A isolates. Previous reports have identified capsular transformation of the Spanish clone (267-Spain-23F-1) from serotype 23F to 19F (6, 7, 20). Therefore, the presence of both serotype 19F and serotype 19A may reflect capsular transformation within serogroup 19. The 23-valent pneumococcal polysaccharide vaccine contains both serotype 19F and serotype 19A and is an underutilized prevention tool for multiresistant S. pneumoniae illness (4, 21). All three of the initial patients, including both fatal cases, were eligible to receive the vaccine because of underlying conditions (i.e., cancer and cardiovascular disease). However, there was no documentation that any of the patients had been vaccinated.
Most of the previous work by others to characterize S. pneumoniae clones by PFGE has used only one enzyme and has allowed up to five-band differences in banding patterns (18). Our laboratory used three enzymes, a three-band difference limit, and the requirement that two enzymes give identical or related PFGE patterns. Our laboratory used a one-band difference limit for both the IS1167 and the mef RFLP patterns (22, 31). Klugman and others have proposed that at least two molecular methods be used in identifying clones, as the PFGE with only one enzyme was not sufficient (27; K. Klugman, Letter). In this study, the PFGE and IS1167 and mef RFLP methods correlated well. This is the first demonstration that mef can be used for RFLP analysis of S. pneumoniae.
A recent report from Spain has described a serogroup 19 isolate that is nonsusceptible to penicillin and resistant to cefotaxime (24). Comparison of the Washington clone to this isolate would be of interest. Surveillance of invasive pneumococcal illness in Washington is continuing, which will allow us to monitor trends in antibiotic resistance and to detect any further increase in multiresistant pneumococcal infections. Based on our results, we suggest that this newly characterized clone has all of the characteristics required to be included in the recently organized Pneumococcal Molecular Epidemiology Network as Washington 19-14 (K. Klugman, Letter).
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ACKNOWLEDGMENTS |
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We thank T. Fritsche at the University of Washington Medical Center Clinical Microbiology Laboratory for some of the S. pneumoniae isolates and A. Parkinson at the AIP of the CDC in Anchorage, Alaska, for the four Alaskan isolates and for subtyping the S. pneumoniae isolates. We also thank Renee Kanan of the University of Washington School of Public Health and Community Medicine Preventive Medicine Residency Program for the epidemiology background. We thank K. Klugman from the South African Institute for Medical Research in Johannesburg, South Africa, for the representative members of the multiresistant clones.
This study was supported in part by Glaxo-Wellcome.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathobiology, Box 357238, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195-7238. Phone: (206) 543-8001. Fax: (206) 543-3873. E-mail: marilynr{at}u.washington.edu.
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Discussion
References
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Journal of Clinical Microbiology, April 2000, p. 1575-1580, Vol. 38, No. 4
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Cousin, S. Jr., Whittington, W. L. H., Roberts, M. C.
(2003). Acquired Macrolide Resistance Genes in Pathogenic Neisseria spp. Isolated between 1940 and 1987. Antimicrob. Agents Chemother.
47: 3877-3880
[Abstract] [Full Text] -
Bogaert, D., Hermans, P. W. M., Grivea, I. N., Katopodis, G. S., Mitchell, T. J., Sluijter, M., de Groot, R., Beratis, N. G., Syrogiannopoulos, G. A.
(2003). Molecular Epidemiology of Penicillin-Susceptible Non-{beta}-Lactam-Resistant Streptococcus pneumoniae Isolates from Greek Children. J. Clin. Microbiol.
41: 5633-5639
[Abstract] [Full Text] -
Luna, V. A., Heiken, M., Judge, K., Ulep, C., Van Kirk, N., Luis, H., Bernardo, M., Leitao, J., Roberts, M. C.
(2002). Distribution of mef(A) in Gram-Positive Bacteria from Healthy Portuguese Children. Antimicrob. Agents Chemother.
46: 2513-2517
[Abstract] [Full Text] -
ROBERTS, M. C., RIEDY, C. A., COLDWELL, S. E., NAGAHAMA, S., JUDGE, K., LAM, M., KAAKKO, T., CASTILLO, J. L., MILGROM, P.
(2002). How xylitol-containing products affect cariogenic bacteria. Journal of the American Dental Association
133: 435-441
[Abstract] [Full Text] -
Berthelot-Herault, F., Marois, C., Gottschalk, M., Kobisch, M.
(2002). Genetic Diversity of Streptococcus suis Strains Isolated from Pigs and Humans as Revealed by Pulsed-Field Gel Electrophoresis. J. Clin. Microbiol.
40: 615-619
[Abstract] [Full Text] -
Haasum, Y., Ström, K., Wehelie, R., Luna, V., Roberts, M. C., Maskell, J. P., Hall, L. M. C., Swedberg, G.
(2001). Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on Substrate Km. Antimicrob. Agents Chemother.
45: 805-809
[Abstract] [Full Text] -
Chiou, C.-S., Hsu, W.-B., Wei, H.-L., Chen, J.-H.
(2001). Molecular Epidemiology of a Shigella flexneri Outbreak in a Mountainous Township in Taiwan, Republic of China. J. Clin. Microbiol.
39: 1048-1056
[Abstract] [Full Text] -
Luna, V. A., Cousin, S. Jr., Whittington, W. L. H., Roberts, M. C.
(2000). Identification of the Conjugative mef Gene in Clinical Acinetobacter junii and Neisseria gonorrhoeae Isolates. Antimicrob. Agents Chemother.
44: 2503-2506
[Abstract] [Full Text]
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