Prevalence of Newer -Lactamases in Gram-Negative Clinical Isolates Col
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《微生物临床杂志》
Center for Research in Anti-Infectives and Biotechnology (CRAB), Department of Medical Microbiology & Immunology, School of Medicine, Creighton University Medical Center, 2500 California Plaza, Omaha, Nebraska 68178
ABSTRACT
Newer -lactamases such as extended-spectrum -lactamases (ESBLs), transferable AmpC -lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC -lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, -lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC -lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC -lactamases were widespread. Clinical laboratories must be able to detect important -lactamases to ensure optimal patient care and infection control.
INTRODUCTION
Pathogens producing extended-spectrum -lactamases (ESBLs) and transferable AmpC -lactamases (often referred to as plasmid-mediated AmpC -lactamases) are an increasing cause of clinical concern. These "newer -lactamases" cause resistance to a wide range of -lactam antibiotics, especially expanded-spectrum cephalosporins. Typically, they are associated with resistance to multiple antibiotics (e.g., aminoglycosides, chloramphenicol, trimethoprim-sulfamethoxazole, and tetracycline), leaving few therapeutic choices (28, 31). Both types of enzymes are associated with potentially fatal laboratory reports of false susceptibility to cephalosporins (26, 28).
Accurate prevalence data are required to evaluate of the success of efforts to control problems associated with these -lactamases. In recent years, many clinical laboratories in the United States have tested for ESBLs in isolates of E. coli and Klebsiella but not other organisms (24) and most laboratories have not attempted to detect transferable AmpC -lactamases (39) or carbapenemases (44). The current approach to -lactamase detection therefore provides an incomplete understanding of the problems posed by these enzymes. For this reason, a study was designed to determine the prevalence in 2001 to 2002 of ESBLs in a wide range of gram-negative species, of transferable AmpC -lactamases in Klebsiella pneumoniae, and of carbapenem-hydrolyzing enzymes in Enterobacteriaceae in United States medical centers.
MATERIALS AND METHODS
Isolates. During the period September 2000 to September 2002, up to 100 consecutive, nonduplicate nosocomial isolates of gram-negative bacilli were tested for susceptibility on-site at each of 63 United States clinical sites in 34 states (listed in Acknowledgments) using custom-made freeze-dried microdilution MIC panels (Dade Behring, Sacramento, CA). The sites were classified as intensive care unit (ICU; n = 42) or non-ICU (n = 21). Nursing homes were included in the latter category. Isolates were screened for the following phenotypic markers. (i) First there was the ESBL screen, which contained any of the following criteria: (a) for all isolates, an eightfold-or-greater decrease in the cefepime MIC in the presence of 10 μg/ml clavulanate (21); (b) for Escherichia spp., Klebsiella spp., Citrobacter koseri, Proteus mirabilis, Kluyvera spp., Salmonella spp., or Shigella spp., a ceftriaxone, ceftazidime, cefepime, or aztreonam MIC of 2 μg/ml (6). (ii) Second there was the AmpC screen, which contained the following criteria: for all K. pneumoniae isolates, a cefoxitin MIC of 16 μg/ml. The screen also included any Klebsiella oxytoca, Proteus mirabilis, or Salmonella isolates that were identified as ESBL or carbapenemase screen positive and also had a cefoxitin MIC of 16 μg/ml (21). (iii). Third, there was the screen for carbapenem-hydrolyzing enzymes, which contained the following parameters: (a) for E. coli, Salmonella, Shigella, or Klebsiella spp., an imipenem MIC of 2 μg/ml; (b) for Enterobacter spp., Serratia spp., and Citrobacter spp., an imipenem MIC of 4 μg/ml (12, 13, 17, 19, 21). Screen-positive isolates were referred to Creighton University for -lactamase analysis. Organism identifications were confirmed where necessary by API 20E, Vitek, or Vitek 2 (bioMerieux Inc., Hazelwood, MO) in combination with any supplementary tests that were appropriate.
Susceptibility testing. An investigational frozen MIC panel (TREK, Westlake, OH) was used to confirm and further investigate phenotypic markers of -lactamase production. The panel contained imipenem, cefoxitin, cefpodoxime, cefpodoxime plus 4 μg/ml clavulanate, ceftazidime, ceftazidime plus 4 μg/ml clavulanate, cefepime, cefepime plus 10 μg/ml clavulanate, and aztreonam. CLSI (formerly NCCLS) methodology and interpretive criteria were used where applicable (6, 23).
-Lactamase investigations. ESBL production was confirmed phenotypically as an eightfold-or-greater reduction in the MIC of cefpodoxime, ceftazidime, or cefepime in the presence of clavulanate with the following exceptions. (i) If the imipenem MIC was elevated sufficiently to yield a positive screen for carbapenem-hydrolyzing enzymes, the possibility of a clavulanate-susceptible class A carbapenem-hydrolyzing enzyme was also investigated. (ii) If the cefepime ± clavulanate test was positive (eightfold reduction in cefepime MIC) but the cefepime MIC was low (0.5 μg/ml) and the other ESBL screening antibiotics (ceftazidime, cefpodoxime, and aztreonam) also had low MICs that would be considered ESBL screen negative, the ESBL status was not considered as confirmed. This was because molecular and biochemical testing indicated that the cefepime ± clavulanate test was overly sensitive for some highly susceptible isolates. AmpC-producing isolates were initially screened for ESBL production with tests involving the three cephalosporins (cefepime, cefpodoxime, or ceftazidime) ± clavulanate. The cefepime ± clavulanate method was included because it had been reported to be effective for ESBL detection in organisms such as Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa (7, 9, 10, 25, 43).
Transferable AmpC -lactamases were initially investigated by the AmpC disk test (3) and three-dimensional test (40). Inducibility with cefoxitin was investigated by disk diffusion and broth-based methodologies (35, 36). Klebsiella pneumoniae was chosen for testing for transferable AmpC -lactamases because this species lacks a chromosomal AmpC -lactamase and is a common host for transferable AmpC -lactamases (1, 21). While screen-positive isolates were being received at Creighton University, it became apparent that additional species lacking a chromosomal AmpC -lactamase also had phenotypes suggestive of AmpC production. These were also investigated for AmpC production. They included Klebsiella oxytoca, Proteus mirabilis, and Salmonella. For these organisms, site occurrence data, not prevalence data, were determined. This was because it was possible that only a subset of all of the isolates of these organisms with a cefoxitin MIC of 16 μg/ml were submitted for analysis.
E. coli, which produces a chromosomally mediated AmpC -lactamase, was not tested for production of transferable AmpC -lactamases. In an organism that may produce both types of -lactamase, it is impossible for phenotypic tests to discriminate between transferable and chromosomal AmpC -lactamases, and the study budget was insufficient for molecular testing for transferable AmpC -lactamases in this species.
All -lactamases were investigated by isoelectric focusing (IEF) overlay procedures to determine the pI(s) of each isolate's -lactamase(s), the capabilities of clavulanate and cloxacillin to inhibit the -lactamase(s), and the capability of the -lactamase(s) to hydrolyze cefotaxime or imipenem (2, 34). Carbapenemase activity was also confirmed by microbiological and spectrophotometric hydrolysis assays (18, 21, 36).
Molecular identification tests utilized PCR primers specific for genes encoding TEM-, SHV-, OXA-, CTX-M-, PSE-, KPC-, NMC-A-, IMI-, and OXY-derived -lactamases (Table 1) (8, 20). blaCTX-M families were differentiated on the basis of PCR primer specificity (32). The CTX-M-1 family includes enzymes such as CTX-M-1, -3, -10, -11, -12, and -22, while the CTX-M-9 family includes enzymes such as CTX-M-9, -13, -14, -15, -16, -17, -19, and -21. AmpC gene identification was investigated by an ampC multiplex PCR using primers specific for blaDHA, blaFOX, blaCMY, blaMOX, blaACC, and blaACT (30), with DNA sequencing done as needed using primers which flanked the gene. Amplified products were sequenced at least two times by automated PCR cycle sequencing with dye terminator chemistry using a DNA stretch sequencer from Applied Biosystems. Sequence alignment and analysis were performed online using the BLAST program of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
RESULTS
During the study period, 6,421 consecutive, nonduplicate, clinical isolates of aerobically growing gram-negative bacilli from patients at 42 ICU and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. Results for all -lactamase types are summarized in Table 2, and limited susceptibility data are provided in Tables 3 and 4.
ESBLs. Two hundred isolates that were confirmed as ESBL producers (i.e., 3.1% of all isolates) were submitted by 40 of the 63 sites (63%) (Table 2). ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae). Seventy-four percent of ICU sites harbored ESBL-producing isolates compared to 43% of non-ICU sites. Thirteen ICU sites and 3 non-ICU sites had least three different types of ESBLs.
The most common ESBL producers were K. pneumoniae (96 isolates, 11.3% of all K. pneumoniae isolates) and E. coli (42 isolates, 2.3%), followed by Enterobacter cloacae (25 isolates, 5.5%) and K. oxytoca (18 isolates, 13.1%). The latter two species were more often ESBL positive in non-ICU sites than in ICUs (14.3% and 23%, respectively, of non-ICU isolates were positive, compared to 4.3% and 9.2% for ICUs). ESBLs were uncommon in other members of the Enterobacteriaceae (19/1,072 isolates, or 1.8% of these organisms overall). Although the numbers were small, ESBL production appeared to be more common in non-ICU isolates of S. marcescens (4/45, or 8.9% prevalence) than in ICU isolates (1/261, 0.4% prevalence), while ESBL-producing P. mirabilis isolates were detected only at ICU sites.
TEM- and SHV-derived ESBLs were identified from isoelectric points, hydrolytic properties, and PCR results, but were not sequenced. SHV types were the most common ESBLs, with 162 isolates producing at least one SHV ESBL (data not shown). These comprised 93 K. pneumoniae, 25 E. cloacae, 17 K. oxytoca, 15 E. coli, 5 S. marcescens, and 2 E. aerogenes isolates and 1 isolate each of C. freundii, C. koseri, Citrobacter sp., Klebsiella sp., and P. mirabilis. Twenty isolates produced at least one TEM ESBL (8 E. coli, 6 K. pneumoniae, 3 P. mirabilis, and 2 K. oxytoca isolates and 1 C. koseri isolate). Seventeen E. coli isolates from 13 states produced a CTX-M ESBL. Nine produced an ESBL from the CTX-M-1 family, and eight produced an ESBL from the CTX-M-9 family. The states with CTX-M-producing isolates were Arizona, Florida, Idaho, Illinois, Kentucky, Ohio, Oklahoma, Pennsylvania, Texas, Utah, Virginia, Washington, and West Virginia.
Eighteen isolates of Klebsiella spp. (17 K. pneumoniae isolates and 1 K. oxytoca isolate) coproduced an SHV ESBL and a transferable AmpC -lactamase. These isolates were predominantly from ICU sites (17/18). One of these isolates also produced a TEM-derived ESBL.
ESBLs were not detected in isolates outside the family Enterobacteriaceae. Four of 1,069 isolates of P. aeruginosa and none of 66 isolates of Alcaligenes spp. were submitted as screen-positive isolates requiring further analysis. The remaining non-Enterobacteriaceae (i) comprised only small numbers of isolates for which all isolates were ESBL negative, or else (ii) the taxa proved to be technically too difficult to evaluate and ESBL detection attempts were eventually abandoned. For the latter organisms, phenotypic tests were consistently positive but other more discriminating tests (primarily IEF overlay tests) did not provide evidence of ESBL production. These organisms were Acinetobacter spp. (n = 210 isolates, of which 56 were submitted as screen positive), Stenotrophomonas maltophilia (n = 140 isolates, of which 47 were submitted as screen positive), and Chryseobacterium spp. (n = 3). For S. maltophilia and Chryseobacterium spp., the apparently false-positive phenotypic tests were attributed to the production of chromosomally encoded, clavulanate-susceptible -lactamases with expanded substrate profiles. The reason for the apparent false-positive tests with Acinetobacter spp. was not determined.
Transferable AmpC -lactamases. Transferable AmpC -lactamases were detected in 28 of 853 isolates of K. pneumoniae (3.3%) at 12 of the 63 sites (19%), with similar prevalences for both ICU and non-ICU sites (3.5% and 2.6%, respectively) (Table 2). Transferable AmpC enzymes were also detected in 5 of 137 K. oxytoca isolates (3.6%), 5 of 359 P. mirabilis isolates (1.4%), and 2 of 4 Salmonella isolates. The latter numbers probably underestimate the true prevalence in these species because, unlike K. pneumoniae, they were not screened for cefoxitin insusceptibility and were only submitted for analysis for other reasons. When all transferable AmpC-producing isolates are considered, a total of 16 of the 63 sites (25%) were positive (29% of ICUs, 19% of non-ICUs). Most of the isolates were from eastern (Florida, Maryland, New York, and New Jersey), southern (Louisiana, Georgia, Texas, and Virginia), and midwestern (Indiana and Illinois) states. The only western state with transferable AmpC-producing isolates was Arizona. Three sites had more than one type of transferable AmpC.
Seventeen of the 28 AmpC-producing K. pneumoniae isolates (61%) coproduced an ESBL. The susceptibilities of the transferable AmpC-producing isolates were as follows: imipenem, 100%; cefepime, 58% (96%, if the CLSI ESBL reporting rule was not applied); aztreonam, 48%; ceftazidime, 15%; and cefoxitin, 5% (Table 3). Only one of these isolates had a cefepime MIC exceeding the CLSI susceptibility breakpoint of 8 μg/ml.
IEF and PCR data indicated that FOX-like enzymes were the predominant transferable AmpC -lactamases. Twenty-six K. pneumoniae isolates produced FOX-like enzymes, while only single isolates of this species produced inducible ACT-1-like and DHA-like enzymes. Four K. oxytoca isolates produced a FOX-like enzyme, and one isolate produced a DHA-like enzyme. Four P. mirabilis isolates produced a CMY-like enzyme, and one produced FOX-5. Two Salmonella isolates produced a CMY-like enzyme. Seven representative FOX-like AmpCs were sequenced and identified as FOX-5.
Carbapenemases. Three of the 63 sites (4.8%) submitted isolates that produced enzymes with carbapenem-hydrolyzing activity that were subsequently confirmed as molecular class A carbapenemases (Table 2). The three isolates comprised 0.05% of all isolates. KPC-2 was produced by a K. pneumoniae isolate from New York, NY, and by an E. cloacae isolate from Boston, MA. An E. cloacae isolate from New York produced an NMC-A- or IMI-1-like enzyme (not sequenced to discriminate between the two enzymes).
Antibiotic susceptibility data. Only limited susceptibility data were determined because the MIC panels used at Creighton University were designed primarily for diagnostic purposes and included drug combinations that lack interpretive criteria. The data for the ESBL- and transferable AmpC-producing isolates are summarized in Table 3, and the data for the three carbapenemase-producing isolates are provided in Table 4. These data indicated that the carbapenem imipenem was more active than cephalosporins, cefoxitin, or aztreonam against isolates that produced ESBLs and/or transferable AmpC -lactamases. None of the -lactam agents tested was active against all three class A carbapenemase-producing isolates.
DISCUSSION
ESBL- and transferable AmpC-producing Enterobacteriaceae were widespread in this study, while class A carbapenemase-producing strains isolates were far less frequently encountered. These enzymes were not detected in taxa outside the Enterobacteriaceae.
Enterobacteriaceae other than E. coli and Klebsiella spp. harbored ESBLs, especially E. cloacae in non-ICU settings (14.3% prevalence). E. coli isolates producing CTX-M ESBLs appear to be spreading. At least three different types of ESBLs were detected at 16 sites, indicating possibly different types of problems from sites where a single ESBL was widespread. Spread of a single ESBL may reflect ineffective infection control within an institution, while the presence of multiple types of ESBLs could either indicate a diversity of sources of ESBLs outside the institution (i.e., different patient populations) or a selection pressure within an institution (i.e., a prescribing problem) favoring the de novo selection of ESBLs. ESBL-producing non-E. coli/Klebsiella isolates were detected at six of these sites. ESBL-producing isolates of E. cloacae were detected at the two sites that shared the highest ESBL prevalence rate: 18% of all gram-negative isolates producing at least one ESBL (data not shown). This indicates that sites with apparently uncontrolled ESBL problems had ESBL-producing isolates that were not E. coli or Klebsiella spp. that may have been undetected ESBL reservoirs that sustained the outbreaks. This possibility provides an additional incentive besides the clinical (therapeutic) one to extend ESBL testing beyond E. coli, Klebsiella spp., and P. mirabilis when laboratories service patient populations with ESBL problems.
Schwaber et al. recently suggested that such testing was unnecessary for cost-benefit reasons because they detected ESBLs in only 15 of 690 isolates (2.2%) of organisms other than E. coli and Klebsiella spp. from 53 United States medical sites (37). While our data were comparable (43 of 1,517 isolates, or 2.8% of non-E. coli/Klebsiella isolates of Enterobacteriaceae were ESBL positive), 16 of the 63 sites had these types of organisms, with some sites having clusters of such isolates and others having large ESBL problems in which these organisms may have been contributing factors. Schwaber et al. concluded that ESBL testing was unnecessary for all of these types of isolates because some of their isolates were highly resistant to ESBL screening drugs, which meant that ESBL detection would not impact susceptibility reporting for these drugs. This conclusion ignores the need to know the ESBL status of a pathogen should cefepime therapy be considered. Seventeen of our 25 ESBL-producing E. cloacae isolates were apparently susceptible to cefepime (MICs of 8 μg/ml), and only 2 had MICs of the three ESBL screening drugs tested below 64 μg/ml. Clearly for these isolates, ESBL detection is vital to avoid inappropriate cefepime therapy. We therefore believe that ESBL testing of such isolates has clinical and epidemiological value and is justified in patient populations where ESBLs are causing problems.
The 3.3% prevalence rate for transferable AmpC-producing K. pneumoniae in this study was less than the 8.5% reported by Alvarez et al., who sampled 70 United States sites during the period 1992 to 2000 (1). The difference in rates probably reflects sampling differences. Our study comprised consecutive, nonduplicate isolates, whereas Alvarez et al. included strains from some previous studies of antibiotic-resistant and/or ESBL-producing isolates, which were probably more resistant isolates and increased their yield of AmpC producers. In our study, if the additional species tested for transferable AmpC -lactamases are included, transferable AmpC -lactamases were detected at 25% of sites. However, we did not test E. coli, a known producer of transferable AmpC -lactamases (9, 27, 31). If E. coli isolates were also tested, the actual percentage of positive sites may have been even higher than 25%. The occurrence of rare isolates of K. pneumoniae and K. oxytoca that produced inducible ACT-1-like and DHA-like enzymes indicates the need for vigilance because inducible transferable AmpC -lactamases may confer a high risk of therapeutic failures with cephalosporins (26).
Clinical laboratories should test for transferable AmpC -lactamases to prevent their spread, to protect patients from therapy with inappropriate antibiotics (14, 26, 39), and to recognize strains in which AmpC -lactamases may mask the detection of an accompanying ESBL (7, 22, 38, 39, 41, 43) and result in fatal, inappropriate cephalosporin therapy (38). In our study, ESBLs were present in 17 of 28 transferable AmpC-producing K. pneumoniae isolates (61%).
The low prevalence of carbapenemases detected in Enterobacteriaceae may underestimate the current status of these enzymes in the United States. Since the completion of this study, there have been several reports of United States isolates of Enterobacteriaceae producing class A carbapenemases (4, 5, 12, 13, 33, 45) and also reports of rare P. aeruginosa isolates producing a metallo--lactamase (11, 16, 42). These are formidable enzymes that clinical laboratories should detect. Major concerns are the multiple antibiotic resistance with which they are associated, the possibility of reporting false in vitro susceptibility to carbapenems, and the possibility that the recent spread of metallo--lactamase-producing pathogens in Asia, Australia, Canada, Europe, and South America may extend to the United States (15, 29, 44).
In conclusion, Enterobacteriaceae producing ESBLs and transferable AmpC -lactamases were found to be widespread in the United States, while carbapenemase-producing isolates were rare. Each of these -lactamases is associated with multiple antibiotic resistance and problems of false in vitro susceptibility. It is therefore vital that clinical laboratories be able to detect these enzymes so that patients can receive appropriate antibiotic therapy and to ensure that infection control efforts are effective.
ACKNOWLEDGMENTS
This study was supported by a grant from Merck & Co., Inc.
We thank T. J. Lockhart and Lloyd B. Olson III for their excellent technical assistance. We gratefully acknowledge the contributing laboratories at the following ICU sites for testing, documenting, and submitting isolates: Arkansas Children's Hospital, Little Rock, AR; Auburn Memorial, Auburn, NY; Bellevue Hospital, New York, NY; Charleston Area Medical Center, Charleston, WV; Children's Hospital, Los Angeles, CA; Children's Memorial Hospital, Chicago, IL; Christus Schumpert Health Systems, Shreveport, LA; Clarian Health Methodist Hospital, Indianapolis, IN; Cleveland Clinic Foundation, Cleveland, OH; Columbian Presbyterian Medical Center, New York, NY; Cook County Hospital, Chicago, IL; Crawford Long Hospital, Atlanta, GA; Emory University Hospital, Atlanta, GA; Fletcher Allen HealthCare, Burlington, VT; Good Samaritan Regional Medical Center, Laboratory Sciences of Arizona, Tempe, AZ; Inova Fairfax Hospital, Falls Church, VA; Jackson Memorial Hospital, Miami, FL; Massachusetts General Hospital, Boston, MA; Roper Hospital, Charleston, SC; Shands Hospital, Gainesville, FL; St. Alphonsus Regional Medical Center, Boise, ID; St. Joseph's Hospital, Atlanta, GA; St. Luke's Hospital of Kansas City, Kansas City, MO; Stanford Hospital and Clinics, Palo Alto, CA; Sunrise Hospital, Las Vegas, NV; Thomason Hospital, El Paso, TX; Tulane University Hospital & Clinic, New Orleans, LA; University of California, San Francisco, Moffitt, CA, University of California Irvine Medical Center, Orange, CA; University of Chicago Hospital, Chicago, IL; University of Colorado Hospital, Denver, CO; University of Kentucky Hospital, Lexington, KY; University of Nebraska, Omaha, NE; University of Pennsylvania Medical Center, Philadelphia, PA; University of Rochester Medical Center, Rochester, NY; University of Washington, Seattle, WA; VA Maryland Healthcare System, Baltimore, MD; VA North Texas Health Care System, Dallas, TX; Vencor Central Hospital, Tampa, FL; Wake Medical Center, Raleigh, NC; and Wishard Health Services, Indianapolis, IN. We also gratefully acknowledge the contributing laboratories at the following non-ICU sites: Acadiana Medical Laboratories, Ltd., Lafayette, LA; Alton Ochsner Medical Foundation, New Orleans, LA; American Medical Labs New Kindred Hospital, Chantilly, VA; Baptist Health System, San Antonio, TX; Baylor University Medical Center, Dallas, TX; Burdette Tomlin Memorial Hospital, Cape May Court House, NJ; Clinical Laboratories, Inc., Throop, PA; Crawford Long Hospital, Atlanta, GA; Dyna Care Laboratories, Oklahoma City, OK; Emory Hospital, Atlanta, GA; Holy Name Hospital, Teaneck, NJ; Inova Fairfax Hospital, Falls Church, VA; Jamaica Hospital Medical Center, Jamaica, NY; LDS Hospital, Salt Lake City, UT; Northwest Medical Specialists, Tacoma, WA; Rochester General Hospital, New York, NY; Temple University Hospital, Philadelphia, PA; University of Colorado Hospital, Denver, CO; University of Kentucky Hospital, Lexington, KY; and Vencor of Arizona Laboratory Sciences of Arizona, Tempe, AZ.
FOOTNOTES
Corresponding author. Mailing address: Center for Research in Anti-Infectives and Biotechnology (CRAB), Department of Medical Microbiology & Immunology, School of Medicine, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-4096. Fax: (402) 280-1875. E-mail: kstaac@creighton.edu.
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ABSTRACT
Newer -lactamases such as extended-spectrum -lactamases (ESBLs), transferable AmpC -lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC -lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, -lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC -lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC -lactamases were widespread. Clinical laboratories must be able to detect important -lactamases to ensure optimal patient care and infection control.
INTRODUCTION
Pathogens producing extended-spectrum -lactamases (ESBLs) and transferable AmpC -lactamases (often referred to as plasmid-mediated AmpC -lactamases) are an increasing cause of clinical concern. These "newer -lactamases" cause resistance to a wide range of -lactam antibiotics, especially expanded-spectrum cephalosporins. Typically, they are associated with resistance to multiple antibiotics (e.g., aminoglycosides, chloramphenicol, trimethoprim-sulfamethoxazole, and tetracycline), leaving few therapeutic choices (28, 31). Both types of enzymes are associated with potentially fatal laboratory reports of false susceptibility to cephalosporins (26, 28).
Accurate prevalence data are required to evaluate of the success of efforts to control problems associated with these -lactamases. In recent years, many clinical laboratories in the United States have tested for ESBLs in isolates of E. coli and Klebsiella but not other organisms (24) and most laboratories have not attempted to detect transferable AmpC -lactamases (39) or carbapenemases (44). The current approach to -lactamase detection therefore provides an incomplete understanding of the problems posed by these enzymes. For this reason, a study was designed to determine the prevalence in 2001 to 2002 of ESBLs in a wide range of gram-negative species, of transferable AmpC -lactamases in Klebsiella pneumoniae, and of carbapenem-hydrolyzing enzymes in Enterobacteriaceae in United States medical centers.
MATERIALS AND METHODS
Isolates. During the period September 2000 to September 2002, up to 100 consecutive, nonduplicate nosocomial isolates of gram-negative bacilli were tested for susceptibility on-site at each of 63 United States clinical sites in 34 states (listed in Acknowledgments) using custom-made freeze-dried microdilution MIC panels (Dade Behring, Sacramento, CA). The sites were classified as intensive care unit (ICU; n = 42) or non-ICU (n = 21). Nursing homes were included in the latter category. Isolates were screened for the following phenotypic markers. (i) First there was the ESBL screen, which contained any of the following criteria: (a) for all isolates, an eightfold-or-greater decrease in the cefepime MIC in the presence of 10 μg/ml clavulanate (21); (b) for Escherichia spp., Klebsiella spp., Citrobacter koseri, Proteus mirabilis, Kluyvera spp., Salmonella spp., or Shigella spp., a ceftriaxone, ceftazidime, cefepime, or aztreonam MIC of 2 μg/ml (6). (ii) Second there was the AmpC screen, which contained the following criteria: for all K. pneumoniae isolates, a cefoxitin MIC of 16 μg/ml. The screen also included any Klebsiella oxytoca, Proteus mirabilis, or Salmonella isolates that were identified as ESBL or carbapenemase screen positive and also had a cefoxitin MIC of 16 μg/ml (21). (iii). Third, there was the screen for carbapenem-hydrolyzing enzymes, which contained the following parameters: (a) for E. coli, Salmonella, Shigella, or Klebsiella spp., an imipenem MIC of 2 μg/ml; (b) for Enterobacter spp., Serratia spp., and Citrobacter spp., an imipenem MIC of 4 μg/ml (12, 13, 17, 19, 21). Screen-positive isolates were referred to Creighton University for -lactamase analysis. Organism identifications were confirmed where necessary by API 20E, Vitek, or Vitek 2 (bioMerieux Inc., Hazelwood, MO) in combination with any supplementary tests that were appropriate.
Susceptibility testing. An investigational frozen MIC panel (TREK, Westlake, OH) was used to confirm and further investigate phenotypic markers of -lactamase production. The panel contained imipenem, cefoxitin, cefpodoxime, cefpodoxime plus 4 μg/ml clavulanate, ceftazidime, ceftazidime plus 4 μg/ml clavulanate, cefepime, cefepime plus 10 μg/ml clavulanate, and aztreonam. CLSI (formerly NCCLS) methodology and interpretive criteria were used where applicable (6, 23).
-Lactamase investigations. ESBL production was confirmed phenotypically as an eightfold-or-greater reduction in the MIC of cefpodoxime, ceftazidime, or cefepime in the presence of clavulanate with the following exceptions. (i) If the imipenem MIC was elevated sufficiently to yield a positive screen for carbapenem-hydrolyzing enzymes, the possibility of a clavulanate-susceptible class A carbapenem-hydrolyzing enzyme was also investigated. (ii) If the cefepime ± clavulanate test was positive (eightfold reduction in cefepime MIC) but the cefepime MIC was low (0.5 μg/ml) and the other ESBL screening antibiotics (ceftazidime, cefpodoxime, and aztreonam) also had low MICs that would be considered ESBL screen negative, the ESBL status was not considered as confirmed. This was because molecular and biochemical testing indicated that the cefepime ± clavulanate test was overly sensitive for some highly susceptible isolates. AmpC-producing isolates were initially screened for ESBL production with tests involving the three cephalosporins (cefepime, cefpodoxime, or ceftazidime) ± clavulanate. The cefepime ± clavulanate method was included because it had been reported to be effective for ESBL detection in organisms such as Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa (7, 9, 10, 25, 43).
Transferable AmpC -lactamases were initially investigated by the AmpC disk test (3) and three-dimensional test (40). Inducibility with cefoxitin was investigated by disk diffusion and broth-based methodologies (35, 36). Klebsiella pneumoniae was chosen for testing for transferable AmpC -lactamases because this species lacks a chromosomal AmpC -lactamase and is a common host for transferable AmpC -lactamases (1, 21). While screen-positive isolates were being received at Creighton University, it became apparent that additional species lacking a chromosomal AmpC -lactamase also had phenotypes suggestive of AmpC production. These were also investigated for AmpC production. They included Klebsiella oxytoca, Proteus mirabilis, and Salmonella. For these organisms, site occurrence data, not prevalence data, were determined. This was because it was possible that only a subset of all of the isolates of these organisms with a cefoxitin MIC of 16 μg/ml were submitted for analysis.
E. coli, which produces a chromosomally mediated AmpC -lactamase, was not tested for production of transferable AmpC -lactamases. In an organism that may produce both types of -lactamase, it is impossible for phenotypic tests to discriminate between transferable and chromosomal AmpC -lactamases, and the study budget was insufficient for molecular testing for transferable AmpC -lactamases in this species.
All -lactamases were investigated by isoelectric focusing (IEF) overlay procedures to determine the pI(s) of each isolate's -lactamase(s), the capabilities of clavulanate and cloxacillin to inhibit the -lactamase(s), and the capability of the -lactamase(s) to hydrolyze cefotaxime or imipenem (2, 34). Carbapenemase activity was also confirmed by microbiological and spectrophotometric hydrolysis assays (18, 21, 36).
Molecular identification tests utilized PCR primers specific for genes encoding TEM-, SHV-, OXA-, CTX-M-, PSE-, KPC-, NMC-A-, IMI-, and OXY-derived -lactamases (Table 1) (8, 20). blaCTX-M families were differentiated on the basis of PCR primer specificity (32). The CTX-M-1 family includes enzymes such as CTX-M-1, -3, -10, -11, -12, and -22, while the CTX-M-9 family includes enzymes such as CTX-M-9, -13, -14, -15, -16, -17, -19, and -21. AmpC gene identification was investigated by an ampC multiplex PCR using primers specific for blaDHA, blaFOX, blaCMY, blaMOX, blaACC, and blaACT (30), with DNA sequencing done as needed using primers which flanked the gene. Amplified products were sequenced at least two times by automated PCR cycle sequencing with dye terminator chemistry using a DNA stretch sequencer from Applied Biosystems. Sequence alignment and analysis were performed online using the BLAST program of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
RESULTS
During the study period, 6,421 consecutive, nonduplicate, clinical isolates of aerobically growing gram-negative bacilli from patients at 42 ICU and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. Results for all -lactamase types are summarized in Table 2, and limited susceptibility data are provided in Tables 3 and 4.
ESBLs. Two hundred isolates that were confirmed as ESBL producers (i.e., 3.1% of all isolates) were submitted by 40 of the 63 sites (63%) (Table 2). ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae). Seventy-four percent of ICU sites harbored ESBL-producing isolates compared to 43% of non-ICU sites. Thirteen ICU sites and 3 non-ICU sites had least three different types of ESBLs.
The most common ESBL producers were K. pneumoniae (96 isolates, 11.3% of all K. pneumoniae isolates) and E. coli (42 isolates, 2.3%), followed by Enterobacter cloacae (25 isolates, 5.5%) and K. oxytoca (18 isolates, 13.1%). The latter two species were more often ESBL positive in non-ICU sites than in ICUs (14.3% and 23%, respectively, of non-ICU isolates were positive, compared to 4.3% and 9.2% for ICUs). ESBLs were uncommon in other members of the Enterobacteriaceae (19/1,072 isolates, or 1.8% of these organisms overall). Although the numbers were small, ESBL production appeared to be more common in non-ICU isolates of S. marcescens (4/45, or 8.9% prevalence) than in ICU isolates (1/261, 0.4% prevalence), while ESBL-producing P. mirabilis isolates were detected only at ICU sites.
TEM- and SHV-derived ESBLs were identified from isoelectric points, hydrolytic properties, and PCR results, but were not sequenced. SHV types were the most common ESBLs, with 162 isolates producing at least one SHV ESBL (data not shown). These comprised 93 K. pneumoniae, 25 E. cloacae, 17 K. oxytoca, 15 E. coli, 5 S. marcescens, and 2 E. aerogenes isolates and 1 isolate each of C. freundii, C. koseri, Citrobacter sp., Klebsiella sp., and P. mirabilis. Twenty isolates produced at least one TEM ESBL (8 E. coli, 6 K. pneumoniae, 3 P. mirabilis, and 2 K. oxytoca isolates and 1 C. koseri isolate). Seventeen E. coli isolates from 13 states produced a CTX-M ESBL. Nine produced an ESBL from the CTX-M-1 family, and eight produced an ESBL from the CTX-M-9 family. The states with CTX-M-producing isolates were Arizona, Florida, Idaho, Illinois, Kentucky, Ohio, Oklahoma, Pennsylvania, Texas, Utah, Virginia, Washington, and West Virginia.
Eighteen isolates of Klebsiella spp. (17 K. pneumoniae isolates and 1 K. oxytoca isolate) coproduced an SHV ESBL and a transferable AmpC -lactamase. These isolates were predominantly from ICU sites (17/18). One of these isolates also produced a TEM-derived ESBL.
ESBLs were not detected in isolates outside the family Enterobacteriaceae. Four of 1,069 isolates of P. aeruginosa and none of 66 isolates of Alcaligenes spp. were submitted as screen-positive isolates requiring further analysis. The remaining non-Enterobacteriaceae (i) comprised only small numbers of isolates for which all isolates were ESBL negative, or else (ii) the taxa proved to be technically too difficult to evaluate and ESBL detection attempts were eventually abandoned. For the latter organisms, phenotypic tests were consistently positive but other more discriminating tests (primarily IEF overlay tests) did not provide evidence of ESBL production. These organisms were Acinetobacter spp. (n = 210 isolates, of which 56 were submitted as screen positive), Stenotrophomonas maltophilia (n = 140 isolates, of which 47 were submitted as screen positive), and Chryseobacterium spp. (n = 3). For S. maltophilia and Chryseobacterium spp., the apparently false-positive phenotypic tests were attributed to the production of chromosomally encoded, clavulanate-susceptible -lactamases with expanded substrate profiles. The reason for the apparent false-positive tests with Acinetobacter spp. was not determined.
Transferable AmpC -lactamases. Transferable AmpC -lactamases were detected in 28 of 853 isolates of K. pneumoniae (3.3%) at 12 of the 63 sites (19%), with similar prevalences for both ICU and non-ICU sites (3.5% and 2.6%, respectively) (Table 2). Transferable AmpC enzymes were also detected in 5 of 137 K. oxytoca isolates (3.6%), 5 of 359 P. mirabilis isolates (1.4%), and 2 of 4 Salmonella isolates. The latter numbers probably underestimate the true prevalence in these species because, unlike K. pneumoniae, they were not screened for cefoxitin insusceptibility and were only submitted for analysis for other reasons. When all transferable AmpC-producing isolates are considered, a total of 16 of the 63 sites (25%) were positive (29% of ICUs, 19% of non-ICUs). Most of the isolates were from eastern (Florida, Maryland, New York, and New Jersey), southern (Louisiana, Georgia, Texas, and Virginia), and midwestern (Indiana and Illinois) states. The only western state with transferable AmpC-producing isolates was Arizona. Three sites had more than one type of transferable AmpC.
Seventeen of the 28 AmpC-producing K. pneumoniae isolates (61%) coproduced an ESBL. The susceptibilities of the transferable AmpC-producing isolates were as follows: imipenem, 100%; cefepime, 58% (96%, if the CLSI ESBL reporting rule was not applied); aztreonam, 48%; ceftazidime, 15%; and cefoxitin, 5% (Table 3). Only one of these isolates had a cefepime MIC exceeding the CLSI susceptibility breakpoint of 8 μg/ml.
IEF and PCR data indicated that FOX-like enzymes were the predominant transferable AmpC -lactamases. Twenty-six K. pneumoniae isolates produced FOX-like enzymes, while only single isolates of this species produced inducible ACT-1-like and DHA-like enzymes. Four K. oxytoca isolates produced a FOX-like enzyme, and one isolate produced a DHA-like enzyme. Four P. mirabilis isolates produced a CMY-like enzyme, and one produced FOX-5. Two Salmonella isolates produced a CMY-like enzyme. Seven representative FOX-like AmpCs were sequenced and identified as FOX-5.
Carbapenemases. Three of the 63 sites (4.8%) submitted isolates that produced enzymes with carbapenem-hydrolyzing activity that were subsequently confirmed as molecular class A carbapenemases (Table 2). The three isolates comprised 0.05% of all isolates. KPC-2 was produced by a K. pneumoniae isolate from New York, NY, and by an E. cloacae isolate from Boston, MA. An E. cloacae isolate from New York produced an NMC-A- or IMI-1-like enzyme (not sequenced to discriminate between the two enzymes).
Antibiotic susceptibility data. Only limited susceptibility data were determined because the MIC panels used at Creighton University were designed primarily for diagnostic purposes and included drug combinations that lack interpretive criteria. The data for the ESBL- and transferable AmpC-producing isolates are summarized in Table 3, and the data for the three carbapenemase-producing isolates are provided in Table 4. These data indicated that the carbapenem imipenem was more active than cephalosporins, cefoxitin, or aztreonam against isolates that produced ESBLs and/or transferable AmpC -lactamases. None of the -lactam agents tested was active against all three class A carbapenemase-producing isolates.
DISCUSSION
ESBL- and transferable AmpC-producing Enterobacteriaceae were widespread in this study, while class A carbapenemase-producing strains isolates were far less frequently encountered. These enzymes were not detected in taxa outside the Enterobacteriaceae.
Enterobacteriaceae other than E. coli and Klebsiella spp. harbored ESBLs, especially E. cloacae in non-ICU settings (14.3% prevalence). E. coli isolates producing CTX-M ESBLs appear to be spreading. At least three different types of ESBLs were detected at 16 sites, indicating possibly different types of problems from sites where a single ESBL was widespread. Spread of a single ESBL may reflect ineffective infection control within an institution, while the presence of multiple types of ESBLs could either indicate a diversity of sources of ESBLs outside the institution (i.e., different patient populations) or a selection pressure within an institution (i.e., a prescribing problem) favoring the de novo selection of ESBLs. ESBL-producing non-E. coli/Klebsiella isolates were detected at six of these sites. ESBL-producing isolates of E. cloacae were detected at the two sites that shared the highest ESBL prevalence rate: 18% of all gram-negative isolates producing at least one ESBL (data not shown). This indicates that sites with apparently uncontrolled ESBL problems had ESBL-producing isolates that were not E. coli or Klebsiella spp. that may have been undetected ESBL reservoirs that sustained the outbreaks. This possibility provides an additional incentive besides the clinical (therapeutic) one to extend ESBL testing beyond E. coli, Klebsiella spp., and P. mirabilis when laboratories service patient populations with ESBL problems.
Schwaber et al. recently suggested that such testing was unnecessary for cost-benefit reasons because they detected ESBLs in only 15 of 690 isolates (2.2%) of organisms other than E. coli and Klebsiella spp. from 53 United States medical sites (37). While our data were comparable (43 of 1,517 isolates, or 2.8% of non-E. coli/Klebsiella isolates of Enterobacteriaceae were ESBL positive), 16 of the 63 sites had these types of organisms, with some sites having clusters of such isolates and others having large ESBL problems in which these organisms may have been contributing factors. Schwaber et al. concluded that ESBL testing was unnecessary for all of these types of isolates because some of their isolates were highly resistant to ESBL screening drugs, which meant that ESBL detection would not impact susceptibility reporting for these drugs. This conclusion ignores the need to know the ESBL status of a pathogen should cefepime therapy be considered. Seventeen of our 25 ESBL-producing E. cloacae isolates were apparently susceptible to cefepime (MICs of 8 μg/ml), and only 2 had MICs of the three ESBL screening drugs tested below 64 μg/ml. Clearly for these isolates, ESBL detection is vital to avoid inappropriate cefepime therapy. We therefore believe that ESBL testing of such isolates has clinical and epidemiological value and is justified in patient populations where ESBLs are causing problems.
The 3.3% prevalence rate for transferable AmpC-producing K. pneumoniae in this study was less than the 8.5% reported by Alvarez et al., who sampled 70 United States sites during the period 1992 to 2000 (1). The difference in rates probably reflects sampling differences. Our study comprised consecutive, nonduplicate isolates, whereas Alvarez et al. included strains from some previous studies of antibiotic-resistant and/or ESBL-producing isolates, which were probably more resistant isolates and increased their yield of AmpC producers. In our study, if the additional species tested for transferable AmpC -lactamases are included, transferable AmpC -lactamases were detected at 25% of sites. However, we did not test E. coli, a known producer of transferable AmpC -lactamases (9, 27, 31). If E. coli isolates were also tested, the actual percentage of positive sites may have been even higher than 25%. The occurrence of rare isolates of K. pneumoniae and K. oxytoca that produced inducible ACT-1-like and DHA-like enzymes indicates the need for vigilance because inducible transferable AmpC -lactamases may confer a high risk of therapeutic failures with cephalosporins (26).
Clinical laboratories should test for transferable AmpC -lactamases to prevent their spread, to protect patients from therapy with inappropriate antibiotics (14, 26, 39), and to recognize strains in which AmpC -lactamases may mask the detection of an accompanying ESBL (7, 22, 38, 39, 41, 43) and result in fatal, inappropriate cephalosporin therapy (38). In our study, ESBLs were present in 17 of 28 transferable AmpC-producing K. pneumoniae isolates (61%).
The low prevalence of carbapenemases detected in Enterobacteriaceae may underestimate the current status of these enzymes in the United States. Since the completion of this study, there have been several reports of United States isolates of Enterobacteriaceae producing class A carbapenemases (4, 5, 12, 13, 33, 45) and also reports of rare P. aeruginosa isolates producing a metallo--lactamase (11, 16, 42). These are formidable enzymes that clinical laboratories should detect. Major concerns are the multiple antibiotic resistance with which they are associated, the possibility of reporting false in vitro susceptibility to carbapenems, and the possibility that the recent spread of metallo--lactamase-producing pathogens in Asia, Australia, Canada, Europe, and South America may extend to the United States (15, 29, 44).
In conclusion, Enterobacteriaceae producing ESBLs and transferable AmpC -lactamases were found to be widespread in the United States, while carbapenemase-producing isolates were rare. Each of these -lactamases is associated with multiple antibiotic resistance and problems of false in vitro susceptibility. It is therefore vital that clinical laboratories be able to detect these enzymes so that patients can receive appropriate antibiotic therapy and to ensure that infection control efforts are effective.
ACKNOWLEDGMENTS
This study was supported by a grant from Merck & Co., Inc.
We thank T. J. Lockhart and Lloyd B. Olson III for their excellent technical assistance. We gratefully acknowledge the contributing laboratories at the following ICU sites for testing, documenting, and submitting isolates: Arkansas Children's Hospital, Little Rock, AR; Auburn Memorial, Auburn, NY; Bellevue Hospital, New York, NY; Charleston Area Medical Center, Charleston, WV; Children's Hospital, Los Angeles, CA; Children's Memorial Hospital, Chicago, IL; Christus Schumpert Health Systems, Shreveport, LA; Clarian Health Methodist Hospital, Indianapolis, IN; Cleveland Clinic Foundation, Cleveland, OH; Columbian Presbyterian Medical Center, New York, NY; Cook County Hospital, Chicago, IL; Crawford Long Hospital, Atlanta, GA; Emory University Hospital, Atlanta, GA; Fletcher Allen HealthCare, Burlington, VT; Good Samaritan Regional Medical Center, Laboratory Sciences of Arizona, Tempe, AZ; Inova Fairfax Hospital, Falls Church, VA; Jackson Memorial Hospital, Miami, FL; Massachusetts General Hospital, Boston, MA; Roper Hospital, Charleston, SC; Shands Hospital, Gainesville, FL; St. Alphonsus Regional Medical Center, Boise, ID; St. Joseph's Hospital, Atlanta, GA; St. Luke's Hospital of Kansas City, Kansas City, MO; Stanford Hospital and Clinics, Palo Alto, CA; Sunrise Hospital, Las Vegas, NV; Thomason Hospital, El Paso, TX; Tulane University Hospital & Clinic, New Orleans, LA; University of California, San Francisco, Moffitt, CA, University of California Irvine Medical Center, Orange, CA; University of Chicago Hospital, Chicago, IL; University of Colorado Hospital, Denver, CO; University of Kentucky Hospital, Lexington, KY; University of Nebraska, Omaha, NE; University of Pennsylvania Medical Center, Philadelphia, PA; University of Rochester Medical Center, Rochester, NY; University of Washington, Seattle, WA; VA Maryland Healthcare System, Baltimore, MD; VA North Texas Health Care System, Dallas, TX; Vencor Central Hospital, Tampa, FL; Wake Medical Center, Raleigh, NC; and Wishard Health Services, Indianapolis, IN. We also gratefully acknowledge the contributing laboratories at the following non-ICU sites: Acadiana Medical Laboratories, Ltd., Lafayette, LA; Alton Ochsner Medical Foundation, New Orleans, LA; American Medical Labs New Kindred Hospital, Chantilly, VA; Baptist Health System, San Antonio, TX; Baylor University Medical Center, Dallas, TX; Burdette Tomlin Memorial Hospital, Cape May Court House, NJ; Clinical Laboratories, Inc., Throop, PA; Crawford Long Hospital, Atlanta, GA; Dyna Care Laboratories, Oklahoma City, OK; Emory Hospital, Atlanta, GA; Holy Name Hospital, Teaneck, NJ; Inova Fairfax Hospital, Falls Church, VA; Jamaica Hospital Medical Center, Jamaica, NY; LDS Hospital, Salt Lake City, UT; Northwest Medical Specialists, Tacoma, WA; Rochester General Hospital, New York, NY; Temple University Hospital, Philadelphia, PA; University of Colorado Hospital, Denver, CO; University of Kentucky Hospital, Lexington, KY; and Vencor of Arizona Laboratory Sciences of Arizona, Tempe, AZ.
FOOTNOTES
Corresponding author. Mailing address: Center for Research in Anti-Infectives and Biotechnology (CRAB), Department of Medical Microbiology & Immunology, School of Medicine, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-4096. Fax: (402) 280-1875. E-mail: kstaac@creighton.edu.
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