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1.  Antimicrobial-Resistant Strains of Clostridium difficile from North America 
A total of 316 toxigenic Clostridium difficile clinical isolates of known PCR ribotypes from patients in North America were screened for resistance to clindamycin, metronidazole, moxifloxacin, and rifampin. Clindamycin resistance was observed among 16 different ribotypes, with ribotypes 017, 053, and 078 showing the highest proportions of resistance. All isolates were susceptible to metronidazole. Moxifloxacin resistance was present in >90% of PCR-ribotype 027 and 053 isolates but was less common among other ribotypes. Only 7.9% of the C. difficile isolates were resistant to rifampin. Multidrug resistance (clindamycin, moxifloxacin, and rifampin) was present in 27.5% of PCR-ribotype 027 strains but was rare in other ribotypes. These results suggest that antimicrobial resistance in North American isolates of C. difficile varies by strain type and parallels rates of resistance reported from Europe and the Far East.
doi:10.1128/AAC.00220-12
PMCID: PMC3370774  PMID: 22411613
3.  Carbapenem Resistance in Klebsiella pneumoniae Not Detected by Automated Susceptibility Testing 
Emerging Infectious Diseases  2006;12(8):1209-1213.
Detecting β-lactamase–mediated carbapenem resistance among Klebsiella pneumoniae isolates and other Enterobacteriaceae is an emerging problem. In this study, 15 blaKPC-positive Klebsiella pneumoniae that showed discrepant results for imipenem and meropenem from 4 New York City hospitals were characterized by isoelectric focusing; broth microdilution (BMD); disk diffusion (DD); and MicroScan, Phoenix, Sensititre, VITEK, and VITEK 2 automated systems. All 15 isolates were either intermediate or resistant to imipenem and meropenem by BMD; 1 was susceptible to imipenem by DD. MicroScan and Phoenix reported 1 (6.7%) and 2 (13.3%) isolates, respectively, as imipenem susceptible. VITEK and VITEK 2 reported 10 (67%) and 5 (33%) isolates, respectively, as imipenem susceptible. By Sensititre, 13 (87%) isolates were susceptible to imipenem, and 12 (80%) were susceptible to meropenem. The VITEK 2 Advanced Expert System changed 2 imipenem MIC results from >16 μg/mL to <2 μg/mL but kept the interpretation as resistant. The recognition of carbapenem-resistant K. pneumoniae continues to challenge automated susceptibility systems.
doi:10.3201/eid1208.060291
PMCID: PMC3291231  PMID: 16965699
carbapenem; imipenem; meropenem; susceptibility testing; Klebsiella; beta-lactamase; carbapenemase; research
4.  Methicillin-Resistant Staphylococcus aureus1 
Emerging Infectious Diseases  2004;10(11):2052-2053.
doi:10.3201/eid1011.040797_10
PMCID: PMC3329056  PMID: 16010747
MRSA; Methicillin-Resistant Staphylococcus aureus,
5.  Characterization of Nasal and Blood Culture Isolates of Methicillin-Resistant Staphylococcus aureus from Patients in United States Hospitals 
A total of 299 nares and 194 blood isolates of methicillin-resistant Staphylococcus aureus (MRSA), each recovered from a unique patient, were collected from 23 U.S. hospitals from May 2009 to March 2010. All isolates underwent spa and staphylococcal cassette chromosome mec element (SCCmec) typing and antimicrobial susceptibility testing; a subset of 84 isolates was typed by pulsed-field gel electrophoresis (PFGE) using SmaI. Seventy-six spa types were observed among the isolates. Overall, for nasal isolates, spa type t002-SCCmec type II (USA100) was the most common strain type (37% of isolates), while among blood isolates, spa type t008-SCCmec type IV (USA300) was the most common (39%). However, the proportion of all USA100 and USA300 isolates varied by United States census region. Nasal isolates were more resistant to tobramycin and clindamycin than blood isolates (55.9% and 48.8% of isolates versus 36.6% and 39.7%, respectively; for both, P < 0.05). The USA300 isolates were largely resistant to fluoroquinolones. High-level mupirocin resistance was low among all spa types (<5%). SCCmec types III and VIII, which are rare in the United States, were observed along with several unusual PFGE types, including CMRSA9, EMRSA15, and the PFGE profile associated with sequence type 239 (ST239) isolates. Typing data from this convenience sample suggest that in U.S. hospitalized patients, USA100 isolates of multiple spa types, while still common in the nares, have been replaced by USA300 isolates as the predominant MRSA strain type in positive blood cultures.
doi:10.1128/AAC.05804-11
PMCID: PMC3294931  PMID: 22155818
6.  Comparison of Strain Typing Results for Clostridium difficile Isolates from North America▿ 
Journal of Clinical Microbiology  2011;49(5):1831-1837.
Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.
doi:10.1128/JCM.02446-10
PMCID: PMC3122689  PMID: 21389155
7.  Complete Nucleotide Sequence Analysis of Plasmids in Strains of Staphylococcus aureus Clone USA300 Reveals a High Level of Identity among Isolates with Closely Related Core Genome Sequences ▿ †  
Journal of Clinical Microbiology  2010;48(12):4504-4511.
A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates—those that comprise the epidemic USA300 clone—were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.
doi:10.1128/JCM.01050-10
PMCID: PMC3008496  PMID: 20943864
8.  Impact of Strain Type on Detection of Toxigenic Clostridium difficile: Comparison of Molecular Diagnostic and Enzyme Immunoassay Approaches ▿  
Journal of Clinical Microbiology  2010;48(10):3719-3724.
A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fisher's exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.
doi:10.1128/JCM.00427-10
PMCID: PMC2953097  PMID: 20702676
9.  Emergence of blaKPC-containing Klebsiella pneumoniae in a long-term acute care hospital: a new challenge to our healthcare system 
Objectives
To characterize isolates of Klebsiella pneumoniae producing KPC carbapenemase (KPC-Kp) associated with an outbreak in a long-term acute care hospital (LTACH) in South Florida.
Methods
During 21 March to 20 April 2008, 241 K. pneumoniae isolates detected at Integrated Regional Laboratories (Ft. Lauderdale, FL) for which the ertapenem MICs were ≥4 mg/L were studied. PCR, cloning and sequence analysis were used to detect blaKPC and to characterize the β-lactamase and outer membrane proteins (Omps). The expression level of KPC enzymes was studied by immunoblotting. Genetic relatedness of isolates was investigated with rep-PCR and PFGE. Clinical records of patients were investigated.
Results
Seven KPC-Kp strains were isolated from different patients located at a single LTACH, with a further three isolates being recovered from patients at different hospitals. All KPC-Kp isolates in patients from the LTACH and from one hospital patient were genetically related and shared PFGE patterns that clustered with known sequence type (ST) 258 strains. These strains were highly resistant to carbapenems (MICs ≥ 32 mg/L) due to an increased level of KPC expression and loss of Omps. Rectal colonization was documented in all LTACH patients with KPC-Kp isolates. Treatment failures were common (crude mortality rate of 69%). Active surveillance and enhanced infection control practices terminated the KPC-Kp outbreak.
Conclusions
The detection of KPC-Kp in an LTACH represents a serious infection control and therapeutic challenge in a new clinical setting. The speed at which the epidemic of KPC-Kp is spreading in our healthcare system mandates urgent action.
doi:10.1093/jac/dkp327
PMCID: PMC2760463  PMID: 19740911
LTCF; porins; carbapenemases; Enterobacteriaceae; outbreak
10.  Characterisation of a Staphylococcus aureus strain with progressive loss of susceptibility to vancomycin and daptomycin during therapy✰ 
Following an initial response to vancomycin therapy, a patient with meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia developed endocarditis, failed a second course of vancomycin and then failed daptomycin therapy. An increase in the vancomycin minimum inhibitory concentrations of four consecutive MRSA blood isolates from 2 μg/mL to 8 μg/mL was shown by Etest. Population analysis of four successive blood culture isolates recovered over the 10-week period showed that the MRSA strain became progressively less susceptible to both vancomycin and daptomycin. Retrospectively, the macro Etest method using teicoplanin indicated a decrease in vancomycin susceptibility in the second blood isolate. The patient improved after treatment with various courses of trimethoprim/sulphamethoxazole, quinupristin/dalfopristin and linezolid. Early detection of vancomycin-heteroresistant S. aureus isolates, which appeared to have clinical significance in this case, continues to be a challenge for the clinical laboratory. Development of suitable practical methods for this should be given priority. Concurrent development of resistance to vancomycin and daptomycin, whilst rare, must be considered in a patient who is unresponsive to daptomycin following vancomycin therapy.
doi:10.1016/j.ijantimicag.2008.12.010
PMCID: PMC2700752  PMID: 19233622
Staphylococci; Vancomycin; Teicoplanin; Heteroresistance
11.  Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA 
Background
The emergence of blaKPC-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed.
Methods
We analysed 42 KPC-Kp recovered during 2006–07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed.
Results
By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem β-lactams (MIC90s ≥ 128 mg/L). Among carbapenems, MIC50/90s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: blaKPC-2 (59.5%), blaKPC-3 (40.5%), blaTEM-1 (90.5%), blaSHV-11 (95.2%) and blaSHV-12 (50.0%). aIEF of crude β-lactamase extracts from these strains supported our findings, showing β-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of β-lactamases was 3.5 (range 3–5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 blaKPC-2 and 12 blaKPC-3) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE.
Conclusions
We demonstrated the complex β-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.
doi:10.1093/jac/dkn547
PMCID: PMC2640158  PMID: 19155227
carbapenemases; ESBLs; Enterobacteriaceae; PFGE; rep-PCR
12.  Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis▿  
Journal of Clinical Microbiology  2009;47(8):2452-2457.
We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.
doi:10.1128/JCM.00476-09
PMCID: PMC2725641  PMID: 19553588
13.  Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus▿  
Journal of Clinical Microbiology  2009;47(7):2013-2017.
We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.
doi:10.1128/JCM.00221-09
PMCID: PMC2708520  PMID: 19420170
14.  Identification of Plasmid-Mediated AmpC β-Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results ▿  
Journal of Clinical Microbiology  2008;47(2):294-299.
The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum β-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC β-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC β-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.
doi:10.1128/JCM.01797-08
PMCID: PMC2643671  PMID: 19036936
15.  Characterization of Staphylococcus aureus Isolates from Nasal Cultures Collected from Individuals in the United States in 2001 to 2004▿  
Journal of Clinical Microbiology  2008;46(9):2837-2841.
This study characterizes methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from nasal cultures of noninstitutionalized individuals in the United States obtained in 2001 to 2004 as part of the National Health and Nutrition Examination Survey. Every tenth MSSA isolate and all MRSA isolates were typed by pulsed-field gel electrophoresis (PFGE), screened for multiple toxin genes, and tested for susceptibility to 14 antimicrobial agents. USA200, USA600, and USA900 were the predominant PFGE types among MSSA isolates in both the 2001 to 2002 and the 2003 to 2004 time periods, although they accounted for only 51.3% of 316 MSSA isolates typed in 2001 and 2002 and only 43.4% of 237 MSSA isolates typed in 2003 and 2004. In contrast, USA100, USA800, and USA700 accounted for 80.0% of the 75 MRSA isolates typed in 2001 and 2002, while USA100, USA800, and USA300 accounted for 78.4% of 134 MRSA isolates typed in 2003 and 2004. The proportion of MRSA isolates that were USA300 increased significantly from the first to the second time period (P = 0.03). Most USA200 isolates (both MSSA and MRSA) carried the gene for toxic shock syndrome toxin; however, carriage of the genes encoding Panton-Valentine leukocidin, while common among MRSA of PFGE type USA300, was rare among MSSA USA300 in both time periods. Most MSSA isolates remained susceptible to all antimicrobial agents except erythromycin (79.1 and 76.0% susceptibilities in the 2001 to 2002 and the 2003 to 2004 periods, respectively). In contrast, the proportions of MRSA isolates that were susceptible to chloramphenicol, clindamycin, and erythromycin were lower in 2003 and 2004 than in 2001 and 2002, although none of these differences was statistically significant.
doi:10.1128/JCM.00480-08
PMCID: PMC2546757  PMID: 18632911
16.  Presence of Plasmid-Mediated Quinolone Resistance in Klebsiella pneumoniae Isolates Possessing blaKPC in the United States▿  
The presence of plasmid-mediated quinolone resistance genes [i.e., qnrA, qnrB, qnrS, aac(6′)-Ib-cr, and qepA] was evaluated among 42 blaKPC-containing Klebsiella pneumoniae isolates collected in the eastern United States. One isolate carried the blaKPC-3 and qnrB19 genes on the same conjugative plasmid, whereas another carried the blaKPC-3 and qnrA1 genes on separate plasmids.
doi:10.1128/AAC.00158-08
PMCID: PMC2443894  PMID: 18426899
18.  Overlapping Population Structures of Nasal Isolates of Staphylococcus aureus from Healthy Dutch and American Individuals▿  
Journal of Clinical Microbiology  2007;46(1):235-241.
To understand Staphylococcus aureus nasal carriage and its relationship with subsequent disease, insight into the natural (nonclinical) bacterial population structure is essential. This study investigated whether the distributions of S. aureus genotypes that cause colonization differ by geographic locales. High-throughput amplified fragment length polymorphism (AFLP) analysis was performed on nasal isolates of S. aureus from healthy American (n = 391) and Dutch (n = 829) volunteers. In total, 164,970 binary outcomes, covering 135 different markers per isolate, were scored. Methicillin resistance was defined for all strains; pulsed-field gel electrophoresis typing was performed for the American isolates. The overall population structures of the American and Dutch S. aureus isolates were comparable. The same four major AFLP clusters (I to IV) and subclusters were identified for both collections. However, the Dutch methicillin-susceptible S. aureus (MSSA) isolates were overrepresented in AFLP cluster III (P = 0.0016). Furthermore, the majority of the American methicillin-resistant S. aureus isolates (90.5%) were located in AFLP cluster I (P < 0.0001). This result identifies differences in the local prevalence of certain S. aureus genotypes. AFLP clusters II and III, which represent multilocus sequence typing clonal complexes 30 and 45, respectively, account for 46.4% of all MSSA isolates in the study, suggesting that these two lineages have evolved as extremely successful pandemic colonizers of humans. In conclusion, the overall population structures of American and Dutch nasal carriage isolates of S. aureus are surprisingly similar, despite subtle geographic differences in the prevalence of certain S. aureus genotypes.
doi:10.1128/JCM.00887-07
PMCID: PMC2224299  PMID: 17977984
19.  High Rate of Mobilization for blaCTX-Ms 
Emerging Infectious Diseases  2008;14(3):423-428.
The blaCTX-Ms have been mobilized to plasmids more frequently than other class A β-lactamases.
We constructed a phylogenetic analysis of class A β-lactamases and found that the blaCTX-Ms have been mobilized to plasmids ≈10 times more frequently than other class A β-lactamases. We also found that the blaCTX-Ms are descended from a common ancestor that was incorporated in ancient times into the chromosome of the ancestor of Kluyvera species through horizontal transfer. Considerable sequence divergence has occurred among the descendents of that ancestral gene sequence since that gene was inserted. That divergence has mainly occurred in the presence of purifying selection, which indicates a slow rate of evolution for blaCTX-Ms in the pre–antimicrobial drug era.
doi:10.3201/eid1403.070405
PMCID: PMC2570810  PMID: 18325257
CTX-M; phylogenetic analysis; plasmid; mobilization; beta-lactamase; evolution; antimicrobial resistance; test for selection; Bayesian inference; class A; research
20.  Accuracy of Six Antimicrobial Susceptibility Methods for Testing Linezolid against Staphylococci and Enterococci▿  
Journal of Clinical Microbiology  2007;45(9):2917-2922.
A challenge panel of enterococci (n = 50) and staphylococci (n = 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers’ protocols. Compared to the results of the broth microdilution method for detecting linezolid-nonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within ±1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolid-nonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.
doi:10.1128/JCM.00913-07
PMCID: PMC2045282  PMID: 17634301
21.  Systems Approach to Improving Antimicrobial Susceptibility Testing in Clinical Laboratories in the United States▿  
Journal of Clinical Microbiology  2007;45(7):2230-2234.
Laboratory practice in the preanalytical phase of antimicrobial susceptibility testing (AST) was evaluated in 102 hospital, reference, physician office-clinic, and public health laboratories in Washington state. Surveys were sent to evaluate (i) use of NCCLS/CLSI (formerly NCCLS) AST performance standards, (ii) technical competence in AST case studies, challenging knowledge of contemporary testing issues, and (iii) choice of antimicrobial agents to test for Streptococcus pneumoniae. Numerous deficiencies were identified in the survey: (i) initially only 40% of the laboratories surveyed used current NCCLS/CLSI AST performance standards, (ii) the rate of accurate responses for three different case studies ranged from 29% to 69%, and (iii) variation was noted in the choice of antimicrobials tested against invasive isolates of S. pneumoniae. These deficiencies could affect therapy and detection of antimicrobial resistance. Several educational programs were implemented to improve AST policies and practices, and a follow-up survey indicated that four intervention strategies were most effective: (i) regional technical workshops, (ii) National Laboratory Training Network teleconferences, (iii) use of the Centers for Disease Control and Prevention (CDC) CD-ROM on AST, and (iv) the CDC Multilevel Antimicrobial Susceptibility Testing Resource website. The interventions could be implemented more widely in the United States to improve AST knowledge and practices.
doi:10.1128/JCM.00184-07
PMCID: PMC1933004  PMID: 17522281
22.  Multiple-Locus Variable-Number Tandem-Repeat Assay Analysis of Methicillin-Resistant Staphylococcus aureus Strains▿  
Journal of Clinical Microbiology  2007;45(7):2215-2219.
Our objective was to determine if a multiple-locus variable-number tandem-repeat assay (MLVA) for Staphylococcus aureus could predict pulsed-field gel electrophoresis (PFGE) types (i.e., USA types), thus allowing us to replace PFGE with a simpler and more rapid typing method. One hundred three well-characterized isolates representing 13 major lineages of S. aureus were tested by both PFGE and MLVA. MLVA was performed using a rapid DNA extraction technique and PCR primers for sdrCDE, clfA, clfB, sspA, and spa. PFGE was performed with genomic DNA fragments generated using SmaI, as per CDC protocols. Banding patterns were analyzed both visually and with BioNumerics software. All isolates were typeable with MLVA and PFGE. MLVA patterns were highly reproducible. PFGE separated the isolates into 13 types with 42 subtypes. Using any band difference to designate a novel MLVA type, MLVA produced 45 types, including 9 clusters containing multiple isolates. Using BioNumerics and a cutoff of >75% relatedness, MLVA produced 28 types, 11 of which contained >1 isolate. Epidemiologically related outbreak isolates of USA300-0114 from five states clustered in one MLVA pattern. USA100 isolates were present in several unrelated (<40%) MLVA types. A cutoff of >80% separated outbreak strains of USA300-0114 into three distinct MLVA types. MLVA did not differentiate community methicillin-resistant S. aureus (MRSA) lineages (USA300, USA400, USA1000, and USA1100) from health care MRSA lineages (USA100, USA200, or USA500). The ability of MLVA to differentiate among strains that are indistinguishable by PFGE may be of epidemiologic value and warrants further study.
doi:10.1128/JCM.02451-06
PMCID: PMC1932968  PMID: 17494714
23.  Epidemiologic Distribution of the Arginine Catabolic Mobile Element among Selected Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates▿  
Journal of Clinical Microbiology  2007;45(6):1981-1984.
We tested 214 Staphylococcus aureus isolates for the arcA locus of the arginine catabolic mobile element (ACME). All USA300 SCCmec IVa isolates, but no isolates containing other SCCmec subtypes, were arcA positive. arcA was also detected in selected methicillin-susceptible USA300 and methicillin-resistant USA100 isolates. DNA sequence analysis confirmed the integration of ACME in orfX.
doi:10.1128/JCM.00273-07
PMCID: PMC1933090  PMID: 17409207
24.  Comparison of Disk Diffusion, VITEK 2, and Broth Microdilution Antimicrobial Susceptibility Test Results for Unusual Species of Enterobacteriaceae▿  
Journal of Clinical Microbiology  2006;45(2):340-346.
We compared the antimicrobial susceptibility testing results generated by disk diffusion and the VITEK 2 automated system with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 61 isolates of unusual species of Enterobacteriaceae. The isolates represented 15 genera and 26 different species, including Buttiauxella, Cedecea, Kluyvera, Leminorella, and Yokenella. Antimicrobial agents included aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, and trimethoprim-sulfamethoxazole. CLSI interpretative criteria for Enterobacteriaceae were used. Of the 12 drugs tested by BMD and disk diffusion, 10 showed >95% categorical agreement (CA). CA was lower for ampicillin (80.3%) and cefazolin (77.0%). There were 3 very major errors (all with cefazolin), 1 major error (also with cefazolin), and 26 minor errors. Of the 40 isolates (representing 12 species) that could be identified with the VITEK 2 database, 36 were identified correctly to species level, 1 was identified to genus level only, and 3 were reported as unidentified. VITEK 2 generated MIC results for 42 (68.8%) of 61 isolates, but categorical interpretations (susceptible, intermediate, and resistant) were provided for only 22. For the 17 drugs tested by both BMD and VITEK 2, essential agreement ranged from 80.9 to 100% and CA ranged from 68.2% (ampicillin) to 100%; thirteen drugs exhibited 100% CA. In summary, disk diffusion provides a reliable alternative to BMD for testing of unusual Enterobacteriaceae, some of which cannot be tested, or produce incorrect results, by automated methods.
doi:10.1128/JCM.01782-06
PMCID: PMC1829079  PMID: 17135429
25.  Rapid Multiplex PCR Assay for Identification of USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Isolates▿  
Journal of Clinical Microbiology  2006;45(1):141-146.
Recent reports have noted a discernible increase in the number of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections in patients without traditional risk factors. In the United States, the most prominent CA-MRSA strain encodes Panton-Valentine leukocidin (PVL) cytotoxin genes, belongs to pulsed field gel electrophoresis type USA300 and multilocus sequence type 8, and carries staphylococcal cassette chromosome mec (SCCmec) type IV. At present, molecular characterization of MRSA strains, such as USA300, can be time-consuming and is often beyond the technical capability of many clinical laboratories, making routine identification difficult. We analyzed the chromosomal regions flanking the SCCmec element in 44 USA300 MRSA isolates and identified a signature “AT repeat” sequence within the conserved hypothetical gene SACOL0058 located 1.4 kb downstream of the 3′ end of the J1-SCCmec chromosomal junction. Only USA300 isolates tested contained a sequence of ≥6 AT repeats in combination with PVL (e.g., related USA500 or Iberian strains had ≥6 AT repeats but were PVL negative). Using a locked nucleic acid primer specific for ≥6 AT repeats in combination with primers to detect PVL, we developed a multiplex PCR assay specific for the identification of USA300 strains. Multiplex results were 100% concordant with DNA sequencing, suggesting that the method has promise as a means of rapidly identifying USA300 isolates.
doi:10.1128/JCM.01228-06
PMCID: PMC1828973  PMID: 17093011

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