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1.  New Insights into Dissemination and Variation of the Health Care-Associated Pathogen Acinetobacter baumannii from Genomic Analysis 
mBio  2014;5(1):e00963-13.
ABSTRACT
Acinetobacter baumannii is a globally important nosocomial pathogen characterized by an increasing incidence of multidrug resistance. Routes of dissemination and gene flow among health care facilities are poorly resolved and are important for understanding the epidemiology of A. baumannii, minimizing disease transmission, and improving patient outcomes. We used whole-genome sequencing to assess diversity and genome dynamics in 49 isolates from one United States hospital system during one year from 2007 to 2008. Core single-nucleotide-variant-based phylogenetic analysis revealed multiple founder strains and multiple independent strains recovered from the same patient yet was insufficient to fully resolve strain relationships, where gene content and insertion sequence patterns added additional discriminatory power. Gene content comparisons illustrated extensive and redundant antibiotic resistance gene carriage and direct evidence of gene transfer, recombination, gene loss, and mutation. Evidence of barriers to gene flow among hospital components was not found, suggesting complex mixing of strains and a large reservoir of A. baumannii strains capable of colonizing patients.
IMPORTANCE
Genome sequencing was used to characterize multidrug-resistant Acinetobacter baumannii strains from one United States hospital system during a 1-year period to better understand how A. baumannii strains that cause infection are related to one another. Extensive variation in gene content was found, even among strains that were very closely related phylogenetically and epidemiologically. Several mechanisms contributed to this diversity, including transfer of mobile genetic elements, mobilization of insertion sequences, insertion sequence-mediated deletions, and genome-wide homologous recombination. Variation in gene content, however, lacked clear spatial or temporal patterns, suggesting a diverse pool of circulating strains with considerable interaction between strains and hospital locations. Widespread genetic variation among strains from the same hospital and even the same patient, particularly involving antibiotic resistance genes, reinforces the need for molecular diagnostic testing and genomic analysis to determine resistance profiles, rather than a reliance primarily on strain typing and antimicrobial resistance phenotypes for epidemiological studies.
doi:10.1128/mBio.00963-13
PMCID: PMC3903280  PMID: 24449752
2.  Novel Bis-Indole Agents Active Against Multidrug-Resistant Acinetobacter baumannii 
The in vitro activity of five novel Microbiotix bis-indole agents (MBXs) against 30 multidrug-resistant (MDR) A. baumannii (including 18 resistant to carbapenems) was evaluated. Overall, MIC90s ranged from 1-8 μg/ml, whereas those for imipenem were > 64 μg/ml. MBX 1196 was the most potent (MIC90 1 μg/ml). MBXs are compounds that are highly effective against MDR A. baumannii.
doi:10.1016/j.diagmicrobio.2010.08.014
PMCID: PMC3725720  PMID: 21146724
bis-indole; multidrug-resistant; MBX; susceptibility; in vitro
3.  In vivo and in vitro activity of the siderophore monosulfactam BAL30072 against Acinetobacter baumannii 
Objectives
New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC β-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need.
Methods
We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time–kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057).
Results
In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2–8-fold. In time–kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2–8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo.
Conclusions
These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.
doi:10.1093/jac/dkr013
PMCID: PMC3058568  PMID: 21393224
MICs; time–kill assays; drug susceptibility testing; multidrug resistant
4.  Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes 
Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.
doi:10.1016/j.ijantimicag.2010.03.024
PMCID: PMC2902549  PMID: 20472405
Streptococcus pneumoniae; Drug-resistant serotypes/serogroups; Transformation frequency; Tn916 transposon; mefE gene
5.  Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination 
Background
Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio.
Methods
Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed.
Results
Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%).
Conclusion
In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.
doi:10.1093/jac/dkq191
PMCID: PMC2904665  PMID: 20513702
LTCF; LTACH; molecular epidemiology; MLST; PFGE; rep-PCR; KPC
6.  Evaluation of Updated Interpretative Criteria for Categorizing Klebsiella pneumoniae with Reduced Carbapenem Susceptibility ▿  
Journal of Clinical Microbiology  2010;48(12):4417-4425.
We studied the accuracy of various susceptibility testing methods, including the 2009, 2010, and updated 2010 CLSI recommendations, to identify Klebsiella pneumoniae isolates with reduced susceptibility to carbapenems associated with different mechanisms of resistance. Forty-three wild-type (WT) strains, 42 extended-spectrum β-lactamase (ESBL) producers, 18 ESBL producers with outer membrane porin protein loss (ESBL/Omp strains), and 42 blaKPC-possessing K. pneumoniae (KPC-Kp) isolates were evaluated. Imipenem (IPM), meropenem (MEM), ertapenem (ERT), and doripenem (DOR) were tested by broth microdilution (BMD), Etest, and disk diffusion (DD), and the modified Hodge test (MHT) was performed using IPM and MEM disks. Results were interpreted according to original as well as recently updated interpretative criteria. MHT was positive for all 42 KPC-Kp isolates and 10 of 18 ESBL/Omp strains and therefore had poor specificity in differentiating between KPC-Kp and ESBL/Omp isolates. Based on the updated CLSI standards, phenotypic susceptibility testing by BMD and DD differentiated most carbapenem-susceptible from carbapenem-nonsusceptible K. pneumoniae isolates without the need for MHT, while the Etest method characterized many KPC-Kp isolates as susceptible, and breakpoints may need to be lowered for this method. However, both the original and updated CLSI criteria do not adequately differentiate between isolates in the KPC-Kp group, which are unlikely to respond to carbapenem therapy, and those in the ESBL/Omp group, which are likely to respond to carbapenem therapy if MICs are within pharmacokinetic/pharmacodynamic targets. Further studies are required to determine if there is a clinical need to differentiate between KPC-Kp and ESBL/Omp groups.
doi:10.1128/JCM.02458-09
PMCID: PMC3008460  PMID: 20881179
7.  Activity of Ceftaroline against Recent Emerging Serotypes of Streptococcus pneumoniae in the United States▿  
The in vitro activity of ceftaroline against 891 pneumococci collected in 2008 from 22 centers in the United States was investigated. Ceftaroline was the most potent agent tested, with the MICs being <0.008 to 0.5 μg/ml and the MIC90s being <0.008 to 0.25 μg/ml against 11 prevailing serotypes. The overall rates of susceptibility were as follows: penicillin G, 86.2%; ceftriaxone, 90.7%; cefuroxime, 70.1%; erythromycin, 61.6%; clindamycin, 79.2%; levofloxacin, 99.4%; and vancomycin, 100%. Serotype 19A isolates were the least susceptible. These results support the use of ceftaroline for the treatment of pneumococcal infections, including those caused by pneumococci resistant to other agents.
doi:10.1128/AAC.01797-09
PMCID: PMC2876371  PMID: 20308374
8.  Risk Factors for Contact Lens Bacterial Contamination During Continuous Wear 
Purpose
Microbial contamination of contact lenses is associated with corneal infection and inflammation. This study determined which microbiological, clinical and demographic factors are associated with bacterial contamination of a silicone hydrogel contact lens when worn for continuous wear.
Methods
Two hundred five normal subjects were enrolled in the Longitudinal Analysis of Silicone Hydrogel (LASH) Contact Lens Study and were fitted with lotrafilcon A lenses for monthly continuous wear and followed for 1 year. Lenses were aseptically removed after 1 week and 4 months of wear and cultured using an agar sandwich technique. Lids and conjunctiva were routinely cultured at baseline, and after 1 week and 4 months of continuous wear. Lenses and ocular sites were considered to have substantial microbial bioburden when they harbored pathogenic organisms or high levels of commensal organisms. Univariate and multivariate logistic regression analyses were conducted to examine whether substantial conjunctival or lid bioburden, subject demographics, lens wearing history, symptoms, and biomicroscopic signs were associated with lens bioburden.
Results
About one-third (32.4%) of subjects had substantial bacterial bioburden in either eye across multiple visits. Over half (53.2%) and about one-tenth (11.7%) of subjects had substantial lid and conjunctival bioburden, respectively, and 11.2% discontinued due to discomfort. The adjusted odds ratios (and 95% confidence intervals) for presence of substantial lens bioburden were 2.49 (1.17–5.30), 4.24 (1.45–12.40), and 4.11 (1.17–14.46) for substantial lid bioburden, substantial conjunctival bioburden, and lens discomfort, respectively.
Conclusions
Bacterial contamination of silicone hydrogel contact lenses is common during continuous wear. Substantial lens bioburden is associated with discomfort precluding successful continuous wear. The presence of substantial lid and conjunctival bioburden are associated with a 2.5 fold and over 4 fold greater risk of substantial lens bioburden and are likely the major routes of contamination.
doi:10.1097/OPX.0b013e3181bbca18
PMCID: PMC2783769  PMID: 19786927
contact lenses; bacterial bioburden; continuous wear
9.  Validation of Factor 6d Antiserum for Serotyping Streptococcus pneumoniae Serotype 6C▿  
Journal of Clinical Microbiology  2010;48(4):1456-1457.
Factor 6d antiserum reacts with the new Streptococcus pneumoniae serotype 6C. Serogroup 6 isolates, consisting of 49 6A, 42 6B and 98 6C strains from the United States and Israel, serotyped in parallel by PCR and capsular swelling methods, were all identified correctly. The new factor 6d antiserum accurately identifies serotype 6C.
doi:10.1128/JCM.01907-09
PMCID: PMC2849593  PMID: 20164277
10.  Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System▿  
The mechanism of colistin resistance (Colr) in Acinetobacter baumannii was studied by selecting in vitro Colr derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Colr clinical isolate. A colistin-susceptible revertant of one Colr mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Colr mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.
doi:10.1128/AAC.00284-09
PMCID: PMC2737849  PMID: 19528270
11.  Occurrence, Distribution, and Origins of Streptococcus pneumoniae Serotype 6C, a Recently Recognized Serotype▿  
The prevalence of Streptococcus pneumoniae serotype 6C, a recently recognized serotype that cross-reacts serologically with serotype 6A, was investigated. Isolates of serotype 6A in various collections were recovered, and serotype 6C was differentiated from 6A by multiplex PCR of DNA extracts by using appropriate primers. Antimicrobial susceptibility was performed by Clinical and Laboratory Standards Institute broth microdilution, and selected isolates were typed by pulsed-field gel electrophoresis, repetitive sequence-based PCR typing, and rapid multilocus sequence typing (MLST) by electrospray ionization mass spectrometry of PCR products. A total of 60 serotype 6C isolates were found: 30 of 122 Cleveland isolates collected from 1979 to 2007, 19 of 39 pediatric isolates collected nationwide in 2005 and 2006, and 11 pediatric isolates from Massachusetts collected in 2006 and 2007. Only four isolates were recovered prior to introduction of the conjugate pneumococcal vaccine in 2000; the earliest isolate was recovered in 1989. The sources of the isolates included blood (n = 5), the lower respiratory tract (n = 27), the sinus (n = 5), the ear (n = 2), and the nasopharynx (n = 18); isolates were recovered from 49 children and 11 adults. Pediatric isolates were found in all six major U.S. geographic regions. Antimicrobial susceptibility showed that 22 isolates were nonsusceptible to penicillin, macrolides, and trimethoprim-sulfamethoxazole, 8 had other resistance patterns, and 30 were fully susceptible. The three typing methods used showed similar clusters of up to eight isolates per cluster. MLST showed five clusters related to serotype 6A, two clusters related to serotype 6B, one cluster related to serotype 3, and one cluster related to serotype 34. This study documents the occurrence, nationwide distribution, diversity, likely origins, and increasing incidence after 2001 of this recently recognized serotype. Serotype 6C warrants consideration for addition to future conjugate pneumococcal vaccines.
doi:10.1128/JCM.01524-08
PMCID: PMC2620864  PMID: 18971356
12.  Changes in Serotypes and Antimicrobial Susceptibility of Invasive Streptococcus pneumoniae Strains in Cleveland: a Quarter Century of Experience▿  
Journal of Clinical Microbiology  2008;46(3):982-990.
The serotypes and susceptibilities to penicillin, macrolides, and clindamycin of 1,655 invasive isolates of Streptococcus pneumoniae recovered between 1979 and 2004 were determined. A precipitous decrease of 61% in the number of isolates was found following 2000, the year of 7-valent protein-conjugated pneumococcal vaccine (PCV7) introduction (139 versus 55 per 2-year period prior to versus after 2000; P < 0.001). This decrease was 84% in children <5 years old (80 versus 13 per 2-year period; P < 0.001) and 18 to 23% in other age groups (P, not significant). PCV7 serotypes decreased by 76% overall (103 versus 25 per 2-year period; P < 0.001) and by 92% in children <5 years old (65 versus 5 per 2-year period; P < 0.001), with significant decreases in six of the seven PCV serotypes. Other serotypes, except for type 19A, decreased from 32 to 22 per 2-year period, while type 19A increased from 4 to 8 per 2-year period, although none of these changes reached significance. Drug resistance emerged slowly, with the first penicillin-intermediate strain isolated in 1980 and the first macrolide/lincosamide-resistant strain isolated in 1984. The first penicillin-resistant strain was isolated in 1993. Resistance increased steadily thereafter until 2003-2004, when 51.1% of isolates were penicillin nonsusceptible and 53.3% were macrolide resistant. Clindamycin resistance remained low until 2003-2004, when 26.7% of strains were resistant; this was associated with the emergence of multidrug-resistant type 19A strains. This study documents the emergence of resistance over a quarter century among invasive pneumococci in the Cleveland area, as well as the reduction in disease caused by PCV7 serotypes following the introduction of PCV7 in 2000.
doi:10.1128/JCM.02321-07
PMCID: PMC2268364  PMID: 18234877
13.  Analysis of Antibiotic Resistance Genes in Multidrug-Resistant Acinetobacter sp. Isolates from Military and Civilian Patients Treated at the Walter Reed Army Medical Center▿  
Antimicrobial Agents and Chemotherapy  2006;50(12):4114-4123.
Military medical facilities treating patients injured in Iraq and Afghanistan have identified a large number of multidrug-resistant (MDR) Acinetobacter baumannii isolates. In order to anticipate the impact of these pathogens on patient care, we analyzed the antibiotic resistance genes responsible for the MDR phenotype in Acinetobacter sp. isolates collected from patients at the Walter Reed Army Medical Center (WRAMC). Susceptibility testing, PCR amplification of the genetic determinants of resistance, and clonality were determined. Seventy-five unique patient isolates were included in this study: 53% were from bloodstream infections, 89% were resistant to at least three classes of antibiotics, and 15% were resistant to all nine antibiotics tested. Thirty-seven percent of the isolates were recovered from patients nosocomially infected or colonized at the WRAMC. Sixteen unique resistance genes or gene families and four mobile genetic elements were detected. In addition, this is the first report of blaOXA-58-like and blaPER-like genes in the U.S. MDR A. baumannii isolates with at least eight identified resistance determinants were recovered from 49 of the 75 patients. Molecular typing revealed multiple clones, with eight major clonal types being nosocomially acquired and with more than 60% of the isolates being related to three pan-European types. This report gives a “snapshot” of the complex genetic background responsible for antimicrobial resistance in Acinetobacter spp. from the WRAMC. Identifying genes associated with the MDR phenotype and defining patterns of transmission serve as a starting point for devising strategies to limit the clinical impact of these serious infections.
doi:10.1128/AAC.00778-06
PMCID: PMC1694013  PMID: 17000742
14.  Relationship between the Original Multiply Resistant South African Isolates of Streptococcus pneumoniae from 1977 to 1978 and Contemporary International Resistant Clones 
Journal of Clinical Microbiology  2005;43(12):6035-6041.
High-level penicillin G-resistant as well as multidrug-resistant Streptococcus pneumoniae isolates were first described in South Africa in 1977. The relationship between these original multidrug-resistant South African isolates and other resistant clones was investigated. Twenty-six representative isolates isolated from initial outbreaks in South Africa from 1977 to 1978 were characterized by multilocus sequence typing and pulsed-field gel electrophoresis. Twenty-one isolates were penicillin resistant and five were penicillin intermediate, with variable susceptibilities to macrolides, clindamycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. Fourteen isolates were serotype 19A, 11 were serotype 6A, and one was serotype 14. Penicillin-resistant serotype 19A isolates belonged to three closely related sequence types (STs), ST 41 (n = 6), ST 1605 (n = 3), and ST 1656 (n = 1). Penicillin-resistant serotype 6A isolates belonged to two closely related STs, ST 1094 (n = 10) and ST 1607 (n = 1), and were not closely related to other international clones. The serotype 14 penicillin-intermediate isolate was not closely related to the other isolates from South Africa but was a predicted founder of a clonal group with 41 different STs. Five new STs, ST 1605, ST 1607, ST 1608, ST 1610, and ST 1656, are described for the first time in this study. New molecular methods have characterized the original multiply resistant South African pneumococcal isolates from 1977 to 1978 and have shown the relationships of these clones to major pneumococcal clones.
doi:10.1128/JCM.43.12.6035-6041.2005
PMCID: PMC1317191  PMID: 16333095
15.  In Vitro Activity of the New Quinolone WCK 771 against Staphylococci 
The activity of WCK 771, an experimental quinolone developed to overcome quinolone resistance in staphylococci and other bacteria, was determined against quinolone-susceptible and -resistant Staphylococcus aureus and S. epidermidis. WCK 771 MICs for 50 and 90% of the strains tested (MIC50 and MIC90, respectively) were 0.008 and 0.015 μg/ml for S. aureus (n = 43) and 0.015 and 0.03 μg/ml for S. epidermidis (n = 44) for quinolone-susceptible isolates, compared to ciprofloxacin values of 0.12 and 0.25 μg/ml and 0.25 and 0.5 μg/ml, respectively. Values for levofloxacin were 0.12 and 0.25 μg/ml and 0.12 and 0.25 μg/ml, those for clinafloxacin were 0.015 and 0.03 μg/ml and 0.015 and 0.03 μg/ml, those for moxifloxacin were 0.03 and 0.06 μg/ml and 0.06 and 0.12 μg/ml, and those for gatifloxacin were 0.06 and 0.12 μg/ml and 0.12 and 0.25 μg/ml, respectively. The WCK 771 MIC50 and MIC90, respectively, were 0.5 and 1 μg/ml for both species of staphylococci (n = 73 for S. aureus, n = 70 for S. epidermidis) for isolates highly resistant to ciprofloxacin (MIC50 and MIC90, >32 and >32 μg/ml, respectively). Values for levofloxacin were 8 and 32 μg/ml and 8 and 32 μg/ml, those for clinafloxacin were 1 and 2 μg/ml and 0.5 and 2 μg/ml, those for moxifloxacin 4 and >4 μg/ml and 4 and >4 μg/ml, and those for gatifloxacin were 4 and >4 μg/ml and 2 and >4 μg/ml, respectively. WCK 771 and clinafloxacin demonstrated MICs of 1 μg/ml against three vancomycin-intermediate strains. WCK 771 showed concentration-independent killing for up to 24 h at 2, 4, and 8 times the MICs against quinolone-resistant staphylococci and was also bactericidal after 8 h for high-density inocula (108 CFU/ml) of quinolone-resistant strains at 5 μg/ml, whereas moxifloxacin at 7.5 μg/ml was bacteriostatic. WCK 771 was not a substrate of the NorA efflux pump as evident from the similar MICs against both an efflux-positive and an efflux-negative strain. Overall, WCK 771 was the most potent quinolone tested against the staphylococci tested, regardless of quinolone susceptibility.
doi:10.1128/AAC.48.9.3338-3342.2004
PMCID: PMC514757  PMID: 15328094
16.  Effects of Various Test Media on the Activities of 21 Antimicrobial Agents against Haemophilus influenzae 
Journal of Clinical Microbiology  2002;40(9):3269-3276.
As considerable variation in the antimicrobial susceptibility of Haemophilus influenzae has been reported, the effects of various test media on the susceptibility of H. influenzae were studied. MICs were determined by three laboratories for 21 antimicrobial agents against a panel of 100 selected isolates. Testing was performed using a reference NCCLS frozen broth microdilution method with Haemophilus test medium (HTM) broth and dried commercial MIC trays rehydrated with the following media: in-house and commercially prepared HTM broth, Mueller-Hinton broth with 2% lysed horse blood and NAD, IsoSensitest broth with 2% lysed horse blood and NAD, and IsoSensitest broth-based HTM. Overall, all results were very reproducible, with the MIC at which 50% of the isolates tested are inhibited (MIC50), MIC90, and geometric mean MIC being within one doubling dilution by all six methods and at all three testing centers for 15 of the 21 agents tested. Interlaboratory differences were more marked than intralaboratory differences or differences among media. Cefprozil, cefaclor, and trimethoprim-sulfamethoxazole results differed the most, while results for ampicillin, amoxicillin-clavulanic acid, cefdinir, cefixime, ceftriaxone, and clarithromycin were the most reproducible. However, these variations in results caused considerable differences in susceptibility rates for agents for which NCCLS susceptible breakpoints were close to the geometric mean MIC, particularly for cefaclor and cefprozil. This was much less of a problem when pharmacokinetic-pharmacodynamic breakpoints were used. Reproducible susceptibility results were obtained for a wide range of agents against H. influenzae in three laboratories using a variety of media that support the growth of this fastidious species.
doi:10.1128/JCM.40.9.3269-3276.2002
PMCID: PMC130805  PMID: 12202564
17.  Susceptibilities of Haemophilus influenzae and Moraxella catarrhalis to ABT-773 Compared to Their Susceptibilities to 11 Other Agents 
The activity of the ketolide ABT-773 against Haemophilus and Moraxella was compared to those of 11 other agents. Against 210 Haemophilus influenzae strains (39.0% β-lactamase positive), microbroth dilution tests showed that azithromycin and ABT-773 had the lowest MICs (0.5 to 4.0 and 1.0 to 8.0 μg/ml, respectively), followed by clarithromycin and roxithromycin (4.0 to >32.0 μg/ml). Of the β-lactams, ceftriaxone had the lowest MICs (≤0.004 to 0.016 μg/ml), followed by cefixime and cefpodoxime (0.008 to 0.125 and ≤0.125 to 0.25 μg/ml, respectively), amoxicillin-clavulanate (0.125 to 4.0 μg/ml), and cefuroxime (0.25 to 8.0 μg/ml). Amoxicillin was only active against β-lactamase-negative strains, and cefprozil had the highest MICs of all oral cephalosporins tested (0.5 to >32.0 μg/ml). Against 50 Moraxella catarrhalis strains, all of the compounds except amoxicillin and cefprozil were active. Time-kill studies against 10 H. influenzae strains showed that ABT-773, at two times the MIC, was bactericidal against 9 of 10 strains, with 99% killing of all strains at the MIC after 24 h; at 12 h, ABT-773 gave 90% killing of all strains at two times the MIC. At 3 and 6 h, killing by ABT-773 was slower, with 99.9% killing of four strains at two times the MIC after 6 h. Similar results were found for azithromycin, with slightly slower killing by erythromycin, clarithromycin, and roxithromycin, especially at earlier times. β-Lactams were bactericidal against 8 to 10 strains at two times the MIC after 24 h, with slower killing at earlier time periods. Most compounds gave good killing of five M. catarrhalis strains, with β-lactams killing more rapidly than other drugs. ABT-773 and azithromycin gave the longest postantibiotic effects (PAEs) of the ketolide-macrolide-azalide group tested (4.4 to >8.0 h), followed by clarithromycin, erythromycin, and roxithromycin. β-Lactam PAEs were similar and shorter than those of the ketolide-macrolide-azalide group for all strains tested.
doi:10.1128/AAC.45.1.67-72.2001
PMCID: PMC90241  PMID: 11120946
18.  Evaluation of Etest for Susceptibility Testing of Mycobacterium tuberculosis 
Journal of Clinical Microbiology  2000;38(10):3834-3836.
The Etest method for susceptibility testing of Mycobacterium tuberculosis was compared to the agar proportion method using four first-line agents and two fluoroquinolones. Catergorical agreement between the methods was 100% for rifampin, ethambutol, streptomycin, and ofloxacin and 98% for isoniazid. Results were obtained in 6 to 10 days by Etest. The Etest method is suitable for testing the agents evaluated against M. tuberculosis.
PMCID: PMC87486  PMID: 11015413
19.  Activities and Postantibiotic Effects of Gemifloxacin Compared to Those of 11 Other Agents against Haemophilus influenzae and Moraxella catarrhalis 
The activity of gemifloxacin against Haemophilus influenzae and Moraxella catarrhalis was compared to those of 11 other agents. All quinolones were very active (MICs, ≤0.125 μg/ml) against 248 quinolone-susceptible H. influenzae isolates (40.7% of which were β-lactamase positive); cefixime (MICs, ≤0.125 μg/ml) and amoxicillin-clavulanate (MICs ≤4.0 μg/ml) were active, followed by cefuroxime (MICs, ≤16.0 μg/ml); azithromycin MICs were ≤4.0 μg/ml. For nine H. influenzae isolates with reduced quinolone susceptibilities, the MICs at which 50% of isolates are inhibited (MIC50s) were 0.25 μg/ml for gemifloxacin and 1.0 μg/ml for the other quinolones tested. All strains had mutations in GyrA (Ser84, Asp88); most also had mutations in ParC (Asp83, Ser84, Glu88) and ParE (Asp420, Ser458), and only one had a mutation in GyrB (Gln468). All quinolones tested were equally active (MICs, ≤0.06 μg/ml) against 50 M. catarrhalis strains; amoxicillin-clavulanate, cefixime, cefuroxime, and azithromycin were very active. Against 10 H. influenzae strains gemifloxacin, levofloxacin, sparfloxacin, and trovafloxacin at 2× the MIC and ciprofloxacin at 4× the MIC were uniformly bactericidal after 24 h, and against 9 of 10 strains grepafloxacin at 2× the MIC was bactericidal after 24 h. After 24 h bactericidal activity was seen with amoxicillin-clavulanate at 2× the MIC for all strains, cefixime at 2× the MIC for 9 of 10 strains, cefuroxime at 4× the MIC for all strains, and azithromycin at 2× the MIC for all strains. All quinolones except grepafloxacin (which was bactericidal against four of five strains) and all ß-lactams at 2× to 4× the MIC were bactericidal against five M. catarrhalis strains after 24 h; azithromycin at the MIC was bactericidal against all strains after 24 h. The postantibiotic effects (PAEs) against four quinolone-susceptible H. influenzae strains were as follows: gemifloxacin, 0.3 to 2.3 h; ciprofloxacin, 1.3 to 4.2 h; levofloxacin, 2.8 to 6.2 h; sparfloxacin, 0.6 to 3.0 h; grepafloxacin, 0 to 2.1 h; trovafloxacin, 0.8 to 2.8 h. At 10× the MIC, no quinolone PAEs were found against the strain for which quinolone MICs were increased. Azithromycin PAEs were 3.7 to 7.3 h.
PMCID: PMC89738  PMID: 10681330
20.  Postantibiotic Effects of Grepafloxacin Compared to Those of Five Other Agents against 12 Gram-Positive and -Negative Bacteria 
The postantibiotic effect (PAE) (10× the MIC) and the postantibiotic sub-MIC effects (0.125, 0.25, and 0.5× the MIC) were determined for six compounds against 12 strains. Measurable PAEs ranged between 0 and 1.8 h for grepafloxacin, 0 and 2.2 h for ciprofloxacin, 0 and 3.1 h for levofloxacin, 0 and 2.2 h for sparfloxacin, 0 and 2.4 h for amoxicillin-clavulanate and 0 and 4.8 h for clarithromycin. Reexposure to subinhibitory concentrations increased the PAEs against some strains.
PMCID: PMC89651  PMID: 10602746
21.  Susceptibilities of Streptococcus pneumoniae and Haemophilus influenzae to 10 Oral Antimicrobial Agents Based on Pharmacodynamic Parameters: 1997 U.S. Surveillance Study 
The susceptibilities of Streptococcus pneumoniae (1,476 strains) and untypeable Haemophilus influenzae (1,676 strains) to various oral β-lactam, macrolide-azalide, and fluoroquinolone antimicrobial agents were determined by broth microdilution. Organisms were isolated from specimens obtained from outpatients in six geographic regions of the United States. MIC data were interpreted according to pharmacodynamically derived breakpoints applicable to the oral agents tested. Among H. influenzae strains, 41.6% were β-lactamase positive. Virtually all H. influenzae strains were susceptible to amoxicillin-clavulanate (98%), cefixime (100%), and ciprofloxacin (100%), while 78% were susceptible to cefuroxime, 57% were susceptible to amoxicillin, 14% were susceptible to cefprozil, 9% were susceptible to loracarbef, 2% were susceptible to cefaclor, and 0% were susceptible to azithromycin and clarithromycin. Among S. pneumoniae isolates, 49.6% were penicillin susceptible, 17.9% were intermediate, and 32.5% were penicillin resistant, with penicillin MICs for 50 and 90% of the isolates tested of 0.12 and 4 μg/ml, respectively. Overall, 94% of S. pneumoniae isolates were susceptible to amoxicillin and amoxicillin-clavulanate, 69% were susceptible to azithromycin and clarithromycin, 63% were susceptible to cefprozil and cefuroxime, 52% were susceptible to cefixime, 22% were susceptible to cefaclor, and 11% were susceptible to loracarbef. Although ciprofloxacin has marginal activity against S. pneumoniae, no high-level fluoroquinolone-resistant strains were found. Significant cross-resistance was found between penicillin and macrolides-azalides among S. pneumoniae isolates, with 5% of the penicillin-susceptible strains being macrolide-azalide resistant, compared with 37% of the intermediate isolates and 66% of the resistant isolates. Resistance was highest in S. pneumoniae isolates from patients younger than 10 years of age, middle ear and paranasal sinus specimens, and the southern half of the United States. With the continuing rise in resistance, judicious use of oral antimicrobial agents is necessary in all age groups.
PMCID: PMC89388  PMID: 10428910
22.  Activity of HMR 3647 Compared to Those of Five Agents against Haemophilus influenzae and Moraxella catarrhalis by MIC Determination and Time-Kill Assay 
Antimicrobial Agents and Chemotherapy  1998;42(11):3032-3034.
The microdilution MICs of HMR 3647, erythromycin A, azithromycin, clarithromycin, roxithromycin, and pristinamycin against 50/90% of 249 Haemophilus influenzae and 50 Moraxella catarrhalis isolates were 2/4, 0.06/0.125; 8/16, 0.25/0.25; 2/4, 0.06/0.125; 16/16, 0.25/0.25; 32/>32, 1/2; and 2/4, 0.5/0.5 μg/ml. Azithromycin was bactericidal against all 10 H. influenzae and 3 of 5 M. catarrhalis isolates and HMR 3647, erythromycin A, clarithromycin, roxithromycin, and pristinamycin were bacteriostatic, against all 15 strains after 24 h at the MIC.
PMCID: PMC105990  PMID: 9797250
23.  Determination of Penicillin MICs for Streptococcus pneumoniae by Using a Two- or Three-Disk Diffusion Procedure 
Journal of Clinical Microbiology  1998;36(1):179-183.
The potential for the use of the disk diffusion method to accurately predict penicillin MICs for Streptococcus pneumoniae was investigated with penicillin (6 μg), methicillin (5 μg), and oxacillin (1 μg) disks. A total of 183 S. pneumoniae isolates were tested by three MIC procedures (agar dilution, microdilution, and E-test). Regression analyses of the geometric mean of the three MIC results against (i) the sum of the zone diameters for methicillin, penicillin, and oxacillin disks; (ii) the sum of the zone diameters for methicillin and penicillin disks; and (iii) each of the three individual zone diameters were performed. Calculated MICs were determined from each of these regression analyses and compared to the mean reference MICs. A high level of correlation was obtained with both the two- and the three-disk procedures (r = 0.97), with essential agreement rates (±1 doubling dilution) between MICs calculated by the three-disk procedure and the two-disk procedure and the mean reference MICs of 98.4 and 98.9%, respectively. No major or very major errors were obtained with the two- or three-disk procedures. The accuracy of the disks used individually was lower (r = 0.84 to 0.93). However, oxacillin and methicillin disk testing remain excellent for screening strains, with all penicillin-susceptible strains having zones of >21 and >22 mm, respectively. The combination disk procedure, which involves the use of three disks (methicillin, oxacillin, and penicillin) or two disks (methicillin and penicillin) for testing S. pneumoniae, can provide accurate penicillin MICs and qualitative category results that are comparable to results obtained by the E-test, agar, and microdilution MIC methods.
PMCID: PMC124830  PMID: 9431943

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