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1.  Characterization of blaOXA-143 Variants in Acinetobacter baumannii and Acinetobacter pittii 
The acquired carbapenem-hydrolyzing oxacillinase (OXA) OXA-143 has thus far been detected only in Acinetobacter baumannii isolates from Brazil. The aim of this study was to characterize three OXA-143 variants: OXA-231 and OXA-253 from carbapenem-resistant A. baumannii isolates and OXA-255 in a carbapenem-susceptible Acinetobacter pittii isolate originating from Brazil, Honduras, and the United States, respectively. The 5′ rapid amplification of cDNA ends (RACE) technique identified the same transcription initiation site for all blaOXA-143-like genes and revealed differences in the putative promoter regions. However, all cloned OXA-143 variants conferred carbapenem resistance on A. baumannii ATCC 17978 and OXA-255 conferred carbapenem resistance on A. pittii SH024, which was correlated with blaOXA-255 gene expression. This is the first description of OXA-143-like outside A. baumannii. Detection of OXA-143-like in the United States and Honduras indicates its dissemination through the American continent.
PMCID: PMC3993222  PMID: 24566181
2.  OXA-235, a Novel Class D β-Lactamase Involved in Resistance to Carbapenems in Acinetobacter baumannii 
We investigated the mechanism of carbapenem resistance in 10 Acinetobacter baumannii strains isolated from the United States and Mexico between 2005 and 2009. The detection of known metallo-β-lactamase or carbapenem-hydrolyzing oxacillinase (OXA) genes by PCR was negative. The presence of plasmid-encoded carbapenem resistance genes was investigated by transformation of A. baumannii ATCC 17978. Shotgun cloning experiments and sequencing were performed, followed by the expression of a novel β-lactamase in A. baumannii. Three novel OXA enzymes were identified, OXA-235 in 8 isolates and the amino acid variants OXA-236 (Glu173-Val) and OXA-237 (Asp208-Gly) in 1 isolate each. The deduced amino acid sequences shared 85% identity with OXA-134, 54% to 57% identities with the acquired OXA-23, OXA-24, OXA-58, and OXA-143, and 56% identity with the intrinsic OXA-51 and, thus, represent a novel subclass of OXA. The expression of OXA-235 in A. baumannii led to reduced carbapenem susceptibility, while cephalosporin MICs were unaffected. Genetic analysis revealed that blaOXA-235, blaOXA-236, and blaOXA-237 were bracketed between two ISAba1 insertion sequences. In addition, the presence of these acquired β-lactamase genes might result from a transposition-mediated mechanism. This highlights the propensity of A. baumannii to acquire multiple carbapenem resistance determinants.
PMCID: PMC3632948  PMID: 23439638
3.  Microarray-Based Genotyping and Clinical Outcomes of Staphylococcus aureus Bloodstream Infection: An Exploratory Study 
PLoS ONE  2013;8(8):e71259.
The clinical course of Staphylococcus aureus bacteremia varies extensively. We sought to determine the relationship between genetic characteristics of the infecting pathogen and clinical outcomes in an exploratory study. In two study centers, 317 blood culture isolates were analyzed by DNA microarray and spa genotyping. By uni- and multivariate regression analyses associations of genotype data with 30-day all-cause mortality, severe sepsis/septic shock, disseminated disease, endocarditis, and osteoarticular infection were investigated. Univariate analysis showed significant association between S. aureus genes/gene-clusters or clonal complexes and clinical endpoints. For example CC15 was associated with 30-day mortality and CC22 with osteoarticular infection. In multivariate analysis methicillin resistance (mecA, OR 4.8 [1.43–16.06]) and the beta-lactamase-gene (bla, OR 3.12 [1.17–8.30]) remained independently associated with 30-day mortality. The presence of genes for enterotoxins (sed/sej/ser) was associated with endocarditis (OR 5.11 [1.14–18.62]). Host factors such as McCabe classification (OR 4.52 [2.09–9.79] for mortality), age (OR 1.06 [1.03–1.10] per year), and community-acquisition (OR 3.40 [1.31–8.81]) had a major influence on disease severity, dissemination and mortality. Individual genotypes and clonal complexes of S. aureus can only partially explain clinical features and outcomes of S. aureus bacteremia. Genotype-phenotype association studies need to include adjustments for host factors like age, comorbidity and community-acquisition.
PMCID: PMC3743874  PMID: 23967176
4.  Molecular Epidemiology of Acinetobacter baumannii Bloodstream Isolates Obtained in the United States from 1995 to 2004 Using rep-PCR and Multilocus Sequence Typing 
Journal of Clinical Microbiology  2012;50(11):3493-3500.
Using a repetitive-sequence-based (rep)-PCR (DiversiLab), we have molecularly typed Acinetobacter nosocomial bloodstream isolates (Acinetobacter baumannii [n = 187], Acinetobacter pittii [n = 23], and Acinetobacter nosocomialis [n = 61]) obtained from patients hospitalized in U.S. hospitals over a 10-year period (1995-2004) during a nationwide surveillance study (Surveillance and Control of Pathogens of Epidemiological Importance [SCOPE]). Patterns of A. baumannii rep-PCR were compared to those of previously identified international clonal lineages (ICs) and were further investigated by multilocus sequence typing (MLST) to compare the two typing methods. Forty-seven of the A. baumannii isolates clustered with the previously defined IC 2. ICs 1, 3, 6, and 7 were also detected. The remaining 81 isolates were unrelated to the described ICs. In contrast, A. pittii and A. nosocomialis isolates were more heterogeneous, as determined by rep-PCR. Our MLST results were in good correlation with the rep-PCR clusters. Our study confirms previous data indicating the predominance of a few major clonal A. baumannii lineages in the United States, particularly IC 2. The presence in the United States of A. baumannii ICs 1, 2, and 3 from as early as 1995 suggests that global dissemination of these lineages was an early event.
PMCID: PMC3486219  PMID: 22895032
5.  Interlaboratory reproducibility of DiversiLab rep-PCR typing and clustering of Acinetobacter baumannii isolates 
Journal of Medical Microbiology  2012;61(Pt 1):137-141.
We have investigated the reproducibility of DiversiLab rep-PCR fingerprints between two laboratories with the aim of determining if the fingerprints and clustering are laboratory-specific or portable. One-hundred non-duplicate Acinetobacter baumannii isolates were used in this study. DNA isolation and rep-PCR were each performed separately in two laboratories and rep-PCR patterns generated in laboratory A were compared with those from laboratory B. Twelve A. baumannii isolates processed in laboratory A showed ≥98 % pattern similarity with the corresponding 12 isolates tested in laboratory B and were considered identical. Sixty-four isolates showed 95–97.9 % similarity with their corresponding isolates. Twenty-three isolates showed 90–94 % similarity with the corresponding isolates, while one isolate showed only 87.4 % similarity. However, intra-laboratory clustering was conserved: isolates that clustered in laboratory A also clustered in laboratory B. While clustering was conserved and reproducible at two different laboratories, demonstrating the robustness of rep-PCR, interlaboratory comparison of individual isolate fingerprints showed more variability. This comparison allows conclusions regarding clonality to be reached independent of the laboratory where the analysis is performed.
PMCID: PMC3347881  PMID: 21903821
6.  Association between β-Lactamase-Encoding blaOXA-51 Variants and DiversiLab Rep-PCR-Based Typing of Acinetobacter baumannii Isolates 
Journal of Clinical Microbiology  2012;50(6):1900-1904.
This study investigated the correlation between blaOXA-51 variants and Acinetobacter baumannii worldwide clonal lineages 1 to 8 (WW1 to -8). The blaOXA-51-like genes of 102 A. baumannii isolates were sequenced. Using DiversiLab repetitive-sequence-based PCR (rep-PCR) typing, 92 of these isolates had previously been assigned to WW1 to -8 and 10 were unclustered. Clustering of DNA sequences was performed using the neighbor-joining method and the Jukes-Cantor phylogenetic correction. blaOXA-51 variants were in good correlation with DiversiLab-defined clonal lineages. Sequence-based typing of blaOXA-51 variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.
PMCID: PMC3372099  PMID: 22422849
7.  The Success of Acinetobacter Species; Genetic, Metabolic and Virulence Attributes 
PLoS ONE  2012;7(10):e46984.
An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.
PMCID: PMC3483291  PMID: 23144699
8.  Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae 
Transcriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes. Klebsiella pneumoniae is a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription of ramA is associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the available Klebsiella genome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded in K. pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568, and Enterobacter cloacae. We show that the overexpression of rarA results in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show that rarA (MGH 78578 KPN_02968) and its neighboring efflux pump operon oqxAB (KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest that rarA overexpression upregulates the oqxAB efflux pump. Additionally, it appears that oqxR, encoding a GntR-type regulator adjacent to the oqxAB operon, is able to downregulate the expression of the oqxAB efflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.
PMCID: PMC3421627  PMID: 22644028
9.  Use of a Simple Criteria Set for Guiding Echocardiography in Nosocomial Staphylococcus aureus Bacteremia 
A set of simple clinical prediction criteria for patients with nosocomial Staphylococcus aureus bacteremia was developed to identify patients at low risk of infective endocarditis in whom transesophageal echocardiography might be dispensable and was validated with two independent cohorts.
(see the editorial commentary and Soriano and Mensa, on pages 10–12.)
Background. Infective endocarditis (IE) is a severe complication in patients with nosocomial Staphylococcus aureus bacteremia (SAB). We sought to develop and validate criteria to identify patients at low risk for the development of IE in whom transesophageal echocardiography (TEE) might be dispensable.
Methods. Consecutive patients with nosocomial SAB from independent cohorts in Europe (Invasive S. aureus Infection Cohort [INSTINCT]) and North America (S. aureus Bacteremia Group [SABG]) were evaluated for the presence of clinical criteria predicting an increased risk for the development of IE (ie, prolonged bacteremia of >4 days' duration, presence of a permanent intracardiac device, hemodialysis dependency, spinal infection, and nonvertebral osteomyelitis). Patients were observed closely for clinical signs and symptoms of IE during hospitalization and a 3-month follow-up period.
Results. IE was present in 13 (4.3%) of 304 patients in the INSTINCT cohort and in 40 (9.3%) of 432 patients in the SABG cohort. Within 14 days after the first positive blood culture result, echocardiography was performed in 39.8% and 57.4% of patients in the INSTINCT and SABG cohorts, respectively. In patients with IE, the most common clinical prediction criteria present were prolonged bacteremia (69.2% vs 90% for INSTINCT vs SABG, respectively) and presence of a permanent intracardiac device (53.8% vs 32.5%). In total, 13 of 13 patients in the INSTINCT cohort and 39 of 40 patients in the SABG cohort with documented IE fulfilled at least 1 criterion (sensitivity, 100% vs. 97.5%; negative predictive value, 100% vs 99.2%).
Conclusions. A simple criteria set for patients with nosocomial SAB can identify patients at low risk of IE. Patients who meet these criteria may not routinely require TEE.
PMCID: PMC3149212  PMID: 21653295
10.  Evaluation of Quantitative Antibiotic Susceptibility Testing by Vitek 2 as a Routine Method To Predict Strain Relatedness of Coagulase-Negative Staphylococci Isolated from Blood Cultures▿ 
Journal of Clinical Microbiology  2011;49(9):3355-3357.
To test the hypothesis that the strain relatedness of coagulase-negative staphylococci (CoNS) recovered from blood cultures can be inferred from automated antibiotic susceptibility testing (AST) results generated by Vitek 2, concordant or discordant AST results were compared with pulsed-field gel electrophoresis (PFGE) typing results for 119 CoNS blood culture isolate pairs. Concordant AST results were highly predictive of the strain relatedness of CoNS isolates.
PMCID: PMC3165565  PMID: 21795506
12.  In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient▿  
Antimicrobial Agents and Chemotherapy  2010;54(12):5021-5027.
The mechanism of stepwise acquired multidrug resistance in Acinetobacter baumannii isolates from a hospitalized patient was investigated. Thirteen consecutive multidrug-resistant isolates were recovered from the same patient over a 2-month period. The Vitek 2 system identified the isolates as meropenem-sensitive Acinetobacter lwoffii; however, molecular identification showed that the isolates were A. baumannii. Etest revealed that the isolates were meropenem resistant. The presence of oxacillinase (OXA)-type enzymes were investigated by sequencing. The clonal relatedness of isolates was assessed by pulsed-field gel electrophoresis (PFGE). Expression of the genes encoding the efflux pumps AdeB and AdeJ was performed by semiquantitative real-time reverse transcription-PCR (qRT-PCR). The adeRS two-component system was sequenced. All isolates had identical PFGE fingerprints, suggesting clonal identity. The first six isolates were positive for the novel blaOXA-164 gene. The following seven isolates, recovered after treatment with a combination of meropenem, amikacin, ciprofloxacin, and co-trimoxazole showed an increase of >7-fold in adeB mRNA transcripts and a missense mutation in blaOXA-164, converting it to blaOXA-58. Sequencing revealed a novel mutation in adeR. These data illustrate how A. baumannii can adapt during antimicrobial therapy, leading to increased antimicrobial resistance.
PMCID: PMC2981280  PMID: 20921306
13.  OXA-134, a Naturally Occurring Carbapenem-Hydrolyzing Class D β-Lactamase from Acinetobacter lwoffii ▿  
Antimicrobial Agents and Chemotherapy  2010;54(12):5372-5375.
Acinetobacter lwoffii, a species whose natural habitat is the human skin, intrinsically possesses a chromosomal gene encoding a carbapenem-hydrolyzing class D β-lactamase, OXA-134. This species may therefore constitute a reservoir for carbapenemase genes that may spread among other Acinetobacter species.
PMCID: PMC2981293  PMID: 20837764
14.  gyrB Multiplex PCR To Differentiate between Acinetobacter calcoaceticus and Acinetobacter Genomic Species 3 ▿  
Journal of Clinical Microbiology  2010;48(12):4592-4594.
A previously established multiplex PCR that identifies to the species level Acinetobacter baumannii and Acinetobacter genomic species 13TU (GS13TU) was expanded to include Acinetobacter calcoaceticus and Acinetobacter genomic species 3.
PMCID: PMC3008493  PMID: 20881170
15.  Biofilm formation at the solid-liquid and air-liquid interfaces by Acinetobacter species 
BMC Research Notes  2011;4:5.
The members of the genus Acinetobacter are Gram-negative cocobacilli that are frequently found in the environment but also in the hospital setting where they have been associated with outbreaks of nosocomial infections. Among them, Acinetobacter baumannii has emerged as the most common pathogenic species involved in hospital-acquired infections. One reason for this emergence may be its persistence in the hospital wards, in particular in the intensive care unit; this persistence could be partially explained by the capacity of these microorganisms to form biofilm. Therefore, our main objective was to study the prevalence of the two main types of biofilm formed by the most relevant Acinetobacter species, comparing biofilm formation between the different species.
Biofilm formation at the air-liquid and solid-liquid interfaces was investigated in different Acinetobacter spp. and it appeared to be generally more important at 25°C than at 37°C. The biofilm formation at the solid-liquid interface by the members of the ACB-complex was at least 3 times higher than the other species (80-91% versus 5-24%). In addition, only the isolates belonging to this complex were able to form biofilm at the air-liquid interface; between 9% and 36% of the tested isolates formed this type of pellicle. Finally, within the ACB-complex, the biofilm formed at the air-liquid interface was almost 4 times higher for A. baumannii and Acinetobacter G13TU than for Acinetobacter G3 (36%, 27% & 9% respectively).
Overall, this study has shown the capacity of the Acinetobacter spp to form two different types of biofilm: solid-liquid and air-liquid interfaces. This ability was generally higher at 25°C which might contribute to their persistence in the inanimate hospital environment. Our work has also demonstrated for the first time the ability of the members of the ACB-complex to form biofilm at the air-liquid interface, a feature that was not observed in other Acinetobacter species.
PMCID: PMC3023692  PMID: 21223561
16.  Activity of the Investigational Fluoroquinolone Finafloxacin against Ciprofloxacin-Sensitive and -Resistant Acinetobacter baumannii Isolates ▿  
This study compared the activity of finafloxacin, a novel fluoroquinolone which shows enhanced activity under acidic pH, and that of ciprofloxacin against Acinetobacter baumannii under standard conditions (pH 7.2) and at a pH of 5.8. Overall, finafloxacin demonstrated superior activity to ciprofloxacin under acidic conditions. Furthermore, finafloxacin showed comparable activity to ciprofloxacin at pH 7.2. Hence, finafloxacin could be a promising new antimicrobial agent for the treatment of A. baumannii infections at acidic body compartments.
PMCID: PMC2849356  PMID: 20100879
17.  OXA-143, a Novel Carbapenem-Hydrolyzing Class D β-Lactamase in Acinetobacter baumannii▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):5035-5038.
A carbapenem-resistant Acinetobacter baumannii strain was isolated in Brazil in 2004 in which no known carbapenemase gene was detected by PCR. Cloning experiments, followed by expression in Escherichia coli, gave an E. coli recombinant strain expressing a novel carbapenem-hydrolyzing class D β-lactamase (CHDL). OXA-143 showed 88% amino acid sequence identity with OXA-40, 63% identity with OXA-23, and 52% identity with OXA-58. It hydrolyzed penicillins, oxacillin, meropenem, and imipenem but not expanded-spectrum cephalosporins. The blaOXA-143 gene was located on a ca. 30-kb plasmid. After transformation into reference strain A. baumannii ATCC 19606, it conferred resistance to carbapenems. Analysis of the genetic environment of blaOXA-143 revealed that it was associated with neither insertion sequences nor integron structures. However, it was bracketed by similar replicase-encoding genes at both ends, suggesting acquisition through a homologous recombination process. This study identified a novel class D β-lactamase involved in carbapenem resistance in A. baumannii. This enzyme is the first member of a novel subgroup of CHDLs whose prevalence remains to be determined.
PMCID: PMC2786334  PMID: 19770279
18.  Differential Expression of ccrA in Methicillin-Resistant Staphylococcus aureus Strains Carrying Staphylococcal Cassette Chromosome mec Type II and IVa Elements▿  
Antimicrobial Agents and Chemotherapy  2009;53(10):4556-4558.
Excision of staphylococcal cassette chromosome mec (SCCmec) is mediated through the ccrA- and -B-encoded recombinases. We investigated the effects of different antimicrobial agents on ccrA expression by using a ccrA::lacZ fusion and reverse transcription-PCR with methicillin (meticillin)-resistant Staphylococcus aureus strains MW2 (SCCmec IVa) and N315 (SCCmec II). Upregulation of ccrA was observed upon exposure to β-lactam antibiotics. Vancomycin increased ccrA expression in MW2 but had no effect on N315. Vancomycin may contribute to the transfer of SCCmec IVa but have no effect in SCCmec II.
PMCID: PMC2764196  PMID: 19596884
19.  Acinetobacter baumannii: Emergence of a Successful Pathogen 
Clinical Microbiology Reviews  2008;21(3):538-582.
Acinetobacter baumannii has emerged as a highly troublesome pathogen for many institutions globally. As a consequence of its immense ability to acquire or upregulate antibiotic drug resistance determinants, it has justifiably been propelled to the forefront of scientific attention. Apart from its predilection for the seriously ill within intensive care units, A. baumannii has more recently caused a range of infectious syndromes in military personnel injured in the Iraq and Afghanistan conflicts. This review details the significant advances that have been made in our understanding of this remarkable organism over the last 10 years, including current taxonomy and species identification, issues with susceptibility testing, mechanisms of antibiotic resistance, global epidemiology, clinical impact of infection, host-pathogen interactions, and infection control and therapeutic considerations.
PMCID: PMC2493088  PMID: 18625687
20.  Activity of Meropenem with and without Ciprofloxacin and Colistin against Pseudomonas aeruginosa and Acinetobacter baumannii▿ † 
Time-kill synergy studies showed that at 24 h, subinhibitory meropenem and ciprofloxacin concentrations of 0.06 to 128 and 0.03 to 32 μg/ml, respectively, showed synergy against 34/51 Pseudomonas aeruginosa strains; subinhibitory concentrations of meropenem (0.06 to 8 μg/ml) and colistin (0.12 to 1 μg/ml) showed synergy against 13 isolates. Subinhibitory meropenem and ciprofloxacin concentrations of 0.25 to 2 and 0.12 to 16 μg/ml, respectively, showed synergy against 18/52 Acinetobacter baumannii strains at 24 h. Subinhibitory meropenem and colistin concentrations of 0.03 to 64 and 0.06 to 8 μg/ml, respectively, showed synergy against 49 strains at 24 h.
PMCID: PMC2223893  PMID: 17967915
22.  Detection of Extended-Spectrum Beta-Lactamases among Enterobacteriaceae by Use of Semiautomated Microbiology Systems and Manual Detection Procedures▿  
Journal of Clinical Microbiology  2007;45(4):1167-1174.
Three commercially available microbiology identification and susceptibility testing systems were compared with regard to their ability to detect extended-spectrum β-lactamase (ESBL) production in Enterobacteriaceae, i.e., the Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD), the VITEK 2 System (bioMérieux, Marcy l'Etoile, France), and the MicroScan WalkAway-96 System (Dade Behring, Inc., West Sacramento, CA), using routine testing panels. One hundred fifty putative ESBL producers were distributed blindly to three participating laboratories. Conventional phenotypic confirmatory tests such as the disk approximation method, the CLSI double-disk synergy test, and the Etest ESBL were also evaluated. Biochemical and molecular characterization of β-lactamases performed at an independent laboratory was used as the reference method. One hundred forty-seven isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, and Morganella morganii were investigated. Of these isolates, 85 were identified as ESBL producers by the reference method. The remaining isolates were identified as non-ESBL producers; they were either hyperproducers of their chromosomal AmpC, Koxy, or SHV enzymes or lacked any detectable β-lactamase activity. The system with the highest sensitivity for the detection of ESBLs was the Phoenix (99%), followed by the VITEK 2 (86%) and the MicroScan (84%); however, specificity was more variable, ranging from 52% (Phoenix) to 78% (VITEK 2). The performance of the semiautomated systems differed widely with the species investigated. The sensitivities of the conventional test methods ranged from 93 to 94%. The double-disk synergy test showed the highest specificity and positive predictive value among all test methods, i.e., 97% and 98%, respectively.
PMCID: PMC1865808  PMID: 17287329
23.  Wound Botulism in Injection Drug Users 
Emerging Infectious Diseases  2007;13(6):942-943.
PMCID: PMC2792833  PMID: 17582887
wound botulism; heroin; abscess; Clostridium botulinum; antitoxin; PFGE; outbreak; letter
25.  Molecular Evolution of Methicillin-Resistant Staphylococcus aureus in the Metropolitan Area of Cologne, Germany, from 1984 to 1998 
Journal of Clinical Microbiology  2005;43(11):5445-5451.
To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.
PMCID: PMC1287791  PMID: 16272468

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