Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate''s clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.In addition to being the single most important genus of toxigenic phytopathogens (40), Fusarium (Hypocreales, Ascomycota) has emerged over the past 3 decades as one of the most important genera of filamentous fungi responsible for deeply invasive, opportunistic infections in humans (83). Clinically, fusarioses in immunocompetent patients typically present as superficial infections, such as onychomycosis and trauma-associated keratitis, or locally invasive infections, such as sinusitis, catheter-associated peritonitis, pneumonia, or diabetic cellulitis (77). The 2005-2006 keratitis outbreaks within the United States and Asia, however, were unusual in that they were linked to the use of a novel soft contact lens cleaning solution, which was subsequently removed from the market (11). In contrast, immunocompromised or immunosuppressed patients who are persistently and profoundly neutropenic may acquire life-threatening angioinvasive, hematogenously disseminated fusarial infections associated with high morbidity and mortality rates (15). The high mortality of immunosuppressed patients is due in part to the broad resistance of most fusaria to the spectrum of antifungals currently available (1, 4-7, 56); liposomal amphotericin B shows the greatest efficacy among the drugs currently in use (3, 17, 66).A series of molecular phylogenetic studies has led to the important conceptual advance that morphological species recognition within Fusarium (22, 38, 47) greatly underestimates its species diversity (49, 50, 53, 54-57, 59, 70, 85). This finding is not too surprising, given that phenotypic methods for identifying fusaria rely on relatively few morphological and cultural characters (75). Based on an extensive literature review, Nucci and Anaissie (48) recently recorded 12 morphospecies associated with fusarial infections within the immunocompromised patient population. However, phylogenetic species recognition based on genealogical concordance of multilocus DNA sequence data (herein referred to as GCPSR) (79) has identified at least 69 clinically important Fusarium species (Table ​(Table1)1) (49, 54, 56, 57, 70, 85). Phylogenetic species in these studies were recognized if they received ≥70% maximum parsimony (MP) bootstrap support (78) from the majority of the individual gene partitions and the combined data set and if their monophyly was not contradicted by analyses of any of the individual single-gene partitions.

TABLE 1.

Fusaria subjected to DNA MLST

Performance of MicroScan WalkAway and Vitek 2 for Detection of Oxacillin Resistance in a Set of Methicillin-Resistant Staphylococcus aureus Isolates with Diverse Genetic Backgrounds

Of 104 genotypically diverse methicillin-resistant Staphylococcus aureus (MRSA) isolates tested with the MicroScan WalkAway (Pos MIC 24 panel) and Vitek 2 (AST-P549 card) systems, 7 and 6 isolates, respectively, showed an oxacillin MIC of ≤2mg/liter. Most of these MRSA isolates were community acquired. However, if the cefoxitin screen of AST-P549 was also considered, MRSA detection failed for only one isolate.The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has increased over the last years. Reliable detection of MRSA is important since a false report of a patient''s isolate as methicillin susceptible would result in inadequate therapy with probably fatal consequences (2). Whereas MRSA infections formerly occurred almost exclusively in hospitalized patients, community-acquired MRSA (cMRSA) isolates have been reported recently in patients without any previous contact with the health care system (7).Many laboratories rely on automatic susceptibility testing methods that use oxacillin MIC testing, oxacillin breakpoint detection in the presence of salt, or cefoxitin MIC testing as markers for the presence of methicillin resistance. Many studies have investigated the detection of MRSA by the Vitek 2 system (3, 4, 8, 11, 12, 13, 15, 17); however, data for the performance of the MicroScan WalkAway system in MRSA detection are scarce (17).Most studies evaluating the performance of Vitek 2 used consecutive clinical strains (3, 8, 11, 12, 15), but this approach may be biased by the overrepresentation of locally predominant clones and may not predict performance in other geographical areas. We therefore used a collection of MRSA strains with distinct pulsed-field gel electrophoresis (PFGE) patterns to study MRSA detection using the MicroScan WalkAway and Vitek 2 systems.From 1998 to 2006, noncopy MRSA isolates (n = 1,516), initially identified by oxacillin screening agar or Vitek, from four hospitals in the Bochum area were collected and typed by PFGE as described previously (5). Of these, 120 isolates with different PFGE patterns were chosen. The patterns were interpreted according to the criteria of Tenover et al. (18), and isolates grouped into PFGE types and subtypes.For susceptibility tests, isolates from frozen storage were subcultured twice on Columbia blood agar at 37°C in 5% CO₂ before being tested with the Vitek 2 system using the AST-P549 card and the MicroScan WalkAway system using the Pos MIC 24 panel.Whenever results for oxacillin in the Vitek 2 or MicroScan WalkAway system or for the cefoxitin screen in the Vitek 2 system were not indicative of MRSA, a mecA PCR was performed from colonies growing on purity control plates of both automatic systems and a S. aureus-specific PCR for SA442 (16) was used as an internal positive control. In addition, the Panton-Valentine leukocidin (PVL)-coding genes lukS-PVL and lukF-PVL were detected by PCR (9). SCCmec typing (10) and spa typing (6) were performed as described previously.Loss of mecA during storage of isolates could be demonstrated in 16 of 120 isolates by PCR (14), a proportion that is similar to that described before (19). Of the remaining 104 true MRSA isolates, 95 were detected as MRSA with both automatic systems.An oxacillin MIC of ≤2 mg/liter was measured for six isolates with the Vitek 2 test and for seven isolates with the WalkAway test (Table ​(Table1);1); thus, those isolates would not have been detected as MRSA based on oxacillin MICs alone. Microdilution performed according to CLSI methods (1) showed resistant oxacillin MICs for all but one of these isolates, whereas by Etest on Mueller-Hinton agar with 2% NaCl, oxacillin MICs of ≥4 mg/liter were found for only two isolates. Microcolonies were the only indication for MRSA in most of the remaining strains, demonstrating the challenge of detecting MRSA in those isolates. The cefoxitin screen incorporated in the Vitek 2 AST-P549 card was positive for five of six isolates not detected by oxacillin MIC. Thus, cefoxitin testing together with oxacillin MIC testing clearly leads to better MRSA detection. Cefoxitin MICs of ≥16 mg/liter and ≥4 mg/liter were also found by microdilution and Etest.

TABLE 1.

All test results for MRSA isolates with negative cefoxitin screen in Vitek 2 or oxacillin MIC of ≤2 mg/liter in Vitek 2 or MicroScan WalkAway assay^a

Multicenter Collaborative Study for Standardization of Candida albicans Genotyping Using a Polymorphic Microsatellite Marker

Microsatellite-based genotyping for Candida albicans can give discrepant results between laboratories when expressed in fragment sizes, because their determination depends on electrophoretic conditions. The interlaboratory reproducibility was assessed in six laboratories provided with an allelic ladder. Despite variations in size determinations, alleles were correctly assigned, making data transportable between laboratories.Candida albicans is a diploid yeast responsible for a wide range of fungal infections in both immunocompetent and immunocompromised individuals. It is the most frequent fungal species involved in recurrent infections, outbreaks, and nosocomial infections (18). Genotyping of C. albicans can be performed using fluorescence-based microsatellite length polymorphism (MLP) analysis of PCR-amplified DNA fragments (2, 4, 5, 9, 11, 20, 21). Microsatellite sequences are defined as tandemly repeated stretches of 1 to 6 nucleotides and are excellent candidates for genetic analysis as they are polymorphic. Microsatellite alleles are often expressed as DNA fragments of different sizes obtained after PCR amplification with primers flanking the microsatellite region (22). Based on the variations in repeat numbers, genetic relatedness between different isolates can be assessed.MLP analysis is a method with a high discriminatory power and reproducibility, and it allows high throughput. It has proved its efficacy in genotyping large collections of samples in epidemiological studies of not only C. albicans but also other yeasts, such as Candida glabrata (10, 13), Candida tropicalis (6), and filamentous fungi, such as Aspergillus fumigatus or Penicillium marneffei (1, 7, 17). However, interlaboratory exchange of microsatellite data can be difficult, because the use of different equipment and diverse capillary electrophoresis conditions can lead to altered assessment of PCR fragment lengths. In the past, calibration by means of allelic ladders was applied for the analysis of population data (12, 14, 19, 23). More recently, de Valk et al. (8) produced locus-specific allelic ladders for the microsatellite-based analysis of Aspergillus fumigatus and tested its utility for assigning allele sizes in an interlaboratory study. In order to normalize C. albicans microsatellite results, we developed an allelic ladder that includes the most frequently occurring alleles of the CDC3 polymorphic microsatellite marker (2). Our objective was to test interlaboratory genotyping results by using the allelic ladder as an internal reference for a given marker.First, the allelic ladder for the tetranucleotide repeat (AGTA) CDC3 marker was constructed. Seven alleles in C. albicans were identified in our previous studies (2, 11). Genomic DNA of isolates known to contain the different alleles was extracted using the High Pure PCR template preparation kit as described by the manufacturer (Roche Applied Biosciences, Germany). Alleles were amplified in single-plex PCRs in a 20-μl reaction volume, consisting of 2 μl DNA, 1× PCR buffer (Roche Diagnostics GmbH, Mannheim, Germany), 0.2 mM each deoxynucleoside triphosphate, 5 mM MgCl₂, and 5 pmol of each CDC3 primer, with the sense primer labeled with hexachlorocarboxyfluorescein (HEX) (2) and 1.25 U of Taq Gold polymerase (Applied Biosystems, les Ulis, France). PCR was performed in an iCycler thermocycler (Bio-Rad, Hercules, CA) and consisted of an initial denaturation step at 95°C for 10 min, followed by 30 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C, with a final extension step of 30 min at 72°C. PCR products were then pooled in equivalent volumes, and 2 μl of this allelic pool was added to 13.5 μl of Hi-Di formamide (Applied Biosystems) and 0.5 μl 6-carboxy-X-rhodamine (ROX)-labeled Geneflo 625 size standard (Eurx, Gdansk, Poland). Capillary electrophoresis of the allelic ladder was performed using the ABI Prism 3730XL sequencer (Fig. ​(Fig.1).1). Sizes of the seven alleles were calculated with GeneMapper (version 4; Applied Biosystems). Reproducibility was tested in 10 separate experiments that resulted in allele size variation of ≤0.08 nucleotides for the seven alleles.Open in a separate windowFIG. 1.Example of allele assignment using the CDC3 allelic ladder for two isolates, numbers 22 and 15. Allele peaks in the ladder (black lines) are marked as p1 to p7. GeneFlo 625 internal size standards (black filled peaks) with sizes in bp are shown beneath each peak. Isolate 15 is p2-p5 heterozygous, and isolate 15 is p4 homozygous.The CDC3 ladder (10 μl) was sent to six participating laboratories with previous experience in MLP analysis. The National Reference Center for Mycoses and Antifungals (NRCMA) also provided five C. albicans DNA samples of known CDC3 genotypes (DNA samples 1 to 5) and the CDC3 primers (2). To test the influence of the DNA extraction method, each participant laboratory selected five isolates from their own strain collection, extracted the genomic DNA, and performed MLP genotyping on these samples (samples 6 to 35). The 35 DNA samples were sent to the NRCMA for blind MLP analysis.Participants were made aware of the number of allele peaks identified in the ladder (one to seven) and were asked to perform MLP using the NRCMA''s PCR amplification/cycling conditions and their own equipment and reagents (Table ​(Table1).1). For each DNA sample, genotyping results were expressed as peak numbers (1 to 7), and fragment sizes were expressed in bp. As shown in Table ​Table2,2, reported fragment sizes varied between the laboratories, with size differences of up to 4.3 bp. However, upon comparing the results to the allelic ladder, all laboratories correctly assigned peak numbers to the five DNA samples.

TABLE 1.

Conditions and equipment used by NRCMA and the six participant teams

In Vitro Activity of Anidulafungin and Other Agents against Esophageal Candidiasis-Associated Isolates from a Phase 3 Clinical Trial

The efficacy of anidulafungin, an echinocandin antifungal agent with potent anti-Candida activity, in treating esophageal candidiasis was tested in a double-blind study versus oral fluconazole. Isolates were identified and tested for susceptibility. Candida albicans represented >90% of baseline isolates. The MIC₉₀ of anidulafungin for all strains was 0.06 mg/liter.Anidulafungin is an echinocandin antifungal agent with broad-spectrum activity against Candida species (2, 12, 13, 17, 19, 20), including fluconazole-resistant strains (10, 16); concentration-dependent fungicidal activity; and a long postantifungal effect in vitro and in animal infection models (1, 6, 7, 10, 16, 18). It is available in the United States for intravenous treatment of esophageal candidiasis, a debilitating opportunistic infection among persons with HIV infection (9) for which cross-resistance among azoles may limit treatment options (4, 14). In patients treated with the anidulafungin dosage regimen for esophageal candidiasis (100-mg loading dose followed by 50 mg daily, half of the dosage used for invasive candidiasis), the steady-state mean maximum and minimum plasma concentrations were 4.2 and 1.6 μg/ml, respectively (Eraxis US package insert). Thus, anidulafungin may be a useful alternative to both amphotericin B and the azole antifungal agents in treating severe oral and esophageal candidiasis in persons with HIV infection and AIDS. We determined the in vitro activity of anidulafungin against clinical isolates of Candida spp. from esophageal candidiasis patients, most of them HIV infected, enrolled in a large (601 patients) phase 3 randomized, comparative, double-blind, double-dummy clinical study. The comparator was oral fluconazole, 200 mg administered on day 1 followed by 100 mg daily for 14 to 21 days.Candida isolates obtained from endoscopic biopsy specimens or brushings (11) were sent to a reference laboratory for identification, using standard methods (8), and susceptibility testing. Standard antifungal powders included anidulafungin (Vicuron, Inc., King of Prussia, PA), fluconazole (Pfizer, New York, NY), voriconazole (Pfizer), caspofungin (Merck, Whitehouse Station, PA), flucytosine (Sigma, St. Louis, MO), amphotericin B (Sigma), and itraconazole (Janssen, Beerse, Belgium). Preparation of stock solutions and broth microdilution susceptibility testing were as detailed in CLSI document M27-A2 (5, 15) for all agents except amphotericin B (tested in antibiotic medium 3). Incubation at 35°C was for 24 h (echinocandins) and 48 h (azoles, amphotericin B, and flucytosine). MICs, determined using a reading mirror, were defined as a prominent decrease in turbidity (ca. 50%), except for amphotericin B (complete growth inhibition).Overall, 96% of patients in both treatment arms were infected with C. albicans at baseline, with or without additional Candida species. A majority of the non-C. albicans species isolated at baseline were present in mixed infection with C. albicans. The predominance of C. albicans is characteristic of esophageal candidiasis (3). A total of 441 unique baseline isolates were received by the reference laboratory, including 411 of Candida albicans, 23 of Candida glabrata, 3 of Candida tropicalis, 2 of Candida krusei, and one isolate each of Candida pelliculosa and Candida lusitaniae.Anidulafungin had potent activity against these isolates (Table ​(Table1).1). Its MIC₉₀ was 0.06 μg/ml, and 99% of strains were inhibited by 0.12 μg/ml. The MIC distribution for caspofungin was similar. Micafungin was not available for testing at the time at which the study was conducted. For all of the azoles, susceptibility was greater than 90%. The MIC_50/90 of fluconazole for the 23 C. glabrata isolates was 8/16 μg/ml, respectively. Fluconazole-resistant strains included 3 of C. albicans and 1 of C. glabrata (MIC, ≥64 μg/ml) as well as the 2 of C. krusei (considered resistant irrespective of MIC). The MIC range of anidulafungin for these 6 isolates was 0.015 to 0.06 μg/ml. As noted previously, there is no cross-resistance between azoles and echinocandins (10, 13, 20).

TABLE 1.

In vitro susceptibilities of 441 esophageal isolates of Candida spp. to anidulafungin and six other systemically active antifungal agents

Single Nucleotide Polymorphisms in the IS900 Sequence of Mycobacterium avium subsp. paratuberculosis Are Strain Type Specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.The insertion sequence IS900 (13) is one of the 20 members of the IS110 family and is considered to be unique to Mycobacterium avium subsp. paratuberculosis, although IS900-like sequences have been found rarely in other environmental mycobacteria (9, 12). IS900 is present in multiple copies (14-18) within the M. avium subsp. paratuberculosis genome (13) and therefore is an ideal target for identification (16, 31). The heterogeneity of M. avium subsp. paratuberculosis isolates based on the number of IS900 copies and their positions within the genome has been exploited for epidemiological purposes. The IS900 sequence has been used in different molecular techniques such as multiplex PCR and restriction fragment length polymorphism analysis with hybridization to IS900, leading to the identification of individual strains and the classification of M. avium subsp. paratuberculosis into strain types (3, 17, 19, 30).M. avium subsp. paratuberculosis strains have been divided into three clusters named type I (also called sheep [S] strains), type II (also called cattle [C] strains), and type III (also called intermediate strains), although some of the studies include types I and III in the S type (8, 35). This classification into three clusters is based on results from molecular characterization techniques such as restriction fragment length polymorphism analysis with hybridization to IS900 (19), pulsed-field gel electrophoresis (PFGE) (11, 29), PCR-restriction enzyme analysis (PCR-REA) of gyrB (5), PCR-REA of inhA (6), PCR and denaturing gradient gel electrophoresis analyses of MAP1506 (14), PCR sequencing of recF (31), and comparative genomic hybridization analyses (4). The genotypic and phenotypic dissimilarities among types of M. avium subsp. paratuberculosis may be reflected in the differences detected in the progression of paratuberculosis or Johne''s disease among infected herds (33, 34).Comparisons of IS900 sequences from different M. avium subsp. paratuberculosis isolates with the published genome sequence of M. avium subsp. paratuberculosis strain K-10 have revealed single nucleotide polymorphisms (SNPs) (2, 20, 21, 23, 24, 27, 28, 35, 36) (Table ​(Table1)1) .

TABLE 1.

Previously described SNPs in the 5′ fragments of IS900 sequences relative to the M. avium subsp. paratuberculosis K-10 genome sequence^a

Novel Use of Tryptose Sulfite Cycloserine Egg Yolk Agar for Isolation of Clostridium perfringens during an Outbreak of Necrotizing Enterocolitis in a Neonatal Unit

Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.Necrotizing enterocolitis (NEC) is the most common acquired disease affecting the gastrointestinal system of neonates, with low-birth-weight babies at highest risk (20, 22). Clinical features of NEC range from mild intestinal signs such as abdominal distension (stage 1), to radiological signs of pneumatosis (stage 2), to advanced disease (stage 3) involving severe abdominal distension, hypotension, and peritonitis (1, 20). The underlying pathophysiology of NEC is poorly understood but is likely to be secondary to multiple injuries to the neonate gut through hypoxia-ischemia, hyperosmolar feeds, and infection (20, 24).No single infectious agent has been consistently identified as a cause of NEC, but Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae), viruses (rotavirus, coronavirus, echovirus, norovirus), and clostridial species have all been implicated (4, 23, 25, 30). The pathology of NEC resembles gas gangrene of the intestine caused by Clostridium perfringens, which produces a range of extracellular toxins (10, 11, 19, 27), and colonization with C. perfringens has been shown to be associated with both sporadic and nursery outbreaks of NEC (2-4). However, it is unclear whether C. perfringens is the causative agent of NEC or a marker of intestinal changes associated with the disease (2, 3, 16).Culture for C. perfringens is not usually undertaken for neonatal feces, as C. perfringens is considered a part of the normal fecal flora, with up to 35% of preterm neonates colonized within the first 2 weeks of life (2, 30). In addition, isolation using conventional media such as horse blood agar (HBA), if required, is difficult without the use of selective supplements (2, 17). However, C. perfringens is also a major cause of human food poisoning, and when implicated in food-borne outbreaks, the causative bacterium can be recovered and enumerated by the use of highly selective media such as tryptose sulfite cycloserine agar, which provides a rapid presumptive morphological identification of C. perfringens (6, 8, 9, 31).The neonatal unit at Monash Medical Centre has the capacity for 50 neonates and includes 18 level III (ventilated) neonatal intensive care unit (NICU) beds. The unit has a stable background NEC rate of about 6/1,000 admissions per year. From 1 January to 30 June 2008, 15 neonates were diagnosed with NEC (modified Bell stage 2 and above), increasing the yearly rate to 32/1,000 admissions and raising concerns of an outbreak. Cases were defined as neonates who met the NEC stage 2 or 3 criteria, and controls were defined as current neonates who were in the unit without NEC at the peak June outbreak period (1).Fecal samples were collected from 11 neonates with NEC and 45 without NEC (current controls) from 6 to 20 June 2008. Microbiological investigations were undertaken for possible bacterial and viral pathogens (28).Fecal samples (n = 56) were also cultured for C. perfringens using HBA (Oxoid CM 0331) and incubated for 48 h at 35°C in anaerobic jars. Colonies were examined for anaerobic hemolytic Gram-positive rods. Presumptive identification of C. perfringens was determined by using the reverse CAMP test (RC) (7, 12, 13). All isolates exhibiting hemolysis were confirmed using the RapID ANA II panel (Remel, Kansas) and by 16S rRNA gene sequencing.Fecal samples were also directly cultured on tryptose sulfite cycloserine with egg yolk agar (TSC-EYA), which consisted of perfringens agar base (CM0587; Oxoid) with 5% egg yolk emulsion and d-cycloserine, for 24 h at 35°C (Media Preparation Unit, University of Melbourne) (5, 9, 14, 15). Black, lecithinase-positive or -negative colonies were presumptively identified as C. perfringens and confirmed as described above. To aid in the recovery of clostridia from a background mixture of bacteria, fecal samples were also heat shocked at 60°C for 20 min and then cultured on TSC-EYA (17). Since some C. perfringens isolates are known to be heat sensitive, a third method was used whereby samples were pretreated with ethanol for 1 h before being cultured on TSC-EYA (17, 18, 21).All phenotypically black colonies that exhibited lecithinase activity and were RC positive underwent 16S rRNA gene sequence analysis and multiplex PCR toxinotyping. The preparation of genomic DNA from clostridial isolates and multiplex PCR including primer pair sequences used for genotyping were done as previously described (26, 29).Direct inoculation onto TSC-EYA, which identified four more culture-positive neonates than did standard culture on HBA, was the most sensitive method examined (Table ​(Table1).1). Both heat shock and ethanol shock were less effective approaches for the isolation of C. perfringens than direct plating on TSC-EYA. No positive results were found by other methods using neonate samples that were negative on TSC-EYA. Direct plating on TSC-EYA was also rapid, saving 24 to 48 h compared to standard culture on HBA. In total, C. perfringens was isolated using TSC-EYA from 10 of 56 (18%) of the study subjects: 3 of 11 (27%) NEC cases and 7 of 45 (16%) controls (odds ratio [OR], 1.69; P = 0.46).

TABLE 1.

Clostridium perfringens isolated from neonatal fecal samples

Disappearance of Vaccine-Type Invasive Pneumococcal Disease and Emergence of Serotype 19A in a Minority Population with a High Prevalence of Human Immunodeficiency Virus and Low Childhood Immunization Rates

We analyzed the epidemiology of invasive pneumococcal disease (IPD) following introduction of pneumococcal conjugated vaccine in an urban population with a 2% human immunodeficiency virus (HIV) prevalence and history of low childhood immunization rates. We observed near-elimination of vaccine-type IPD. Substantial disease remains due to non-vaccine-type pneumococci, highlighting the need to increase pneumococcal immunization among HIV-infected adults.Following the introduction of 7-valent pneumococcal conjugate vaccine (PCV7) in mid-2000, declines in invasive pneumococcal disease (IPD) were documented across all age groups in the United States (1, 4, 20) and in vulnerable populations (4, 7, 17). Subsequently, increases in IPD caused by nonvaccine serotypes, particularly 19A, were observed (4, 7, 8, 9, 13, 14, 17).Newark, NJ, is a mid-sized U.S. city with a predominantly black and Hispanic population (19), a high human immunodeficiency virus (HIV)/AIDS prevalence (2%) (11), and a history of low childhood immunization rates (2). Potential PCV7-related direct and indirect effects in such populations have not been fully studied. Statewide, passive surveillance of IPD began in mid-2003. We previously described a single Newark medical center''s experience with IPD (18). In the current study, we conducted active, population-based surveillance to complement these efforts and to better understand the contemporary epidemiology of IPD in Newark, specifically, PCV7''s impact and the roles of HIV/AIDS and race/ethnicity in IPD incidence.Multicenter, active surveillance of all Newark IPD cases was conducted from 1 December 2007 through 30 November 2008. Cases were identified at the clinical microbiology laboratories of the four major hospitals serving Newark residents. Case ascertainment was augmented by comparisons with passive reports to New Jersey''s Communicable Disease Reporting and Surveillance System. A case was defined as any Newark resident during the study period with Streptococcus pneumoniae isolated from either blood or cerebrospinal fluid (CSF).Patient demographics and medical information were abstracted from hospital medical charts. Collected isolates were serotyped with sequential multiplex PCR molecular methods developed by the Centers for Disease Control and Prevention (3, 12). DNA sequencing was used for resolution of serotypes 6A, 6B, and 6C. Vaccine serotypes (VT) were defined as those included in PCV7: 4, 6B, 9V, 14, 18C, 19F, and 23F. All others, including vaccine-related serotypes, were defined as nonvaccine serotypes (NVT). We separately considered the additional serotypes included in the 10- and 13-valent vaccines currently under development (6, 21).Race/ethnicity was dichotomized as black, not Hispanic (abbreviated as “black”) versus “all others.” The “all others” category included “Hispanic, all races,” “not Hispanic, white,” “not Hispanic, other,” and “unknown.” HIV status was categorized as either HIV infected or HIV uninfected/status unknown. For most incidence analyses, results are presented using the age intervals in the U.S. Census 2000 (19): <5, 5 to 17, 18 to 44, 45 to 64, and 65 years or older. For HIV-stratified analyses, results are presented using the age intervals in Newark''s HIV/AIDS prevalence reports (11): <13, 13 to 54, and 55 years or older. The χ² test of independence and Fisher''s exact test were used, as appropriate, in univariate analyses (16).During the study period 87 cases of IPD were identified among Newark residents. A total of 81 (93%) occurred at study centers, of which 72 (89%) were collected for serotyping and are described in detail in this report. Three isolates had no extractable DNA, and of the remaining 69, 5 were nontypeable. Ten cases that had not been reported through passive surveillance were identified using the study''s active surveillance methods.Considering all 87 cases during the study period, the reported 1-year incidence of IPD per 100,000 in Newark was 32 (95% confidence interval [CI], 25 to 38). The age distribution was bimodal with peaks in the under-5-years age group and the 45- to 64-years age group (Fig. ​(Fig.1).1). Considering the 72 cases for which full data were available, the relative risk (RR) of IPD for black cases versus all other cases was 2.2 (95% CI, 1.4 to 3.7) and was highest among persons 18 to 44 years old (6.4; 95% CI, 1.4 to 29) (Fig. ​(Fig.1).1). The RR of IPD for HIV-infected patients was 24 (95% CI, 15 to 38), with an incidence of 414 per 100,000 (95% CI, 268 to 611) among HIV-infected patients versus 18 (95% CI, 13 to 24) among HIV-uninfected/status unknown cases and was highest among persons 18 to 54 years old (RR, 47; 95% CI, 25 to 89). The RRs for HIV infected versus HIV uninfected/status unknown, stratified by black and all other race/ethnicity groups, were 19 (95% CI, 11 to 33) and 31 (95% CI, 12 to 85), respectively.Open in a separate windowFIG. 1.Age distribution of invasive pneumococcal disease incidence (per 100,000) by race/ethnicity, Newark, NJ, December 2007 to November 2008. Age- and race-stratified incidences were calculated based on the 72 cases for which full data were available.Table ​Table11 describes the demographic and clinical characteristics of the 72 collected cases. Of the adult cases, 25/63 (40%) were known to be HIV infected. There were no documented HIV-infected pediatric cases. The case fatality ratio was 19% (12/63) in adults and 11% (1/9) in children. Among HIV-infected adults for whom vaccination status was known, 13/21 (62%) had received the 23-valent pneumococcal polysaccharide vaccine (PPV23). Among children whose PCV7 status was known, 3/5 (60%) were up-to-date with PCV7.

TABLE 1.

Demographic and clinical characteristics of Newark IPD cases by HIV status

Testing Susceptibility of Multidrug-Resistant Mycobacterium tuberculosis to Second-Line Drugs by Use of Blood Agar

In this study, the susceptibilities of 35 multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates to second-line drugs, including kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH), were investigated on blood agar according to CLSI recommendations. Compared with the results of the Bactec 460 TB system, agreement was 100, 100, 97, 100, 100, 100, and 86% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively. Compared with the results of the proportion method, agreement was 100, 100, 97, 100, 97, 100, and 77% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively.Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are major public health problems, especially in developing countries (13, 18, 21). Rapid susceptibility testing is critical for early diagnosis of MDR- and XDR-TB and the initiation of effective regimens (14). The agar proportion method performed on Middlebrook 7H10 and 7H11 agars is the reference method for susceptibility of Mycobacterium tuberculosis according to CLSI recommendations (16). The Bactec 460 TB system (Becton Dickinson Diagnostic Systems, Sparks, MD), Bactec MGIT 960 (Becton Dickinson Diagnostic Systems, Sparks, MD), and Versa TREK system (formerly known as ESP II; Trek Diagnostic Systems, West Lake, OH) are cleared for used by the U.S. FDA for testing M. tuberculosis susceptibility to first-line drugs (16). It has recently been demonstrated that blood agar can be used for routine culture and testing of M. tuberculosis susceptibility to first-line drugs (1-4, 6, 7, 15).In this study, the susceptibilities of 35 MDR M. tuberculosis clinical isolates against the second-line drugs kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH) were investigated on blood agar, and results were compared to those obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar, performed according to CLSI recommendations.Clinical MDR-TB strains were obtained from the Istanbul Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Istanbul, Turkey. Isolates were identified by the Bactec NAP test as M. tuberculosis complex. Susceptibility to first-line drugs was determined by the radiometric method (Bactec 460 TB system) (19, 20). M. tuberculosis H37Rv was used as a control strain. All drugs were obtained from the manufacturers in a chemically pure form. Stock antibiotic solutions and subsequent dilutions were prepared. All stock solutions except CFM, which was stored in the dark at room temperature, were stored at −70°C in small aliquots.The agar proportion method using Middlebrook 7H10 and blood agar was performed with concentrations of 10, 5, 5, 1, 2, 1, and 2 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).Middlebrook 7H12 broth (Bactec 12B; Becton Dickinson Microbiology Systems) was used for radiometric testing (5, 11, 16, 19, 20). Second-line drugs (KM, RBU, PAS, CAP, CFM, OFX, and ETH) were tested by using single critical concentrations. Concentrations of second-line drugs tested in the Bactec 460 TB system were 1.25, 1.25, 5, 0.5, 2, 0.5, and 4 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).The inoculum was prepared from freshly grown colonies on Löwenstein-Jensen medium. The supernatant of each isolate was adjusted to a 1 McFarland standard. The agar proportion method was performed on both Middlebrook 7H10 agar and blood agar (infusion agar, Becton Dickinson) separately according to CLSI recommendations (16). The plates were monitored twice a week by the naked eye. Resistance was defined as growth of a colony count of >1% on drug-containing quadrants in comparison to drug-free quadrants (16).The Bactec 460 TB system revealed that all isolates were susceptible to KM and CFM. RBU, OFX, PAS, CAP, and ETH resistance was detected in 27, 1, 3, 2, and 17 isolates, respectively. All isolates were susceptible to KM and CFM on Middlebrook 7H10 agar; resistance was detected in 27, 1, 3, 1, and 14 isolates for RBU, OFX, PAS, CAP, and ETH, respectively. On blood agar, all isolates were susceptible to KM and CFM; resistance was detected in 27, 2, 3, 2, and 22 isolates for RBU, OFX, PAS, CAP, and ETH, respectively.Blood agar results were compared with the results obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar. Agreement, sensitivity, specificity, positive predictive value, and negative predictive value are shown in Tables ​Tables11 and ​and2.2. The results of susceptibility testing were obtained on the 21st day of incubation by the proportion method on blood and Middlebrook 7H10 agar as recommended by the CLSI. The results of susceptibility testing by the Bactec 460 TB system were obtained on the 5th day of incubation for 31 isolates and on the 8th day for 4 isolates.

TABLE 1.

Comparison of blood agar results and Bactec 460 TB system results^a

In Vitro Activity of Seven Systemically Active Antifungal Agents against a Large Global Collection of Rare Candida Species as Determined by CLSI Broth Microdilution Methods

Five Candida species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) account for over 95% of invasive candidiasis cases. Some less common Candida species have emerged as causes of nosocomial candidiasis, but there is little information about their in vitro susceptibilities to antifungals. We determined the in vitro activities of fluconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against invasive, unique patient isolates of Candida collected from 100 centers worldwide between January 2001 and December 2007. Antifungal susceptibility testing was performed by the CLSI M27-A3 method. CLSI breakpoints for susceptibility were used for fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin, while a provisional susceptibility breakpoint of ≤1 μg/ml was used for amphotericin and posaconazole. Of 14,007 Candida isolates tested, 658 (4.7%) were among the less common species. Against all 658 isolates combined, the activity of each agent, expressed as the MIC₅₀/MIC₉₀ ratio (and the percentage of susceptible isolates) was as follows: fluconazole, 1/4 (94.8%); voriconazole, 0.03/0.12 (98.6%); posaconazole, 0.12/0.5 (95.9%); amphotericin, 0.5/2 (88.3%); anidulafungin, 0.5/2 (97.4%); caspofungin, 0.12/0.5 (98.0%); and micafungin, 0.25/1 (99.2%). Among the isolates not susceptible to one or more of the echinocandins, most (68%) were C. guilliermondii. All isolates of the less common species within the C. parapsilosis complex (C. orthopsilosis and C. metapsilosis) were susceptible to voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Over 95% of clinical isolates of the rare Candida species were susceptible to the available antifungals. However, activity did vary by drug-species combination, with some species (e.g., C. rugosa and C. guilliermondii) demonstrating reduced susceptibilities to commonly used agents such as fluconazole and echinocandins.More than 95% of Candida bloodstream infections (BSI) are caused by five species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei (83). The in vitro activities of available antifungal agents against these five species have been documented extensively (21, 25, 64, 83, 91), whereas very little is known regarding the susceptibility profiles of the less frequently isolated Candida species (8, 40, 75-77, 94).Among the Candida strains reported to cause BSI, more than 17 different species have been identified (31, 32, 84). Aside from the five most common species noted above, the remaining species include C. lusitaniae, C. guilliermondii, C. kefyr, C. pelliculosa, C. famata, C. lipolytica, and C. rugosa (Table ​(Table1)1) (31, 77, 84). Many of these species have been observed to occur in nosocomial clusters and/or to exhibit innate or acquired resistance to one or more established antifungal agents (6, 9, 23, 27, 36, 48, 49, 79, 80, 87, 88, 106). In addition, the use of molecular identification methods has resulted in the identification of new species within larger species complexes (e.g., C. dubliniensis within the C. albicans complex and C. orthopsilosis and C. metapsilosis within the C. parapsilosis complex) (43-46, 100). In vitro susceptibility data specific to those newly described species groups are also lacking.

TABLE 1.

Distribution of 14,007 isolates of Candida spp. from blood and other normally sterile sites, 2001 to 2007

Streptococcus gallolyticus Subspecies pasteurianus (Biotype II/2), a Newly Reported Cause of Adult Meningitis

We report the first case of adult meningitis confirmed to be due to Streptococcus gallolyticus subsp. pasteurianus. Phenotypically reported as Streptococcus bovis biotype II/2, 16S rRNA sequencing revealed S. gallolyticus subsp. pasteurianus. Because of taxonomic uncertainties, S. gallolyticus subsp. pasteurianus may be an underrecognized agent of systemic infections.The group D nonenterococcal streptococci include Streptococcus bovis, with two biotypes (I and II) that cause human infections. Biotype I (Streptococcus gallolyticus) is associated with colonic carcinoma and endocarditis (20). Biotype II/1 (Streptococcus infantarius) has been associated with noncolonic cancers (5). These clinical implications make accurate species identification critical. However, the S. bovis group is genetically diverse, and organisms previously classified as S. bovis now represent multiple species with unique clinical manifestations (8, 9, 22). S. gallolyticus subsp. pasteurianus, also named Streptococcus pasteurianus, was proposed to replace S. bovis II/2 (19, 22). Clinicians and laboratory staff do not recognize this taxonomy and its associated clinical implications. We report a case of S. gallolyticus subsp. pasteurianus meningitis.A 75-year-old man presented to the emergency room 2 days after the onset of headache, fever, and photophobia. He had a history of prostate cancer 8 years previously, which was treated with pelvic irradiation, with subsequent radiation proctitis. He denied intravenous drug abuse. Physical exam revealed a temperature of 38.3°C, photophobia, and nuchal rigidity. His peripheral white blood cell count (WBC) was 11,400/mm³ (with 65% neutrophils, 15% bands, and 10% lymphocytes), and his glucose was 160 mg/dl. The patient was given 1 g ceftriaxone, and 2 hours later lumbar puncture showed clear, colorless cerebrospinal fluid (CSF), with a WBC of 112/mm³ (62% neutrophils), glucose of 38 mg/dl, and protein of 282 mg/dl; no organisms were seen on Gram stain. HIV testing and three stool specimens for ova and parasites were negative.The patient was treated for bacterial meningitis with ampicillin, vancomycin, ceftriaxone, and dexamethasone (0.15 mg/kg of body weight). A group D nonenterococcal streptococcus was identified from blood and CSF cultures. The API Rapid Strep kit (bioMérieux, Marcy l''Etoile, France) identified the organism as S. bovis biotype II/2, and RapID Strep (Remel, Lenexa, KS) identified it as S. bovis variant group D (also known as biotype II). As the cultures were sensitive to ceftriaxone, clindamycin, erythromycin, levofloxacin, linezolid, penicillin, and vancomycin, both ampicillin and vancomycin were discontinued. A transesophageal echocardiogram showed no evidence of endocarditis, and colonoscopy was negative. He received intravenous antibiotics for 10 days, and as of January 2010 has not had recurrence of illness after 54 months of follow-up.After incubation on tryptic soy blood agar (TSBA) plates, colonies were tested for catalase production and failed growth in 6.5% NaCl. Lancefield typing was determined by using Streptex (Remel). Carbohydrate fermentation analysis was performed using the API 20 Strep (ID 7650450; bioMérieux) and RapID Strep (ID 22301; Remel) kits. See Table ​Table11 for the results of phenotypic testing.

TABLE 1.

Phenotypic characteristics of S. bovis biotype II/2 (S. gallolyticus subsp. pasteurianus)

Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.Otitis media remains a significant health problem for children in this country and elsewhere in the world (10, 11). Most children in the United States have had at least one episode of otitis by their third birthdays, and one-third have had three or more episodes (34). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (33, 41). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for the prevention of otitis media in young children (20). Although the total prevention of disease will be a difficult goal to achieve, the prevention of even a portion of cases would be beneficial, given the magnitude and costs of the problem.Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of the cases of acute otitis media (16, 35). Streptococcus pneumoniae is the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (16, 35). Nontypeable Haemophilus influenzae (NTHI) is the second-most-common bacterium recovered and accounts for 20% to 30% of the cases of acute otitis media and a larger percentage of the cases of chronic and recurrent disease (26). Interestingly, since the introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccine schedule, NTHI has become an even more common cause of acute and recurrent middle ear disease, often surpassing S. pneumoniae in the frequency of recovery from middle ear specimens (12, 26). Many different antigens have been suggested as possible NTHI vaccine candidates (1, 3, 18, 29, 30, 42). Outer membrane proteins appear to be the principal targets of bactericidal and protective antibodies (22), and as a group, they have been the major focus of vaccine development efforts. Table ​Table11 summarizes the relevant characteristics of some of the leading vaccine candidates currently under active investigation.

TABLE 1.

Potential vaccine antigens of NTHI

Use of a New Single Multiplex PCR-Based Assay for Direct Simultaneous Characterization of Six Neisseria meningitidis Serogroups

We developed a new Neisseria meningitidis multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping N. meningitidis from cerebrospinal fluid that could be useful in Africa.Meningitis is one of the various forms of meningococcal diseases, which may occur as epidemics and severe sepsis, often with a fatal outcome (4). An etiologic diagnosis is confirmed by the isolation of Neisseria meningitidis from cerebrospinal fluid (CSF), blood, or other body fluids. Characterization of meningococci by serogroup is compulsory to investigate disease outbreaks and before starting vaccination campaigns. Twelve meningococcal serogroups identified by the capsular polysaccharide antigens are distinguished and chemically defined. Patient strains are nearly always encapsulated, but only six of these groups (A, B, C, W135, Y, and more recently X) (3) have significant pathogenic potential, causing more than 90% of the invasive disease worldwide (12).Vaccines are available against strains of N. meningitidis belonging to serogroups A, C, W135, and Y but not against strains of serogroups B or X. Hence, serogrouping is really necessary to better apply preventive measures (14). Recent outbreaks due to serogroup X have been described in Niger and Ghana (3, 5, 8); thus, the detection of serogroup X becomes essential in African countries and a low-cost assay needs to be developed.Standard PCR-based assays are routinely used for the detection and identification of serogroups A, B, C, W135, and Y. Simultaneous identification of serogroups A, B, C, W135, and Y has been described in one previous report (14) but a subsequent PCR was necessary to distinguish between W135 and Y. Recently, a separate standard multiplex PCR has also been described for the detection of serogroups 29E, X, and Z (1). Alternatively, a real-time quantitative PCR analysis has also been developed for simultaneous serogroup determination (10), but such an assay might be too expensive for African laboratories.Here we report for the first time a standard multiplex PCR-based assay for rapid simultaneous identification of the six serogroups A, B, C, W135, Y, and X, with direct discrimination between W135 and Y. We used oligonucleotides in orf-2 of a gene cassette required for the biosynthesis of the capsule of serogroup A (13) in the synD and synE genes encoding the polysialyltransferase responsible for the polymerization of polymers of sialic acid containing polysaccharides in strains belonging to serogroups B and C, respectively, and in the synF and synG genes, encoding proteins that catalyze the linkage of sialic acid with other sugars in the polysaccharide chains of serogroups Y and W135, respectively (6, 10); these primers were those described by Taha (14). The sizes of the expected amplicons are 450 bp (B), 400 bp (A), 250 bp (C), and 120 bp (W135). For direct identification of serogroup Y, the reverse primer in the synF gene was identical to that previously described (14), but a new forward primer in the synF gene was designed to amplify a Y-specific 75-bp product, distinct from the 120-bp W135-specific amplicon. The 3′ end of the crtA gene, which encodes an outer membrane protein involved in capsule transport, is highly conserved among meningococci irrespective of serogroups, but the 5′ end is very variable (7). Both forward and reverse primers specific to serogroup X were then designed within this variable region, giving rise to a serogroup X-specific 190-bp amplification product (Table ​(Table11).

TABLE 1.

Oligonucleotides used in this study

Incidence of Acinetobacter Species Other than A. baumannii among Clinical Isolates of Acinetobacter: Evidence for Emerging Species

Six hundred ninety nonduplicate isolates of Acinetobacter species were identified using a combination of detection of bla_OXA-51-like and rpoB sequence cluster analysis. Although most isolates were identified as A. baumannii (78%), significant numbers of other species, particularly A. lwoffii/genomic species 9 (8.8%), A. ursingii (4%), genomic species 3 (1.7%), and A. johnsonii (1.7%), were received, often associated with bacteremias.The Acinetobacter genus consists of more than 30 species, of which A. baumannii, and to a lesser extent genomic species 3 and 13TU, are most associated with the clinical environment and nosocomial infections. Identification within the genus is difficult and requires molecular methods, and these organisms are rarely identified to the species level using appropriate methods (3, 4, 6, 24). While A. baumannii can relatively readily be identified by detection of bla_OXA-51-like, the intrinsic carbapenemase gene in this species (22), the use of rpoB sequencing has facilitated identification across the genus (5, 8), and it is becoming clear that other species, such as A. ursingii (which has also been called A. septicus [13]) and A. haemolyticus, are also important nosocomial pathogens in some cases (3, 4, 5, 6, 9, 24). rpoB sequencing has advantages over such techniques as amplified ribosomal DNA restriction analysis (ARDRA), based on 16S rRNA gene sequences, and those based on the 16S-23S intergenic spacer region, since there is a relatively high degree of polymorphism in this gene among the Acinetobacter species, and sequences are available for all the currently described species (8), including those more recently described (11-14).Our laboratory provides a typing and identification service for hospitals in the United Kingdom and Republic of Ireland for this organism, and here we describe the species found among 690 isolates of Acinetobacter, each from a different patient, submitted over a 20-month period during 2008/2009, from some 135 hospitals. While A. baumannii, which is frequently associated with outbreaks, is still by far the most common of the Acinetobacter species among clinical isolates, it is clear that lesser-known species, such as A. lwoffii, A. ursingii, and A. parvus, are regularly encountered, have been associated with serious infections, and may represent emerging pathogens.The majority of isolates were received as Acinetobacter species for typing, identification, and/or susceptibility determinations and were subjected to a multiplex PCR for detection of bla_OXA-58-like, bla_OXA-23-like, bla_OXA-51-like, bla_OXA-40-like, and class 1 integrase genes, as described by Turton et al. (22), with the addition of primers for bla_OXA-58-like (27). Detection of bla_OXA-51-like was regarded as a positive identification of A. baumannii; the identity of a proportion of such isolates was also checked by rpoB sequence cluster analysis; in addition, many were shown by pulsed-field gel electrophoresis (PFGE) to be further representatives of strains previously identified as A. baumannii, and all gave amplicons in a PCR to amplify variable-number tandem repeat loci found in A. baumannii (23). The remaining isolates were identified by rpoB sequence cluster analysis using primers described by La Scola et al. (8). Briefly, a 903-bp portion of the rpoB gene covering two variable regions was amplified using the primers Ac696F and Ac1598R. Amplicons were treated with Exo-SAP-IT (USB Corporation, Cleveland, OH) according to the manufacturer''s instructions, and four sequencing reactions were carried out, using the primers Ac696F, Ac1055F, Ac1093R, and Ac1598R, respectively. The resulting fragments were separated on a Beckman-Coulter CEQ8000 genetic analysis system or an Applied Biosystems 3730 DNA analyzer and aligned, and sequences of a 765-bp fragment corresponding to nucleotides (nt) 2964 to 3728 of the coding sequence were compared using the BioNumerics software program; a phylogenetic tree was constructed using the MEGA software program (http://www.megasoftware.net/mega41.html) (19) (Fig. ​(Fig.1).1). Sequences of reference isolates were also included, and isolates were identified both by BLAST searches and according to which species they clustered most closely with; as more isolates were added to the database, a measure of the extent of sequence diversity associated with each species was obtained, allowing determination of whether isolates clustered closely enough to be assigned to that species. Susceptibilities to at least 16 antibiotics were determined by agar dilution and interpreted using British Society of Antimicrobial Chemotherapy (BSAC) breakpoints (http://www.bsac.org.uk). Pulsed-field gel electrophoresis (PFGE) of ApaI-digested genomic DNA was carried out as described previously (21).Open in a separate windowFIG. 1.Phylogenetic tree of sequences corresponding to nt 2964 to 3728 of the rpoB coding sequence of isolates of Acinetobacter species. Clinical and reference isolates were included, with GenBank accession numbers being included with the latter. Phylogenetic analyses were conducted in MEGA4 (19) using the neighbor-joining method. One thousand replicates were used for bootstrap analysis.Using this method, we were able to identify most isolates to the species level, although isolates of A. lwoffii and genomic species 9 clustered too closely to be distinguished from one another, as did those of A. baylyi and genomic species 11 (A. guillouiae). Similarly, as has been observed by others (26), A. grimontii and A. junii could not be distinguished and are likely to be a single species. Three isolates (UA1 to -3), two of which had highly similar rpoB sequences, did not cluster closely enough with any of the described species and may represent new species. For all three, the closest currently described species is A. towneri. Isolates that were identified as A. radioresistens were PCR positive for bla_OXA-23-like, the naturally occurring carbapenemase gene in this species (17), consistent with the identification. Detection of OXA carbapenemase genes among species other than A. baumannii was rare, with the only other examples being two isolates, one of genomic species 3 and the other of genomic species 16, with bla_OXA-58-like.As expected, the majority (78.0%) of isolates were identified as A. baumannii; with A. lwoffii/genomic species 9 (8.8%), A. ursingii (4.0%), genomic species 3 (1.7%), A. johnsonii (1.7%), and A. parvus (1.3%) accounting for most of the rest (Table ​(Table1).1). In most cases, these non-A. baumannii isolates were from blood and were associated with bacteremia or septicemia. Of note is that some isolates (of A. johnsonii, genomic species 13, and A. beijerinckii) were implicated in endocarditis; this has been described for other Acinetobacter species (7, 18, 28) but not these and provides further evidence that these organisms can cause life-threatening infections. The relatively high incidence of A. ursingii, which exceeded those of both genomic species 3 and 13TU, was unexpected but agrees with observations from hospitals in the Netherlands and Northern Ireland (2, 24). It has previously been documented that this organism has the capacity to cause bloodstream infections in hospitalized patients (4, 9, 11), and it has been associated with a nosocomial outbreak of bloodstream infections in a neonatal intensive care unit, in which two babies died (6). The isolates in the present study were from 28 patients in 24 centers, suggesting that they were not epidemiologically related; nevertheless, three isolates, each from different centers, formed a cluster by PFGE (see Fig. S1 in the supplemental material). Similarly, A. lwoffii has previously been linked with catheter-related bloodstream infections (20), as has A. parvus (12).

TABLE 1.

Submissions of Acinetobacter sp. other than A. baumannii received during the study period and associated clinical information^a

In Vivo Acquisition of a Plasmid-Mediated blaKPC-2 Gene among Clonal Isolates of Serratia marcescens

Three patients admitted to a Greek hospital were infected with Serratia marcescens isolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniae carbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescens isolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescens that acquired a plasmid-mediated bla_KPC-2 gene during the hospitalization.The emergence of organisms producing class A β-lactamases of Klebsiella pneumoniae carbapenemase (KPC) types is a major clinical and public health concern (2, 10). They are typically transposon-encoded determinants and therefore have the potential to disseminate between plasmids and across bacterial species (9). K. pneumoniae remains the species most likely to harbor bla_KPC genes (2, 8, 10, 13). Nevertheless, other species of Enterobacteriaceae, as well as species of nonfermenters, have been occasionally reported to exhibit this pattern of resistance (3, 15, 16, 19). In Serratia marcescens carbapenemase production is mostly attributed to class B metallo-β-lactamases (MBLs) as well as to the class A SME family of carbapenemases (14). Only recently has carbapenem-hydrolyzing activity in S. marcescens been attributed to the production of a KPC in China and the United States (3, 17, 23). We report the spread of three bla_KPC-possessing S. marcescens isolates in a Greek intensive care unit and give in vitro and in vivo evidence of the potential acquisition of such plasmid-borne resistance genes.In December 2008 a 77-year-old woman was admitted to the unit following a neurosurgical procedure. Ampicillin-sulbactam was administered postoperatively. Two months after her admission, the patient developed pneumonia, and bronchial lavage samples grew a S. marcescens isolate (S53) that exhibited reduced carbapenem susceptibility. The patient was successfully treated with tigecycline and inhaled colistin.Approximately 5 months later, in April 2009, a 49-year-old man was admitted following the surgical removal of a subcranial hematoma. He remained febrile while receiving empirical prophylactic antibiotic treatment with ampicillin-sulbactam, vancomycin, and amikacin. Bronchial lavage samples produced a carbapenem-susceptible S. marcescens isolate (S51) and a carbapenem-resistant K. pneumoniae isolate (K72). Antibiotic therapy was changed to meropenem and colistin. A second episode of pneumonia occurred approximately 2 weeks later, and a new S. marcescens isolate (S54) with reduced susceptibility to carbapenems was recovered from the bronchial lavage cultures. The patient was successfully treated with tigecycline and colistin.Finally, in April 2009 a 33-year-old woman was admitted following extensive surgery to the spine. The patient received ampicillin-sulbactam postoperatively. Approximately a week after her admission she presented with bacteremia due to a carbapenem-susceptible S. marcescens isolate (S52) and was treated with ciprofloxacin. Three weeks later the patient had an episode of pneumonia. Bronchial lavage sample cultures produced a new S. marcescens isolate (S55) that exhibited reduced susceptibility to carbapenems. Administration of ciprofloxacin in combination with gentamicin led to the successful treatment of this episode.The isolates that were recovered from the aforementioned patients were evaluated. Species identification was performed with the Vitek 2 system (bioMérieux, Marcy l''Étoile, France) and confirmed with API 20E (bioMérieux). MICs for several β-lactams, aminoglycosides, ciprofloxacin, tigecycline, and colistin were further determined by agar dilution according to CLSI recommendations (4). The MBL Etest (AB Biodisk, Solna, Sweden) and the combined disk test with imipenem and EDTA (5) were used to screen for MBL production. The phenotypic detection of KPC-possessing isolates was evaluated with the boronic acid potentiation disk test using meropenem as an antibiotic substrate (20). Extended-spectrum β-lactamase (ESBL) production was tested with the CLSI confirmatory test and in the KPC-possessing isolates with the modified CLSI ESBL confirmatory test, using clavulanate in combination with boronic acid (21).Isolates were screened for β-lactamase genes by PCR amplification using a panel of primers for the detection of all types of MBLs (6), KPCs (8), plasmid-mediated AmpCs in single PCRs for each gene (11), and ESBLs (22). PCR products were subjected to direct sequencing. Pulsed-field gel electrophoresis (PFGE) of SpeI- and of XbaI-digested genomic DNA of the S. marcescens isolates was performed with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, United Kingdom), and PFGE patterns were compared visually following previously described criteria (18). The potential for conjugational transfer of carbapenem resistance was examined in biparental matings using LB broth cultures and Escherichia coli 26R764 (lac⁺ Rif^r) as the recipient strain. Transconjugant clones were screened on MacConkey agar plates containing rifampin (150 μg/ml) and amoxicillin (40 μg/ml) or ertapenem (0.5 μg/ml). MICs were determined by agar dilution (4). All β-lactamase genes were detected by PCR amplification. Plasmid extraction was performed by using an alkaline lysis protocol with E. coli 39R861 as a control strain (7).The susceptibility patterns of the S. marcescens and K. pneumoniae isolates are shown in Table ​Table1.1. S. marcescens isolates S51 and S52 were susceptible to all carbepenems and to most β-lactam antibiotics. Phenotypic tests were negative for carbapenemase and ESBL production, amplification of the β-lactamase genes confirmed the presence solely of the bla_TEM gene, and DNA sequencing identified the gene in both isolates as bla_TEM-1.

TABLE 1.

Antimicrobial susceptibility patterns of the study''s clinical isolates, their transconjugants, and the recipient strain, E. coli 26R764

Cooccurrence of Predominant Panton-Valentine Leukocidin-Positive Sequence Type (ST) 152 and Multidrug-Resistant ST 241 Staphylococcus aureus Clones in Nigerian Hospitals

Ninety-six clinical isolates of Staphylococcus aureus from Nigeria were characterized phenotypically and genetically. Twelve multidrug-resistant methicillin (meticillin)-resistant S. aureus (MRSA) isolates carrying a new staphylococcal cassette chromosome mec element and a high proportion of Panton-Valentine leukocidin (PVL)-positive methicillin-susceptible S. aureus (MSSA) isolates were observed. The cooccurrence of multidrug-resistant MRSA and PVL-positive MSSA isolates entails the risk of emergence of a multidrug-resistant PVL-positive MRSA clone.Staphylococcus aureus is a major cause of both hospital- and community-acquired infections. In particular, methicillin (meticillin)-resistant S. aureus (MRSA) strains have been detected worldwide (15), and the prevalence of MRSA varies among countries and health institutions (2, 4, 27). The emergence of MRSA strains resistant to glycopeptides, as well as the increasing prevalence in the community (7), highlights the need for worldwide epidemiological studies of this pathogen. However, data about the epidemiology and prevalence of staphylococcal infections in Africa are scarce compared to information about such infections in the rest of the world. Studies have indicated low prevalences of MRSA in Nigeria, Somalia, and Tanzania (1), but high prevalences in South Africa, Zimbabwe, Kenya, Ethiopia, Egypt, Senegal, and the Ivory Coast have been reported (2, 9, 18). In addition, a recent study of the genetic diversity of S. aureus strains in a carriage population from Mali showed a high frequency of a Panton-Valentine leukocidin (PVL)-positive clone (25). The mechanisms for the emergence and spread of S. aureus clones in Africa are largely unknown; hence, the characterization of isolates may provide baseline information needed in establishing effective infection control measures in Nigeria.In this study, a total of 96 S. aureus isolates obtained between January and December 2007 from clinical specimens in six tertiary-care hospitals located in northeastern Nigeria were characterized. The isolates were identified based on standard bacteriological procedures (i.e., Gram staining and catalase, tube coagulase, and DNase testing), and susceptibilities to 12 antibiotics (Table ​(Table1)1) were determined by the disk diffusion method according to the CLSI guidelines. All the isolates were susceptible to vancomycin, fusidic acid, and mupirocin, and 12 (12.5%) were resistant to methicillin (i.e., oxacillin and cefoxitin resistant) (Table ​(Table1).1). The MRSA isolates were multidrug resistant (i.e., resistant to beta-lactams, along with at least three other classes of antibiotics), a finding similar to previously reported findings in other African countries like Morocco, Kenya, Cameroon, and South Africa (17). MRSA resistance to non-beta-lactams may further increase the medical expenses and the complexity of patient management, as well as morbidity and mortality rates since alternative antibiotics may not be affordable in many African countries.

TABLE 1.

Frequency of resistance of S. aureus (MSSA and MRSA) isolates to antibiotics

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the Hap_S passenger domain, leaving the Hap_β C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.Haemophilus influenzae is a gram-negative coccobacillus that typically colonizes the nasopharynxes of children and adults. In addition, this organism is an important cause of localized respiratory tract and invasive disease. Nonencapsulated strains cause otitis media, sinusitis, conjunctivitis, and exacerbations of respiratory symptoms in individuals with underlying lung disease, bronchiectasis, and cystic fibrosis (21, 29). Encapsulated strains are an important cause of bacteremic diseases, including sepsis and meningitis (29). Colonization of the upper respiratory tract represents an early step in the pathogenesis of all Haemophilus disease and requires adherence to respiratory epithelium (19). Adherence is facilitated by a number of adhesins, including Hap, Hia, Hsf, HMW1/HMW2, P5, pili, and lipooligosaccharide (2, 18, 21, 26, 27).The Hap adhesin is ubiquitous among isolates of H. influenzae and is a member of the autotransporter family of virulence factors that have been recognized among many gram-negative bacteria (10). Autotransporters are synthesized as precursor proteins with three functional regions, namely, an N-terminal signal sequence, an internal passenger domain, and a C-terminal β-barrel domain (11). The signal sequence targets the precursor protein to the inner membrane and is then cleaved. The C-terminal β-barrel domain inserts into the outer membrane and facilitates presentation of the passenger domain on the bacterial cell surface. Depending upon the protein, the passenger domain remains covalently attached to the β-barrel domain, is cleaved but remains loosely attached to the β-barrel domain, or is cleaved and released entirely from the cell surface (10-12). Although diverse autotransporters share a similar structural organization and a common secretion mechanism, they vary widely in function, possibly reflecting adaptations to particular bacterial pathogenic niches. Autotransporters may function as adhesins mediating tissue tropism, as proteases involved in tissue degradation, as toxins causing host tissue damage, or as mediators of serum resistance (11).Hap is synthesized as a 155-kDa preprotein encompassing a 110-kDa passenger domain, Hap_S, and a 45-kDa β-barrel domain, Hap_β. The Hap_S passenger domain harbors adhesive activity that has been shown to promote interactions with human respiratory cells, as well as with extracellular matrix proteins such as fibronectin, laminin, and collagen IV (7). Hap_S is also responsible for bacterial aggregation via Hap-Hap interactions, contributing to microcolony formation (5). Adherence to epithelial cells and bacterial aggregation are mediated by the C-terminal 311 amino acids of Hap_S, whereas interaction with extracellular matrix proteins is mediated by the C-terminal 511 amino acids of Hap_S (7).Beyond possessing adhesive activities, the Hap_S passenger domain functions as a protease that directs the autoproteolytic cleavage of Hap_S from Hap_β, resulting in release of Hap_S from the bacterial cell surface (6). Hap autoproteolysis has been determined to occur at least partly through intermolecular cleavage on the surface of the bacterium and involves a catalytic triad consisting of residues His98, Asp140, and Ser243. Ser243 is part of the GDSGS motif that is characteristic of many serine proteases (6, 13). In wild-type Hap, cleavage occurs most abundantly at the L1036-N1037 peptide bond, which is referred to as the primary cleavage site (13). Site-directed mutagenesis of this site and N-terminal sequencing of the resulting cleaved Hap fragments has identified three additional cleavage sites, including L1046-T1047, F1077-A1078, and F1067-S1068, termed the secondary, tertiary, and quaternary cleavage sites, respectively (see Table ​Table2)2) (6). Alignment of the amino acid sequences at these cleavage sites has revealed a consensus target sequence motif that consists of (Q/R)(A/S)X(L/F) at the P4 through P1 positions (see Table ​Table2)2) (6).

TABLE 2.

Alignment of sequences at the Hap cleavage sites

MIC Quality Control Guidelines and Disk Diffusion Test Optimization for CEM-101, a Novel Fluoroketolide

The development of diagnostic susceptibility tests for CEM-101, a new fluoroketolide, was addressed by structured studies to determine the optimal disk diffusion test concentration and effects of various testing conditions or supplements and to establish the quality control (QC) ranges for reference broth microdilution tests. The 15-μg CEM-101 disk was selected, and MIC ranges for a total of four QC organisms were proposed, with only three doubling dilutions each that included 95.6 to 99.7% of values reported from the eight-laboratory investigation.CEM-101 is an investigational fluoroketolide antimicrobial agent with spectrum features superior to existing macrolides and most similar to telithromycin (1, 6, 8-16). This agent has potential clinical applications against community-acquired bacterial pneumonia (CABP) and uncomplicated skin and skin structure infections (uSSSI) caused by Staphylococcus aureus, Streptococcus spp., some Enterococcus spp., and fastidious Gram-negative pathogens such as Haemophilus influenzae and Moraxella catarrhalis (12, 15, 16). To prepare for CEM-101 in vitro susceptibility testing during clinical trials, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) standard broth microdilution susceptibility method (3) was performed with various testing parameters. A disk content ranging study was initiated to optimize the discrimination of susceptible and resistant isolate populations (2-5). Using the selected 15-μg CEM-101 disk concentration, a pilot MIC/disk diffusion zone diameter scattergram experiment was performed (4). In addition, a CEM-101 broth microdilution quality control (QC) study utilized the CLSI M23-A3 guideline (4) design. The QC study employed eight laboratories, three different manufacturers of media, and several antimicrobial control agents. The results are presented as proposed QC ranges in μg/ml for four ATCC strains (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247) (4, 5).The CLSI M07-A8 method (3) was utilized for susceptibility testing of CEM-101 under standard conditions as well as with the following test alterations (12 organisms) to assess the impact of changing test conditions: anaerobic and CO₂ incubation environment; concentrations of 5 × 10³ and 5 × 10⁷ CFU/ml inoculum; lysed horse blood (LHB) and Haemophilus test medium (HTM) broths; medium pHs at 5, 6, and 8; 5, 10, and 20% added pooled human serum; trace and 50-mg/liter calcium ion contents; and polysorbate 80 (P-80) supplementation (0.000002 to 2.0%). CLSI standardized disk diffusion method M02-A10 (2) was performed with investigator-prepared 2-, 5-, 10-, 15-, and 30-μg CEM-101 disks to determine the optimal disk content to discriminate zone diameters for the susceptible or wild-type population from the resistant organism population. A total of 50 additional isolates, including staphylococci, enterococci, H. influenzae, and Enterobacteriaceae, were selected to calculate the correlation of zone diameters produced with the optimized 15-μg CEM-101 disk content when plotted against reference MIC test results (2-5).Eight laboratories were recruited to provide sufficient information for the MIC QC investigation (4). Four cation-adjusted Mueller-Hinton broth (CAMHB) media included lots produced by Difco Laboratories (Detroit, MI), Becton Dickinson (BD, Sparks, MD), and Oxoid (Hampshire, United Kingdom). Four CAMHB lots supplemented with 2 to 5% lysed horse blood or HTM were also supplied by Difco, BD, and Oxoid. CEM-101 was provided by Cempra Pharmaceuticals, Inc. (Chapel Hill, NC), and azithromycin, erythromycin, and clarithromycin were acquired from Sigma-Aldrich (St. Louis, MO). Panels were prepared by a certified good manufacturing practice (GMP) laboratory source (Trek Diagnostics, Cleveland, OH).Internal QC was established throughout all phases of these studies by using erythromycin tested against S. aureus and E. faecalis as a “peer drug” comparator agent and azithromycin and/or clarithromycin for S. pneumoniae and H. influenzae testing (4). Appropriate inoculum concentrations were monitored by performing colony counts with the inoculated broth microdilution trays, which were subcultured onto drug-free agar plates. The average colony counts among the participating centers ranged from 2.6 × 10⁵ to 5.7 × 10⁵ CFU/ml, which is an acceptable performance (3-5).The CEM-101 reference MIC results for 12 selected bacterial strains were tested under various conditions. Compared to standardized testing conditions (3), CEM-101 MICs were significantly increased (≥4-fold) only with elevated inoculum concentrations at 5 × 10⁷ CFU/ml (two S. aureus strains) and when the medium pH was less than 7. An extended experiment of a combined effect of medium pH and 10% human serum protein on five S. aureus strains illustrated that the adverse influences of low medium pH on CEM-101 were moderated by human serum protein (data not shown). In fact, one Klebsiella pneumoniae strain was 4-fold more susceptible to CEM-101 when tested in CAMHB with pooled human serum.Recent standardization of large-molecule lipoglycopeptides (dalbavancin, oritavancin, and telavancin) requires the use of 0.002 to 0.02% polysorbate 80, a surfactant, to minimize drug binding to plastic panels (3, 5). When CEM-101 was tested in the presence of 0.000002 to 0.02% polysorbate 80, no change in potency against five S. aureus strains was observed (Fig. ​(Fig.1).1). However, when the surfactant concentrations were increased to 0.2 or 2%, an antagonistic result was detected (the CEM-101 MIC increased to 1 μg/ml, a 16-fold elevation). Clearly, this new fluoroketolide will not require the adjunctive use of polysorbate 80.Open in a separate windowFIG. 1.Average CEM-101 MICs for five tested S. aureus strains when combined with various concentrations of a surfactant (polysorbate 80 at 0.000002 to 2%).An optimization of the disk diffusion test was performed in two phases, where CEM-101 disks were prepared at concentrations of 2, 5, 10, 15, and 30 μg, and organisms with CEM-101 MICs representing the indicated wild-type species (staphylococci, streptococci, enterococci, and H. influenzae) were tested and compared to generally resistant pathogens (10 total strains). The best discrimination of susceptible wild-type strains from resistant (MIC, ≥16 μg/ml) organisms was achieved with CEM-101 disks with a ≥10-μg content. The disk concentration routinely used for macrolides (azithromycin, clarithromycin, and erythromycin) and telithromycin (also a ketolide) has been 15 μg (5); therefore, this disk content was recommended for further development. A total of 50 strains were tested in duplicate (average zone and MIC results are presented in Fig. ​Fig.2)2) to confirm initial findings. Figure ​Figure22 illustrates the positions of various CEM-101 zone diameter and MIC results (r = 0.92), leading to the potential zone diameter breakpoint for H. influenzae of ≥17 mm (MIC, ≤4 μg/ml), which is most similar to that of telithromycin or azithromycin. Larger zones and lower MIC breakpoints could be considered for the staphylococcus and streptococcus (including S. pneumoniae) isolates during clinical trial development (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Proposed CEM-101 breakpoints to be applied to the clinical trials and further diagnostic test development (susceptible at ≥17 mm [≤4 μg/ml] and resistant at ≤13 mm [≥16 μg/ml]). Alternative susceptibility breakpoints are illustrated by the broken horizontal and vertical lines.The CEM-101 MIC QC trial followed the design dictated by the CLSI M23-A3 document (4) for the M07-A8 method (3). Table ​Table11 shows the remarkable consistency of CEM-101 MICs generated for the four studied QC strains. All QC organisms produced clear modal and median MICs, and the following three log₂ dilution MIC QC ranges were proposed for each organism: 0.015 to 0.06 μg/ml for E. faecalis ATCC 29212 (95.6% of results in the calculated range), 0.03 to 0.12 μg/ml for S. aureus ATCC 29213 (96.6%), 0.004 to 0.015 μg/ml for S. pneumoniae ATCC 49619 (99.4%) (also see Fig. ​Fig.3),3), and 1 to 4 μg/ml for H. influenzae ATCC 49247 (99.7%). All internal quality assurance QC range results for azithromycin, clarithromycin, and erythromycin were within previously reported limits (5).Open in a separate windowFIG. 3.CEM-101 MIC distribution for S. pneumoniae QC strain ATCC 49619 (320 results).

TABLE 1.

Inter- and intralaboratory comparisons of CEM-101 MIC results for four ATCC control strains from an eight-laboratory protocol conforming to study design guidelines of the CLSI^e