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

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

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

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

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

Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.Enterocytozoon bieneusi is an opportunistic organism causing diarrhea in human immunodeficiency virus (HIV)-positive patients, in whom it has a prevalence of 2 to 50% (5). The infection not only has been reported to occur in immunocompromised hosts but also has been found in healthy individuals (14, 20). This organism can infect a broad range of animals (4, 12, 16, 18, 22, 23). Genotypes of E. bieneusi in humans and animals are differentiated using the polymorphisms of the internal transcribed spacer (ITS) sequence of the rRNA gene (4, 11). To date, at least 70 ITS genotypes have been reported to infect humans and animals (2, 6). The zoonotic nature of E. bieneusi was confirmed because ITS genotypes found in domestic and wild animals had been reported to occur in immunosuppressed hosts (22). In Thailand, we reported genotypes E, O, and PigEBITS 7, which have previously been identified in pigs (3, 4) and in Thai HIV-infected patients (9). This study aimed to identify the ITS genotypes of E. bieneusi in pigs and humans who worked in or lived near pig farms to investigate the transmission of E. bieneusi among these host species.A cross-sectional study of E. bieneusi infection was conducted in a community in Nakorn Pathom Province, Central Thailand, January 2005. This community is composed primarily of four pig farms, a residential area, and a school. The residential area, but not the school, was near the pig farms. Fecal specimens were collected from school children and those who were living in this community, including pig farmers. Fecal specimens were also collected from pigs of four farms and examined for E. bieneusi. The study was approved by the Ethics Committee of the Royal Thai Army, Medical Department. Informed consent was obtained from each adult individual and from parents of school children before enrollment in the study.Fecal specimens from pigs and humans were examined for microsporidian spores using gram-chromotrope staining under light microscopy (13). DNA was prepared from water-ethyl acetate-concentrated stool specimens using FTA filter paper (Whatman Bioscience, United Kingdom) as previously described (21). Amplification of the ITS region of the small-subunit rRNA gene was performed using primers under conditions described by Katzwinkel-Wladarsch et al. (8). For specimens with PCR-negative results, PCR amplification was repeated at least twice. DNA sequencing was conducted by Macrogen Inc., Seoul, Republic of Korea. Nucleotide sequences were determined using the Sequencher program (Gene Codes Corporation, Inc.), and multiple alignment was performed using Clustal X 1.83 for Windows (24). The genotype of each specimen was confirmed by determining the homology of the sequenced PCR product with the published sequence.A total of 268 pig fecal samples were collected. Pigs aged between 21 days and 22 months were examined for E. bieneusi infection. Microsporidial spores were found in 0.7% of pig fecal samples using gram-chromotrope staining, while a greater prevalence of E. bieneusi infection, 15.7%, was detected by PCR. The prevalences of E. bieneusi infection among the four farms and different age groups are presented in Table ​Table1.1. A significantly higher prevalence of E. bieneusi was found in pigs aged 2 to 3.9 months than in pigs of other age groups (chi-square test, P < 0.001). Multivariate analysis confirmed that pigs aged 2 to 3.9 months had a 5.3-times-greater risk of infection than pigs in other age groups (95% confidence interval, 2.6 to 10.6; P < 0.001). Of these 42 E. bieneusi-positive samples, 21 (50%) were successfully characterized by sequencing analysis, and the organism was identified as being of genotypes E (12 samples [57.1%]), O (8 samples [38.1%]), and H (1 sample [4.8%]).

TABLE 1.

Prevalence of E. bieneusi positivity in pig specimens as determined by PCR

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

Genetic Characterization of Genogroup I Norovirus in Outbreaks of Gastroenteritis

In this study, we demonstrate that differences within the P2 domain of norovirus genogroup I (GI) strains can be used to segregate outbreaks which are unrelated, whereas complete conservation within this region allows tracking of strains that are part of a single outbreak and likely to have a common source.Noroviruses (NoVs) are members of the Caliciviridae family (7) and the leading cause of outbreaks of acute gastroenteritis worldwide (14). NoV outbreaks are frequently associated with semiclosed or closed institutions such as hospitals and homes for the elderly (11, 22), but outbreaks also occur in other settings, including eating establishments, cruise ships, concert halls (2, 10, 20), and schools (16). Transmission of NoVs is usually person to person (15), although food and water (1, 3, 5, 9, 17, 18, 21) and environmental or airborne contamination (6, 19) have all been implicated in transmission.Human NoVs are genetically diverse, and three distinct genogroups (GI, GII, and GIV) and many genotypes/genetic clusters exist (8, 13, 26). Diversity among NoVs is generated through the accumulation of point mutations associated with the error-prone nature of RNA replication and genetic recombination involving the exchange of sequences between related RNA viruses.The NoV capsid is divided into the S domain, which constitutes the 5′ end (amino acids [aa] 1 to 225), and the P (protruding) domain (aa 226 to 530) (23). The P domain can be further subdivided into two subdomains, P1 and P2. The P2 domain is the hypervariable region of the capsid and corresponds to the most exposed area likely to be involved in immune recognition and attachment. Due to the high diversity within this region, it is not possible to design a single cross-reactive primer pair capable of amplifying all genotypes within a genogroup; therefore, genotype-specific primers are required in order to amplify this region, as previously seen for NoV genogroup II (25).Fecal samples were collected from genogroup I outbreaks of gastroenteritis as part of the ongoing National Surveillance Programme of the molecular epidemiology of norovirus genotypes.Outbreaks were defined as including two or more cases of gastroenteritis linked in place and time. A new outbreak was arbitrarily defined as occurring at least 7 days after the last case in a previous outbreak or as occurring in a different patient care unit such as a ward or hospital.Outbreak 714124 occurred in a nursing home in Blackburn, Lancashire, United Kingdom, in August 2007; outbreak 414003 occurred in a restaurant in Chorley, Lancashire, in January 2004 (4); outbreak 512057 occurred in a bar/restaurant in Liverpool, United Kingdom, in November 2005; outbreak 612057 occurred in a nursing home and outbreak 612058 occurred in a hotel in Liverpool in November 2006; outbreak E/2005/UK occurred in a cruise ship in December 2007; outbreak Q1/2007/US occurred in a cruise ship in the United States in February 2007; and outbreak Newquay/2008/UK occurred in Newquay, Cornwall, United Kingdom, in August 2008.Fecal specimens were prepared as previously described, and nucleic acid extraction, Norovirus detection through amplification of a small region spanning the open reading frame 1/2 (Orf1/2) junction, and genotyping through sequence analysis of the S domain were all performed as previously described (4).Oligonucleotide primers for the amplification of a region encompassing the P2 domain of NoV GI genotypes 1 to 7 were designed from alignments of complete Orf2 nucleotide sequence data. See Table ​Table11 for primer sequences and positions and amplification conditions. Separate monoplex reactions were carried out for each of the genotypes, and amplicons were separated by agarose gel electrophoresis and sequenced directly after purification using the same P domain genotype-specific primers.

TABLE 1.

Norovirus genogroup I genotype-specific primers for amplification of a region encompassing the P2 domain^a

Outbreak of Febrile Respiratory Illness Associated with Adenovirus 11a Infection in a Singapore Military Training Camp

Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China.Due to unique risk factors that include crowding and increased physical and psychological stress, military recruits in training are highly susceptible to outbreaks of acute respiratory disease (ARD), which are most often caused by viruses (26, 29). Human adenovirus (HAdV) infections have been recognized for decades as being important causes of ARD among military trainees in North America, Europe, and Asia (5, 8, 10, 15, 25, 27, 30, 32). The HAdV serotypes most frequently associated with ARD in both military and civilian communities include subspecies B1 HAdV-3, HAdV-7, and HAdV-21 and species E HAdV-4. The association of subspecies B2 HAdV infection with ARD has historically been rarely reported and restricted mostly to closed community outbreaks, some of which were documented among military trainees (1, 2, 8, 24, 36).From 11 January 2005 to 14 October 2005, a total of 226 male participants aged 18 to 24 years were enrolled in a study designed to detect and characterize viral agents responsible for ARD in the Singapore military recruit population. Analysis of laboratory data collected during this period identified influenza virus as the primary causative viral agent of respiratory illness among Singapore recruits in training (51 influenza virus-positive isolates out of 226 tested cases [22.5%]). HAdV-associated ARD cases were detected sporadically between January and October 2005. Thirty symptomatic trainees (13.3%) tested positive for HAdV during this period by a PCR assay described previously by Echavarria and colleagues (6). The temporal distribution of confirmed HAdV-associated cases of ARD is shown in Fig. ​Fig.1.1. Cases of adenovirus-associated ARD were detected between February and June 2005 and in October 2005.Open in a separate windowFIG. 1.Temporal distribution of HAdV-associated cases of ARD diagnosed among Singapore military recruits during 2005.One case of coinfection with influenza A virus was detected among these patients. Recruits testing positive for HAdV were between 19 and 21 years old. The examination of clinical characteristics of the HAdV-positive cases of ARD showed that 23.3% of the recruits reported shortness of breath, 50% reported nasal congestion, 80% reported a headache, 80% reported body ache, and 13.3% reported signs of nausea or vomiting. The identified influenza A virus coinfection did not increase the severity of the respiratory symptoms. One patient presented with additional symptoms of conjunctivitis, but no eye swab samples were collected. Adenovirus isolation was accomplished for 27 of the 30 positive clinical specimens. All virus isolates were initially typed as species B HAdVs by PCR as described previously by Metzgar and colleagues (22). Isolates were further characterized by restriction enzyme analysis and sequencing of the hexon and fiber genes as described previously by Kajon and Erdman (13). Digestion with BamHI identified 26 of 27 isolates as belonging to subspecies B2 HAdVs and one isolate as being HAdV-3. Digestion of viral DNAs with BclI, BglII, BstEII, DraI, HindIII, PstI, SmaI, and XbaI determined that the 26 subspecies B2 isolates were identical and identified them as corresponding to genome type 11a (Fig. ​(Fig.2A)2A) (17). The HAdV-3 isolate was identified as belonging to genome type 3a2 (Fig. ​(Fig.2B)2B) (16).Open in a separate windowFIG. 2.(A) Restriction enzyme analysis of a representative HAdV-11a isolate (SNG1222). (B) Restriction enzyme analysis of HAdV-3a2 isolate SNG1206. M, 1-kbp and 100-bp molecular markers.By using the primer sets described in Table ​Table1,1, identical hexon and fiber sequences were obtained for three randomly selected HAdV-11a isolates (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607010","term_id":"241992696","term_text":"FJ607010"}}FJ607010 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603103","term_id":"284808702","term_text":"FJ603103"}}FJ603103 for isolate SNG1218, accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607011","term_id":"241992698","term_text":"FJ607011"}}FJ607011 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603104","term_id":"284808704","term_text":"FJ603104"}}FJ603104 for isolate SNG1222, and accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607012","term_id":"241992700","term_text":"FJ607012"}}FJ607012 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603105","term_id":"284808706","term_text":"FJ603105"}}FJ603105 for isolate SNG1223). Alignment of the sequences of the hexon gene corresponding to hypervariable regions 1 to 7 using the Basic Local Alignment Search Tool (BLAST) optimized for highly similar sequences (megablast) against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) showed the three examined viruses to correspond to serotype 11 (19, 28). By using ClustalW implemented in Lasergene (DNASTAR, Inc., Madison, WI), the sequences for both the hexon and the fiber genes of the Singapore HAdV-11a strain were found to be highly similar to those reported for Asian and Middle Eastern HAdV-11 strains circulating over the last 2 decades in association with respiratory disease (Table ​(Table2).2). In addition, the complete genomic sequence obtained at the Walter Reed Army Institute of Research for isolate SNG1222 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ597732","term_id":"257122613","term_text":"FJ597732"}}FJ597732) was found to be 99.9% identical to that reported previously for strain QS-DLL, isolated in China in 2006 during an outbreak of ARD (33) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ643676","term_id":"256028986","term_text":"FJ643676"}}FJ643676).The identified differences between these two genomes are listed in Table ​Table2.2. The results of this study suggest that, in contrast to other geographic locations where HAdV-3, -4, -7, and -21 rank among the most prevalent serotypes, HAdV-11 may be a relatively more important causative agent of ARD in military training facilities in Singapore. The circulation of HAdV-11 in South East Asia has been documented since the early 1960s in association with conjunctivitis, pharyngoconjuctival fever, ARD, and hemorrhagic cystitis among immunocompromised individuals (7, 9, 14, 17, 24, 31, 34-36). The work of Wadell and colleagues demonstrated the existence of two main clusters of relative homology for HAdV-11 genome types: the cluster of prototype-like genomic variants with a tropism for the renal epithelium, and the a-like cluster, with a distinct tropism for the respiratory tract. In addition to their unique restriction site maps, the p-like and a-like genomes differ in the sequences of the fiber gene involving the receptor binding domains (17, 20, 21). As noted by others previously (33), the fiber of the 11a-like genomes is more closely related to the fiber of the prototype strain of HAdV-14, de Wit (99.5% identity), than to the fiber of the prototype strain of HAdV-11, Slobitski (94.4% identity), suggesting that this genomic variant of HAdV-11 is an intertypic recombinant 11-14.

TABLE 1.

Primers used for amplification and sequencing of hexon and fiber genes of Singapore HAdV-11 isolates^a

Clostridium difficile Strains from Community-Associated Infections

Clostridium difficile isolates from presumed community-associated infections (n = 92) were characterized by toxinotyping, pulsed-field gel electrophoresis, tcdC and cdtB PCR, and antimicrobial susceptibility. Nine toxinotypes (TOX) and 31 PFGE patterns were identified. TOX 0 (48, 52%), TOX III (18, 20%), and TOX V (9, 10%) were the most common; three isolates were nontoxigenic.Clostridium difficile infection (CDI) is an important cause of health care-associated diarrhea, especially in patients receiving antibiotics. C. difficile also causes diarrhea among patients in community settings (1, 6, 13, 19, 20), although diarrheic outpatients are not routinely tested for CDI. Community-associated CDI (CA-CDI) appears to be increasing (3, 11, 22), but little is known about strains that cause it. We collected and characterized isolates from patients with presumed CA-CDIs.Surveillance for CA-CDI was conducted in 2006 for 3 months at 19 clinical laboratories in nine states participating in the Food-Borne Disease Active Surveillance Network (FoodNet; California, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York, Oregon, and Tennessee). Stool specimens from patients with positive physician-ordered C. difficile toxin tests were stored at −20°C until medical record review confirmed study eligibility. Presumed CA-CDI was defined as a C. difficile toxin-positive stool specimen from an outpatient (or within 72 h of hospital admission) without a previous positive result in the same laboratory for >8 weeks or an overnight health care stay in the preceding 3 months. Diagnostic assays varied and included toxin A-only or toxin A and B enzyme immunoassays. The sensitivity and specificity of diagnostic assays were not evaluated.C. difficile toxin-positive stool samples were submitted frozen to the state''s public health laboratory or the Durham Veterans Affairs Medical Center laboratory (Durham, NC) for culture. Thawed stool samples were cultured by alcohol shock (12) on anaerobe blood agar plates (BD, Franklin Lakes, NJ), direct inoculation of cycloserine-cefoxitin fructose agar (4), or both and then incubated anaerobically at 35°C for 72 to 96 h. Plates were examined daily for characteristic colonies (4), p-cresol odor, and yellow-green fluorescence under UV light. Chopped meat broth (BD) was inoculated with a single colony and shipped to the Centers for Disease Control and Prevention (CDC) for confirmatory identification by the characteristics noted above, a negative indole reaction, and a positive l-proline-aminopeptidase (Remel, Lenexa, KS) reaction. Isolates were characterized by toxinotyping, pulsed-field gel electrophoresis (PFGE), PCR evaluation of tcdC (10) and cdtB (20), and antimicrobial susceptibility testing.Of 175 specimens obtained from patients with presumed CA-CDIs, 162 were cultured; 103 isolates were submitted to the CDC, and 92 were confirmed as C. difficile. The C. difficile recovery rate from 162 toxin-positive stool samples was 57% (range, 29 to 100% by laboratory).Toxinotyping was based on restriction fragment length polymorphisms in the A3 and B1 fragments of the pathogenicity locus, as described previously (17). Three isolates were nontoxigenic. We tested only one isolate per specimen, so it is unknown whether nontoxigenic isolates represent coinfection or false-positive diagnostic tests.Nine toxinotypes were identified among 89 toxigenic C. difficile isolates. Most (n = 48; 54%) of the isolates were TOX 0; the remaining 41 (46%) had variant toxinotypes. TOX 0 strains (15) are a common cause of health care-associated CDI (HA-CDI) (6, 15, 20), whereas variant toxinotypes were rare prior to the emergence of the epidemic strain (6, 15, 20). TOX III was the most common variant (n = 18; 20%) identified, followed by TOX V (n = 9; 10%) and TOX IX (n = 7; 8%). Excepting TOX VIII and TOX XII, variant toxinotypes correlated with cdtB presence (n = 39; 42%), as expected (16, 20). The pathogenic contribution of binary toxin is unclear (5, 18), but binary toxin-positive strains are increasingly isolated from human CDIs in Europe and the United States (7, 8, 13, 18, 19).Isolates were characterized by SmaI PFGE and analyzed with BioNumerics v5.01 (Applied Maths, Austin, TX), as described previously (10), and then compared to the entire CDC C. difficile database. North American pulsed-field (NAP) types were assigned to patterns ≥80% similar to established NAP clusters. Thirty-one PFGE types were identified among 92 CA-CDI study isolates, 65% of which were assigned established NAP types. The 32 isolates not assigned NAP types belonged to 21 unnamed patterns (Fig. ​(Fig.1).1). Nontoxigenic isolates yielded unnamed patterns distinct from toxigenic isolates.Open in a separate windowFIG. 1.PFGE patterns and characteristics of 92 isolates from patients with CA-CDI in nine FoodNet states in 2006. Neg, negative; Pos, positive.NAP1 was the most common PFGE type identified (n = 19; 21%) and was uniformly binary toxin positive. Fourteen isolates were typical of the epidemic strain (TOX III, 18-bp tcdC deletion); five were unusual (TOX IX, wild-type tcdC [Fig. ​[Fig.1]).1]). Four isolates were typical of NAP1 (TOX III, 18-bp tcdC deletion, binary toxin positive) but <80% related to NAP1 by PFGE and thus classified as NAP1 related. Other recognized PFGE types included NAP4 (10%), NAP7 (8%), NAP11 (6%), NAP2 (5%), and others (each less than 5% of the isolates [Fig. ​[Fig.1]).1]). NAP2 (restriction endonuclease type J; PCR ribotype 001; TOX 0), a previous epidemic strain in the United States and Europe (9, 21), and NAP4 (restriction endonuclease type Y; PCR ribotype 077; TOX 0) were previously common in health care settings (1) but have been uncommon in recent years (CDC, unpublished data). NAP11 has not been associated with outbreaks in the United States.MICs of moxifloxacin, levofloxacin, ciprofloxacin, clindamycin, erythromycin, metronidazole, and vancomycin were determined for 87 isolates by broth microdilution (2). This method is not approved for C. difficile reference testing and was used for surveillance only. Five isolates received after broth microdilution panels had been exhausted were evaluated by Etest (AB Biodisk, Piscataway, NJ) as recommended (EAS 007; AB Biodisk). The frequencies at which the MICs for NAP1/TOX III isolates and non-NAP1/TOX III isolates were above the MICs for 50 and 90% of the entire isolate population tested (MIC₅₀s and MIC₉₀s, respectively) were compared by using Fisher''s exact t test.NAP1/TOX III isolates displayed different MIC distributions than others and thus are presented together and separately (Table ​(Table1).1). MICs for NAP1/TOX III isolates were more likely than those for non-NAP1/TOX III isolates to be above the aggregate MIC₅₀ of all of the antimicrobials tested (P < 0.05), except clindamycin and vancomycin, and above the aggregate MIC₉₀ of ciprofloxacin (P < 0.05). All of the isolates were susceptible to metronidazole and vancomycin (Table ​(Table11).

TABLE 1.

Ranges of MICs for NAP1/TOX III and non-NAP1/TOX III C. difficile isolates from patients with presumed CA-CDIs in nine FoodNet states in 2006

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

Prevalence of ST9 Methicillin-Resistant Staphylococcus aureus among Pigs and Pig Handlers in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.Methicillin-resistant Staphylococcus aureus (MRSA) primarily causes human diseases and has recently been identified in pigs and pig handlers. This raised concerns about the role of this porcine reservoir in human infections. In Malaysia, several studies reported the prevalence and characteristics of MRSA isolates from clinical and community settings (15, 20). However, no data have yet been presented on MRSA in pigs. Here, we determined the prevalence of MRSA colonization in pigs and pig handlers in Malaysia.Thirty randomly selected farms in the district of Kuala Langat from the Selangor state of Malaysia were sampled for MRSA. All farms were within a 5-km radius. The farm sizes ranged from 2,000 to 10,000 pigs. Twelve samples (three each from four different age groups, sows, piglets, weanlings, and grower-finishers) were taken at each farm. Nares of the pigs were swabbed by a trained veterinarian. In addition, the three pig handlers per farm (the maximum number of workers in a farm varied from six to eight) who had the highest level of exposure to pigs provided nasal swabs. Employees filled out a questionnaire regarding possible risk factors for MRSA colonization (11). Risk factors included the number of years of work with pigs on that farm, number of hours working with pigs per week, contact with other animals, recent hospitalization, recent treatment with antibiotics, personal or familial skin and soft tissue infection in the last 3 months, and participation in team sports.MRSA was isolated according to methods described previously (13). The antibiotic susceptibility of the strains against the antibiotics mentioned below was tested by using the Kirby-Bauer disk diffusion method. The results of susceptibility testing were interpreted according to accepted guidelines (4). MRSA isolates were PCR tested for the mecA gene and subjected to staphylococcal protein A gene (spa) sequencing (http://spaserver.ridom.de), staphylococcal cassette chromosome mec (SCCmec) typing (22), and multilocus sequence typing (http://www.mlst.net). All isolates were screened for the virulence genes pvl (14), fnb and cna (1), sea, seb, sec, sed, see, seg, tsst, eta, and etb (8).MRSA was isolated from one or more pigs on 30% (9/30) of the farms. The overall prevalence of MRSA among pigs was found to be 1.38% (5/360), with 5.3% (4/75) in weanlings and 1.3% (1/75) in grower-finishers. None of the piglets or sows was colonized. Except for farm 1, in all other eight farms, only one animal was colonized. The prevalence of MRSA colonization in humans was 5.5% (5/90). None of the risk factors identified in the questionnaire were found to have a significant association with human colonization. MRSA was not isolated from both pigs and pig handlers on any of the farms. When MRSA-positive animals were tested a second time, no MRSA was isolated. Unfortunately, no secondary samples were taken from humans. This indicates that MRSA is only transiently present, similar to what we found in a study with healthy Malaysians (16).Susceptibility testing revealed 100% resistance to erythromycin, ceftriaxone, cefoxitin, ciprofloxacin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, clindamycin, and quinupristin-dalfopristine, while tigecycline, cephalexin (cefalexin), and fusidic acid showed 80%, 70%, and 20% resistance, respectively. No resistance was observed for mupirocin, amikacin, linezolid, vancomycin, and netilmicin. The surprising pattern of resistance to clindamycin, quinupristin-dalfopristine, and tigecycline was confirmed after three repeats of the test.Molecular typing showed that MRSA isolates belonged to two sequence types: ST9 (spa type t4358) and ST1 (spa type t1784). Except for strains from handlers on farm 12 and 29 (ST1), all other isolates were ST9 (Table ​(Table1).1). All isolates carried SCCmec V. Virulence gene analysis revealed enterotoxin genes, such as seb (60%), see (10%), and seg (90%), and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that include Cna (20%, only in ST1 isolates) and Fnb (100%). None of the MRSA isolates carried pvl, eta, etb, tsst, or enterotoxin genes other than seb, see, and seg (Table ​(Table11).

TABLE 1.

Characteristics of MRSA isolates from pigs and pig handlers in Malaysia

High Seroprevalence of Neutralizing Capacity against Human Metapneumovirus in All Age Groups Studied in Bonn,Germany

Human metapneumovirus (hMPV) infections occur frequently despite high rates of perpetual seroprevalence for all age groups. Analyses of ∼2,000 archived, randomly selected serum samples demonstrated that neutralizing capacities remain high, with a minor decrease for individuals over 69 years of age, leading to the hypothesis that reinfections occur because humoral immune responses play minor roles in the clearance of hMPV infections.The human metapneumovirus (hMPV) was first isolated in 2001 from children hospitalized with acute respiratory infections (ARIs) (18). hMPV belongs to the Paramyxoviridae family and is one of the major causes of upper and lower ARIs. By the age of five, nearly every child has been infected with hMPV (18). Since hMPV was first described, it has been reported from all over the world, with prevalences of infection ranging from 3.9 to 43% (12, 13). hMPV seems to have a seasonal distribution, like respiratory syncytial virus (RSV) and influenza virus. Infections occur mainly during the winter months (2, 14, 20). Up to now, two genotypes (A and B), each with two subgroups (A1 and A2 and B1 and B2, respectively), have been identified (11), but it is not known if the two genotypes represent two serotypes and if they lead to variations in the severity of clinical symptoms (19). Symptoms associated with a hMPV infection range from mild infections of the upper respiratory tract to severe lower respiratory tract infections like bronchiolitis and pneumonia. Wheezing, coughing, fever, and dyspnea are frequently observed (2, 9, 18). More-severe hMPV infections primarily affect infants and children, while otherwise healthy adults suffer solely from influenza-like illnesses. However, immunocompromised adults show exacerbated courses of asthma and chronic obstructive pulmonary diseases (8, 10, 21). For the elderly, only a few studies have been released, but it has been stated that hMPV infections often lead to hospitalizations and are associated with high mortality in the elderly (3-5). The aim of the present study was to analyze patient sera for the ability to neutralize hMPV and to investigate whether there are any differences among the different age groups.Serum samples from a total of 2,000 patient were randomly collected from the archives of the Institute of Virology of the University Hospital Bonn (which includes a large trauma center for the geographic area and a large obstetrics unit, resulting in many patients in the 20- to 50-year-old age range) and screened for neutralizing capacity, using the XTT-based neutralization test described previously (17).In brief, 5 × 10⁴ genome equivalents (geq) of hMPV cells in 50 μl of Dulbecco''s modified Eagle''s medium (DMEM) or 50 μl of DMEM without the virus was applied to the wells of a 96-well plate (Nunc, Karlsruhe, Germany). Afterward, 25 μl of sera was added to each well. Finally, 5 × 10⁴ HepG2 cells in 125 μl of medium were added to each well and preincubated for 30 min. The DMEM formulation was clear DMEM with 4.5 g liter⁻¹ glucose, 3% (vol/vol) fetal calf serum (FCS), 1% (vol/vol) 100× penicillin-streptomycin mixture (10,000 U/ml of penicillin and 10 mg/ml of streptomycin), 1% nonessential amino acids, 1% l-glutamine, and 1% sodium pyruvate (all from PAA, Austria).The cells were incubated for 7 days at 33.4°C and 5.0% CO₂. The confluence and morphology of the cells were controlled daily under an inverse microscope. At day 7, 150 μl of supernatant was removed from each well and discarded. The prewarmed (37°C) XTT test kit solutions were mixed by pipetting the coupling reagent into the yellow tetrazolium salt. Fifty microliters of the solution was added to each well, and the plate was incubated for 1 h at 33.4°C and 5.0% CO₂ before extinction was measured at 456 nm, with 650 nm as the reference measurement, in a 96-well plate reader. For additional verification of the results, cells were counterstained with crystal violet. To investigate the neutralizing capacity of the tested patients'' sera, the results of the XTT test of the cells infected with hMPV and treated with patients'' sera were compared to a reference dilution series and the results for the corresponding noninfected cells. The optical density (OD) value quotients for the infected and corresponding noninfected cells were calculated. A value less than 1 indicated that the sera had a neutralizing effect on the virus.For calibration purposes and as a quality control for each test, serial virus dilutions were run in parallel on each plate to ensure that the virus concentration in the inoculum was reciprocal to cell viability, as previously shown (17). As expected, the absorption rate increased with decreasing virus concentrations (Fig. ​(Fig.1a).1a). A reduction in half of the virus concentration was comparable to an increase in OD of approximately 0.04.Open in a separate windowFIG. 1.(a) Dilution series were performed for quality control. Cells were infected with decreasing amounts of hMPV and incubated for 7 days before an XTT test was performed. Data are given as means ± standard errors of the means (SEMs). (b) Evaluation of neutralizing capacity, using an XTT-based neutralization assay for children. Sera were grouped by age in order to elucidate putative breast-feeding-derived neutralization capacity. Data are given as means ± SEMs. (c) Sera from a total of 1,953 patients were screened for neutralizing capacity, using an XTT-based neutralization assay. Data are given as means ± SEMs. AU, arbitrary units; *, results significantly different from those for 0- to 9-year-old group (P ≤ 0.05).In total, 2,000 serum samples were initially included in the study. The sera were collected routinely as retained samples and archived for up to 10 years. Collection and archiving of the sera were in concordance with a vote by the local ethical committee. The serum samples were randomly selected and represent a cross section of the patient cohort admitted to our hospital during the last 10 years and reflect the demographic distribution of patients admitted to our hospital.Of the 2,000 serum samples tested, 49 were toxic per se for the cultured cells and could not be used for further evaluations. Of the remaining 1,951 serum samples, 1,879 (96.21%) showed neutralizing effects (Table ​(Table1).1). The majority of the negative serum samples were from cohorts of patients who were 0 to ≤2, 60 to 69, and 70 to 79 years old. The sera of the group of patients who were >2 to 9 years old showed the least virus neutralization capacity. Neutralizing capacity increased in patients 10 to 29 years old before it reached a plateau. On average, the neutralizing effect was ∼10% higher for patients 29 to 69 years old than for patients 0 to ≤2 years old and ∼20% higher than for patients >2 to 9 years old. Starting with patients 70 to 79 years old, a trend of decreasing neutralizing ability of the sera was seen (Fig. ​(Fig.1b).1b). Admittedly, the standard deviation for the older-patient groups is very high due to the small group size, but the results indicate that it may be worth conducting more-specific surveys on the elderly concerning neutralization potency.

TABLE 1.

Age distribution of patients and number of samples per group that tested positive or negative for neutralizing ability^a

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)

Carriage Rate of Enterocytozoon bieneusi in an Orphanage in Bangkok,Thailand

This study was performed to evaluate the incidence of and risk factors for Enterocytozoon bieneusi carriage in an orphanage in Bangkok, Thailand. E. bieneusi has been identified by PCR every 2 consecutive months since June 2003. The incidence ranged between 0.6 and 4.7/100 person-months. Person-to-person transmission was indicated by risk factor analysis and genotyping information.Enterocytozoon bieneusi, the most common microsporidial organism infecting humans, causes chronic diarrhea, especially in AIDS patients (4, 12). It can also cause diarrhea in immunocompetent individuals (15, 17). In Thailand, E. bieneusi is one of the most common causes of diarrhea in both adults and children with AIDS (9, 24, 25). It is assumed that E. bieneusi is transmitted by the fecal-oral route; however, the sources of infection and the modes of transmission remain unclear (3). Recently, knowledge about this infection has been increased because of PCR-based detection methods which have higher sensitivity and can identify the organisms'' species and genotypes (16, 19). Genotyping of E. bieneusi is determined based on the polymorphic sequences of the internal transcribed spacer (ITS) of the rRNA gene (2, 11, 18). Recent epidemiological studies have indicated the transmission modes of E. bieneusi including person-to-person, zoonotic, waterborne, and food-borne routes (2, 5-7, 10).We previously reported that ∼4% of human immunodeficiency virus (HIV)-negative children in an orphanage in Bangkok were positive for E. bieneusi (10). To develop effective control strategies, it is essential to understand the epidemiology of this infection. Thus, we conducted a 1-year longitudinal study of E. bieneusi infection in this orphanage. This study was approved by the Ethical Committee, Royal Thai Army Medical Department. A total of 540 orphans and 81 child care workers were enrolled in the study during June 2003 to April 2004. The orphanage consisted of 12 rooms (10 rooms for orphans and 2 rooms for milk and food preparation). Orphans within specific groups were assigned to 10 different rooms (Table ​(Table1).1). Each room accommodated 30 to 40 orphans with 3 child care workers. The child care workers in each room were asked to collect stool samples and complete standardized questionnaires for the orphans for whom they were responsible every 2 months from June 2003 to April 2004. The information, including age, sex, weight, height, HIV status, and present illness, was recorded. The numbers of enrolled subjects during each consecutive round of survey were 338, 337, 321, 286, 340, and 306, respectively. Of 540 orphans, 318 (58.9%) were males. The median age of the orphans was 13 months (0.26 months to 11 years). Seventy-seven orphans (14.3%) were HIV positive (47 males and 30 females). Information on CD4⁺ T-lymphocyte count was not available. All HIV-positive orphans were prescribed antiretroviral therapy (i.e., zidovudine and didanosine). Child care workers who participated in this study had a median age of 38 years (19 to 55 years).

TABLE 1.

Characteristics of 75 orphans with intestinal microsporidiosis