West Sacramento Research Articles

Antimicrobial Drug Resistance in Peru

To the Editor: In Latin American countries, rates of antimicrobial drug resistance among bacterial pathogens are high. Data on these rates in Peru are incomplete (1), and no institution in Peru has participated in multinational surveillance studies (2–4). To document the antimicrobial drug resistance profile of key pathogens, we organized a surveillance network of clinical laboratories from 9 hospitals (public, general, tertiary care, and quaternary care) in Lima, the capital of Peru. Over a 12-month period (April 2008–March 2009), we consecutively collected positive bacterial blood culture isolates (other than coagulase-negative staphylococci) from each of the 9 hospitals. Only the first isolate per patient was included. Patients’ age and hospital ward were recorded. Identification and susceptibility testing were performed at the Institute of Tropical Medicine Alexander von Humboldt (Lima, Peru). Staphylococcus aureus was identified by conventional methods, and susceptibility testing was conducted by oxacillin salt agar screening and disk diffusion (5). For gram-negative bacilli, including extended-spectrum β-lactamases (ESBL), identification and susceptibility testing were performed by conventional techniques and by MicroScan NC50 panels (Dade-Behring, West Sacramento, CA, USA) (5). American Type Culture Collection strains were used as controls. During the study period, we collected 1,681 unique isolates. We report the first 934 isolates tested from the more common species collected (375 Staphylococcus aureus, 321 Klebsiella pneumoniae, 125 Escherichia coli, and 113 Pseudomonas aeruginosa). Overall, S. aureus was the most frequently recovered species, accounting for 22.0% of organisms. Of 375 S. aureus isolates tested, 244 (65.0%) were methicillin resistant (MRSA) and 131 were methicillin susceptible. MRSA frequency was highest among isolates from intensive care units (ICUs) (61 [68.5%] of 89 isolates), but it was also high among isolates from emergency wards (55 [57.3%] of 96 isolates); this difference did not reach statistical significance. Among the 244 MRSA isolates, 170 (69.6%) were also co-resistant to the combination of ciprofloxacin, gentamicin, and clindamycin; rates of co-resistance did not differ significantly among MRSA isolates from patients in the emergency ward (32/55, 58.2%) and those from patients in ICUs and hospital wards (133/184, 72.3%, p = 0.67). Among the 131 methicillin-susceptible isolates, resistance rates were as follows: ciprofloxacin (5.3%), gentamicin (10.7%), clindamycin (14.5%), and erythromycin (14.5%). All S. aureus isolates were susceptible to linezolid, teicoplanin, and vancomycin; clindamycin-inducible resistance was found in 10 (38.5%) of 26 isolates resistant to erythromycin and apparently susceptible to clindamycin. K. pneumoniae was the second most frequently recovered organism, accounting for 19.1% of organisms collected. Among 321 K. pneumoniae isolates tested, 241 (75.1%) produced ESBL, 207 (64.5%) showed resistance to ciprofloxacin, and 233 (72.6%) were resistant to trimethoprim-sulfamethoxazole; proportions did not differ among age groups, wards, or hospitals. Of the 241 ESBL-producing isolates, 136 (56.4%) showed co-resistance to ciprofloxacin and gentamicin and 66 (27.4%) were also resistant to amikacin. Of the 80 non–ESBL-producing isolates, 37 (46.3%) were resistant to ciprofloxacin. All K. pneumoniae isolates retained susceptibility to imipenem and meropenem. A large proportion of K. pneumoniae infections were suspected to have been hospital acquired because most (280/321, 87.2%) were recovered from patients already hospitalized, including one third (96/321, 29.9%) of those from the neonatal ward. Although K. pneumoniae occasionally caused microepidemics in neonatal wards, most isolates were recovered randomly over time and from different hospitals. Among 125 E. coli isolates tested, 96 (76.8%) produced ESBL, 107 (85.6%) were resistant to ciprofloxacin, and 108 (86.4%) were resistant to trimethoprim-sulfamethoxazole. The resistance rate to ciprofloxacin was higher among adults than children (90.5% vs. 60.0%, p = 0.002). Of 96 ESBL-positive isolates, 59 (61.5%) were co-resistant to gentamicin and ciprofloxacin but only 9 (9.4%) were resistant to amikacin. Among 29 non–ESBL-producing E. coli isolates, 19 (65.5%) were resistant to ciprofloxacin. All isolates were susceptible to imipenem and meropenem. We hypothesized that the high level of E. coli resistance to ciprofloxacin may be related to community overuse of fluorquinolones for common infections, such as acute diarrhea. Among 113 P. aeruginosa isolates tested, 62 (54.8%) came from patients in ICUs and 73 (64.0%) were isolated from adults. Multidrug-resistance (defined as resistance to at least 3 of the following: ciprofloxacin, imipenem, amikacin, ceftazidime) was found for 67 (59.3%) of the 133 isolates, more among adults (65.7%) than among children (43.2%, p = 0.024). Overall, 34.5% were resistant to piperacilin-tazobactam. Our main study limitation was not having complete clinical and epidemiologic information to define which isolates were acquired in the hospital and which were acquired in the community. Overall, rates of antimicrobial drug resistance among common pathogens in hospitals of Lima, Peru, were high.

16S rRNA gene sequence analysis of a Brucella melitensis infection misidentified as Bergeyella zoohelcum

Misidentification of Brucella species from clinical specimens using commercial bacterial identification systems is a recurring problem. An isolate from a bacterimic patient was identified as Bergeyella zoohelcum by MicroScan Walk-Away (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA) and as Brucella melitensis by Vitek 2 system (bioMérieuxInc., Durham, NC, USA). Because of this identification ambiguity by the two automated bacterial identification systems we performed 16S rRNA sequencing and serotyping of the isolate and confirmed it as a Brucella spp. Combining the sequence data with the Vitek 2 system data we conclude that the infection was caused by B. melitensis.

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.

Detection of Vancomycin-IntermediateStaphylococcus aureusWith the Updated Trek-Sensititre System and the MicroScan System: Table 1

Vancomycin-intermediate Staphylococcus aureus (VISA) organisms have minimum inhibitory concentrations (MICs) of 4 to 8 microg/mL and are often associated with vancomycin treatment failure. Detection of VISA has remained problematic. A comparison of 4 methods to detect VISA was done. Of the 20 VISA isolates, the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method yielded susceptible end points of 2 microg/mL for 7, MicroScan (Siemens Healthcare Diagnostics, West Sacramento, CA) for 2, Trek Sensititre (Trek Diagnostic Systems, Cleveland, OH) for 1, and Etest (AB Biodisk North America, Piscataway, NJ) for none. Comparison with the CLSI method showed essential agreement for 95% or more for the Etest, MicroScan, and Trek methods; categorical agreement was as follows: Etest, 60%; MicroScan, 65%; and Trek, 60%. Reliance on a single automated method for determining vancomycin MICs could lead to misclassification of some VISA isolates as vancomycin susceptible. At least 2 methods, including the Etest, should be used when confirming VISA because of slight differences in results from different methods around the end points of 2 and 4 microg/mL .

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover

Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Hybrid El TorVibrio choleraeO1, Kuwait

To the Editor: The traditional causative agent of cholera, Vibrio cholerae O1, has 2 biotypes, classical and El Tor. The current seventh pandemic that began in 1961 and has spread to much of the world is caused by the El Tor biotype. This biotype has replaced the classical biotype responsible for the previous pandemics. The classical and El Tor biotypes are differentiated by phenotypic tests (1), and several nucleotide base differences occur at positions 115 and 203 in the ctxB gene (C in both positions in the classical and T in both positions in the El Tor biotype). These differences translate to histidine at amino acid position 39 and threonine at amino acid position 68 for the B subunit of cholera toxin (CT) in the classical biotype and tyrosine and isoleucine, respectively, for the corresponding amino acids in the El Tor biotype (2). Recently, 3 variants of the El Tor biotype have been found. These are the Matlab variants, which could not be biotyped because they have a mixture of classical and El Tor traits (1); the Mozambique variant, which has a typical El Tor genome but a tandem repeat of the classical CTX prophage located in the small chromosome (3); and the hybrid El Tor variant, which has a typical El Tor biotype and an El Tor CTX prophage but produces CT of the classical type (4). This hybrid El Tor variant has replaced the El Tor biotype in Dhaka and other parts of Bangladesh (4) and in India (5), Japan, Hong Kong, Zambia, the People’s Republic of China, Sri Lanka, and Vietnam (6). Kuwait was affected by cholera in the mid 1960s during the current seventh pandemic. Subsequently, cholera disappeared from Kuwait as living standards improved. Screening of ≈5,000 acute-phase diarrheal stool samples in the mid-1980s in a major hospital in Kuwait did not yield V. cholerae O1 (7). The occasional cholera cases detected in Kuwait are imported, mainly from Asia through expatriate workers. Two adult men, one who had just arrived from India and one who had just arrived from the Philippines, were admitted with severe watery diarrhea, vomiting, and dehydration to the Al-Adan Hospital, Kuwait, in November and December 2008, respectively. The patient from India reported eating in restaurants, and the patient from the Philippines had consumed fish soup just before the journey. Both patients were initially rehydrated with intravenous fluids. Stool cultures were performed by using a battery of media, including thiosulfate citrate bile salt sucrose (TCBS) agar. Yellow colonies from TCBS agar were tested by using MicroScan WalkAway 96 (Dade Behring, West Sacramento, CA, USA) panel NBPC 34 and API 20E strip (bioMerieux, Marcy l’Etoile, France), which suggested V. cholerae. In slide agglutination tests, the colonies agglutinated with V. cholerae O1 polyvalent antiserum and Ogawa serotype antiserum (Denka-Seiken, Tokyo, Japan). The isolate from the patient from India was susceptible to tetracycline and ampicillin and resistant to co-trimoxazole, but the isolate from the patient from the Philippines was susceptible to all 3 of these antimicrobial agents in MicroScan (Dade Behring) and disk diffusion tests. Both patients were successfully treated with rehydration therapy and intravenous vibramycin (a semisynthetic tetracycline). Both V. cholerae O1 Ogawa isolates showed positive results in Vogues-Proskauer, chicken cell agglutination, and tube hemolysin tests and were resistant to polymyxin B (50 international units), results that suggest the El Tor biotype (1). Both isolates were positive for the ctxA gene and El Tor–specific tcp gene but negative for classical-specific tcp gene by PCR (8). The genotype of the ctxB gene was determined by a mismatch amplification mutation assay PCR that detects polymorphism at nucleotide position 203; both isolates yielded a 186-bp amplicon with the classical biotype-specific primers and no amplicon with the El Tor biotype-specific primers (9). The classical ctxB genotype of the isolates was further confirmed by sequencing the ctxB gene by using the BigDye termination method (Applied Biosystems, Foster City, CA, USA) with specific primers (10). This sequencing showed that both isolates had histidine at position 39 and threonine at position 68 in CT-B subunit. Thus, the isolates are hybrid variants that are phenotypically El Tor but genotypically classical for the ctxB gene. Our findings suggest that cholera caused by the hybrid variant is present in the Philippines. The 2 cholera cases reported here were imported into Kuwait by travelers from cholera-endemic regions and were not endemic illnesses. The hybrid variant could possibly initiate the next cholera pandemic. Also, because CT is related to the major clinical sign of the disease, genetic changes in the molecule could result in alteration in the manifestation of the disease. The changes could also influence the effectiveness of cholera vaccines that contain CT-B as a component (6).

The Concurrent Validity of a Hand-held versus a Stationary Dynamometer in Testing Isometric Shoulder Strength

Study design Clinical Measurement-Validity. Introduction Validity of the JTech PowerTrack II hand-held dynamometer (JTech; JTech Medical, Salt Lake City, UT) for measuring shoulder strength has yet to be established. Purpose of the Study To examine the concurrent validity of isometric strength scores obtained with the JTech PowerTrack II, and on a stationary dynamometer, the LIDO WorkSET (LIDO; LoredanBiomedical, West Sacramento, CA). Methods Thirty-eight subjects performed three maximal efforts of shoulder flexion, abduction, and external rotation on a single occasion on the two dynamometers. Two testers were randomly assigned to administer the tests. Results Pearson correlations between the scores on the two dynamometers (r.0.81) indicated a good concurrent validity. Correlations were similar when the results were subdivided by tester or gender. Conclusions This study suggests that either the JTech PowerTrack II or LIDOWorkSET provide comparable scores for shoulder strength. Although not interchangeable because of the differences in units of measurement, the relative conclusions about strength should be similar, regardless of which instrument is used. Level of Evidence Not applicable.

Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-ras(Ha) transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser(280). Since Akt phosphorylates Chk1-Ser(280), the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism.

Accuracy of Detecting Resistance to Carbapenems among Gram Negative Rods: Comparison of Three Methods

Abstract Objective To compare the results of imipenem and meropenem susceptibility testing among multi-drug resistant (MDR) isolates of Acinetobacter spp., Pseaudomonas aeruginosa (P.aeruginosa) and members of the Enterobacteriacae. Methods Three methods used for susceptibility testing of 210 isolates: disk diffusion (a reference method), MicroScan (MicroScan Walk Away 96 System, Dade Behring Inc. West Sacramento CA 95691, USA) and Etest (AB Biodisk Solna, Sweden). Results Of the 210 isolates, Acinetobacter spp. accounted for the majority of isolates [110(52.4%)] followed by P.aeruginosa, 79 (37.6%). These isolates were more prevalent from respiratory specimens 98 (46.7%), Acinetobacter spp. 60(28.6%) and P.aeruginosa 34(16.2%). The study has demonstrated discrepant results for carbapenems tested by MicroScan and Etest. For imipenem, the MicroScan exhibited 2.8 % very major error, major error was 10.1% but 3.9% by Etest for Acinetobacter spp., Other discrepant results (minor errors) were 28.7% and 33% for MicroScan and Etest, respectively. For meropenem, minor errors were higher by MicroScan (13.6%) and Etest (21%). For P.aeruginosa, very major error (1.6%) was exhibited by imipenem Etest but major errors were 23% and 30.5% for both drugs by MicroScan, respectively. Minor errors were higher for both drugs by both methods (MicroScan: 15.3% to 20.8% and Etest : 34.9% to 34.2%). Conclusion Microbiology laboratories should consider the use of an additional confirmatory test for carbapenem susceptibility testing of clinical isolates of Acinetobacter spp. and P.aeruginosa and members of the Enterobacteriacae.

Archaeology and Architecture of the Overseas Chinese: A Bibliography

In the mid-1990s, SHA published a series of bibliographies that documented archaeological references concerning the immigrant experience in North America. This bibliography is intended to be an extension of that series. It is inevitable that any bibliographic effort involving an active area of research will be out of date immediately. For that reason, in addition to this published version, the authors will periodically update the information and publish it on SHA's website. The focus of this bibliography is the archaeo logical and architectural record of Overseas Chinese immigrants and communities. It includes published and unpublished reports of investiga tions of archaeology and architecture. While the vast majority of the references included here deal with sites in the western United States and Australia, similar investigations of Chinese mate rial history have been included, regardless of geographical location. The authors have gathered copies of all references listed here, and placed them in the California Department of Parks and Recreation, Archaeology, History, and Museums Division library in West Sacramento, Califor nia. The purpose of this archive was to verify all citations; there may be additional related references, but unless authors have provided a corresponding hard copy, the reference is not included here. Authors who know of additional related materials are encouraged to forward those hard copies to Peter Schulz. In the interest of maintaining specific focus for this bibliography, it does not include obvi ously relevant studies from China nor strictly historical investigations of the overseas com munities. The bibliography also does not include references to the China Trade, even though some investigations deal with Gold Rush era discover ies in California. Likewise, the references omit studies of material culture, ceramic production, and architecture from southeastern China. Read

Urinary Tract Infection Caused by CapnophilicEscherichia coli

Urinary Tract Infection Caused by Capnophilic<i>Escherichia coli</i>

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process.

Antimicrobial Susceptibility Profile of Streptococcus pseudopneumoniae Isolated from Sputum

Streptococcus pseudopneumoniae is a recently described member of the Streptococcus mitis/oralis group of viridans streptococci that shares some characteristics with Streptococcus pneumoniae (1). Key characteristics of S. pseudopneumoniae are the absence of pneumococcal capsule, insolubility in bile, resistance or indeterminate susceptibility to optochin when incubated in 5% CO2 but susceptibility to optochin when incubated in ambient air, and positive reactions in DNA probe hybridization and antigen detection tests (1, 3). The clinical relevance of S. pseudopneumoniae has not yet been established, although it may be associated with chronic obstructive pulmonary disease (3). As yet, the antibiotic susceptibility profile of S. pseudopneumoniae has not been reported in detail. The aim of this study was to determine the antibiotic susceptibility profile of a large number of S. pseudopneumoniae isolates recovered from clinical specimens. Ninety-five isolates of S. pseudopneumoniae collected between 2000 and 2007 were studied. All isolates had been recovered as the predominant organism from good-quality sputum specimens containing >25 leukocytes and ≤10 squamous epithelial cells per 100× field. The isolates were identified on the basis of phenotypic characteristics as previously described (1, 3). MICs were determined by broth microdilution with the MicroScan Micro STREP plus 1 system (Dade Behring, West Sacramento, CA). The MICs of penicillin, ampicillin, ceftriaxone, cefotaxime, cefepime, meropenem, chloramphenicol, clindamycin, erythromycin, azithromycin, tetracycline, vancomycin, gatifloxacin, levofloxacin, and trimethoprim-sulfamethoxazole were determined. Strains for which the MICs were greater than the highest dilution included on the MicroScan panel were retested with the Etest (AB Biodisk, Solna, Sweden) to determine the precise MIC. Susceptibility breakpoints were based on CLSI guidelines (2). Strains showing resistance to erythromycin but susceptibility to clindamycin were further tested to determine the presence of macrolide-lincosamide-streptogramin B resistance (MLS phenotype). Discs containing erythromycin (15 μg) and clindamycin (2 μg) were placed 20 mm apart on the plate. After incubation, the plates were examined for flattening of the clindamycin zone, which is indicative of the phenotype. The MICs of the 15 antibiotics are shown in Table ​Table1.1. No isolate was resistant to any β-lactam agent, although about one-third showed reduced susceptibility to penicillin and ampicillin. About one-third of the isolates were resistant to macrolides, almost half were resistant to tetracycline, and 71% were resistant to tetracycline and/or macrolide antibiotics. Reduced susceptibility to the macrolide antibiotics only (M phenotype) occurred in seven isolates, with an additional isolate being resistant to clindamycin (intrinsic MLS resistance). Inducible MLS resistance was noted in 3 of 28 erythromycin-resistant, clindamycin-susceptible isolates. In each case, the MIC was within a 1-dilution difference from susceptible. All azithromycin-resistant isolates were resistant to erythromycin. TABLE 1. In vitro activities of antimicrobial agents against 95 isolates of S. pseudopneumoniae One limitation of our study is that all of the isolates were collected from respiratory samples from patients attending a tertiary care center in one geographical area of New Zealand. As such, they may not be representative of the isolates found in the wider community or other regions of the world. Further studies are needed in other geographic regions, and possibly with other sample types, in order to better clarify the antibiotic susceptibility profile of S. pseudopneumoniae.

Direct Comparison of the BD Phoenix System with the MicroScan WalkAway System for Identification and Antimicrobial Susceptibility Testing of Enterobacteriaceae and Nonfermentative Gram-Negative Organisms

The Phoenix automated microbiology system (BD Diagnostics, Sparks, MD) is designed for the rapid identification (ID) and antimicrobial susceptibility testing (AST) of clinically significant human bacterial pathogens. We evaluated the performance of the Phoenix instrument in comparison with that of the MicroScan WalkAway system (Dade Behring, West Sacramento, CA) in the ID and AST of gram-negative clinical strains and challenge isolates of Enterobacteriaceae (n = 150) and nonfermentative gram-negative bacilli (NFGNB; 45 clinical isolates and 8 challenge isolates). ID discrepancies were resolved with the API 20E and API 20NE conventional biochemical ID systems (bioMerieux, Durham, NC). The standard disk diffusion method was used to resolve discordant AST results. The overall percentages of agreement between the Phoenix ID results and the MicroScan results at the genus and species levels for clinical isolates of Enterobacteriaceae were 98.7 and 97.7%, respectively; following resolution with conventional biochemical testing, the accuracy of the Phoenix system was determined to be 100%. For NFGNB, the levels of agreement were 100 and 97.7%, respectively. Both systems incorrectly identified the majority of the uncommon nonfermentative nonpseudomonal challenge isolates recovered from cystic fibrosis patients; these isolates are not included in the databases of the respective systems. For AST of Enterobacteriaceae, the rate of complete agreement between the Phoenix results and the MicroScan results was 97%; the rates of very major, major, and minor errors were 0.3, 0.2, and 2.7%, respectively. For NFGNB, the rate of complete agreement between the Phoenix results and the MicroScan results was 89.1%; the rates of very major, major, and minor errors were 0, 0.5, and 7.7%, respectively. Following the confirmatory testing of nine clinical isolates initially screened by the MicroScan system as possible extended-spectrum-beta-lactamase (ESBL)-producing organisms (seven Klebsiella pneumoniae isolates and two Escherichia coli isolates), complete agreement was achieved for eight isolates (one ESBL positive and seven negative); one false positive was obtained with the Phoenix instrument. The MicroScan system correctly detected the 10 ESBL challenge isolates, versus the 6 detected by the Phoenix system. Overall, there was good correlation between the Phoenix instrument and the MicroScan system for the ID and AST of Enterobacteriaceae and common NFGNB. The Phoenix system is a reliable method for the ID and AST of the majority of clinical strains encountered in the clinical microbiology laboratory. Until additional performance data are available, results for all Klebsiella pneumoniae or Klebsiella oxytoca and E. coli isolates screened and confirmed as ESBL producers by any automated system should be confirmed by alternate methods prior to the release of final results.

The β1 Integrin Activates JNK Independent of CagA, and JNK Activation Is Required for Helicobacter pylori CagA+-induced Motility of Gastric Cancer Cells

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.

Prevalência da contaminação bacteriana em concentrados de plaquetas do serviço de hemoterapia de um hospital universitário em Goiânia-GO

Abstract Although the transmission of microorganisms by bloodtransfusion has been described for more than half a century, bacterialcontamination of blood components, mainly platelet concentrates,remains a public health problem.This study aimed at evaluating the prevalence ofmicrobiological contamination and also isolate and identify themicroorganisms found in random donor platelets (RDP) in thehemotherapy service of a university hospital of Goiânia, Brazil,between November 2004 and May 2005.Two thousand samples of RDP were collected with thestorage time ranging from the day of the donation (Dzero) to thefifth day of storage (D5). Cultures were performed in pools of twoto ten units in bottles with brain heart infusion (BHI), incubated at37oC for up to 7 days and sub cultivated in chocolate agar to isolatethe microorganisms. In cases of a pool bring positive, the culturewas individually performed for all the samples of that pool. Theisolated bacteria were identified by enzymatic and biochemical testsand by a MicroScan® automated bacterial detection system (DadeBehring - West Sacramento, California, USA), using Pos-Combo 21and Neg-Combo 32 kits and the reader autoSCAN4® reader.Eight positive samples were detected, resulting in a prevalenceof 0.4% (95%CI = 0.31 < µ < 0.49). Isolated microorganisms were:five (62.5%) Gram-negative rods (three

Polyunsaturated fat in the methionine-choline-deficient diet influences hepatic inflammation but not hepatocellular injury

Methionine-choline-deficient (MCD) diets that cause steatohepatitis in rodents are typically enriched in polyunsaturated fat. To determine whether the fat composition of the MCD formula influences the development of liver disease, we manufactured custom MCD formulas with fats ranging in PUFA content from 2% to 59% and tested them for their ability to induce steatohepatitis. All modified-fat MCD formulas caused identical degrees of hepatic steatosis and resulted in a similar distribution of fat within individual hepatic lipid compartments. The fatty acid composition of hepatic lipids, however, reflected the fat composition of the diet. Mice fed a PUFA-rich MCD formula showed extensive hepatic lipid peroxidation, induction of proinflammatory genes, and histologic inflammation. When PUFAs were substituted with more saturated fats, lipid peroxidation, proinflammatory gene induction, and hepatic inflammation all declined significantly. Despite the close relationship between PUFAs and hepatic inflammation in mice fed MCD formulas, dietary fat had no impact on MCD-mediated damage to hepatocytes. Indeed, histologic apoptosis and serum alanine aminotransferase levels were comparable in all MCD-fed mice regardless of dietary fat content. Together, these results indicate that dietary PUFAs promote hepatic inflammation but not hepatotoxicity in the MCD model of liver disease. These findings emphasize that individual dietary nutrients can make specific contributions to steatohepatitis.

Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction–restriction fragment length polymorphism analysis of a partial groEL gene sequence

A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.

Accuracy of Six Antimicrobial Susceptibility Methods for Testing Linezolid against Staphylococci and Enterococci

A challenge panel of enterococci (n = 50) and staphylococci (n = 50), including 17 and 15 isolates that were nonsusceptible to linezolid, respectively, were tested with the Clinical and Laboratory Standards Institute broth microdilution and disk diffusion reference methods. In addition, all 100 isolates were tested in parallel by Etest (AB Biodisk, Solna, Sweden), MicroScan WalkAway (Dade, West Sacramento, CA), BD Phoenix (BD Diagnostic Systems, Sparks, MD), VITEK (bioMérieux, Durham, NC), and VITEK 2 (bioMérieux) by using the manufacturers' protocols. Compared to the results of the broth microdilution method for detecting linezolid-nonsusceptible staphylococci and enterococci, MicroScan results showed the highest category agreement (96.0%). The overall categorical agreement levels for VITEK 2, Etest, Phoenix, disk diffusion, and VITEK were 93.0%, 90.0%, 89.6%, 88.0%, and 85.9%, respectively. The essential agreement levels (results within +/-1 doubling dilution of the MIC determined by the reference method) for MicroScan, Phoenix, VITEK 2, Etest, and VITEK were 99.0%, 95.8%, 92.0%, 92.0%, and 85.9%, respectively. The very major error rates for staphylococci were the highest for VITEK (35.7%), Etest (40.0%), and disk diffusion (53.3%), although the total number of resistant isolates tested was small. The very major error rate for enterococci with VITEK was 20.0%. Three systems (MicroScan, VITEK, and VITEK 2) provided no interpretations of nonsusceptible results for staphylococci. These data, from a challenge panel of isolates, illustrate that the recent emergence of linezolid-nonsusceptible staphylococci and enterococci is providing a challenge for many susceptibility testing systems.