Oligonucleotide Array and VITEK Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Species Identification of Blood Yeast Isolates.

We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry using VITEK MS (IVD database) and an oligonucleotide array based on the internal transcribed spacer-1 (ITS-1) and ITS-2 sequences of rRNA genes for the identification of Candida spp. from blood cultures. Five-hundred and twelve consecutive bloodstream yeast isolates were collected daily and initially identified by the phenotypic automated method (VITEK YBC or VITEK2 YST card). Inconsistent results were confirmed by D1-D2 region of 28S rRNA genes and ITSs. Excluding two unidentified yeast isolates, the oligonucleotide array and VITEK MS correctly identified 99.6% (508) and 96.9% (494) of 510 yeast isolates, respectively. The oligonucleotide array and VITEK MS demonstrated high correct identification rates for four major Candida species (C. albicans 100%, 98.4%; C. glabrata 100%, 100%; C. parapsilosis 100%, 93.3%; C. tropicalis 100%, 97.3%), but lower correct identification rates for other Candida species (91.7 and 87.5%, respectively). In conclusion, the identification performance of the oligonucleotide array is comparable to that of VITEK MS, and can serve as a supplemental tool for the identification of Candida species.

Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

OBJECTIVE: The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. RESULTS: The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). CONCLUSION: Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues.

Aeromonas stool isolates from individuals with or without diarrhea in southern Taiwan: Predominance of Aeromonas veronii.

BACKGROUND: Although aeromonads are important pathogens causing invasive infections in southern Taiwan, Aeromonas-associated intestinal infections have been rarely mentioned. PURPOSE: The aim of this study was to understand the frequency of isolation and clinical significance of aeromonads recovered from adult stool samples in southern Taiwan. METHODS: During a 15-month study period, 514 adults with diarrhea and 167 asymptomatic controls were prospectively screened for the presence of aeromonads in stools. The identity of Aeromonas species was determined by the rpoD sequencing. Clinical information was retrieved from medical records, and in vitro cytotoxicity assay and polymerase chain reaction detection of putative virulent genes were performed. RESULTS: Thirteen (2.5 %) of 514 diarrheal patients and six (3.6%) of 167 asymptomatic controls had Aeromonas isolates in their stools. Of 11 diarrheal patients with available clinical information, Aeromonas veronii, the predominant species, was noted in six patients, and another potential enteropathogen was present in four patients. The cytotoxicity of A. veronii isolates to the HT-29 cell line was more potent in the isolates from diarrheal patients than those from asymptomatic controls (p = 0.015). The cytotoxicity of A. veronii isolates was more potent than that of A. caviae from symptomatic patients (p = 0.001). Putative virulence markers, including AHCYTONE, ascV, ascF-ascG, and aexT, were detected exclusively in A. veronii. The presence of the ascV gene was associated with cytotoxicity in A. veronii isolates. All Aeromonas isolates were susceptible to varied antimicrobial agents, except ampicillin/sulbactam. CONCLUSION: A. veronii is the predominant species in stools from individuals with or without diarrhea in southern Taiwan.

Incidence of Aeromonas bacteremia in Southern Taiwan: vibrio and Salmonella bacteremia as comparators.

The aim of the investigation was to describe the incidence of Aeromonas bacteremia in a city with a population of about 1.87 million inhabitants, located in southern Taiwan, between 2008 and 2010. Such data were compared with the incidences of Vibrio and Salmonella bacteremia in the same period and the incidence of Aeromonas bacteremia in other countries in the literature. The data revealed the average annual incidences of bacteremia due to Aeromonas, Vibrio, and Salmonella species were 76, 38, and 103 cases/million inhabitants, respectively. The incidence of Aeromonas bacteremia was higher than those in Western countries.

Acrophialophora fusispora brain abscess in a patient with acquired immunodeficiency syndrome: a case report and review of the literature.

We reported a fatal case of brain abscess caused by Acrophialophora fusispora in a patient with acquired immunodeficiency syndrome. Identification of the fungus was based on microscopic morphology and sequence analyses of the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of ribosomal RNA gene from the isolate recovered from brain abscess. Four published cases were reviewed as well.

Characterization of the modified Hodge test-positive isolates of Enterobacteriaceae in Taiwan.

BACKGROUND/PURPOSE: The modified Hodge test is a phenotypic test to detect KPC-type carbapenemase producers among Enterobacteriaceae, as recommended by the Clinical Laboratory Standards Institute. However, false positive results were reported. In this study, we aimed to large-scale investigate the characterization of the modified Hodge test-positive isolates of Enterobacteriaceae collected between 2006 and 2010 in Taiwan. METHODS: Fifty-six isolates, including 24 Enterobacter cloacae, 17 Escherichia coli, 10 Klebsiella pneumoniae, and 5 Citrobacter freundii, tested positive with the modified Hodge test. The in vitro activities of 10 antimicrobial agents were determined by the agar dilution method. Boronic acid combined-disk test was used to further confirm the KPC producers. Phenotype of ESBL, AmpC, class B carbapenemases, and profile of outer membrane proteins were investigated by the confirmatory test, boronic acid disk method, 2-mercaptopropionic acid double-disk method, and urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. ß-lactamase genes were examined by PCR and sequencing. RESULTS: These isolates were resistant to ceftazidime (100%), aztreonam (82.1%), ertapenem (64.3%), gentamicin (53.6%), ciprofloxacin (50%), levofloxacin (48.2%), cefepime (19.6%), imipenem (16.1%), meropenem (12.5%), and amikacin (8.9%). Phenotypic testing among isolates revealed the production of ESBLs, metallo-ß-lactamases (MBLs), and AmpC in 10 (17.9%), 16 (28.6%), and 12 (44.4%) isolates, respectively. Carbapenemase and non-carbapenemase ß-lactamase genes bla(TEM-1), bla(SHV), bla(CTX-M), bla(IMP-8), bla(CMY-2), and bla(DHA-1) were found in 32 (57.1%), 19 (33.9%), 4 (7.1%), 16 (28.6%), 14 (25%), and 5 (8.9%) of the strains, respectively. No class A and D carbapenemase genes were detected. Outer membrane protein profile showed obviously decreased expression in 49 (87.5%) isolates with positive result of modified Hodge test. CONCLUSIONS: Our data show that ESBLs, AmpC, and imipenemase-8 (IMP-8) carbapenemase coupled with decreased expression of outer membrane protein were prevalent in Enterobacteriaceae isolates testing positive for the modified Hodge test in Taiwan.

Screening of antibiotic susceptibility to ß-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis.

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the ß-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 µm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to ß-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

Metallo-ß-lactamase-producing Enterobacteriaceae isolates at a Taiwanese hospital: lack of distinctive phenotypes for screening.

We investigated the prevalence of metallo-ß-lactamases (MBLs) among 1,827 Enterobacteriaceae isolates collected in 2006 and evaluated the VITEK 2 microbiology system, modified Hodge test, and 2 combined disk tests as the screening tools for MBLs by using these isolates and 77 previously characterized IMP-8 producers. The IMP-8 MBL was identified in 18 isolates of 2006, and the IMP-8-positive isolates represented 0.2%, 1.1%, and 5.0% of all Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates, respectively. Only one-third of all MBL producers could be recognized by either VITEK 2 or the Hodge test. MBL production could be identified in 38 (40%) of the 95 IMP-8-producing isolates by the combined disk test using meropenem disks supplemented by phenylboronic acid and EDTA, and only 2 (2.1%) isolates gave positive results in the combined disk test using meropenem disks supplemented with dipicolinic acid. Of all IMP-8 producers, 37.9%, 50.5%, and 32.6% were nonsusceptible to tigecycline, fluoroquinolones, and both, respectively. In conclusion, this study demonstrated the lack of distinct phenotypes that could be easily identified among the IMP-8-producing Enterobacteriaceae isolates at a Taiwanese hospital. Continuous surveillance and monitoring are needed because the widespread of tigecycline- and fluoroquinolone-coresistant MBL producers may become a serious therapeutic problem.

Prevalence and characteristics of ertapenem-resistant Klebsiella pneumoniae isolates in a Taiwanese university hospital.

This study was conducted to investigate the prevalence and characteristics of ertapenem-resistant (ETP-R) Klebsiella pneumoniae isolates at a Taiwanese hospital. The disk-diffusion tests revealed that the rate of ertapenem resistance among all isolates collected in 2008 was 13.5%, and the resistance rate among bloodstream isolates increased from 0% to 13.6% between 2001 and 2008. Eighty-two nonduplicate ETP-R isolates collected in 2008 were examined. Seventy-four (90.2%) isolates of them had extended-spectrum ß-lactamases (CTX-M- and SHV-type), AmpC enzymes (DHA-1 and CMY-2), and IMP-8 metallo-ß-lactamase alone or in combination, and an extremely high prevalence of fluoroquinolone resistance (95.1%) and plasmid-mediated quinolone resistance determinants (90.2%) were also observed. Eighteen ETP-R but imipenem-susceptible isolates were selected and compared with 18 imipenem-nonsusceptible isolates collected before 2008. Sequence analyses revealed genetic disruptions of OmpK36 in 11 imipenem-nonsusceptible and 6 imipenem-susceptible isolates, respectively, and OmpK35 disruptions in 10 isolates for both groups. For the isolates with intact ompK36, sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests decreased expression of OmpK36 in 5 of 7 imipenem-nonsusceptible isolates and 3 of 12 imipenem-susceptible isolates. In conclusion, the increasing prevalence of ertapenem resistance that was predominantly attributed to noncarbapenemase-mediated resistance mechanisms in K. pneumoniae is becoming a serious treat to patients in Taiwan.

Effect of overnight storage of blood culture bottles on bacterial detection time in the BACTEC 9240 blood culture system.

BACKGROUND/PURPOSE: Identifying the pathogens present in blood stream infections is crucial to initiate appropriate antimicrobial therapy and avoid morbidity and mortality. The aim of this study was to evaluate the effect of overnight storage of aerobic and anaerobic BACTEC 9240 blood culture bottles on the detection time for common pathogens. METHODS: From November 2007 to July 2008, a total of 2,105 isolates were positively detected using the BACTEC 9240 system. The time to positive detection (TTD) was calculated by subtracting the time of receipt in the laboratory from the time required to detect a positive culture. The mean TTD values were calculated using the TTD value of the first positive culture bottle only. Overnight delay at the National Cheng Kung University Hospital, Taiwan was 15 hours (from 5 pm to 8 am). RESULTS: Of the 2,105 total isolates, 972 (46.1%) were Gram-positive bacteria, 1,024 (48.6%) were Gram-negative bacteria and 109 (5.1%) were fungi. Among the top 10 pathogens, 24.7% grew only in the aerobic bottle and 15.1% in the anaerobic bottle, including Staphylococcus spp., Enterococcus faecium, Enterobacteriaceae, and Gram-positive bacilli. Due to the overnight delay in loading a blood culture bottle into the instrument, for most of the pathogens (including Staphylococcus spp. and Enterobacteriaceae), a decrease in TTD by

Identification of clinically important anaerobic bacteria by an oligonucleotide array.

Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.

Emergence of Qnr determinants in human Salmonella isolates in Taiwan.

OBJECTIVES: The aim of this study was to determine the prevalence and characteristics of qnr-carrying Salmonella isolates from humans in southern Taiwan. METHODS: A total of 446 Salmonella isolates collected between 2003 and 2006 were screened for qnrA, qnrB and qnrS by PCR experiments. Genetic structures of qnr were determined by PCR-based methods or direct sequencing of plasmid DNA. RESULTS: qnrB2 and qnrS1 were detected in two serovar Enteritidis isolates and two serovar Typhimurium isolates, respectively. One qnrS1-positive isolate was found to produce the CMY-2 AmpC enzyme. qnrS1 was identified on a 10 kb plasmid, which exhibited >99% nucleotide sequence identity with plasmid TPqnrS-1a reported from the UK. qnrB2 was found in a complex sul1-type class 1 integron on a >100 kb plasmid. CONCLUSIONS: This study demonstrated the occurrence of qnrB2 and qnrS1 in Salmonella for the first time in Taiwan and characterized their genetic structures.

Prevalence of extended-spectrum beta-lactamases in Proteus mirabilis in a Taiwanese university hospital, 1999 to 2005: identification of a novel CTX-M enzyme (CTX-M-66).

A total of 1574 nonduplicate Proteus mirabilis isolates collected at a Taiwanese hospital during 1999 to 2005 were analyzed for production of extended-spectrum beta-lactamases (ESBLs). Forty-four ESBL-producing isolates including 22 CTX-M-14, 18 CTX-M-3, 2 CTX-M-24, and 2 CTX-M-66 producers were detected, and the proportion of ESBL producers increased from 0.7% in 1999 to approximately 6% after 2002. CTX-M-66 is a novel variant of CTX-M ESBLs that differs from CTX-M-3 by a Ser to Asn change at amino acid position 23. Coresistances to aminoglycosides and ciprofloxacin were very common in the CTX-M-3 producers. The presence of ArmA-type or RmtB-type 16S rRNA methylase that confers high-level aminoglycoside resistance was detected in 12 CTX-M-3 producers and 4 CTX-M-14 producers. Twenty-four clones including an endemic CTX-M-14-producing clone were observed among the 44 ESBL producers by pulsed-field gel electrophoresis, suggesting that both horizontal transfer and clonal spread contributed to the increased prevalence of bla(CTX-M) in P. mirabilis.

Comparison of CPS ID 3 and CHROMagar Orientation chromogenic agars with standard biplate technique for culture of clinical urine samples.

BACKGROUND AND PURPOSE: Chromogenic agars have been developed to recognize frequently occurring microorganisms directly on primary cultures, thus reducing the daily workload in a clinical microbiology laboratory. We compare two chromogenic agars, CHROMagar Orientation (CO) and CPS ID 3 (CPS3), with routine media (biplate technique using trypticase soy blood agar and eosin methylene blue agar) for the isolation, enumeration and identification of organisms in urinary tract infection (UTI). METHODS: The clinical significance of the urine samples was categorized as probable UTI, possible UTI, no UTI (negative), or contaminated according to the culture result. Discrepancy analysis with the categories of minor error, major error and very major error was used to compare the culture media. RESULTS: Of 1386 urine specimens, the consistencies in clinical significance of CO and CPS3 to routine media were 90.7% and 89.8%, respectively. For the enumeration of microorganisms, 524, 514, and 521 clinically significant isolates were isolated on routine media, CO, and CPS3, respectively. Of the 524 significant isolates on routine media, results for 473 and 474 isolates agreed on CO and CPS3, respectively. Approximately 91.9% of Escherichia coli and 100.0% of Enterococcus spp. could be identified directly on CO media, while 97.5% of E. coli and 94.4% of Enterococcus spp. could be identified on CPS3 media. CONCLUSION: The use of CO and CPS3 as single media is promising for clinical urine culture.

Prosthetic valve endocarditis caused by Streptobacillus moniliformis: a case of rat bite fever.

We report a case of rat bite fever caused by Streptobacillus moniliformis in Taiwan. It manifested as prosthetic valve endocarditis, which was cured by cardiac valve replacement and antimicrobial therapy. The DNA sequence of the 16S rRNA gene of S. moniliformis was detected in valve specimens by PCR and nucleotide sequencing.

Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S-23S intergenic spacer region.

The feasibility of sequence analysis of the ribosomal 16S-23S intergenic spacer region (ITS) was evaluated for identification of 24 species of Streptococcus, one species of Abiotrophia, 18 species of Enterococcus and three species of Granulicatella. As GenBank currently lacks ITS sequence entries for many species of these four genera, the ITS sequences of 38 type strains were first sequenced and submitted to GenBank to facilitate species identification of these genera. Subsequently, the ITS sequences of 217 strains (84 reference strains and 133 clinical isolates) were determined and species identification was made by blast search for homologous sequences in public databases. Species other than Streptococcus contained multiple ITS fragments and only the shortest fragment was analysed. A total of 25 isolates (11.5 %) produced discrepant identification by ITS sequencing. The 25 discordant strains were analysed further by sequencing of the 16S rRNA gene for species clarification, and 21 were found to be identified correctly by ITS sequence analysis. The correct identification rate by ITS sequencing was 98.2 % (213/217). Several closely related enterococcal and streptococcal species/subspecies contained specific ITS signature sequences that were useful for differentiating these bacteria. In conclusion, ITS sequencing provides a useful approach towards identifying this group of pathogens on a molecular platform alongside 16S rRNA gene sequencing.

Inducible clindamycin resistance in Staphylococcus aureus isolates causing bacteremia at a university hospital in southern Taiwan.

A major concern while prescribing clindamycin to treat infections caused by inducible macrolide, lincosamide, and group B streptogramin (iMLS(B))-resistant strains is clinical therapy failure. In this study, we determined the prevalence, mechanism, and clonality of the iMLS(B) phenotype in oxacillin-resistant Staphylococcus aureus (ORSA) and oxacillin-susceptible S. aureus (OSSA). Among the 729 OSSA isolates collected from July 1995 to March 2006, 72 (10%) were clindamycin sensitive (Cli(s)) and erythromycin resistant (Erm(r)), and 55 (8%) had the iMLS(B) phenotype. In the 709 ORSA isolates collected from January 1997 to March 2006, 31 (4%) were Cli(s) and Erm(r), and 29 (4%) isolates demonstrated the iMLS(B) phenotype. In OSSA, ermC was the predominant (51 of 55 isolates) genetic determinant responsible for the iMLS(B) phenotype, whereas in ORSA, ermA was predominant (27 of 29). Pulsed-field gel electrophoresis showed that 8 pulsed types (RA to RH) were present in ORSA isolates (n = 27), and pulsed type RC was predominant in 17 isolates with 5 identifiable subtypes (RC1 to RC5); this type was prevalent from November 1997 to June 2004. In the OSSA (n = 24) isolates, 14 different pulsed types (SA to SN) were identified, but none was predominant. These results indicate that the incidence of iMLS(B) resistance phenotype is higher in OSSA than ORSA in Taiwan, and the genetic determinants responsible for the iMLS(B) phenotype vary in OSSA and ORSA.

Array-based identification of species of the genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus.

Some species of enterococci and streptococci are difficult to differentiate by phenotypic traits. The feasibility of using an oligonucleotide array for identification of 11 viridans group streptococci was previously established. The aim of this study was to expand the array to identify species of Abiotrophia (1 species), Enterococcus (18 species), Granulicatella (3 species), and Streptococcus (31 species and 6 subspecies). The method consisted of PCR amplification of the ribosomal DNA intergenic spacer (ITS) regions, followed by hybridization of the digoxigenin-labeled PCR products to a panel of oligonucleotide probes (16- to 30-mers) immobilized on a nylon membrane. Probes could be divided into three categories: species specific, group specific, and supplemental probes. All probes were designed either from the ITS regions or from the 3' ends of the 16S rRNA genes. A collection of 312 target strains (162 reference strains and 150 clinical isolates) and 73 nontarget strains was identified by the array. Most clinical isolates were isolated from blood cultures or deep abscesses, and only those strains having excellent species identification with the Rapid ID 32 STREP system (bioMérieux Vitek, Taipei, Taiwan) were used for array testing. The test sensitivity and specificity of the array were 100% (312/312) and 98.6% (72/73), respectively. The whole procedure of array hybridization took about 8 h, starting from isolated colonies, and the hybridization patterns could be read by the naked eye. The oligonucleotide array is accurate for identification of the above microorganisms and could be used as a reliable alternative to phenotypic identification methods.