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A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCC fusC , which was integrated into rlmH and located upstream from SCC mec . The SCC fusC element contained speG , which may contribute to the polyamine resistance.

To understand the high prevalence of fusB genes in fusidic acid-resistant Staphylococcus epidermidis , analysis of resistance elements in 34 isolates was performed. First, sequence analysis of the aj1 -LP- fusB region indicated that at least three types were present. Type I contained full-length aj1 , type II contained a partial aj1 truncated from nucleotide position 93 to 421, and type III contained a more truncated aj1 that retained only the last 37 bp. Isolates with type I or type II aj1 displayed slightly higher levels of resistance to fusidic acid (MICs, 8 to 32 μg/ml) than did those with type III aj1 (MICs, 4 to 16 μg/ml). Subsequent sequencing of the flanking regions of fusB from four selected isolates carrying different types of aj1 -LP- fusB regions revealed that the fusB genes were all located on phage-related resistance islands (RIs), referred to as SeRI fusB -2793 , SeRI fusB -704 , SeRI fusB -5907 , and SeRI fusB -7778 , respectively. Among them, three islands (SeRI fusB -2793 , SeRI fusB -704 , and SeRI fusB -5907 ) were located downstream of groEL (corresponding to the 44-min position based on Staphylococcus aureus whole genomic sequences), and one (SeRI fusB -7778 ) was located downstream of rpsR (corresponding to the 8-min position). All of the RIs were inserted into integrase-recognized att sites. Among 34 isolates, the insertion sites of fusB RIs were mostly (28/34, 82%) located downstream of groEL and two were located downstream of rpsR , but four remained unidentified. The pulsotype distribution indicated that fusB -containing S. epidermidis isolates were heterogeneous. In conclusion, the fusB resistance determinant in S. epidermidis was highly associated with phage-related RIs. This is the first report of fusB RI in S. epidermidis .

Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn 5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn 5 inserted into the P pmrI ( Proteus pmrI ) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the P pmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the P pmrI knockout mutant. Moreover, the P pmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the P pmrI mutant with the full-length P pmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis . Here we showed that RppA could mediate the induction of P pmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative P pmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis .

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains, which often produce Panton-Valentine leucocidin (PVL), are increasingly noted worldwide. In this study, we examined 42 MRSA strains (25 PVL-positive [PVL + ] strains and 17 PVL-negative [PVL − ] strains) isolated in Taiwan for their molecular characteristics. The PVL + MRSA strains included CA-MRSA strains with multilocus sequence type (ST) 59 (major PVL + MRSA in Taiwan), its variants, and worldwide CA-MRSA ST30 strains. The PVL − MRSA strains included the pandemic Hungarian MRSA ST239 strain, the Hungarian MRSA ST239 variant, MRSA ST59 (largely hospital-acquired MRSA strains) and its variants, the pandemic New York/Japan MRSA ST5 strain (Japanese type), and the MRSA ST8 strain. The major PVL + CA-MRSA ST59 strain possessed a tetracycline resistance-conferring ( tetK positive) penicillinase plasmid and a drug resistance gene cluster (a possible composite transposon) for multidrug resistance. Moreover, it carried a novel staphylococcal cassette chromosome mec (SCC mec ) with two distinct ccrC genes ( ccrC2 - C8 ). This SCC mec (previously named SCC mec type V T ) was tentatively designated SCC mec type VII. Sequencing of the PVL genes revealed the polymorphisms, and the PVL + CA-MRSA ST59 strain possessed the ST59-specific PVL gene sequence. The data suggest that a significant amount of clonal spread is occurring in Taiwan and that the major PVL + CA-MRSA ST59 Taiwan strain exhibits unique genetic characteristics, such as a novel SCC mec type and an ST59-specific PVL gene sequence.

We describe the structure of a transposon-like element named Tn 6001 , which contains a bla VIM-3 -harboring integron In450, which was derived from a multidrug-resistant Pseudomonas aeruginosa clinical isolate in Taiwan. The transposon backbone structure is most closely related to those of Tn 1404 * and Tn 1403 . Tn 6001 was inserted into the chromosome of the clinical isolate.

We investigated the sequence and the genetic context of the erm (T) gene in six inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus (formerly S. bovis biotype II.2) isolates. In all isolates, the erm (T) genes were flanked by two IS 1216V -like elements with the same polarity and were found to be inserted in the chromosome.

The susceptibilities of nonduplicate isolates to six antifungal agents were determined for 391 blood isolates of seven Candida species, 70 clinical isolates (from blood or cerebrospinal fluid) of Cryptococcus neoformans , and 96 clinical isolates of four Aspergillus species, which were collected in seven different hospitals in Taiwan (as part of the 2003 program of the study group Surveillance of Multicenter Antimicrobial Resistance in Taiwan). All isolates of Candida species other than C. glabrata and C. krusei were susceptible to fluconazole. Among the 59 C. glabrata isolates, 16 (27%) were not susceptible to fluconazole, and all were dose-dependently susceptible or resistant to itraconazole. For three (5.1%) C. glabrata isolates, voriconazole MICs were 2 to 4 μg/ml, and for all other Candida species isolates, voriconazole MICs were ≤0.5 μg/ml. The proportions of isolates for which amphotericin B MICs were ≥2 μg/ml were 100% (3 isolates) for C. krusei , 11% (23 of 207 isolates) for Candida albicans , 3.0% (2 of 67 isolates) for Candida tropicalis , 20% (12 of 59 isolates) for C. glabrata , and 0% for both Candida parapsilosis and Candida lusitaniae . For three (4%) Cryptococcus neoformans isolates, fluconazole MICs were ≥16 μg/ml, and two (3%) isolates were not inhibited by 1 μg of amphotericin B/ml. For four (4.2%) of the Aspergillus isolates, itraconazole MICs were 8 μg/ml. Aspergillus flavus was less susceptible to amphotericin B, with the MICs at which 50% (1 μg/ml) and 90% (2 μg/ml) nsrsid417869\delrsid7301351 of isolates were inhibited being twofold greater than those for Aspergillus fumigatus and Aspergillus niger . All Aspergillus isolates were inhibited by ≤1 μg of voriconazole/ml, including isolates with increased resistance to amphotericin B and itraconazole. This study revealed the emergence in Taiwan of decreased susceptibilities of Candida species to amphotericin B and of C. neoformans to fluconazole and amphotericin B. Voriconazole was the most potent agent against the fungal isolates tested, including fluconazole- and amphotericin B-nonsusceptible strains.

Nucleotide sequencing of the fusB -flanking regions in two fusidic acid-resistant Staphylococcus epidermidis isolates with the type IV aj1 -leader peptide (LP)- fusB structure (lacking aj1 ) revealed that their fusB gene was located on novel phage-related islands inserted downstream of smpB and are here referred to as SeRI fusB -3692 and SePI fusB -857 . The novel SePI fusB -857 structure was followed by SeCI 857 , forming a composite pathogenicity island which contained a putative virulence gene, vapE . The linkage of fusB and vapE may contribute to bacterial adaption.

A rapid emergence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infection (from 26.3% in 1986 to 77% in 2001) was found. The susceptibility of 200 nonduplicate blood isolates of MRSA and 100 MRSA isolates causing refractory bacteremia to 22 antimicrobial agents disclosed that glycopeptides, quinupristin-dalfopristin, and linezolid remained the most active agents.

This study evaluated the current status of antimicrobial resistance in clinical isolates of Streptococcus pyogenes in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. In 2001, 419 different isolates of S. pyogenes , including 275 from respiratory secretions, 87 from wound pus, and 31 from blood, were collected from nine hospitals in different parts of Taiwan. MICs of 23 antimicrobial agents were determined at a central location by the agar dilution method. All of the isolates were susceptible to penicillin (MIC at which 90% of the isolates were inhibited [MIC 90 ], ≤0.03 μg/ml), cefotaxime (MIC 90 , ≤0.03 μg/ml), cefepime (MIC 90 , 0.06 μg/ml), meropenem (MIC 90 , ≤0.03 μg/ml), moxifloxacin (MIC 90 , 0.25 μg/ml), vancomycin (MIC 90 , 0.5 μg/ml), and linezolid (MIC 90 , 1 μg/ml). Overall, 78% of isolates were not susceptible to erythromycin (54% were intermediate, and 24% were resistant), and 5% were not susceptible to clindamycin. Of the 101 erythromycin-resistant isolates, 80.2% exhibited the M phenotype ( mefA gene positive), 18.9% exhibited the cMLS (constitutive resistance to macrolides-lincosamides-streptogramin B [MLS]) phenotype ( ermB gene positive), and 1% exhibited the iMLS (inducible resistance to MLS) phenotype ( ermB gene positive). Fluoroquinolones (sitafloxacin > moxifloxacin > ciprofloxacin = levofloxacin = gatifloxacin > gemifloxacin) demonstrated potent activity against nearly all of the isolates of S. pyogenes tested. Thirty-two isolates (8%) were not susceptible to quinupristin-dalfopristin. Seventeen percent of isolates had telithromycin MICs of ≥1 μg/ml, and all of these isolates exhibited erythromycin MICs of ≥32 μg/ml. The high prevalence of resistance to telithromycin (which is not available in Taiwan) limits its potential use in the treatment of S. pyogenes infections, particularly in areas with high rates of macrolide resistance.

There is a high prevalence of β-lactam- and macrolide-resistant Streptococcus pneumoniae in Taiwan. To understand the in vitro susceptibilities of recent isolates of S. pneumoniae to fluoroquinolones and telithromycin (which is not available in Taiwan), the MICs of 23 antimicrobial agents for 936 clinical isolates of S. pneumoniae isolated from different parts of Taiwan from 2000 to 2001 were determined by the agar dilution method. Overall, 72% of isolates were not susceptible to penicillin (with 61% being intermediate and 11% being resistant) and 92% were resistant to erythromycin. Telithromycin MICs were ≥1 μg/ml for 16% of the isolates, and for 99% of these isolates the MICs of all macrolides tested were ≥256 μg/ml; all of these isolates had the constitutive macrolide-lincosamide-streptogramin B phenotype. Eighty-eight percent of the isolates were resistant to three or more classes of drugs. The ciprofloxacin MICs were ≥4 μg/ml for six (0.6%) isolates from five patients collected in 2000 and 2001, and the levofloxacin MICs were ≥8 μg/ml for five of these isolates. Seven isolates for which ciprofloxacin MICs were ≥4 μg/ml, including one isolate recovered in 1999, belonged to three serotypes (serotype 19F, five isolates; serotype 23A, one isolate; and serotype 23B, one isolate). The isolates from the six patients for which ciprofloxacin MICs were ≥4 μg/ml had different pulsed-field gel electrophoresis profiles and random amplified polymorphic DNA patterns, indicating that no clonal dissemination occurred over this time period. Despite the increased rate of fluoroquinolone use, the proportion of pneumococcal isolates for which ciprofloxacin MICs were elevated (≥4 μg/ml) remained low. However, the occurrence of telithromycin resistance is impressive and raises concerns for the future.

An increasing number of clinical isolations of rapidly growing mycobacteria (RGM) at the National Taiwan University Hospital were noted from 1992 to 2001. Broth microdilution MICs of 15 antimicrobial agents were determined for 200 clinical isolates of RGM, including the Mycobacterium fortuitum group (69 isolates), M. chelonae (39 isolates), and M. abscessus (92 isolates). Our results showed that the resistance rates of these isolates to the currently available agents were remarkably high. Amikacin was active against nearly all RGM isolates. Clarithromycin was usually active against M. abscessus (79% susceptibility) and the M. fortuitum group (65% susceptibility). The majority of M. fortuitum group isolates were susceptible to ciprofloxacin (62%) and imipenem (61%). The susceptibilities to other conventional anti-RGM agents of these isolates were poor but differed markedly by species. The newer fluoroquinolones (levofloxacin, moxifloxacin, and gatifloxacin) and meropenem showed better in vitro activities against the M. fortuitum group isolates than against the other two species of RGM. Linezolid had fairly good activity against these RGM isolates, particularly against M. chelonae isolates (82% susceptible). Telithromycin had poor activity against these RGM isolates (the MICs at which 50% of the isolates tested are inhibited [MIC 50 s] were 32 to 64 μg/ml, and the MIC 90 s were >64 μg/ml).

The in vitro susceptibilities of 266 isolates of Streptococcus agalactiae determined by the agar dilution method showed that 6% of isolates were nonsusceptible to penicillin and 46% was resistant to erythromycin. Of the erythromycin-resistant isolates, 86.3% had the macrolide-lincosamide-streptogramin (MLS) resistance phenotype (constitutive MLS, 85.5%; inducible MLS, 0.8%) and 13.7% had the M phenotype.

To understand quinupristin-dalfopristin resistance among clinical isolates of gram-positive bacteria in Taiwan, where this agent is not yet available for clinical use, we evaluated 1,287 nonduplicate isolates recovered from January 1996 to December 1999 for in vitro susceptibility to quinupristin-dalfopristin and other newer antimicrobial agents. All methicillin-susceptible Staphylococcus aureus (MSSA) isolates were susceptible to quinupristin-dalfopristin. High rates of nonsusceptibility to quinupristin-dalfopristin (MICs, ≥2 μg/ml) were demonstrated for the following organisms: methicillin-resistant S. aureus (MRSA) (31%), coagulase-negative staphylococci (CoNS) (16%), Streptococcus pneumoniae (8%), viridans group streptococci (51%), vancomycin-susceptible enterococci (85%), vancomycin-resistant Enterococcus faecalis (100%), vancomycin-resistant Enterococcus faecium (66%), Leuconostoc spp. (100%), Lactobacillus spp. (50%), and Pediococcus spp. (87%). All isolates of MSSA, MRSA, S. pneumoniae , and viridans group streptococci were susceptible to vancomycin and teicoplanin. The rates of nonsusceptibility to vancomycin and teicoplanin were 5 and 7%, respectively, for CoNS, ranging from 12 and 18% for S. simulans to 0 and 0% for S. cohnii and S. auricularis . Moxifloxacin and trovafloxacin had good activities against these isolates except for ciprofloxacin-resistant vancomycin-resistant enterococci and methicillin-resistant staphylococci. In Taiwan, virginiamycin has been used in animal husbandry for more than 20 years, which may contribute to the high rates of quinupristin-dalfopristin resistance.

We studied the antimicrobial activity of fosfomycin against 960 strains of commonly encountered bacteria associated with urinary tract infection using standard agar dilution and disk diffusion methods. Species studied included 3 common species of Enterobacteriaceae , Pseudomonas aeruginosa , Acinetobacter baumannii , and Stenotrophomonas maltophilia ; methicillin-susceptible and -resistant Staphylococcus aureus ; and vancomycin-susceptible and resistant Enterococcus faecalis and E. faecium . MICs and inhibition zone diameters were interpreted in accordance with both the currently recommended Clinical and Laboratory Standards Institute (CLSI) criteria for urinary tract isolates of Escherichia coli and Enterococcus faecalis and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria for Enterobacteriaceae . Tentative zone diameter interpretive criteria were developed for species not currently published by CLSI or EUCAST. Escherichia coli was uniformly susceptible to fosfomycin, as were most strains of Klebsiella pneumoniae and Enterobacter cloacae. A. baumannii was resistant to fosfomycin, while the prevalence of resistance in P. aeruginosa and S. maltophilia was greatly affected by the choice of MIC breakpoint. New tentative zone diameter criteria for K. pneumoniae , E. cloacae , S. aureus , and E. faecium were able to be set, providing some interim laboratory guidance for disk diffusion until further breakpoint evaluations are undertaken by CLSI and EUCAST.

A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible) isolates were examined for the presence of resistance determinants. Among 45 fusidic acid-resistant methicillin-resistant S. aureus (MRSA), isolates, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (the MIC was ≥128 μg/ml for 22/38), none had fusB , and 7 (16%) had fusC . For 26 fusidic acid-resistant methicillin-susceptible S. aureus (MSSA), only 3 possessed fusA mutations, but 15 (58%) had fusB and 8 (31%) had fusC . Low-level resistance to fusidic acid (MICs ≤ 32 μg/ml) was found in most fusB - or fusC -positive isolates. For 41 isolates (38 MRSA and 3 MSSA), with fusA mutations, a total of 21 amino acid substitutions in EF-G ( fusA gene) were detected, of which R76C, E444K, E444V, C473S, P478S, and M651I were identified for the first time. The nucleotide sequencing of fusB and flanking regions in an MSSA isolate revealed the structure of partial IS 257 - aj1 -LP- fusB - aj2 - aj3 -IS 257 -partial blaZ , which is identical to the corresponding region in pUB101, and the rest of fusB -carrying MSSA isolates also show similar structures. On the basis of spa and staphylococcal cassette chromosome mec element (SCC mec ) typing, two major genotypes, spa type t037-SCC mec type III (t037-III; 28/45; 62%) and t002-II (13/45; 29%), were predominant among 45 MRSA isolates. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. Taken together, the distribution of fusidic acid resistance determinants ( fusA mutations, fusB , and fusC ) was quite different between MRSA and MSSA groups.

Susceptibilities to 13 antimicrobial agents were determined by measurement of MICs for 60 isolates of Streptococcus bovis from blood cultures. Thirty-eight isolates (63.3%) had high-level resistance to erythromycin (MICs, ≥128 μg/ml). Among the 38 erythromycin-resistant strains, 21 isolates (55%) had inducible resistance to macrolides-lincosamides-streptogramin B (iMLS isolates) and 17 (45%) had constitutive resistance to macrolides-lincosamides-streptogramin B (cMLS isolates). Tetracycline resistance was also found among all of the erythromycin-resistant strains. None of the strains displayed resistance to penicillin, chloramphenicol, or vancomycin. Detection of erythromycin resistance genes by PCR and sequencing indicated that all 17 cMLS isolates were positive for the ermB gene and that 7 of 21 iMLS isolates carried the ermB gene and the remaining 14 iMLS isolates carried the ermT gene. Sequence analysis of amplified partial ermB fragments (594 bp) from S. bovis isolates revealed a 99.8% nucleotide identity and a 100% amino acid homology compared with the sequences from gene banks. The sequences of amplified fragments with primers targeted for ermC were shown to be very similar to that of ermGT ( ermT ) from Lactobacillus reuteri (98.5% nucleotide identity). This is the first report to describe the detection of the ermT class of erythromycin resistance determinants in S. bovis . The high rate of inducible erythromycin resistance among S. bovis isolates in Taiwan was not reported before. The iMLS S. bovis isolates were shown to be heterogeneous by randomly amplified polymorphic DNA analysis. These results indicate that the prevalence of inducible erythromycin resistance in S. bovis in Taiwan is very high and that most of the resistant strains carry the ermT or the ermB gene.