Hsiao-Jan Chen

Fusidic Acid Resistance Determinants in Staphylococcus aureus Clinical Isolates

ABSTRACT 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 IS257-aj1-LP-fusB-aj2-aj3-IS257-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 (SCCmec) typing, two major genotypes, spa type t037-SCCmec 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.

The erm(T) Gene Is Flanked by IS1216V in Inducible Erythromycin-Resistant Streptococcus gallolyticus subsp. pasteurianus

ABSTRACT 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 IS1216V-like elements with the same polarity and were found to be inserted in the chromosome.

PCR-RFLP assay for species and subspecies differentiation of the Streptococcus bovis group based on groESL sequences

The sequence diversity of groESL genes among Streptococcus bovis group isolates was analysed, including five reference strains and 36 clinical isolates. Phylogenetic analysis of the groES and groEL sequences showed that the isolates that belonged to the same species or subspecies usually clustered together. The intergenic spacer region between groES and groEL was variable in size (67-342 bp) and sequence and appeared to be a unique marker for species or subspecies determination. Sequence similarities of the groESL genes among species and subspecies ranged from 84.2 to 99.0 % in groES, and from 88.0 to 99.0 % in groEL. Based on the sequences determined, a Streptococcus bovis group-specific PCR assay was developed, which may provide an alternative means of distinguishing the bovis group from other viridans streptococci. Restriction digestion of the amplicon with AclI further differentiated the species and subspecies.

A Novel Staphylococcal Cassette Chromosomal Element, SCCfusC, Carrying fusC and speG in Fusidic Acid-Resistant Methicillin-Resistant Staphylococcus aureus

ABSTRACT 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, SCCfusC, which was integrated into rlmH and located upstream from SCCmec. The SCCfusC element contained speG, which may contribute to the polyamine resistance.

Use of groESL as a Target for Identification of Abiotrophia, Granulicatella, and Gemella Species

ABSTRACT We determined the groESL sequences of three species of nutritionally variant streptococci (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and three Gemella species (Gemella morbillorum, Gemella haemolysans, and Gemella sanguinis). The nucleotide sequence similarities between the groES and groEL genes of the above genera were 41.7 to 85.9% and 63.7 to 84.3%, respectively. The intraspecies similarities of groESL sequences for the isolates of Abiotrophia and Granulicatella species were 94.4 to 97.8% for groES and 94.0 to 98.2% for groEL. For Ge. morbillorum and Ge. sanguinis, all strains showed the same groESL spacer length (8 bp), and sequence identities within species were >97.8% for groES and >96.1% for groEL. However, higher intraspecies heterogeneity was observed in Ge. haemolysans. Phylogenetic analysis of groEL sequences separated the 6 isolates of Ge. haemolysans into two subgroups. Among these isolates, three isolates with the same groESL spacer region length (45 bp) clustered together but were distant from the ATCC reference strain (with a spacer length of 8 bp). The remaining three isolates, with a spacer length of 50 or 8 bp, clustered together. Although 16S rRNA gene sequence analysis did not provide enough discrimination for the 6 Ge. haemolysans isolates, rpoB gene sequence analysis supported the subgrouping. Based on the obtained groESL sequences, we developed a multiplex PCR that enables simple, rapid, and accurate identification of Abiotrophia, Granulicatella, and Gemella at the genus level. This assay would be helpful for identifying these fastidious and slow-growing organisms in clinical laboratories.

Distribution of emm Types and Genetic Characterization of the mgc Locus in Group G Streptococcus dysgalactiae subsp. equisimilis from a Hospital in Northern Taiwan

ABSTRACT A total of 274 Streptococcus dysgalactiae subsp. equisimilis isolates was analyzed by emm typing and by determining the organization of their mgrC loci. Three of the most frequent emm types were stG485.0 (45/274, 16.4%), stG6.1 (43/274, 15.7%), and stC839.0 (32/274, 11.7%), in decreasing order. The cpdB-positive mgrC locus appears to be predominant in some emm types.

Emergence of a small colony variant of vancomycin-intermediate Staphylococcus aureus in a patient with septic arthritis during long-term treatment with daptomycin

OBJECTIVES Small colony variants (SCVs) of Staphylococcus aureus are associated with persistent and drug-resistant infections. We demonstrated for the first time the emergence of SCVs in a patient with vancomycin-intermediate S. aureus (VISA) infection during long-term treatment with daptomycin. METHODS A 73-year-old man with septic arthritis was infected with VISA. The patient was treated with daptomycin; however, the patient remained infected with VISA, with continuous isolation of VISA from his blood during long-term treatment. Five VISA isolates were characterized by: PFGE; genotyping including staphylococcal cassette chromosome mec (SCCmec), spa and MLST; antimicrobial susceptibility testing; and scanning and transmission electron microscopy. WGS and fatty acid analysis were also performed. RESULTS The five VISA isolates were from a single clone of ST239/spa3(t037) and, of these, the first three were SCCmecIII positive and daptomycin susceptible, whereas the last two were SCCmecIII negative and daptomycin resistant and exhibited the characteristics of SCVs. The first and last isolates showed 13 remarkable genetic differences in SCCmec and the mprF, cls2, clpX and fabF genes. Of these, mutation of fabF (encoding the fatty acid synthase) seemed to be partially responsible for the slow growth and ultrastructural features, including an abnormal intercellular substance, and for the daptomycin resistance of SCVs. CONCLUSIONS For the first time, we identified SCVs of VISA in a patient with septic arthritis during long-term treatment with daptomycin. Daptomycin-resistant SCVs of VISA were evolved in a stepwise manner and the mutation of fabF is likely responsible for the physical and ultrastructural characteristics and daptomycin resistance.

Identification of fusB-Mediated Fusidic Acid Resistance Islands in Staphylococcus epidermidis Isolates

ABSTRACT 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 SeRIfusB-2793, SeRIfusB-704, SeRIfusB-5907, and SeRIfusB-7778, respectively. Among them, three islands (SeRIfusB-2793, SeRIfusB-704, and SeRIfusB-5907) were located downstream of groEL (corresponding to the 44-min position based on Staphylococcus aureus whole genomic sequences), and one (SeRIfusB-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.

New Structure of Phage-Related Islands Carrying fusB and a Virulence Gene in Fusidic Acid-Resistant Staphylococcus epidermidis

ABSTRACT 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 SeRIfusB-3692 and SePIfusB-857. The novel SePIfusB-857 structure was followed by SeCI857, forming a composite pathogenicity island which contained a putative virulence gene, vapE. The linkage of fusB and vapE may contribute to bacterial adaption.

Genotypes and phenotypes of Staphylococcus lugdunensis isolates recovered from bacteremia

BACKGROUND Staphylococcus lugdunensis is a member of coagulase-negative staphylococci, which has the potential to cause serious infections, such as endocarditis, bone and joint infections, and septicemia. Differences in phenotypic/genotypic characterization may be linked to different diseases. METHODS Genotypes of 11 S. lugdunensis isolates from bacteremia were determined by pulsed field gel electrophoresis and accessory gene regulator (agr) typing. The SCCmec elements in two oxacillin-resistant isolates were sequenced. Phenotypes were tested by antimicrobial susceptibility testing, biofilm formation assessments, and virulence factor analysis (hemolytic and protease activities). RESULTS Among the 11 isolates, six pulsotypes were found, and seven isolates belonged to two major pulsotypes. Two agr types (agr-1sl or agr-2sl) were found. The 11 isolates were susceptible to most antimicrobial agents tested. The SCCmec elements in two oxacillin-resistant isolates belonged to the SCCmec type V, but with additional ccrAB2 genes. The agr-2sl isolates (n = 7) displayed higher hemolytic and protease activities than the agr-1sl isolates. All isolates contained the icaA gene but with variable biofilm activities. The results suggest that protein might play an important part in S. lugdunensis biofilms, possibly through an ica-independent pathway. Of the 11 patients with S. lugdunensis bacteremia, one patient had a community-onset infection, and others had a hospital-acquired infection, which were mostly central venous catheter-related infections. CONCLUSION The 11 S. lugdunensis bacteremia isolates displayed various genotypes and phenotypes. Two oxacillin-resistant isolates contained SCCmec type V and carried additional ccrAB2 genes. Correlation of genotypes and phenotypes with infections needs further studies.