Staphylococcus aureus is one of most common pathogens in humans. Methicillin-resistant S. aureus (MRSA) accounts for 64 % of S. aureus bacteremia isolated in intensive care units (ICUs), and heteroresistant vancomycin-intermediates S. aureus (hVISA) is a phenotype of MRSA. However, studies focusing on the hVISA impact on critically ill patients are scarce. This was a retrospective study conducted in a tertiary medical center from January 2009 to December 2010. All adult patients in ICUs with MRSA bloodstream infection were eligible. A modified population analysis profile and area under the curve method was applied to all isolates to confirm hVISA phenotype. Multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and the accessory gene regulator (agr) typing were performed individually. Clinical outcomes including in-hospital mortality, length of stay in intensive care unit and hospital after MRSA bacteremia of the patients were also analyzed. A total of 48 patients were enrolled and 14 patients were confirmed to have the hVISA phenotype. The prevalence of hVISA was 29.2 %. There was no difference in the age, sex, comorbidity, Charlson’s comorbidity score and previous vancomycin therapy between the hVISA and VSSA groups. The hVISA group had a significantly higher in-hospital mortality than the VSSA group (13/14 versus 22/34; p = 0.046). All of the 14 hVISA patients had an MIC = 2 mg/L by E-test and this represented a significant association between high MIC and the development of hVISA (p < 0.001). MLST analysis showed all the isolates in the hVISA group were ST239, while ST239 (14/34; 41.2 %) and ST5 (12/34; 35.3 %) were predominant in the VSSA group (p = 0.007). A comparison of the survivor and non-survivor group showed that the hVISA phenotype (OR 11.8; 95 % CI 1.1–126.99; p = 0.042) and sequential organ failure assessment (SOFA) score (OR 1.39; 95 % CI 1.07–1.81; p = 0.014) were independent factors significantly associated with in-hospital mortality. Patients in ICUs with MRSA bacteremia may have a higher in-hospital mortality if they have the hVISA phenotype. SOFA score is also predictor of mortality.

Prolonged vancomycin usage may cause methicillin-resistant Staphylococcus aureus to become vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). Mechanisms of vancomycin resistance of VISA and hVISA are still unclear. In this study, analyses of nucleotide sequence variations in 30 vancomycin-sensitive S. aureus (VSSA), 41 hVISA and 16 VISA isolates revealed 29 single-nucleotide variations in 12 genes (fmtC, graR, graS, htrA, mecA, pbp2, pbp4, srtA, tcaA, upps, vicK and vraR) that are related to cell wall synthesis or the two-component system. Six of these 29 single-nucleotide variations were novel and resulted in the following amino acid changes: Q692E in FmtC; T278I, P306L and I311T in HtrA; and I63V and K101E in Upps. Since P306L and I311T in HtrA and I63V in Upps were present in the majority (76.7%–86.7%) of VSSA isolates, these three amino acid variations may not be associated with vancomycin resistance. The other three amino acid variations (T278I in HtrA, K101E in Upps and Q692E in FmtC) were present in the majority (87.5%–93.8%) of hVISA and VISA isolates, but only in a small number (22.9%–25.7%) of VSSA isolates, suggesting that they are associated with vancomycin resistance.

Background: Antibiotic-resistant type III/ST-17 Streptococcus agalactiae (group B Streptococcus, GBS) strain is predominant in neonatal invasive GBS diseases. We aimed to investigate the antibiotic resistance profiles and genetic characteristics of type III/ST-17 GBS strains. Methods: A total of 681 non-duplicate GBS isolates were typed (MLST, capsular types) and their antibiotic resistances were performed. Several molecular methods (WGS, PCR, sequencing and sequence analysis) were used to determine the genetic context of antibiotic resistant genes and pili genes. Results: The antibiotic resistant rates were significantly higher in type Ib (90.1%) and type III (71.1%) GBS isolates. WGS revealed that the loss of PI-1 genes and absence of ISSag5 was found in antibiotic-resistant III/ST-17 GBS isolates, which is replaced by a ~75-kb integrative and conjugative element, ICESag37, comprising multiple antibiotic resistance and virulence genes. Among 190 serotype III GBS isolates, the most common pilus island was PI-2b (58.4%) alone, which was found in 81.3% of the III/ST-17 GBS isolates. Loss of PI-1 and ISSag5 was significantly associated with antibiotic resistance (95.5% vs. 27.8%, p < 0.001). The presence of ICESag37 was found in 83.6% of all III/ST-17 GBS isolates and 99.1% (105/106) of the antibiotic-resistant III/ST-17 GBS isolates. Conclusions: Loss of PI-1 and ISSag5, which is replaced by ICESag37 carrying multiple antibiotic resistance genes, accounts for the high antibiotic resistance rate in III/ST-17 GBS isolates. The emerging clonal expansion of this hypervirulent strain with antibiotic resistance after acquisition of ICESag37 highlights the urgent need for continuous surveillance of GBS infections.

Introduction: Staphylococcus haemolyticus is an acquired opportunistic pathogen causing nosocomial infections. Our previous studies of S. haemolyticus showed a group of isolates that produced a significantly higher disease severity than the others. Further molecular typing showed that the sequence type (ST) 42 was the major clone among the isolates. The main aim of this study was to characterize ST42. Materials and Methods: Sixty-one and 36 isolates were collected from burn and nonburn patients, respectively. Molecular typing, antibiotic susceptibility assays, and phenotypic characterizations were performed. Results: Thirteen STs, including seven new STs, were established (ST42 to ST48). ST42 was prevalent in burn and nonburn patients, and all the pulsotype C isolates were ST42. Four of the novel STs originated from ST3, suggesting that these clonal lineages evolved locally. ST3 and ST42 showed a significant difference in clindamycin susceptibility; molecular typing showed only one MLST locus variation among seven loci in SH1431 , which has been reportedly involved in the regulation of biofilm formation through Zn ²⁺ binding affinities. Conclusions: Seven novel S. haemolyticus STs were identified; phylogenetic analysis suggested the presence of locally evolved clonal lineages. The predominant ST42 showed weak biofilm formation abilities; other factors that cause the clonal lineage change still need further investigation.

Group B Streptococcus (GBS) is an important pathogen of neonatal infections, and the clonal complex (CC)-17/serotype III GBS strain has emerged as the dominant strain. The clinical manifestations of CC17/III GBS sepsis may vary greatly but have not been well-investigated. A total of 103 CC17/III GBS isolates that caused neonatal invasive diseases were studied using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) loci and restriction fragment length polymorphism (RFLP) analyses. All spacers of CRISPR loci were sequenced and analyzed with the clinical presentations. After CRISPR-RFLP analyses, a total of 11 different patterns were observed among the 103 CRISPR-positive GBS isolates. GBS isolates with the same RFLP patterns were found to have highly comparable spacer contents. Comparative sequence analysis of the CRISPR1 spacer content revealed that it is highly diverse and consistent with the dynamics of this system. A total of 29 of 43 (67.4%) spacers displayed homology to reported phage and plasmid DNA sequences. In addition, all CC17/III GBS isolates could be categorized into three subgroups based on the CRISPR-RFLP patterns and eBURST analysis. The CC17/III GBS isolates with a specific CRISPR-RFLP pattern were more significantly associated with occurrences of severe sepsis (57.1% vs. 29.3%, p = 0.012) and meningitis (50.0% vs. 20.8%, p = 0.009) than GBS isolates with RFLP lengths between 1000 and 1300 bp. Whole-genome sequencing was also performed to verify the differences between CC17/III GBS isolates with different CRISPR-RFLP patterns. We concluded that the CRISPR-RFLP analysis is potentially applicable to categorizing CC17/III GBS isolates, and a specific CRISPR-RFLP pattern could be used as a new biomarker to predict meningitis and illness severity after further verification.

Multi-drug resistant Staphylococcus haemolyticus is a frequent nosocomial invasive bacteremia pathogen in hospitals. Our previous analysis showed one of the predominant strains, ST42 originated from ST3, had only one multilocus sequence typing (MLST) variation among seven loci in SH1431; yet no significant differences in biofilm formation observed between ST42 and ST3, suggesting that other factors influence clonal lineage change. Whole genome sequencing was conducted on two isolates from ST42 and ST3 to find phenotypic and genotypic variations, and these variations were further validated in 140 clinical isolates. The fusidic acid- and tetracycline-resistant genes (fusB and tetK) were found only in CGMH-SH51 (ST42). Further investigation revealed consistent resistant genotypes in all isolates, with 46% and 70% of ST42 containing fusB and tetK, respectively. In contrast, only 23% and 4.2% ST3 contained these two genes, respectively. The phenotypic analysis also showed that ST42 isolates were highly resistant to fusidic acid (47%) and tetracycline (70%), compared with ST3 (23% and 4%, respectively). Along with drug-resistant genes, three capsule-related genes were found in higher percentage distributions in ST42 than in ST3 isolates. Our findings indicate that ST42 could become endemic in Taiwan, further constitutive surveillance is required to prevent the spread of this bacterium.