A 20-Year Study of Capsular Polysaccharide Seroepidemiology, Susceptibility Profiles, and Virulence Determinants of Klebsiella pneumoniae from Bacteremia Patients in Taiwan.

In this study, we selected bacteremic Klebsiella pneumoniae isolates from the Taiwan Surveillance of Antimicrobial Resistance program. A total of 521 isolates were collected over a period of 2 decades, including 121 from 1998, 197 from 2008, and 203 from 2018. Seroepidemiology showed that the top five capsular polysaccharide types were serotypes K1, K2, K20, K54, and K62, constituting 48.5% of the total isolates, and the respective ratios at each time point have remained similar over the past 2 decades. The antibacterial susceptibility tests showed that K1, K2, K20, and K54 were susceptible to most antibiotics, while K62 was relatively resistant compared to other typeable and nontypeable strains. In addition, six virulence-associated genes, clbA, entB, iroN, rmpA, iutA, and iucA, were predominant in K1 and K2 isolates of K. pneumoniae. In conclusion, serotypes K1, K2, K20, K54, and K62 of K. pneumoniae are the most prevalent serotypes and carry more virulence determinants in bacteremia patients, which may indicate their invasiveness. If further serotype-specific vaccine development is performed, these five serotypes should be included. Since the antibiotic susceptibility profiles were stable over a long duration, empirical treatment may be predicted according to serotype if rapid diagnosis from direct clinical specimens is available, such as PCR or antigen serotyping for serotype K1 and K2. IMPORTANCE This is the first nationwide study to examine the seroepidemiology of Klebsiella pneumoniae using blood culture isolates collected over a period of 20 years. The study found that the prevalence of serotypes remained consistent over the 20-year period, with high-prevalence serotypes associated with invasive types. Nontypeable isolates had fewer virulence determinants than other serotypes. With the exception of serotype K62, the other high-prevalence serotypes were highly susceptible to antibiotics. If rapid diagnosis using direct clinical specimens, such as PCR or antigen serotyping, is available, empirical treatment can be predicted based on serotype, particularly for K1 and K2. The results of this seroepidemiology study could also help the development of future capsule polysaccharide vaccines.

Cross-protection induced by highly conserved outer membrane proteins (Omps) in mice immunized with OmpC of Salmonella Typhi or OmpK36 of Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Outer membrane proteins (Omps) are a family of proteins that are highly conserved throughout the evolution of Enterobacteriaceae. Previous studies using sequence comparisons have found a high degree of sequence homology between OmpK36 of Klebsiella pneumoniae and OmpC of Salmonella enterica serovar Typhi. Whether highly conserved OmpC can be directly extrapolated as a common vaccine candidate against K. pneumoniae or other Enterobacteriaceae remains to be verified. METHODS: OmpK36 and OmpC were purified and used to immunize BALB/c mice. After immunization, five mice from each group were injected intraperitoneally with a cell suspension of K. pneumoniae or S. Typhi, and the mice were monitored daily for 14 days to measure the severity of illness and assess their survival. RESULTS: Cross-reacting OmpK36 and OmpC antibodies were identified in the mice immunized with OmpK36 or OmpC. No cross-protection was observed in the mice immunized with OmpC in the presence of K. pneumoniae infection. CONCLUSION: Although a high degree of similarity was observed for the amino acid sequences between OmpK36 and OmpC, our results suggested that no cross-protection occurred in the mice challenged with other species.

Rapid identification of capsular serotype K1/K2 Klebsiella pneumoniae in pus samples from liver abscess patients and positive blood culture samples from bacteremia cases via an immunochromatographic strip assay.

BACKGROUND: In Asia, serotype K1/K2 Klebsiella pneumoniae are the major capsular serotypes that cause liver abscess or bacteremia in patients. The purpose of this study was to compare novel immunochromatographic strips (ICSs), which can rapidly detect K. pneumoniae serotypes K1/K2 in clinical samples, to conventional capsular serotyping methods. METHODS: Pus drainage samples from 16 patients with a liver abscess caused by K. pneumoniae, blood samples from 112 positive flagged blood culture bottle and a subsequent single colony in the medium were tested with the ICS. The results were then compared to findings of capsular swelling tests. Samples subjected to the polymerase chain reaction (PCR) analysis were used as reference. RESULTS: The identification of K. pneumoniae via the traditional bacterial culture from pus samples took 3.4 days on average (ranging from 2.2 to 5.5 days). Further capsular serotyping of K. pneumoniae by the capsular swelling test of pure isolates lasted 5-10 min, and the PCR method took ~ 4 h. As for ICSs, the time for direct identification of the K. pneumoniae capsular serotype K1/K2 in pus was < 4 min (ranging from 2 to 4 min). The results of ICSs were consistent with capsular swelling tests and PCR methods. Testing of 112 blood culture samples and subsequent single colonies in the medium with ICSs yielded consistent results for most samples. CONCLUSIONS: This study indicates that ICSs can rapidly detect K. pneumoniae serotypes K1 and K2 in pus or positive flagged blood culture broth samples within 5 min. Their accuracy is comparable to that of the conventional capsular serotyping methods such as a serum agglutination assay or PCR.

Important Mutations Contributing to High-Level Penicillin Resistance in Taiwan19F-14, Taiwan23F-15, and Spain23F-1 of Streptococcus pneumoniae Isolated from Taiwan.

Penicillin-resistant Streptococcus pneumoniae is a serious concern worldwide. In this study, we analyzed the cause of ß-lactam resistance in pandemic multidrug-resistant clones. A total of 41 penicillin-nonsusceptible clinical isolates were collected from 1996 to 2012. Sero- and molecular typing confirmed that these isolates were clonal types of Taiwan19F-14, Taiwan23F-15, and Spain23F-1. Sero-switching was found in four isolates. All isolates were multidrug resistant. Sequencing analysis of the penicillin binding proteins (PBPs) was performed on PBP1a, 2b, and 2x, and a large number of mutations were identified in comparing to clinical penicillin-susceptible isolates and the recipient strain R6 used for homologous recombination. The T451A substitution was the key amino acid in PBP2b that contributed to penicillin resistance. T338A in PBP2x played a role in resistance and reached the highest level of resistance when combined with other mutations in PBP2x. High-level penicillin resistance could not be obtained without the combination of mutations in PBP1a with PBP2b and 2x. The amino acid substitutions in PBP1a, 2b, and 2x were the crucial factors for ß-lactam resistance.

Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae.

BACKGROUND: Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are common techniques for studying gene function, and have been applied to pneumolysin (ply), a virulence gene in Streptococcus pneumoniae (D39). Discrepancies in virulence between the two techniques were observed in both the previous and present studies. This phenomenon was also observed during mutation analysis of autolysin (lytA). RESULTS: Our data showed that target gene restoration (TGR) occurred in IDM mutants, even in the presence of antibiotics, while the IFD mutants were stable. In PCR result, TGR occurred later in IDM-ply and -lytA mutants cultured in non-supplemented medium (4-5 h) compared with those grown in medium supplemented with erythromycin (erm)/chloramphenicol (cat) (3-4 h), but plateaued faster. Real-time PCR for detecting TGR had been performed. When compared with 8-h culture, TGR detection increased from Day 1 and Day 2 of IDM mutant's culture. erm-sensitive clones from IDM mutant were found. Southern blot hybridization and Western blotting also confirmed the phenomenon of TGR. The median survival of mice following intraperitoneal (IP) injection with a 3-h culture of IDM-mutants was significantly longer than that with an 8-h culture, irrespective of antibiotic usage. The median survival time of mice following IP injection of a 3-h culture versus an 8-h culture of IDM-ply in the absence of antibiotics was 10 days versus 2 days (p = 0.031), respectively, while in the presence of erm, the median survival was 5 days versus 2.5 days (p = 0.037), respectively. For an IDM-lytA mutant, the corresponding values were 8.5 days versus 2 days (p = 0.019), respectively, for non-supplemented medium, and 2.5 versus 2 days (p = 0.021), respectively, in the presence of cat. A comparable survival rate was observed between WT D39 and an 8-h IDM culture. CONCLUSION: TGR in IDM mutants should be monitored to avoid inconsistent results, and misinterpretation of data due to TGR could lead to important biological meaning being overlooked. Therefore, based on these results, IFD is preferable to IDM for disruption of target genes.