Chang-Phone Fung

A global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an important factor for complicated endophthalmitis?

Background and aims: Over the past two decades in Taiwan, pyogenic liver abscess has usually been caused by a single microorganism, Klebsiella pneumoniae, and is frequently associated with the serious complication of endophthalmitis, especially in diabetic patients. However, the relationship between the clinical presentation and bacterial factors remains unclear. The aim of this study was to investigate the clinical features of patients and the serotype and ribotype of K pneumoniae liver abscess. Methods: From July 1991 to June 1998, a total of 134 cases of K pneumoniae liver abscess with 248 K pneumoniae isolates from the same patients were collected from two large medical centres in northern Taiwan. Clinical data were collected from medical records. Serotyping and ribotyping were performed using the countercurrent immunoelectrophoresis method and automated Riboprinter. Results: Serotyping revealed that the most common serotypes were K1 (63.4%) and K2 (14.2%). K1 isolates occurred at a significantly higher frequency (p<0.01) than all other serotypes. Among 134 patients, 105 (78.4%) had suffered from diabetes mellitus for 3–15 years. Fourteen patients (10.4%) had metastatic infection to the eye causing septic endophthalmitis. Liver aspirates, and blood and vitreous pus cultures yielded the same serotype of K pneumoniae in all patients. Among patients with septic endophthalmitis, 92.3% (13/14) were diabetic, and 85.7% (12/14) of the isolates belonged to serotype K1. For molecular typing, different degrees of genetic polymorphism among isolates with the same K1 serotype suggested no particular prevalence of any one strain in K pneumoniae liver abscess. Conclusion: K pneumoniae serotype K1 was significantly associated with liver abscess and the complication of endophthalmitis, especially in diabetic patients. Physicians should request an immediate report of serotyping and susceptibility test results simultaneously if a diagnosis of pyogenic liver abscess has been made so that early and appropriate management for possible complications will not be delayed. The use of ceftriaxone because of its higher concentration in the aqueous humor is suggested to decrease the chance of septic endophthalmitis.

Klebsiella pneumoniae liver abscess: a new invasive syndrome.

Klebsiella pneumoniae is a well known human nosocomial pathogen. Most community-acquired K pneumoniae infections cause pneumonia or urinary tract infections. During the past two decades, however, a distinct invasive syndrome that causes liver abscesses has been increasingly reported in Asia, and this syndrome is emerging as a global disease. In this Review, we summarise the clinical presentation and management as well the microbiological aspects of this invasive disease. Diabetes mellitus and two specific capsular types in the bacterium predispose a patient to the development of liver abscesses and the following metastatic complications: bacteraemia, meningitis, endophthalmitis, and necrotising fasciitis. For patients with this invasive syndrome, appropriate antimicrobial treatment combined with percutaneous drainage of liver abscesses increases their chances of survival. Rapid detection of the hypervirulent strain that causes this syndrome allows earlier diagnosis and treatment, thus minimising the occurrence of sequelae and improving clinical outcomes.

Association between rmpA and magA Genes and Clinical Syndromes Caused by Klebsiella pneumoniae in Taiwan

BACKGROUND The association of the magA gene with the hypermucoviscosity phenotype relevant to the pathogenesis of Klebsiella pneumoniae liver abscess has been reported in Taiwan. Similarly, the rmpA gene, known as a positive regulator of extracapsular polysaccharide synthesis that confers a mucoid phenotype, may be another candidate gene causing hypermucoviscosity. However, the association of rmpA with K. pneumoniae clinical syndromes is unreported. We aimed to investigate the clinical correlation between rmpA and primary Klebsiella abscess, focusing on sites other than the liver. METHODS From July 2003 through December 2004, a total of 151 K. pneumoniae isolates recovered from 151 patients with bacteremia were collected from 2 large medical centers in southern Taiwan. Clinical data were collected from medical records. The genes rmpA and magA were amplified by polymerase chain reaction using specific primers. RESULTS The prevalences of hypermucoviscosity, rmpA, and magA were 38%, 48%, and 17%, respectively. As determined by statistical multivariate analysis, strains carrying rmpA were significantly associated with the hypermucoviscosity phenotype, and there was a significant correlation with purulent tissue infections, such as liver abscess and lung, neck, psoas muscle, or other focal abscess. CONCLUSION Our data support a statistical correlation between the rmpA gene and virulence in terms of abscess formation for these hypermucoviscous K. pneumoniae strains. Hypermucoviscosity associated with rmpA, together with a thorough physical examination, may be helpful as a guide to carry out appropriate diagnostic tests on patients with an initially unknown source of K. pneumoniae bacteremia, particularly when looking for the occurrence of an underlying abscess.

Capsular Serotype K1 or K2, Rather than magA and rmpA, Is a Major Virulence Determinant for Klebsiella pneumoniae Liver Abscess in Singapore and Taiwan

ABSTRACT Capsular serotypes, magA, and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

Klebsiella pneumoniae Outer Membrane Porins OmpK35 and OmpK36 Play Roles in both Antimicrobial Resistance and Virulence

ABSTRACT OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (ΔompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (ΔompK35) had no significant effect. A double deletion of ompK35 and ompK36 (ΔompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ΔompK35 mutation resulted in a significant (≥4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ΔompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ΔompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ΔompK36 and ΔompK35/36 mutants. In a mouse peritonitis model, the ΔompK35 mutant showed no change in virulence, and the ΔompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ΔompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

Molecular Typing and Virulence Analysis of Serotype K1 Klebsiella pneumoniae Strains Isolated from Liver Abscess Patients and Stool Samples from Noninfectious Subjects in Hong Kong, Singapore, and Taiwan

ABSTRACT Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <102 CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.

Emergence and Distribution of Plasmids Bearing the blaOXA-51-Like Gene with an Upstream ISAba1 in Carbapenem-Resistant Acinetobacter baumannii Isolates in Taiwan

ABSTRACT The blaOXA-51-like gene with an upstream ISAba1 (ISAba1-blaOXA-51-like gene) was originally found on the chromosomes of carbapenem-resistant or -susceptible Acinetobacter baumannii isolates. However, a plasmid-borne ISAba1-blaOXA-51-like gene has recently been identified in Acinetobacter genomic species 13TU and several A. baumannii isolates in Taiwan, and all of the isolates are carbapenem resistant. This study aimed to characterize the plasmids bearing the ISAba1-blaOXA-51-like gene and their significance in A. baumannii. Among the 117 ISAba1-blaOXA-51-like-harboring isolates collected from 10 hospitals in Taiwan, 58 isolates (49.6%) from 24 clones had the genes located on plasmids that likely originated from a common progenitor. Among the 58 isolates, four had additional copy of the ISAba1-blaOXA-51-like gene on their chromosomes. Based on the analysis of these four isolates, the plasmid-located ISAba1-blaOXA-51-like gene appeared to be acquired via one-ended transposition (Tn6080). The isolates with a plasmid bearing the ISAba1-blaOXA-51-like gene had higher rates of resistance to imipenem (98% versus 46.6%; P < 0.001) and meropenem (98% versus 69%; P = 0.019) than those with the genes chromosomally encoded, which is most likely due to increased gene dosage provided by the higher copy number of associated plasmids. Transformation with a recombinant plasmid harboring only the ISAba1-blaOXA-51-like gene was enough to confer a high level of carbapenem resistance to A. baumannii, eliminating the possible contribution of other factors on the original plasmids. This study demonstrated that the carbapenem resistance-associated plasmids carrying the ISAba1-blaOXA-51-like gene are widespread in A. baumannii strains in Taiwan.

Clinical correlates of antifungal macrodilution susceptibility test results for non-AIDS patients with severe Candida infections treated with fluconazole.

ABSTRACT Although the clinical correlates of the reference antifungal susceptibility test results in hematogenous and deep-seatedCandida infection are still controversial, we evaluated the clinical correlates of this test in deep-seatedCandida infections in non-AIDS patients. Thirty-two non-AIDS patients with hematogenous or deep-seated Candidainfections were treated with intravenous fluconazole (400 mg a day), and the clinical outcomes were evaluated. Coexisting bacterial infections were treated with appropriate antibiotics, superinfection or reinfection was excluded, inadequate fluconazole therapy was avoided, and essential surgical intervention was performed. The MICs of fluconazole for these 32 Candida isolates were determined according to the M27-A procedure approved by the National Committee on Clinical Laboratory Standards. MICs were interpreted as susceptible (≤8 μg/ml), dose-dependent susceptible (16 to 32 μg/ml), and resistant (≥64 μg/ml) according to the criteria of the M27-A standard. The success rates were 79% (19 of 24; 95% confidence interval [CI], 59 to 93%) in the susceptible category, 66% (4 of 6; 95% CI, 19 to 95%) in the dose-dependent susceptible category, and 0% (0 of 2; 95% CI, 0 to 84%) in the resistant category. We conclude that the clinical correlation of the reference antifungal susceptibility test results is high in hematogenous and deep-seatedCandida infections.

A 5-Year Study of the Seroepidemiology of Klebsiella pneumoniae: High Prevalence of Capsular Serotype K1 in Taiwan and Implication for Vaccine Efficacy

Seroepidemiology of Klebsiella pneumoniae was determined for 1000 nonrepetitive K. pneumoniae isolates collected by a medical center in Taiwan during 1993-1997. Of these, 630 isolates (63%) were from community-acquired infections; the rest were from hospital-acquired infections. The isolates were serotyped according to capsular antigen by countercurrent immunoelectrophoresis. About 77% were typeable. Serotypes K1 and K2 accounted for 21.7% and 9.3% of the isolates, respectively, followed by K57 (5.1%), K54 (4.2%), K21 (3. 3%), and K16 (3%). The frequency of serotype K1 among bacteremic isolates (30.8%) far exceeded that reported by other investigators worldwide. Molecular typing of random K1 isolates by pulsed-field gel electrophoresis revealed several different pulsotypes, suggesting a nonclonal spread. This study indicates that a Klebsiella vaccine developed in Europe is not optimal for use in Taiwan because it does not contain the most predominant serotypes-K1, K54, and K57.

An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii

ABSTRACT We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, blaOXA-51-like genes were found to exist in all 23 clinical strains; however, the transcript levels of blaOXA-51-like in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of blaOXA-51-like in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of blaOXA-66 (a blaOXA-51-like gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the blaOXA-66/blaOXA-51-like gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.