Induction of alloimmune tolerance in heart transplantation through gene silencing of TLR adaptors.

Toll-like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4(+) CD25(+) FoxP3(+) regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.

Targeted gene silencing of TLR4 using liposomal nanoparticles for preventing liver ischemia reperfusion injury.

RNAi-based therapy is a promising strategy for the prevention of ischemia-reperfusion injury (IRI). However, systemic administration of small interfering RNA (siRNA) may cause globally nonspecific targeting of all tissues, which impedes clinical use. Here we report a hepatocyte-specific delivery system for the treatment of liver IRI, using galactose-conjugated liposome nanoparticles (Gal-LipoNP). Heptocyte-specific targeting was validated by selective in vivo delivery as observed by increased Gal-LipoNP accumulation and gene silencing in the liver. Gal-LipoNP TLR4 siRNA treatment resulted in a significant decrease of serum alanine transferase (ALT) and aspartate transaminase (AST) in a hepatic IRI model. Histopathology displayed an overall reduction of the injury area in the Gal-LipoNP TLR4 siRNA treated mice. Additionally, neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by MPO, MDA and ROS respectively, were attenuated after Gal-LipoNP TLR4 siRNA treatment. Moreover, therapeutic effects of Gal-LipoNP TLR4 siRNA were associated with suppression of the inflammatory cytokines IL-1 and TNF-α. Taken together, this study is the first demonstration of liver IRI treatment using liver-specific siRNA delivery.

Molecular characterization of beta-lactamase genes and their genetic structures in Acinetobacter genospecies 3 isolates in Taiwan.

The genetic structure of beta-lactamases in Acinetobacter genospecies 3 (AG3) isolates in Taiwan was studied to analyze their high rates of resistance to beta-lactams, including carbapenems (57.9%). bla(IMP-1) and bla(IMP-8) were located in a class 1 integron. bla(OXA-58) was bracketed by ISAba3. A novel TnpF-like integrase gene was identified upstream of bla(VEB-3). Adjacent to the 5' sequence of the bla(ADC) gene, folE was identified. Four new Acinetobacter-derived cephalosporinase (ADC) enzymes were found, which clustered phylogenetically with published AG3 ADC proteins.

Contribution of outer membrane protein K36 to antimicrobial resistance and virulence in Klebsiella pneumoniae.

OBJECTIVES: Loss of outer membrane protein (Omp) is commonly encountered in multidrug-resistant Klebsiella pneumoniae. However, little is known about the association between Omp loss and virulence. In the present study, this association was investigated in K. pneumoniae. METHODS: An OmpK36-deficient mutant (DeltaOmpK36) was derived from a virulent clinical isolate by targeted gene insertion. Antimicrobial susceptibility was tested by microbroth dilution and disc diffusion. Virulence was assessed by serum resistance, phagocytosis, clearance of viable bacteria in the liver and lethality in mice following inoculation with bacteria. RESULTS: Susceptibility tests showed that DeltaOmpK36 contributed to the resistance to cefazolin and cefoxitin but not to resistance to late-generation cephalosporins. In vitro assays demonstrated that loss of OmpK36 decreased the resistance to neutrophil phagocytosis and increased the resistance to serum killing during the first hour of the assay, but did not influence the growth rate when compared with the parental strain. Intraperitoneal injection of similar doses ( approximately 4 x 10(4) cfu) of the parental strain and DeltaOmpK36 led to significantly fewer viable bacteria in the liver 24 h post-inoculation in DeltaOmpK36-inoculated mice. In the mice LD(50) (the bacterial dose that caused 50% death) assay, the parental strain was approximately 100-fold more lethal ( approximately 10(3) cfu) than the DeltaOmpK36 mutant ( approximately 10(5) cfu). CONCLUSIONS: Loss of OmpK36 in K. pneumoniae resulted in increased antimicrobial resistance, increased susceptibility to neutrophil phagocytosis, increased resistance to serum killing and reduced virulence.

Clinical and microbiological characteristics of community-acquired thoracic empyema or complicated parapneumonic effusion caused by Klebsiella pneumoniae in Taiwan.

Klebsiella pneumoniae is the major cause of community-acquired pyogenic infections in Taiwan and is becoming an increasing problem in acute thoracic empyema. This study evaluated the clinical and microbiological characteristics of community-acquired thoracic empyema or complicated parapneumonic effusion caused by K. pneumoniae in Taiwanese adults treated during the period 2001-2008 at a tertiary medical center. All clinical isolates were examined for capsular serotypes K1/K2, and pulsed-field gel electrophoresis (PFGE) was performed on strains of the same serotype. K. pneumoniae was the most frequent cause of community-acquired thoracic empyema or complicated parapneumonic effusion. It was associated with high mortality (32.4%) and was an independent risk factor for fatal outcome. Diabetes mellitus, liver cirrhosis, and bronchogenic carcinoma were independent risk factors for K. pneumoniae infection. Serotypes K1 (9/37, 24.3%) and K2 (13/37, 35.1%) were the prevalent strains but did not predispose patients to poor outcome compared with other non-K1/K2 serotypes. There was no major cluster of isolates found among serotype K1/K2 strains. In summary, physicians should be aware of the risk factors for thoracic empyema or complicated parapneumonic effusion caused by K. pneumoniae and the associated high mortality, and monitor these patients more closely.

Widespread dissemination of aminoglycoside resistance genes armA and rmtB in Klebsiella pneumoniae isolates in Taiwan producing CTX-M-type extended-spectrum beta-lactamases.

Among 235 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL) isolates collected from a nationwide surveillance performed in Taiwan, 102 (43.4%) were resistant to amikacin. Ninety-two of these 102 (90.2%) isolates were carrying CTX-M-type beta-lactamases individually or concomitantly with SHV-type or CMY-2 beta-lactamases. The armA and rmtB alleles were individually detected in 44 and 37 of these 92 isolates, respectively. One isolate contained both armA and rmtB. The coexistence of the aac(6')-Il and rmtB genes was detected in three isolates. CTX-M-type beta-lactamase genes belonging to either group 1 (CTX-M-3 and CTX-M-15) or group 9 (CTX-M-14) were found in all armA- or rmtB-bearing ESBL-producing K. pneumoniae isolates, and all were conjugatively transferable. All except one of the isolates bearing armA produced CTX-M enzymes of group 1, and the remaining isolate bearing armA produced a group 9 CTX-M-type beta-lactamase. On the contrary, in the majority of rmtB carriers, the CTX-M-type beta-lactamase belonged to group 9 (62.2%). Molecular typing revealed that the amikacin-resistant ESBL-producing K. pneumoniae isolates were epidemiologically unrelated, indicating that the acquisition of resistance was not through the spread of a resistant clone or a resistance plasmid. A tandem repeat or multiple copies of bla(CTX-M-3) were found in some armA-bearing isolates. An ISEcp1 insert was found in all CTX-M ESBL-producing K. pneumoniae isolates carrying armA or rmtB. In conclusion, the concomitant presence of a 16S rRNA methylase gene (armA or rmtB) and bla(CTX-M) among amikacin-resistant ESBL-producing K. pneumoniae isolates is widespread in Taiwan.

Recurrent Klebsiella pneumoniae liver abscess: clinical and microbiological characteristics.

Recurrent Klebsiella pneumoniae liver abscesses (KLAs) are rarely reported. Six cases of recurrent KLAs are characterized. Most of the patients had diabetes and K1 serotype KLAs. All of the isolates were uniformly susceptible to cefazolin. Distinct molecular fingerprints were found for the strains isolated from both primary and recurrent KLAs.

Methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii on computer interface surfaces of hospital wards and association with clinical isolates.

BACKGROUND: Computer keyboards and mice are potential reservoirs of nosocomial pathogens, but routine disinfection for non-water-proof computer devices is a problem. With better hand hygiene compliance of health-care workers (HCWs), the impact of these potential sources of contamination on clinical infection needs to be clarified. METHODS: This study was conducted in a 1600-bed medical center of southern Taiwan with 47 wards and 282 computers. With education and monitoring program of hand hygiene for HCWs, the average compliance rate was 74% before our surveillance. We investigated the association of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Acinetobacter baumannii, three leading hospital-acquired pathogens, from ward computer keyboards, mice and from clinical isolates in non-outbreak period by pulsed field gel electrophoresis and antibiogram. RESULTS: Our results revealed a 17.4% (49/282) contamination rate of these computer devices by S. aureus, Acinetobacter spp. or Pseudomonas spp. The contamination rates of MRSA and A. baumannii in the ward computers were 1.1% and 4.3%, respectively. No P. aeruginosa was isolated. All isolates from computers and clinical specimens at the same ward showed different pulsotypes. However, A. baumannii isolates on two ward computers had the same pulsotype. CONCLUSION: With good hand hygiene compliance, we found relatively low contamination rates of MRSA, P. aeruginosa and A. baumannii on ward computer interface, and without further contribution to nosocomial infection. Our results suggested no necessity of routine culture surveillance in non-outbreak situation.

Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes.

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.

Methicillin-resistant Staphylococcus aureus carriage, infection and transmission in dialysis patients, healthcare workers and their family members.

BACKGROUND: Carriage and subsequent infection with methicillin resistant S. aureus (MRSA) and its transmission between hospital and community settings have not been studied in dialysis patients and their contacts. METHODS: Surveillance for nasal MRSA carriage and infection among dialysis patients, healthcare workers (HCWs) and their family members in a dialysis centre was prospectively undertaken during three time periods within 1 year. Molecular typing was used to determine epidemiological relationship. RESULTS: Among 1687 samples collected, MRSA colonization rates were 2.41% (2/83) for peritoneal dialysis patients and 2.36% (12/509) for haemodialysis patients. Five (5/14) subjects subsequently had MRSA infection. The clinical MRSA isolates had the same molecular type as the colonized strains of the same person, indicating MRSA colonization preceded clinical infection. Significantly higher MRSA nasal carriage rates were observed among family members of HCWs than family members of dialysis patients (P = 0.0024). Only three major clones were observed. Pulmonary diseases (OR: 4.873, 95% CI: 1.668-14.235), recent admission to a hospital (OR: 2.797, 95% CI: 1.291-6.059) and recent antibiotics usage (OR: 2.319, 95% CI: 1.053-5.104) were also significantly associated with MRSA carriage. CONCLUSION: Transmission of MRSA among dialysis patients, HCWs and their family members in a dialysis unit could be inferred. Monitoring and eradication of MRSA from patients, HCWs and their family members should be considered to prevent continuous spread between healthcare facilities and the community.

How carbapenem-resistant Acinetobacter spp. established in a newly constructed hospital.

Annually increasing rates of carbapenem-resistant Acinetobacter spp. were observed in a Taiwan hospital since its establishment in November 1998 to March 2005. Increasing consumption of carbapenems was also noticed. Carbapenem-resistant Acinetobacter spp. from 33 patients carried a class 1 integron. Twenty-eight isolates were Acinetobacter baumannii harbouring both ISAba1 and an OXA-51-like gene. Twenty-four of the 28 A. baumannii isolates had ISAba1 upstream of the OXA-51-like gene. Four A. baumannii isolates harboured the OXA-24-like gene and one isolate had the VIM-11 gene. Regarding the five non-baumannii Acinetobacter spp., three Acinetobacter genomic species 3 isolates and one Acinetobacter radioresistens isolate had both IMP-1 and OXA-58-like genes. One A. radioresistens isolate had an OXA-23-like gene. One major clone of A. baumannii (25/28; 89.3%) was identified by ribotyping. Three ribotypes were identified as being brought into the hospital by patient transfer from other hospitals. In conclusion, an insidious clonal dissemination with various resistance mechanisms contributed to the spread of carbapenem-resistant Acinetobacter spp. in a hospital setting, with increasing usage of carbapenems as the possible selection pressure. Notification of carbapenem-resistant Acinetobacter spp. infection when patients are transferred between hospitals is important to control the spread of carbapenem resistance.

Influence of ethanol concentration in the phagocytic function of neutrophils against Klebsiella pneumoniae isolates in an experimental model.

BACKGROUND/PURPOSE: Although the prevalence of pneumonia or other extrapulmonary infections is higher in people with alcoholism or acute alcohol intoxication, the possible relationship of acute alcohol intoxication to phagocytic function has not been investigated. Our aim was to determine whether acute alcohol intoxication suppresses phagocytic function in human neutrophils. METHODS: Twenty healthy individuals were enrolled for isolating neutrophils to evaluate the neutrophil phagocytic function at different alcohol concentrations. Klebsiella pneumoniae was isolated from clinical specimens of liver abscesses. The rate of K. pneumonia phagocytosis (K2 and non-K1/K2 isolates) by neutrophils was determined using flow cytometry and compared among the nine groups with different alcohol concentrations. RESULTS: The rate of phagocytic uptake decreased significantly with increasing alcohol concentration in both the K2 and non-K1/K2 K. pneumonia groups (r = -0.866, p = 0.03 vs. r = -0.975, p < 0.001). Moreover, the percentage of K. pneumoniae ingested by neutrophils decreased with age. CONCLUSION: The ability of neutrophils to phagocytose virulent K2 K. pneumoniae was suppressed by ethanol at high concentrations. This finding may account for the higher prevalence of pneumonia or other extrapulmonary infection in people with acute alcohol intoxication.

Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a serious cause of nosocomial infections. Rapid identification of this pathogen is required so that appropriate therapy can be given and outbreaks controlled. This study evaluated a multiplex PCR and an automated ribotyping system for the rapid identification of Acinetobacter baumannii. In total, 22 different reference strains and 138 clinical isolates of Acinetobacter spp., identified by 16S-23S rRNA intergenic spacer (ITS) sequence analysis, were evaluated. All A. baumannii isolates (82 clinical isolates and one reference strain) were identified by the multiplex PCR method (specificity 100%). The sensitivity and specificity of the ribotyping system for identification of A. baumannii were 85.5% (71/83) and 93.5% (72/77), respectively. An additional 100 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex were used to compare these two methods for identification of A. baumannii, and this comparison revealed a level of disagreement of 14% (14 isolates). The accuracy of the multiplex PCR was 100%, which was confirmed by sequence analysis of the ITS and recA gene of these isolates. Thus, the multiplex PCR method dramatically increased the efficiency and speed of A. baumannii identification.

CMY-2 beta-lactamase-carrying community-acquired urinary tract Escherichia coli: genetic correlation with Salmonella enterica serotypes Choleraesuis and Typhimurium.

Forty-six cephamycin-resistant Escherichia coli isolates from patients diagnosed with community-acquired urinary tract infection were selected in order to study their resistance mechanism. With the exception of one isolate producing CMY-4, all isolates produced a CMY-2 beta-lactamase. Molecular typing showed that the CMY-2-producing isolates were not related. Cephamycin resistance was plasmid encoded and conjugatively transferred. Plasmid digest profiles suggested that the plasmids were different. Thirty-nine of the 45 CMY-2-producing isolates harboured a plasmid containing a specific DNA fragment, ISEcp1-bla(CMY-2)-blc-sugE, which was identical to those previously published in CMY-2-producing Salmonella enterica serotype Choleraesuis (SCB67) and Salmonella enterica serotype Typhimurium (pNF1358) from Taiwan and the USA, respectively. Among the remaining six isolates, insertion of IS1294 and IS1 at different positions was observed in one and five isolates, respectively. The regions surrounding bla(CMY-2) of the six isolates were identical to the other 39 isolates as well as to SCB67 and pNF1358. Since the present identical transmissible bla(CMY-2)-carrying element was observed in food animal sources both in the USA and Taiwan, its possible transmission to humans, as revealed in this study, is of great concern. Awareness of this mobile resistance element is required to prevent introduction into hospitals and to reduce the spread of this emerging resistance within the community.

Comparative antimicrobial susceptibility of aerobic and facultative bacteria from community-acquired bacteremia to ertapenem in Taiwan.

BACKGROUND: Ertapenem is a once-a-day carbapenem and has excellent activity against many gram-positive and gram-negative aerobic, facultative, and anaerobic bacteria. The susceptibility of isolates of community-acquired bacteremia to ertapenem has not been reported yet. The present study assesses the in vitro activity of ertapenem against aerobic and facultative bacterial pathogens isolated from patients with community-acquired bacteremia by determining and comparing the MICs of cefepime, cefoxitin, ceftazidime, ceftriaxone, ertapenem, piperacillin, piperacillin-tazobactam, ciprofloxacin, amikacin and gentamicin. The prevalence of extended broad spectrum beta-lactamases (ESBL) producing strains of community-acquired bacteremia and their susceptibility to these antibiotics are investigated. METHODS: Aerobic and facultative bacteria isolated from blood obtained from hospitalized patients with community-acquired bacteremia within 48 hours of admission between August 1, 2004 and September 30, 2004 in Chang Gung Memorial Hospital at Keelung, Taiwan, were identified using standard procedures. Antimicrobial susceptibility was evaluated by Etest according to the standard guidelines provided by the manufacturer and document M100-S16 Performance Standards of the Clinical Laboratory of Standard Institute. Antimicrobial agents including cefepime, cefoxitin, ceftazidime, ceftriaxone, ertapenem, piperacillin, piperacillin-tazobactam, ciprofloxacin, amikacin and gentamicin were used against the bacterial isolates to test their MICs as determined by Etest. For Staphylococcus aureus isolates, MICs of oxacillin were also tested by Etest to differentiate oxacillin-sensitive and oxacillin-resistant S. aureus. RESULTS: Ertapenem was highly active in vitro against many aerobic and facultative bacterial pathogens commonly recovered from patients with community-acquired bacteremia (128/159, 80.5 %). Ertapenem had more potent activity than ceftriaxone, piperacillin-tazobactam, or ciprofloxacin against oxacillin-susceptible S. aureus (17/17, 100%)and was more active than any of these agents against enterobacteriaceae (82/82, 100%). CONCLUSION: Based on the microbiology pattern of community-acquired bacteremia, initial empiric treatment that requires coverage of a broad spectrum of both gram-negative and gram-positive aerobic bacteria, such as ertapenem, may be justified in moderately severe cases of community-acquired bacteremia in non-immunocompromised hosts.

Impaired phagocytosis of capsular serotypes K1 or K2 Klebsiella pneumoniae in type 2 diabetes mellitus patients with poor glycemic control.

CONTEXT: Diabetes mellitus (DM) and capsular serotypes K1 and K2 Klebsiella pneumoniae have been identified as risk factors for liver abscess and complicated endophthalmitis. OBJECTIVE: The objective of this study was to determine whether poor glycemic control contributes to the development of capsular serotype K1 or K2 K. pneumoniae liver abscess. DESIGN AND SETTING: Neutrophil phagocytosis in patients with type 2 DM and nondiabetic controls was compared with isolates from liver abscess. Phagocytic rates of 18 K1/K2 and nine non-K1/K2 K. pneumoniae strains were evaluated by flow cytometry and electron microscopy. PATIENTS OR STUDY PARTICIPANTS: Forty patients with type 2 diabetes, 14 with good glycemic control, 26 with poor glycemic control, and 13 age-matched healthy normal subjects, were studied. MAIN OUTCOME MEASURES: Phagocytic rate of K. pneumoniae was measured. RESULTS: Phagocytosis of serotype K1/K2 isolates by neutrophils from diabetics was significantly less than normal controls (P < 0.01). Further analysis revealed that, in type 2 DM patients with poor glycemic control, phagocytosis of K1/K2 was remarkably impaired at 10 min (25.2 +/- 1.7 vs. 42.4 +/- 1.8%) and persisted until 60 min (51 +/- 1.2 vs. 59.4 +/- 1.4%; P < 0.01), but in type 2 DM patients with good glycemic control were similar at 10 min (38.2 +/- 1.7% vs. 42.4 +/- 1.8%) and at 60 min (57 +/- 0.3% vs. 59.4 +/- 1.4%; P = 0.2). No significant difference in the phagocytosis of non-K1/K2 K. pneumoniae among all subjects was observed. CONCLUSIONS: Poor glycemic control plays a role in impairing neutrophil phagocytosis of K1/K2 K. pneumoniae, but does not significantly affect the phagocytosis of non-K1/K2 K. pneumoniae. This study identifies poor glycemic control as a risk factor for susceptibility to serotype K1/K2 K. pneumoniae liver abscess and complicated endophthalmitis.

Secondary Aeromonas peritonitis is associated with polymicrobial ascites culture and absence of liver cirrhosis compared to primary Aeromonas peritonitis.

Aeromonas peritonitis remains a rare condition. In this study we describe the clinical features of primary and secondary Aeromonas peritonitis, and compare the differences between these two diseases entities. Patients with Aeromonas peritonitis were identified from microbiological and medical records during the period between March 1994 and March 2003. Clinical characteristics, laboratory data, microbiological results, treatment and outcome of patients were obtained by retrospective chart review. 22 and 27 patients with primary or secondary peritonitis caused by Aeromonas species were identified. All except two of these patients were adults, with a median age of 62.4 (31-76) vs 65.8 (8-85) years, respectively. Males were predominant (82 vs 78%). Peritonitis was community acquired in 73% and 56% of patients in these two groups, respectively. Significantly higher prevalence of underlying liver cirrhosis (96 vs 7%, p<0.001), which was Child-Pugh class C in 91% of cases, in primary peritonitis was noted. Primary peritonitis was more likely to be monomicrobial (100 vs 15%, p<0.001) and complicated by bacteremia (50 vs 7%, p=0.011). A source of intraabdominal infection should be sought when Aeromonas peritonitis occurs in a patient who has no history of liver cirrhosis or who has a polymicrobial result of ascites culture.

Regulator of the mucoid phenotype A gene increases the virulent ability of extended-spectrum beta-lactamase-producing serotype non-K1/K2 Klebsiella pneumonia.

BACKGROUND: To determine whether the presence of a capsule regulator gene [i.e., regulator of mucoid phenotype A (rmpA) gene] contributes to virulence on extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) with serotype non-K1/K2 strains. METHODS: Twenty-eight ESBL-KP and non-ESBL-KP isolates were collected from the Tri-Service General Hospital (Taipei, Taiwan). The impact of the virulent rmpA gene in different capsular polysaccharide serotypes on ESBL-KP and non-ESBL-KP isolates was studied by a neutrophil phagocytosis reaction, a serum bactericidal assay, and an animal survival model. RESULTS: Resistance to broad spectrum antibiotics was more prevalent in ESBL-KP strains than in non-ESBL-KP strains (p < 0.01). The ESBL-KP strains had different molecular patterns from non-ESBL-KP strains, based on pulsed-field gel electrophoresis. The frequency of serum-resistant isolates was the highest among ESBL-KP strains with rmpA (i.e., rmpA(+)) [71.4% (5/7)] than among of non-ESBL-KP rmpA(+) strains [42.8% (6/14)], ESBL-KP strains without rmpA (rmpA(-)) [33.3% (7/21)], and non-ESBL-KP rmpA(-) strains [14.2% (2/14)]. The most significant increase in neutrophil resistance occurred in the ESBL-KP rmpA(+) strains in comparison to the non-ESBL-KP rmpA(+), ESBL-KP rmpA(-), and non-ESBL-KP rmpA(-) strains (p < 0.01). The results of the animal survival model were compatible with the neutrophil phagocytosis reaction and serum bactericidal assay. CONCLUSION: We conclude that the pathogenic potential is greater in rmpA(+) ESBL-KP strains than in rmpA(-) ESBL-KP and non-ESBL-KP strains.

Quantification and comparison of virulence and characteristics of different variants of carbapenemase-producing Klebsiella pneumoniae clinical isolates from Taiwan and the United States.

BACKGROUND/PURPOSE: The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing strains is a challenge for clinicians. The characteristics and virulence of variants of KPC-producing K. pneumoniae isolates were evaluated. METHODS: Five clinical isolates-three KPC subtypes from Taiwan (KPC2-TW, KPC3-TW, and KPC17-TW) and two clinical strains from the United States (US; KPC2-US, KPC3-US)-were included. Virulent traits and capsular serotypes were analyzed by Polymerase Chain Reaction (PCR). Serum killing, neutrophil phagocytosis, and mice lethargy studies were performed to evaluate virulence. RESULTS: Multilocus sequence typing (MLST) demonstrated that KPC2-TW and KPC17-TW belonged to sequence type (ST)11, and KPC2-US, KPC3-US, and KPC3-TW to ST258. KPC3-TW expressed capsular serotype K1, whereas the others were non-K1/K2/K5 isolates. MLST analysis indicated that ST11 strains were serum resistant, whereas ST258 isolates were serum sensitive. ST11 isolates exhibited significantly higher 15-minute phagocytic rates than ST258 isolates (70.28 ± 16.68% vs. 34.88 ± 10.52%, p < 0.001). The capsular serotype K1 strain was more resistant to neutrophil phagocytosis than non-K1/K2/K5 isolates (27.1 ± 10.23% vs. 54.46 ± 20.94%, p = 0.050). All KPC-producing strain variants from Taiwan and the US demonstrated less virulence in a mouse lethality study, where the LD50 ranged from approximately 10(6) colony-forming units (CFU) to >10(7) CFU. Immunological responses were not significantly correlated with KPC subtype; however, responses were associated with MLST and capsular serotype. CONCLUSION: Production of KPC itself was not associated with increased virulence despite different variants of KPC. The ST11 KPC-producing strain was resistant to serum killing, whereas capsular ss K1 was associated with resistance to neutrophil phagocytosis.

Variety of TEM-, SHV-, and CTX-M-type beta-lactamases present in recent clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae from Taiwan.

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) of > or =2 microg/ml for ceftazidime or cefotaxime were evaluated for the production of beta-lactamases. PCR amplification with specific primers for the bla (SHV), bla (TEM), and bla (CTX) genes revealed that a total of 53, 81, and 43 of these genes were amplified, respectively. Sequencing results confirmed that TEM-1, CTX-M-3 and -14, SHV-1, -5, -11, -12, and -33, OXY-1a, and LEN-1 were presented among these isolates. No specific large cluster of isolates carried the same beta-lactamases, indicating the wide diversity of the collected strains. Plasmid spread between E. coli and K. pneumoniae was identified in few isolates. Combinations of TEM, SHV, and CTX beta-lactamase genes, including extended-spectrum beta-lactamase genes, were observed in all three species.