Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Placental mesenchymal stem cells boost M2 alveolar over M1 bone marrow macrophages via IL-1ß in Klebsiella-mediated acute respiratory distress syndrome.

RATIONALE: Acute respiratory distress syndrome (ARDS) is a lethal complication of severe bacterial pneumonia due to the inability to dampen overexuberant immune responses without compromising pathogen clearance. Both of these processes involve tissue-resident and bone marrow (BM)-recruited macrophage (MΦ) populations which can be polarised to have divergent functions. Surprisingly, despite the known immunomodulatory properties of mesenchymal stem cells (MSCs), simultaneous interactions with tissue-resident and recruited BMMΦ populations are largely unexplored. OBJECTIVES: We assessed the therapeutic use of human placental MSCs (PMSCs) in severe bacterial pneumonia with elucidation of the roles of resident alveolar MΦs (AMΦs) and BMMΦs. METHODS: We developed a lethal, murine pneumonia model using intratracheal infection of a clinically relevant Klebsiella pneumoniae (KP) strain with subsequent intravenous human PMSC treatment. Pulmonary AMΦ and recruited BMMΦ analyses, histological evaluation, bacterial clearance and mice survival were assessed. To elucidate the role of resident AMΦs in improving outcome, we performed AMΦ depletion in the KP-pneumonia model with intratracheal clodronate pretreatment. MEASUREMENTS AND MAIN RESULTS: Human PMSC treatment decreased tissue injury and improved survival of severe KP-pneumonia mice by decreasing the presence and function of recruited M1 BMMΦ while preserving M2 AMΦs and enhancing their antibacterial functions. Interestingly, PMSC therapy failed to rescue AMΦ-depleted mice with KP pneumonia, and PMSC-secreted IL-1ß was identified as critical in increasing AMΦ antibacterial activities to significantly improve pathogen clearance-especially bacteraemia-and survival. CONCLUSIONS: Human PMSC treatment preferentially rescued resident M2 AMΦs over recruited M1 BMMΦs with overall M2 polarisation to improve KP-related ARDS survival.

A Novel Deletion Mutation in pmrB Contributes to Concurrent Colistin Resistance in Carbapenem-Resistant Escherichia coli Sequence Type 405 of Clinical Origin.

We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

[Differences in Patient Safety Reporting Attitudes and Knowledge Among Different Hospital Levels].

BACKGROUND: Establishing a positive reporting culture, which helps medical and healthcare workers learn from errors and reduce the risks of future adverse events, is essential to fostering a culture of patient safety. PURPOSE: The objectives of this study were to investigate the differences among the three levels of hospitals in terms of the knowledge and attitudes of hospital staff regarding the patient safety reporting system and to identify the potential factors affecting these differences. METHODS: This cross-sectional study was carried out in six hospitals, including two academic medical centers, two regional hospitals, and two district hospitals. The subjects were physicians, nurses, medical technicians, and administrative staffs. Data were collected using a patient safety reporting questionnaire. RESULTS: Three hundred and forty-eight participants were recruited, with 348 valid questionnaires returned (response rate: 100%). The average score for knowledge of patient safety reporting was 12.76 (total possible score: 14). Age, work position, and work experience were significantly associated with knowledge of patient safety reporting (p < .01). The patient safety reporting attitudes questionnaire comprised 21 items, each of which was scored using a five-point Likert scale. The mean score for each item was 3.92 ± 0.50. Gender, age, work position, work experience, and job discipline were significantly associated with attitude toward reporting (p < .01). The level of hospital was found to significantly impact attitudes toward patient safety reporting (p = .01), with participants working at medical centers scoring the highest. In addition, participants who were older and in more-senior positions scored higher and more positively for both knowledge and attitudes. CONCLUSIONS: The key factors to successfully fostering a strong patient safety reporting culture are staff security, a reliable reporting system, and a user-friendly interface. Improving attitudes toward reporting requires more resources and time than improving knowledge of reporting, which may be improved using education and promotion. Regional hospitals may invest more resources to enhance positive attitudes toward reporting and increase the willingness of staff to report.

Roles of ramR and tet(A) Mutations in Conferring Tigecycline Resistance in Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates.

Tigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniae isolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the mechanisms underlying tigecycline resistance in 16 tigecycline- and carbapenem-resistant K. pneumoniae (TCRKP) isolates. Mutations in tigecycline resistance determinant genes [ramR, acrR, oqxR, tet(A), tet(L), tet(X), tet(M), rpsJ] were assessed by PCR amplicon sequencing, and mutations in ramR and tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramR mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant ramR Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility or the upregulation of ramA and acrA Two tet(A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 tet(A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 tet(A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no ramR gene and a mutated tet(A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated tet(A) being seen. Overall, mutations in the ramR and tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a ramR deficiency and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.

Developing a measure of medication-related quality of life for people with polypharmacy.

PURPOSE: To develop a measure of medication-related quality of life (MRQoL) and to validate the measure in a hospital-based population of patients with polypharmacy. METHODS: The Medication-Related Quality of Life Scale version 1.0 (MRQoLS-v1.0) included 14 items developed on the basis of interviews with elderly patients with polypharmacy, defined as taking five or more medications simultaneously. This scale was tested in 219 outpatients (99 with polypharmacy and 120 without polypharmacy). Two measures were used to establish construct validity the Psychological Distress Checklist, for convergent validity, and the Medication Adherence Behavior Scale (MABS), for discriminant validity. RESULTS: The 14-item scale was found to be both reliable and valid. Internal consistency reliability evaluated using Cronbach's alpha for this scale was 0.91. Scores on the MRQoLS-v1.0 correlated statistically significantly and negatively with those on the Psychological Distress Checklist. Discriminant validity was demonstrated by low correlation with MABS, indicating that the MRQoLS-v1.0 measured concepts different from medication adherence. Significant differences in the MRQoLS-v1.0 between patients with polypharmacy and those without polypharmacy provided evidence for known-group validity. CONCLUSIONS: The study presents a psychometric evaluation of a measure used to assess MRQoL of patients with polypharmacy. The instrument is practical to administer in clinics and provides a valuable adjunct to the outcome measurement for patients with polypharmacy. Further research on the sensitivity of this instrument to medication change in multi-medicated patients is warranted.

Successful treatment of a patient with ventriculoperitoneal shunt-associated meningitis caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae.

Bacterial meningitis is responsible for significant morbidity and mortality worldwide, despite that modern antibiotics effectively penetrate cerebrospinal fluid to eradicate bacteria. A clinical suspicion of bacterial meningitis should be recognized early for the rapid diagnostic workup. Bacterial meningitis associated with ventriculoperitoneal shunt (VPS) is not uncommon and infrequently presents as abdominal symptoms and signs. Infections of the central nervous system caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are extremely rare, and such multiple drug-resistant pathogens frequently cause inappropriate treatments and mortality. ß-Lactamases are bacterial enzymes that inactivate ß-lactam antimicrobial agents. The increased prevalence of ESBL-producing organism infections has become a worldwide problem. Timely and appropriate treatment is important to reduce mortality and morbidity of infections caused by ESBL-producing organisms. Here, we report a 61-year-old male patient who underwent VPS implantation for consequent hydrocephalus following spontaneous intracranial hemorrhage six months before this presentation. He was admitted for intermittent fever and right lower quadrant abdominal pain, and he was initially managed as acute appendicitis with its typical presentation. Finally, he was diagnosed VPS-associated meningitis caused by ESBL-KP. This patient was successfully treated with the combination of meropenem, a carbapenem antibiotic that is the drug of choice for treating ESBL-producing organisms, and high-dose fosfomycin, a phosphonic acid derivative antibiotic that is effective in treating some drug-resistant pathogens. In the present report, we emphasize the clinical presentations of catheter-related meningitis and risk factors for infections caused by ESBL-producing pathogens. Antibiotic combination therapy can provide synergistic effect and maximize anti-bacterial activity in ESBL-KP meningitis.

Molecular epidemiology and resistance patterns of blaOXA-48Klebsiella pneumoniae and Escherichia coli: A nationwide multicenter study in Taiwan.

BACKGROUND: We describe the molecular epidemiology and resistance patterns of blaOXA-48Klebsiella pneumoniae and Escherichia coli in Taiwan. METHODS: In this multicenter surveillance study from January 2012 to August 2015, the identified blaOXA-48Enterobacteriaceae isolates were subjected to antibiotics susceptibility testing. PCR method was used for detecting concomitant other beta-lactamases. Outer membrane porins were analyzed. Genetic relatedness and molecular epidemiology of the isolates were determined through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid incompatibility was determined using PCR-based replicon typing. RESULTS: Forty-three blaOXA-48K. pneumoniae and two E. coli isolates were analyzed. The annual incidence of blaOXA-48K. pneumoniae isolates from 2012 to 2015 were 0%, 1.1%, 2.4%, and 7.6%, respectively. Forty-three (95.5%) of 45 isolates were non-susceptible to broad-spectrum beta-lactams (ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam), Forty-two (93.3%) of the 45 isolates showed resistance against all tested carbapenems (imipenem, meropenem, doripenem, and ertapenem). Molecular characterization revealed that they co-produced at least one extended-spectrum beta-lactamases or AmpC beta-lactamases, with at least one outer membrane porin loss. Thirty-eight (88.3%) of the 43 K. pneumoniae isolates belonged to ST11. PFGE analysis of 43 K. pneumoniae isolates revealed dissemination of multiple clones. Six of the 12 tested K. pneumoniae representatives of different pulso-types belonged to IncA/C. CONCLUSION: Concomitant loss of porins and production of other beta-lactamases renders the blaOXA-48-producing isolates in Taiwan a high-level carbapenem resistance and broad resistance against many beta-lactam antibiotics. Following dissemination of multiple clones of blaOXA-48 K pneumoniae ST 11, a trend of increased blaOXA-48 prevalence was noted.

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.

Human Placental MSC-Secreted IL-1ß Enhances Neutrophil Bactericidal Functions during Hypervirulent Klebsiella Infection.

Hypervirulent Klebsiella pneumoniae (hvKP) causes severe infections even in healthy individuals by escaping surveillance and killing from polymorphonuclear neutrophils (PMNs), the first-line leukocytes in bacterial infections; moreover, the emergence of multidrug-resistant strains further limits treatment options. We therefore assess whether multilineage mesenchymal stem cells (MSCs), best known for immunomodulation toward T cells, could be therapeutic for highly virulent bacterial infections via modulation of PMNs. We find that both bone marrow MSCs and placental MSCs (PMSCs) preserve in vitro PMN survival, but only PMSCs significantly enhance multiple PMN bactericidal functions, including phagocytosis, through secretion of interleukin-1ß (IL-1ß). PMSC treatment of hvKP-infected mice suppresses T and natural killer (NK) cell responses as expected but can preferentially recruit PMNs and enhance antibacterial functions to allow for disease survival; IL-1ß knockdown in PMSCs significantly decreases hvKP clearance, worsening survival and resulting in 100% lethality. Our data strongly implicate the possible use of PMSCs for infections of PMN-resistant hvKP strains.

Acinetobacter baylyi as a pathogen for opportunistic infection.

There are no previous reports of human infection due to Acinetobacter baylyi. In this study, we report on six patients with bacteremia due to A. baylyi, based on analysis of the 16S-23S rRNA intergenic spacer and the 16S rRNA gene. All six patients had multiple underlying diseases. The infection was nosocomially acquired in five patients. The six clinical isolates had similar ribopatterns, suggesting a clonal relationship. Compared to the reference strain, the clinical isolates were more resistant to antimicrobial agents, especially beta-lactam antibiotics. In three of the isolates, they may have undetermined plasmid mediated class C type beta-lactamases because of the positive results in a double-disk synergy test using 3-aminophenylboronic acid. Two of the clinical isolates retained a level of natural transformability similar to that of the reference strain. None of the patients died, although only three of them received appropriate antimicrobial therapy. This study demonstrates that A. baylyi is a potential human pathogen that can cause nosocomial infection in immunocompromised patients.

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.

Effect in virulence of switching conserved homologous capsular polysaccharide genes from Klebsiella pneumoniae serotype K1 into K20.

The capsular polysaccharides in different serotypes of Klebsiella pneumoniae (KP) coded by the (CPS) gene cluster are characterized by a conserved and a hyper-variable region. We performed a virulence study by switching genes in the highly conserved region of the CPS cluster between strains. Six genes in the CPS conserved region in serotype K20, including galF, acidPPc, wzi, wza, wzb and wzc, were knocked out and replaced by the homologous genes from serotype K1. Compared to the parental K20 strain, the mutants showed a decline in lethality (LD50) in mice from 10-fold to > 105-fold and were categorized in terms of the effect on virulence as low (L) for galF and acidPPC, moderate (M) for wzi, and high (H) for wza, wzb and wzc. Although substituting the acidPPC gene from K1 for acidPPC in the K20 strain fully restored virulence, substitution with the wzi, wza, wzb or wzc homologs from K1 did not. The restoration with wzi from K1 led to a partial restoration of virulence, with the LD50 in mice changing from 104 to 103 CFU. For the wza, wzb and wzc genes, Complementation of K20 wza, wzb and wzc from K1 resulted in varied degrees of lethality in mice. Variable improvement in serum killing and phagocytosis was observed when the knockout mutants were compared with the gene-switched strains. In conclusion, homologous genes for capsule synthesis failed to exhibit the same functionality when switched between serotypes and virulence was decreased in different degree in according to the genes' homology.

Outbreak of imipenem-resistant Acinetobacter baumannii in different wards at a regional hospital related to untrained bedside caregivers.

BACKGROUND: This study describes an outbreak caused by imipenem-resistant Acinetobacter baumannii (IRAB) involving 2 general wards at the Penghu branch of Tri-Service General Hospital. METHODS: Clinical data obtained from the patients with IRAB during an outbreak from May 2014-October 2014 were reviewed. Microbiologic sampling from the environment and the hands of health care workers (HCWs) was performed. Clinical isolates from case patients were genotyped using pulsed-field gel electrophoresis (PFGE). RESULTS: During the outbreak period, 12 patients were colonized or infected with IRAB. The hospital room environments of the case patients were contaminated with IRAB. Hands of nurses and physicians were not colonized with IRAB, but the hands of 2 bedside caregivers of case patients were colonized with IRAB. The PFGE analysis revealed that at least 2 major genetically distinct strains disseminated between 2 different wards. After implementation of infection control measures with a cohort of nursing patients, hand hygiene education for caregivers who had not received instructions before the outbreak, and a critical value alert system to notify case patients, the outbreak was controlled successfully. CONCLUSIONS: This outbreak study highlights the importance of adherence to hand hygiene by all HCWs to prevent the dissemination of multidrug-resistant organisms.

Tigecycline resistance among carbapenem-resistant Klebsiella Pneumoniae: Clinical characteristics and expression levels of efflux pump genes.

OBJECTIVES: Tigecycline is a treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Emerging tigecycline resistance in CRKP represents a growing threat. Knowledge of the clinical, microbiological, and molecular characteristics of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) is limited. METHODS: Patients infected with TCRKP were identified from a Taiwanese national surveillance study. Clinical data were collected from medical records. We performed susceptibility tests, carbapenemase gene detection, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) analyses to assess the expression levels of the efflux pump genes acrB and oqxB. RESULTS: We identified 16 patients infected with TCRKP, with urinary tract infection (UTI) being the most common type of infection (63%). The all-cause 30-day mortality rate was 44% in patients with TCRKP infection. Patients with a site of infection other than the urinary tract had a significantly higher mortality rate than patients with UTIs (83% vs. 20%, p = 0.035). PFGE and MLST revealed no dominant clone or sequence type. Using qRT-PCR, overexpression of both the acrB and oqxB genes was identified in seven isolates, and overexpression of the oqxB gene was observed in another seven. There was poor correlation between acrB or oqxB expression and tigecycline MICs (r = -0.038 and -0.166, respectively). CONCLUSIONS: The mortality rate in patients infected with TCRKP in this study was 44% and this is an important subset of patients. The absence of a linear relationship between efflux pump genes expression and MICs indicates that tigecycline resistance may be mediated by other factors. Continuous monitoring of tigecycline resistance among CRKP isolates and resistance mechanisms are necessary.

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

BACKGROUND: The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. METHOD: To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. RESULTS: Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. CONCLUSION: We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

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.

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess.

To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.