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Background/purposeThe 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.MethodsFive 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.ResultsMultilocus 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 107 CFU. Immunological responses were not significantly correlated with KPC subtype; however, responses were associated with MLST and capsular serotype.ConclusionProduction 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.

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

Repeated isolation of multidrug-resistant Acinetobacter baumannii (MDRAB) from respiratory secretions poses a great challenge for infection control. We conducted a retrospective case-control study to evaluate the efficacy and adverse effect of inhaled colistin methanesulfonate (CMS) in the eradication of MDRAB from the respiratory tract. Patients who were admitted to Taipei Veterans General Hospital between February 2009 and June 2010, had at least two sets of monomicrobial culture of MDRAB from respiratory secretions, and remained in hospital for at least 14 days after the first isolation of MDRAB (index day) were included. Patients who received intravenous CMS were excluded. Patients who received CMS inhalation for ≥3 days were selected as cases whereas the controls were matched for age and Acute Physiology and Chronic Health Evaluation II score. Thirty-nine cases and controls were identified. The duration of CMS inhalation was 10.9 ± 3.6 days. The use of inhaled CMS was the only independent factor associated with the eradication of MDRAB within 14 days after the index day (OR 266.33; 95% CI 11.26–6302.18, p

BackgroundKlebsiella pneumoniae liver abscess (KLA) is an emerging infectious disease caused by the virulent K pneumoniae strains of capsular serotype K1 and commonly associated with diabetes mellitus. Recurrent KLA is rarely reported and the mechanism of recurrence is uncertain. In this study we evaluated both phagocytosis by neutrophils and serum killing ability of serum from recurrent K1 KLA patients compared to normal healthy subjects (NHS).MethodsThis prospective study included six cases of recurrent K1 KLA consisting of three male and three female patients with a mean age of 67.2 years (range, 56-88 years). The different serotypes of K pneumoniae were reacted with serum from patients with recurrent KLA and NHS. Subsequent phagocytosis by neutrophils was determined using flow cytometry and serum killing assays were performed.ResultsThe most common underlying disease in patients with recurrent KLA was diabetes mellitus, occurring in about 83.3% (5/6) of patients. The antibiogram of the strains associated with recurrent KLA remained uniquely resistant to ampicillin. The average percentage derived from the serum killing assays showed serotype K1 and K2 resistance to serum from NHS (1281% and 621%, respectively); however, serum susceptibly was observed in the serum of patients with recurrent K1 KLA (0.3% and 1.1%, respectively). A significant increase in neutrophil phagocytosis of serotype K1 was observed following opsonisation with serum from patients with recurrent KLA compared with serum from NHS (p = 0.008). No significant difference in the phagocytic rate of non-K1/K2 or K2 serotypes was observed between NHS and patients with recurrent KLA (p = 0.76 and p = 0.132, respectively).ConclusionThese preliminary results showed possible immunologic protection in patients with recurrent KLA due to increasing opsonization and serum killing.

Extensively drug-resistant Acinetobacter baumannii (XDRAb) emerges as an important pathogen of health care–associated infections and outbreaks worldwide. During January and February 2006, there was a hospital-wide outbreak of XDRAb at a medical center in Taiwan. Without limiting the usage of carbapenems or the closure of any ward, this outbreak was effectively controlled. We investigated the molecular epidemiology and reported the infection control experiences. XDRAb is defined as A baumannii that is resistant to multiple antibiotics but susceptible to tigecycline and polymyxin B. During the outbreak, the clinical and environmental XDRAb isolates were collected and studied by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and polymerase chain reaction for Verona integron-encoded metallo-beta-lactamases, imipenemases, and oxacillinases (OXA). Our measures to control the outbreak included private room isolation of patients until there were three successive negative cultures, reinforcement of contact precautions, daily environmental cleansing with room-dedicated cleaning tools and sodium hypochlorite, and careful auditing of adherence. During the outbreak, 32 clinical XDRAb isolates came from 13 patients who were hospitalized in four intensive care units and three wards. Most (7 of 13, 53.8%) cases were associated with a surgical intensive care unit. The results from pulsed-field gel electrophoresis study indicated that all isolates were of one genotype. All 32 isolates harbored IS Aba 1- bla OxA-51-like and bla OxA-72 genes. After this outbreak till August 2010, further incidences of XDRAb were sporadic cases of XDRAb with different clones and did not reach the level of outbreak. To our knowledge, this is the first reported hospital-wide outbreak caused by OXA-72 carbapenemase–producing A baumannii in the Asia-Pacific region, with successful and sustained control. Although the source or vehicle of the outbreak was not identified, our results suggest that a hospital-wide outbreak can be successfully managed with strict infection control measures, and that the limitation of the use of carbapenems and closure of wards may not be necessary.

Outer membrane porin (OMP) expression has been shown to play an important role in antimicrobial resistance. In this study, we observed that OmpK35 of Klebsiella pneumoniae had varied expression profiles in different nutrient broths. the potential factors that could influence protein expression were assessed. K. pneumoniae (ATCC 13883) was cultured into two commercial available nutrient broths and also into solutions of the individual ingredients. To ensure that OmpK35 was detected, an OmpK35 deficient mutant was generated as control. When OmpK protein expression profiles were analyzed by SDS-PAGE, OmpK35 exhibited two different isoforms. Expression of an additional isoform-like OmpK35 protein was identified in one of the broths. No OmpK35 isoforms were observed when the individual ingredients of beef extract, casein or gelatin were used as culture medium. OmpK35 isoform expression could be repressed by adding more beef extract. In summary, OmpK can exhibit varied protein expression profiles when growing in different nutrient broths. The isoform-like protein expression of OmpK35 may lead to confusion in OmpK protein analysis.

The bla OXA-51 -like gene with an upstream IS Aba1 (IS Aba1 - bla OXA-51 -like gene) was originally found on the chromosomes of carbapenem-resistant or -susceptible Acinetobacter baumannii isolates. However, a plasmid-borne IS Aba1 - bla OXA-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 IS Aba1 - bla OXA-51 -like gene and their significance in A. baumannii . Among the 117 IS Aba1 - bla OXA-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 IS Aba1 - bla OXA-51 -like gene on their chromosomes. Based on the analysis of these four isolates, the plasmid-located IS Aba1 - bla OXA-51 -like gene appeared to be acquired via one-ended transposition (Tn 6080 ). The isolates with a plasmid bearing the IS Aba1 - bla OXA-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 IS Aba1 - bla OXA-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 IS Aba1 - bla OXA-51 -like gene are widespread in A. baumannii strains in Taiwan.

The contribution of the bla OXA-58 gene and its promoter to β-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii . We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which bla OXA-58 was the only detected carbapenemase gene. The bla OXA-58 gene was plasmid located, flanked by IS Aba3 (downstream) and an IS Aba3- like element (upstream). An IS 1006 element was inserted into IS Aba3 -like (IS 1006 -ΔIS Aba3 -like) to generate a hybrid promoter for bla OXA-58 , with a −35 promoter located in IS 1006 and a −10 promoter in IS Aba3 -like. The reference strain of Acinetobacter genomic species 13TU, ATCC 17903, revealed higher MICs of amoxicillin, ticarcillin, and piperacillin and heteroresistance to imipenem and meropenem when it was transformed with a shuttle vector containing a fragment encompassing ΔIS Aba3 -like- bla OXA-58 , compared to the same host containing only bla OXA-58 . When the fragment was changed from ΔIS Aba3 -like- bla OXA-58 to IS 1006 -ΔIS Aba3 -like- bla OXA-58 , the ATCC 17903 transformant revealed a markedly higher level of bla OXA-58 transcription (12-fold), increased cefuroxime and piperacillin-tazobactam MICs, and homoresistance to imipenem and meropenem. Different roles of the insertion elements preceding the bla OXA-58 gene in Acinetobacter genomic species 13TU are demonstrated. The IS Aba3 -like- -bla OXA-58 construct can mediate resistance to penicillin derivatives but only heteroresistance to carbapenems. The insertion of IS 1006 into IS Aba3 -like, generating a hybrid promoter, could further enhance the transcription of bla OXA-58 and mediate homoresistance to carbapenems and also enhanced resistance to piperacillin-tazobactam.

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.

Background Mucoviscosity-associated gene A (magA) is proposed to play a decisive role in the pathogenesis of liver abscesses due to Klebsiella pneumoniae. Although some investigators consider MagA to be a putative O-antigen ligase, it is also reportedly associated with the K1 antigen. Methods Using magA-positive serotype K1 K. pneumoniae STL43 isolated from a patient with liver abscess, we constructed 3 bacterial mutants by targeting genes within the same transcription unit, including magA, wcaG, and rfbP. The virulence of these mutants was determined by neutrophil phagocytosis and inoculation of mice. Transmission electron microscopy and Western blot analysis were used to define their surface polysaccharides. Results STL43 was resistant, and all 3 mutants were highly susceptible, to phagocytosis. None of the mutant strains caused death in mice at the lethal dose of STL43. In contrast to previous reports, transmission electron microscopy revealed that all 3 mutants were nonencapsulated. Analysis of surface polysaccharides revealed that all 3 mutants retained their O antigen but lost their K antigen/capsule. Furthermore, amino acid analysis showed that MagA shared a conserved domain of Wzy, the serotype-specific capsular polysaccharide polymerase. Conclusions In accordance with the bacterial polysaccharide gene nomenclature (BPGN) scheme, MagA should be renamed Wzy(KpK1), the capsular polymerase specific to K. pneumoniae serotype K1.

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.

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.

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.

A 68-y-old male had necrotizing fasciitis and bacteremia due to Pasteurella multocida. Saliva culture from his dog grew P. multocida and Pseudomonas aeruginosa. The human and dog P. multocida strains were of the same antibiogram but not identical tested with ribotyping. The wound licked by his dog was the only risk factor.

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

Diabetes mellitus (DM) and capsular serotypes K1 and K2 Klebsiella pneumoniae have been identified as risk factors for liver abscess and complicated endophthalmitis.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.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.Phagocytic rate of K. pneumoniae was measured.Phagocytosis of serotype K1/K2 isolates by neutrophils from diabetics was significantly less than normal controls (P0.01). 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%; P0.01), 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.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.

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) ofor =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.

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.

Background. A 1400-bed tertiary medical center in northern Taiwan was used to conduct an epidemiological study of children hospitalized with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection during a 5-year period. Methods. Nineteen previously healthy children with predominantly skin and soft-tissue CA-MRSA infections were enrolled into the study. Seventeen CA-MRSA isolates were examined for antimicrobial susceptibility and molecular typing. Results. A comparison of our results with the reported resistance rates among CA-MRSA isolates from other countries showed uniformly high macrolide resistance (100%). Of the 17 MRSA isolates in our study, all had the macrolide-lincosamide-streptogramin-constitutive phenotype and the ermB gene. Moreover, on the basis of molecular typing results, 11 (65%) of 17 CA-MRSA isolates were genetically related (as determined by pulsed-field gel electrophoresis), and multilocus sequence typing revealed a sequence type of 59 in all isolates. Staphylococcal toxin genes lukS-PV and lukF-PV were detected in all isolates. However, staphylococcal cassette chromosome mec type IV was only detected in 3 (17.6%) of 17 isolates; the remaining 14 isolates were untypeable. Conclusions. Analysis of our data suggests the predominance of a single endemic CA-MRSA strain with high macrolide resistance in our community. Clinical improvement with incision and drainage was noted for most patients, despite treatment with an ineffective antibiotic, so the need for a change in treatment guidelines should be addressed.

Objective To determine the mechanism of rifampicin resistance in Streptococcus pneumoniae in Taiwan. Methods Rifampicin resistance was investigated with respect to the rpoB gene in 23 invasive S. pneumoniae isolates collected from 1996 to 2001. PCR and molecular typing were used for genetic and epidemiological analyses. Transformation was used to determine the functional gene for resistance. Results Twenty-two of 23 isolates carried at least one mutation at either cluster I or III of rpoB; the most frequent mutation found in 21 isolates (91%) was histidine (H499) to asparagine or tyrosine at position 499, followed by isoleucine to valine (I624V) at position 624 in 16 isolates (70%), tyrosine to phenylalanine (Y589F) at position 589 in 14 isolates (60.9%) and isoleucine to valine (I608V) at position 608 in 13 isolates (56.5%). Less-frequent mutations were also identified: D489V, R597F, N623E, N623S, N669D, Q671K, Y674F and A683V. High-level rifampicin resistance was observed in isolates with a mutation at position 499 or 489. Mutations other than at position 499 or 489 played little role in or had no relation to rifampicin resistance. No dominant epidemic strain was observed with ribotyping, multilocus sequence typing, or serotyping. Conclusions Rifampicin resistance among multidrug-resistant S. pneumoniae in Taiwan was mostly caused by rpoB mutations.

Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC β-lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G→A at position −28) and insertion of thymidine between positions −20 and −19 of the ampC promoter gene (GenBank accession no. AE000487 ). The single nucleotide insertion of T between positions −19 and −20 created an optimal distance (17 bp) in the Pribnow box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G→A at position −28 and C→T at position +58) and a 2-base (GT) insertion between positions −14 and −15. Although the insertion of GT between positions −14 and −15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original −35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at −1, +1, +2, and +3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the −35 and −10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC β-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting.

From July, 1998, to June, 1999, pneumococcal isolates from 288 patients with invasive disease in Taiwan were serogrouped and tested for their susceptibility to various antibiotics. Automated ribotyping was used to study their molecular epidemiology. The mortality rate among thoseor = 65 years was higher than those 18 or 19-64 years (p0.001). The total incidence of infection was significantly higher during the cooler season than the warmer season (p = 0.017). Among strains isolated from children agedor = 18 years, 76% were not susceptible to penicillin, a rate that was significantly higher (p0.001) than that for adults (45%), as was the susceptibility to azithromycin, erythromycin, and trimethoprim-sulfamethoxazole (p0.005). The most prevalent serogroup encountered in the invasive isolates was 23, followed by 6, 14, 19, and 3. Isolation of Streptococcus pneumoniae in cerebrospinal fluid was at high rate in children under 5 years (p = 0.00012). Molecular typing revealed a high degree of polymorphism among the isolates. Among serogroup 23 and 19 isolates, a high proportion had the same ribotypes, the Taiwan23F-15 and Taiwan19F-14 isolates, suggesting the circulation of a Taiwanese epidemic strain. In Taiwan, S. pneumoniae isolates should be tested for their resistance profile for childrenor = 18 years old, as these are more likely to harbor high-level resistance. Control of pneumococcal infection with the 7-valent-conjugated vaccine should also be considered because it is estimated that it would cover nearly 90% of the serotypes among pediatric invasive disease.

A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM b-lactamase production. blaSHV was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of blaSHV-1 were found at positions T18A (ThrACC3AlaGCC), L35Q (LeuCTA3GluCAA), and M129V (MetATG3ValGTG) for SHV-25 and at position A187T (AlaGCC3ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillinclavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV b-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum b-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the b-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV b-lactamases in Taiwan. Extended-spectrum b-lactamases (ESBLs) are predominantly derived from plasmid-mediated TEM or SHV b-lactamases through a mutation or mutations that lead to one or more amino acid changes and that result in the alteration of the binding to and hydrolysis of specific substrates at the active site (6; G. A. Jacoby and K. Bush, http://www.lahey.org/studies /webt.htm). These enzymes, especially TEM and SHV, are most commonly found in Klebsiella pneumoniae and Escherichia coli and have been reported worldwide (6, 17, 19; Jacoby and Bush, http://www.lahey.org/studies/webt.htm). ESBLs have also recently been found in other members of the enteric bacteria (2, 8, 16). Most of these ESBLs are associated with nosocomial infections and treatment with antibiotics prior to the infection. Selective pressure has been suggested to enhance stepwise mutations in the nucleotides of classic b-lactamases (9).

An outbreak caused by rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) in an intensive care unit for cardiovascular surgery was investigated by phenotypic and genotypic methods. Fourteen isolates were collected during a 2-month period from clinical and environmental specimens in the unit recently re-opened after reconstruction. The isolates were tested for antibiotic susceptibility patterns and genotyped by automated ribotyping, randomly amplified polymorphic DNA-PCR (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Automated ribotyping applying EcoRI digestion proved to be of no value in separating the isolates. In contrast, PFGE grouped the isolates into four clusters different from the reference strain. These results fully correlated with the antibiograms. Twelve of the isolates were grouped into two clonally related clusters. RAPD analyses grouped the isolates into five clusters. Except for two isolates of one patient, which had different RAPD patterns, PFGE and RAPD analyses presented very similar results. The results verified the usefulness of PFGE in studies of MRSA epidemics. A combination of these two methods reduces the time to identification of an outbreak and increases the accuracy in detection of intraspecies differences.

Sixty-five ciprofloxacin-resistant clinical Escherichia coli isolates were collected from a Taiwan Medical Center from December 1998 to February 1999. All 65 clinical isolates were resistant (MICsor = 4 microg/mL) to the following fluoroquinolones: ofloxacin, levofloxacin, sparfloxacin, and trovafloxacin. These isolates were cross-resistant to chloramphenicol (65 isolates, 100%), tetracycline (65 isolates, 100%), cefuroxime (64 isolates, 98.5%), ampicillin (57 isolates, 87.7%), gentamicin (53 isolates, 81.5%), and cephalothin (24 isolates, 36.9%). Pulsed-field gel electrophoresis (PFGE) revealed a high diversity among the genomes of these isolates and indicated that clonal spread was not responsible for the prevalence of ciprofloxacin resistance in the hospital. Sequencing of the polymerase chain reaction (PCR) amplified products of the quinolone resistance determining regions (QRDRs) of gyrA and parC showed that all isolates carrying double mutations in gyrA at codon 83 and 87 and at least one parC mutation at codon 80 and/or 84. The mutation at codon 83 of GyrA from serine to leucine (S83L) was present in all the clinical isolates. The most prevalent pattern was the S83L mutation and the mutation at codon 87 from an aspartate to an asparagine (D87N) of GyrA plus a mutation from a serine to an isoleucine (S80I) at codon 80 of ParC (63.2%). This indicated that the presence of high-level resistance to quinolones in clinical E. coli isolates were associated with mutations at hot spots, codon 83 and 87 in GyrA and followed by subsequent mutation in either codon 80 and/or 84 in ParC.

Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were Escherichia coli and 12 were Klebsiella pneumoniae , and these isolates harbored extended-spectrum β-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum β-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four K. pneumoniae SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum β-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.

Epidemiological data suggests that ethnic groupsusing chopsticks for eating have a higher prevalence ofH. pylori infection. This study investigated thecarriage of H. pylori in chopsticks after eating. Used chopsticks and saliva were collected fromasymptomatic individuals whose H. pylori status wasdetermined by [13C]urea breath test andserology. Both the saliva specimens and chopsticks werecultured and processed by polymerase chain reaction(PCR) for the detection of H. pylori . Furthermore,chopsticks used by hospital staff in the cafeteria werepooled for the detection of H. pylori by bacteriologic culture and PCR. Sixty-nine volunteers wererecruited in the first study and 45 (65%) were diagnosedto have H. pylori infection. While all cultures werenegative, H. pylori was detected by PCR in the saliva from 15 (33%) infected subjects and in thechopsticks from one (2%). Among the 12 sets of pooledchopstick-washing studied, H. pylori was detected by PCRin two sets. This study showed that H. pylori was rarely detected in chopsticks after eating andhence, the risk of contracting this infection via theuse of chopsticks is low.

Correlation between in vitro susceptibility results for amoxicillin-clavulanate (AMC) and ampicillin-sulbactam (SAM) was studied using 136 clinical and control strains of Enterobacteriaceae harboring TEM-1, SHV-1 or OXA-1-like beta-lactamases. Determination of minimal inhibitory concentration of antibiotics was performed by agar dilution. The beta-lactamases were initially characterized using isoelectric focusing. Further identification was done by DNA hybridization with or without prior PCR amplification. All strains sensitive to SAM were found to be sensitive also to AMC. In contrast, among those susceptible to AMC, only 50% were sensitive to SAM while 36% gave intermediate results and 14% were resistant. Major differences were found solely among SHV-producers while minor differences occurred mostly among TEM-producers. This phenomenon is probably related to the differential activities of clavulanate and sulbactam against various beta-lactamases. In conclusion, testing of Enterobacteriaceae isolates for susceptibility to AMC and SAM should be performed and reported individually to avoid erroneous designation of susceptibility.

An in vivo development of Pantoea agglomerans mutants (isolates PA2 to PA4) with reduced ertapenem susceptibility from that of isolate PA1 was associated with an inadequate clinical response to ertapenem therapy. All four isolates harbored the bla ACT-9 AmpC β-lactamase gene. However, a loss-of-function mutation in the ampD gene in PA2 to PA4, but not PA1, led to derepressed ACT-9. The reduced ertapenem susceptibility caused by derepressed ACT-9 was confirmed with an ampD knockout mutant of PA1.

Between 2003 and 2009, the prevalence of extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) increased significantly in northern Taiwan from 1.0% to 2.1%. Molecular methods were used to investigate the genetic relatedness and carbapenem resistance mechanisms of a collection of 203 non-repetitive XDR-PA isolates available for study. Using pulsed-field gel electrophoresis (PFGE), 52 genotypes were observed; one predominant genotype (pulsotype 1) was found in 57.6% of the isolates. Polymerase chain reaction (PCR), sequencing and quantitative reverse-transcriptase PCR analyses demonstrated that one horizontally acquired mechanism [metallo-β-lactamase (MBL) genes] and two mutational mechanisms (efflux and porins) accounted for the carbapenem resistance. The most predominant horizontally acquired mechanism was carriage of bla(VIM-3), which was found in 61.1% of isolates. Decreased expression of oprD was the most prevalent mutational mechanism and was found in 70.0% of the XDR-PA isolates, whereas overexpression of mexA was found in 27.6% of the isolates. The highlight of this study was the discovery of statistically significant relationships between certain horizontally acquired and mutational resistance mechanisms and their contribution to carbapenem susceptibility. MBL-producers expressed significantly lower MexAB and higher OprD than non-MBL-producers. Amongst isolates without an acquired β-lactamase gene, oprD expression was significantly reduced, whilst expression of efflux pumps was increased. Reduced OprD expression alone or the production of VIM-type MBLs showed similar contributions to a low to intermediate MIC(50) (minimum inhibitory concentration for 50% of the organisms) for carbapenems. Isolates with reduced OprD expression that simultaneously harboured bla(VIM) exhibited high levels of resistance to carbapenems, which implied that these two mechanisms had a synergistic effect on the MICs.

BackgroundAlthough the prevalence of K pneumoniae liver abscess is higher in patients older than 55 years, the possible relationship of age with decreased phagocytic function of the patients with Klebsiella pneumoniae liver abscess has not been investigated. Our aim was to determine whether susceptibility to K pneumoniae infection depended on age-related impairment of phagocytic function.MethodsThe study enrolled 42 subjects in three age groups: younger than 40 years (n=10), 40–65 years (n=12), and older than 65 years (n=20). Seventy-five strains of K pneumoniae were investigated, including liver abscess isolates (n=25) and blood isolates from the patients without liver abscesses (n=50). The rate of phagocytosis of K1/K2 (n=36) and non-K1/K2 (n=39) K pneumoniae by neutrophils was determined using flow cytometry and compared among the three age groups.ResultsThe rate of phagocytosis of serotype K1/K2 isolates was significantly lower in the middle-aged group than that in the younger group (p=0.015) and significantly lower in the older group than those in the middle-aged and younger groups (p=0.025 and p

The genetic structure of β-lactamases inAcinetobactergenospecies 3 (AG3) isolates in Taiwan was studied to analyze their high rates of resistance to β-lactams, including carbapenems (57.9%).blaIMP-1andblaIMP-8were located in a class 1 integron.blaOXA-58was bracketed by ISAba3. A novel TnpF-like integrase gene was identified upstream ofblaVEB-3. Adjacent to the 5′ sequence of theblaADCgene,folEwas identified. Four newAcinetobacter-derived cephalosporinase (ADC) enzymes were found, which clustered phylogenetically with published AG3 ADC proteins.

Bla(OXA-51-like), the intrinsic carbapenemase gene in Acinetobacter baumannii previously found only in this species, was detected in a clinical isolate of Acinetobacter genomic species 13tU. this study aimed to characterize this gene in the isolate. Genomic species identification was confirmed by amplified ribosomal DNA restriction analysis and sequence analysis of 16S-23S ribosomal DNA intergenic spacer, rpoB and recA. The bla(OXA-51-like) gene, with an upstream ISAba1 insertion, was plasmid-encoded and the surrounding sequences suggested that its origin was from A. baumannii. Transformation of Acinetobacter genomic species 13TU AtCC 17903 with recombinant plasmid bearing ISAba1-bla(OXA-51-like) from the isolate increased the minimum inhibitory concentrations (MICs) of meropenem and imipenem 256-fold. This is the first report of bla(OXA-51-like) in an organism other than A. baumannii. This plasmid-borne bla(OXA-51-like) gene with an upstream ISAba1 insertion confers a high level of carbapenem resistance to Acinetobacter genomic species 13TU.

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.

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.

In this study, a rapid and simple one-tube multiplex PCR (M-PCR) method was developed for Staphylococcal Cassette Chromosome mec (SCCmec), primary screening. One hundred and eight methicillin-resistant Staphylococcus aureus (MRSA) isolates with SCC mec typing results using the Oliveira and de Lencastre method (Oliveira and de Lencastre. Antimicrob. Agents Chemother. 46:2155-2161, 2002), were evaluated for the efficacy of our method. Typing results were also compared to the recently published PCR protocol by Kondo et al., (Kondo et al., Antimicrob Agents Chemother. 51:264-74, 2007). The one-tube M-PCR method showed comparable efficacy to the other two methods. None of the isolates were classified as non-typable in Kondo's and our method. Two formerly assigned SCCmec type I isolates by Oliveira and de Lencastre were determined as SCCmec type IV by Kondo's and the method in this study. In conclusion, the one-tube M-PCR method developed in this study increased the rapidity and simplicity for primary screening SCCmec type I to V MRSA.

In this study, we investigated the distribution of genes encoding various carbapenemases as well as their association with carbapenem resistance in clinical isolates of Acinetobacter genomic species from Taiwan. A total of 129 imipenem-non-susceptible and 79 imipenem-susceptible isolates were examined, of which 185 (88.9%) were Acinetobacter baumannii. Among the 185 A. baumannii isolates, imipenem non-susceptibility was more common in isolates with IS Aba1–bla OXA-51-like (72/75; 96%), bla OXA-58-like (33/33; 100%) or bla OXA-24-like (7/7; 100%) than in isolates with only bla OXA-51-like (4/72; 5.6%). A metallo-β-lactamase (MBL) gene was present in two isolates of imipenem-resistant A. baumannii , and bla OXA-58-like was also present in these isolates. A total of 18% and 1% of imipenem-non-susceptible isolates of A. baumannii were resistant to tigecycline and colistin, respectively. Among the 23 isolates of non- baumannii Acinetobacter spp., bla OXA-58-like and MBL genes were widely disseminated in the imipenem-resistant isolates, and isolates with bla OXA-58-like and MBL genes had higher imipenem minimum inhibitory concentrations than those with bla OXA-58-like alone. Although the rate of non-susceptibility to colistin was 26.7% among the imipenem-non-susceptible isolates of non- baumannii Acinetobacter , 93.3% and 100% were susceptible to ciprofloxacin and tigecycline, respectively. In conclusion, different isolates of imipenem-non-susceptible A. baumannii and non- baumannii Acinetobacter contained different carbapenemases and had different antimicrobial susceptibilities.

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.

Since the means of culturing Helicobacter pylori may not be available in some laboratories, prolonging the survival of this organism during transportation is a major concern in terms of improving detection rates. A selective transport medium was evaluated for the preservation of H. pylori from 254 gastric biopsy specimens collected from a rural area in China where culturing is not feasible. Gastric biopsy specimens were inoculated in sterile broth consisting of brain heart infusion (BHI) broth, horse serum, and yeast extract supplemented with vancomycin, amphotericin B, and nalidixic acid (VAN). Of the 254 biopsy specimens, 238 were identified by histology to have H. pylori infection. Total rates of recovery of H. pylori from the H. pylori -positive gastric biopsy specimens stored in the BHI-VAN broth ranged from 76 to 46% after storage of specimens for 5 to 9 days. In conclusion, the selective medium is useful for prolonging the survival of H. pylori in gastric biopsy specimens for which immediate culture is not feasible.

Hyperproductionof b-lactamaseswasfoundtobeanimportant cause of resistance to b-lactam‐b-lactamase inhibitors among clinical isolates of the family Enterobacteriaceae. For example, hyperproduction of the plasmid-encoded b-lactamase TEM-1 was reported to be the predominant mechanism of resistance to amoxicillin-clavulanate in about 60% ofEscherichia coli strains (13, 19). Three mechanisms may lead to hyperproduction. First, an increase in the gene dosage as a result of repetitions of b-lactamase genes; e.g., repetition of thewild-typeallelesofthegeneencodingAmpC(17),hasbeen known to confer a high level of resistance to amoxicillin-clavulanate. Second, multiple copies of the blaTEM gene due to the presence of multiple copies of plasmid per cell have been reported to cause hyperproduction of b-lactamase (21). Third, hyperproduction of enzyme due to various genetic events affecting the promoter region has been widely reported in various bacteria (2, 3, 10), although this mechanism was much less studied for b-lactamases. We have isolated in Hong Kong twoShigellaflexneristrains resistant to ampicillin-sulbactam due to the hyperproduction of TEM-1 b-lactamase, the gene of which was encoded on a large 120-kb plasmid and was transferable toE. coliby conjugation. The purpose of the present study was to find the molecular basis of TEM-1 hyperproduction by isolating and analyzingtheplasmidDNAandsequencingthe b-lactamasegenes from the two strains. The results thus obtained were compared with those for a TEM-1-producing, ampicillin-sulbactam-susceptible S. flexneri strain in which the blaTEM gene was also found to be encoded on a 120-kb conjugatively transferable plasmid. The strains used in this study and their characteristics are listedinTable1.TheMICsofampicillin,ampicillin-sulbactam, amoxicillin, and amoxicillin-clavulanate were determined by the agar dilution method (15). The ampicillin-sulbactam and amoxicillin-clavulanate combinations were tested at a fixed ratio of 2:1. Antibiotic powders were kindly provided by Pfizer Corporation, Hong Kong (ampicillin and sulbactam), and SmithKline Beecham, Hong Kong (amoxicillin and clavulanate). Susceptibility testing of sulfamethoxazole-trimethoprim, chloramphenicol, tetracycline, and rifampin were performed by the Kirby-Bauer disk diffusion method (16). A rifampin-resistantE. colistrain, strain JP-995 (kindly supplied

Salmonella enterica serotype Panama is capable of causing severe infection in children and is often transmitted via contaminated food. Here, we present the first documented case of serotype Panama infection that was acquired through the consumption of contaminated breast milk. The mother excreted the organism asymptomatically for at least 2 weeks.

Objectives To describe the immunological responses and clinical outcome of coronavirus (SARS) infected healthcare workers (HCW) who had been administered with convalescent plasma as a treatment. Methods Convalescent plasma (500 mL) was obtained from each of three SARS patients and transfused into the three infected HCW. Donors were blood type O and seronegative for hepatitis B and C, HIV, syphilis and human T-cell lymphotropic virus types I and II (HTLV-I and -II). Serum antibody (IgG) titre was >640. Apharesis was performed with a CS 3000 plus cell separator followed by the forming of the convalescent phase plasma. As part of the routine check with donated plasma, the convalescent plasma was confirmed free of residual SARS-CoV by RT-PCR. Serial serum samples obtained from the recipients of the convalescent plasma were collected to undertake real-time quantitative RT-PCR for SARS-CoV for direct measurement of viral concentration. Specific immunoglobulin IgM and IgG concentrations were titrated using an antigen microarray developed in-house. Results Viral load dropped from 495 x 10(3), 76 x 10(3) or 650 x 10(3) copies/mL to zero or 1 copy/mL one day after transfusion. Anti-SARS-CoV IgM and IgG also increased in a time-dependent manner following transfusion. All three patients survived. One HCW became pregnant subsequently, delivering 13 months after discharge. Positive anti-SARS-CoV IgG was detected in the newborn. Passive transfer of anti-SARS-CoV antibody from the mother was considered as a possibility. Conclusions All infected HCW whose condition had progressed severely and who had failed to respond to the available treatment, survived after transfusion with convalescent plasma.

A new SHV-derived extended-spectrum β-lactamase, SHV-57, that confers high-level resistance to ceftazidime but not cefotaxime or cefazolin was identified from a national surveillance study conducted in Taiwan in 1998. An Escherichia coli isolate resistant to ampicillin, cephalothin, and ceftazidime but sensitive to cefoxitin, ceftriaxone, cefotaxime, imipenem, and a narrow-spectrum cephem (cefazolin) was isolated from the urine of a patient treated with β-lactam antibiotics. Resistance to β-lactams was conjugatively transferred with a plasmid of about 50 kbp. The pI of this enzyme was 8.3. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 861 bases (GenBank accession number AY223863 ). Kinetic parameters showed that SHV-57 had a poor affinity to cefazolin. The K m value toward cefazolin (5.57 × 10 3 μM) was extremely high in comparison to those toward ceftazidime (30.9 μM) and penicillin G (67 μM), indicating its low affinity to cefazolin. Although the K m value of the β-lactamase inhibitor was too high for the study of catalytic activity ( k cat ), indicating the low k cat of SHV-57, the SHV-57 carrier was highly susceptible to a β-lactam-β-lactamase inhibitor combination. Comparison of the three-dimensional molecular model of SHV-57 with that of the SHV-1 β-lactamase suggests that the substitution of arginine for leucine-169 in the Ω loop is important for the substrate specificity.

Between 1998 and 2000, we characterized 91 nosocomial isolates of Acinetobacter baumannii by antibiotyping and genotyping. A total of 25 ribotypes were obtained among these 91 isolates. When the isolates from surgical intensive care units (ICUs) and other wards were compared, multiresistant A. baumannii isolates with the same ribotype 25 (R-25) were significantly more prevalent in nosocomial infections among the surgical patients. Further subtyping of the strains by pulsed-field gel electrophoresis confirmed that strains of the same ribotype in surgical ICUs and a few isolates from other wards were identical or clonally related. Different antibiotic resistance profiles were observed among these R-25 isolates. All R-25 isolates contained intI1 integrase, and two clusters of integron cassettes were found. These clusters of cassettes were encoded by an open reading frame (ORF) of -5'CS-aac(3)Ia-aadA1a-unknown or f-3'CS or 5'CS-aacA4-aadA1-catB8-3'CS, indicating the involvement of different resistant genes. Two isolates contained bla (IMP-1), which was acquired from a conjugatively transferable plasmid and did not involve integron-associated resistance. In conclusion, an epidemic of nosocomial infections associated with A. baumannii strains that have different resistance profiles was identified. Resistance profiles can change by a combination of plasmid- and integron-associated acquisition, especially in a unit with high antibiotic selective pressures. Infectious control personnel should be alert to the change in resistance profiles during routine monitoring.

Four Escherichia coli isolates harboring CTX-M-14, with a single Ala231→Val substitution compared to CTX-M-9, had three different ribotypes. Cefotaxime resistance was plasmid encoded and conjugatively transferable. Three isolates had the same plasmid restriction enzyme digestion profile, suggesting clonal spread of a resistant plasmid. A high k cat / K m value for cefotaxime (20.3 μM −1 s −1 ) but low values for ceftazidime and aztreonam (−1 s −1 ) were observed in hydrolysis assays, indicating resistance to cefotaxime (MIC ⩾ 64 μg/ml) but susceptibility to ceftazidime (MIC ⩽ 2 μg/ml).

Although several epidemiological surveys of Klebsiella clinical isolates have been performed, few studies have correlated the clinical isolate with disease.To compare the clinical and bacteriological characteristics of Klebsiella pneumoniae bacteremia acquired as community or nosocomial infections.We prospectively enrolled 158 consecutively hospitalized patients with K pneumoniae bacteremia. Clinical data were reviewed. Antimicrobial susceptibility testing and capsular serotyping were performed. We used the chi(2) test, the Fisher exact test, or the t test for statistic analysis.Underlying diabetes mellitus was more common in community-acquired than in nosocomial infection (46/94 [49%] vs. 8/64 [12%]; P.001). On the other hand, neoplastic disease (34/64 [53%] vs. 13/94 [14%]; P.001) and antibiotic resistance (P.01) were more frequent in patients with nosocomial compared with community-acquired infections. Klebsiella pneumoniae liver abscesses, which were all community acquired, accounted for the source of 22 (23%) of 94 community-acquired K pneumoniae infections. No attributable source of infection was found for 37 (58%) of the 64 nosocomial infections vs. 15 (16%) of the 94 community-acquired infections. Only 58 isolates (36.7%) could be serotyped; of these, capsular serotypes K1, K2, and K28 accounted for 37 (23.4%), 8 (5.1%), and 6 (3.8%), respectively, of all strains. However, typeable isolates were significantly more common among community-acquired than nosocomial isolates (42/94 [45%] vs. 16/64 [25%]; P =.01), especially for serotype K1 (28/94 [30%] vs. 9/64 [14%]; P =.02). Significant risk factors for mortality included nosocomial infection, lung infection, thrombocytopenia, leukopenia, ceftazidime resistance, inappropriate antimicrobial therapy, and septic shock.Significant differences were identified between community-acquired and nosocomial K pneumoniae bacteremia. Ceftazidime resistance in nosocomial K pneumoniae bacteremia carried a high risk for mortality, and serotype K1 in K pneumoniae was more prevalent in community-acquired infection, suggesting more virulence.

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.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.Serotyping revealed that the most common serotypes were K1 (63.4%) and K2 (14.2%). K1 isolates occurred at a significantly higher frequency (p0.01) 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.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.