Objectives
The global spread and increasing incidence of carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) has made its treatment difficult, increasing the mortality. To establish nationwide data on CnSKP spread and carbapenem-resistance mechanisms, we conducted a national surveillance study in Taiwanese hospitals.

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National Surveillance Study on Carbapenem Non-Susceptible Klebsiella pneumoniae in Taiwan: The Emergence and Rapid Dissemination of KPC-2 Carbapenemase

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases, and the prevalence of multidrug-resistant K. pneumoniae (MDRKP) is rapidly increasing. Here we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains by wzc sequencing and investigated the presence of carbapenemases and integrons among CRKP strains. Ten CRKP strains (12%) were positive for carbapenemase (imipenemase, 6/85 strains; K. pneumoniae carbapenemase, 3/85 strains; Verona integron-encoded metallo-β-lactamase, 1/85 strains). Capsular type K64 accounted for 32 CRKP strains (38%), followed by K62 (13%), K24 (8%), KN2 (7%), and K28 (6%). Sequence types (STs) were determined by multilocus sequence typing (MLST), and the results indicated that ST11, which accounted for 47% of these CRKP strains (40/85 strains), was the major ST. We further isolated a K64-specific capsule depolymerase (K64dep), which could enhance serum and neutrophil killing in vitro and increase survival rates for K64 K. pneumoniae-inoculated mice. The toxicity study demonstrated that mice treated with K64dep showed normal biochemical parameters and no significant histopathological changes of liver, kidney, and spleen, indicating that enzyme treatment did not cause toxicity in mice. Therefore, the findings of capsular type clustering among CRKP strains and effective treatment with capsule depolymerase for MDRKP infections are important for capsule-based vaccine development and therapy.

Identification of Capsular Types in Carbapenem-Resistant Klebsiella pneumoniae Strains by wzc Sequencing and Implications for Capsule Depolymerase Treatment

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

Widespread Dissemination of Aminoglycoside Resistance Genes armA and rmtB in Klebsiella pneumoniae Isolates in Taiwan Producing CTX-M-Type Extended-Spectrum β-Lactamases

Nosocomial infections caused by Acinetobacter baumannii often prove difficult to treat owing to their multiple drug resistance. Carbapenems play a pivotal role in the management of severe Acinetobacter infections. However, reports of carbapenem resistance have been increasing alarmingly due to production of a variety of carbapenemases including metallo-beta-lactamases (MBLs). This study investigated by both phenotypic and genotypic assays the prevalence of MBLs in a total of 55 A. baumannii strains isolated from a South Indian tertiary care hospital. Random amplified polymorphic DNA (RAPD) genotyping and antimicrobial susceptibility testing for nine clinically relevant antibiotics was done for characterization of isolates. Phenotypic expression of MBLs was examined by a simple double disc synergy (DDS) test, and the presence of the most frequent MBL coding genes, bla(IMP1) and bla(VIM2), was checked by PCR. RAPD analysis generated six clusters of isolates and there was very little correlation between RAPD clusters and resistant profiles. Most of the isolates showed complete or high resistance to imipenem (100 %), meropenem (89 %), amikacin (80 %), cefotaxime (89 %) and ciprofloxacin (72 %). In addition, 44 % of isolates showed a high MIC level (> or =16 microg ml(-1)) for meropenem. Thirty-nine isolates (70.9 %) were positive for MBL production by the DDS test while bla(IMP1) gene amplification was seen only in 23 isolates (42 %). Interestingly, none of the isolates showed amplification of bla(VIM2). Further investigations on DDS-positive/PCR-negative isolates by spectrophotometric assay showed MBL activity in most of the isolates, suggesting involvement of other genes. The high incidence of isolates possessing MBL activity in the present study represents an emerging threat of complete resistance to carbapenems among Acinetobacter spp. in India.

Phenotypic and genotypic assays for detecting the prevalence of metallo-β-lactamases in clinical isolates of Acinetobacter baumannii from a South Indian tertiary care hospital

Objective:To evaluate three different DNA techniques for typing nonfermentative gram-negative bacilli isolated from Latin American hospitals.Design:One hundred twenty-six nonfermentative gram-negative bacilli were typed.Participants:Pseudomonas aeruginosa (n = 64) and Acinetobacter baumannii (n = 42) samples were obtained from blood cultures of patients admitted to 10 medical centers in Latin America during 1998 and Stenotrophomonas maltophilia (n = 20) samples were obtained from patients admitted to the Hospital Sao Paulo between 1999 and 2001.Methods:All samples were typed using automated ribotyping, PFGE, and ERIC-PCR. The discriminatory power for each technique was calculated using Hunter's generalized formula.Results:All strains could be typed by automated ribotyping and ERIC-PCR, but two strains (1.6%) were not typeable by PFGE. All three techniques showed 100% reproducibility. The time to obtain the results was shorter for automated ribotyping and ERIC-PCR compared with PFGE. Likewise, the costs for ERIC-PCR and PFGE were lower than those for automated ribotyping. The interpretation of results was more complicated and more difficult with ERIC-PCR than with both PFGE and automated ribotyping. All techniques presented excellent discriminatory power for P. aeruginosa (0.98). PFGE presented the highest discriminatory power (0.94) for A. baumannii, and both PFGE and ERIC-PCR showed higher discriminatory power (0.90 for both) than automated ribotyping (0.82) for S. maltophilia.Conclusions:PFGE showed the highest discriminatory power for typing these nonfermentative gram-negative bacilli. However, automated ribotyping and ERIC-PCR can provide results in a shorter time period with similar discriminatory power.

Evaluation of three molecular typing techniques for nonfermentative Gram-negative bacilli.

Objective:To compare molecular typing by amplified fragment-length polymorphism (AFLP) analysis with pulsed-field gel electrophoresis (PFGE) with respect to the ability to differentiate between epidemiologically related and unrelated isolates of common nosocomial pathogens recovered during a period of endemicity.Design:Retrospective laboratory analysis.Setting:Tertiary-care institution.Methods:17 isolates of Acinetobacter baumannii, 22 isolates of Pseudomonas aeruginosa, and 22 vancomycin-resistant Enterococcus faecium (VRE) were typed by both methods.Results:AFLP generated comparable results to PFGE for A baumannii and P aeruginosa isolates; both methods identified epidemiologically related and unrelated isolates. However, strain typing of VRE isolates produced discordant results between the two methods. PFGE identified 10 different strain types and differentiated between all epidemiologically related and unrelated isolates. In contrast, AFLP generated only five different strain types, three of which contained both epidemiologically related and unrelated isolates.Conclusion:Molecular typing by AFLP is comparable to PFGE for A baumannii and P aeruginosa isolates. For VRE isolates, however, PFGE remains the method of choice.

Comparison of pulsed-field gel electrophoresis and amplified fragment-length polymorphism for epidemiological investigations of common nosocomial pathogens

Monique M. Gerrits

Papers

Comparison of pulsed-field gel electrophoresis and amplified fragment-length polymorphism for epidemiological investigations of common nosocomial pathogens