Use of inverse PCR for analysis of class 1 integrons carrying an unusual 3' conserved segment structure.

By using inverse PCR and DNA sequencing, 13 sul3-associated mutational integrons, 2 defective class 1 integrons, and 1 qnrB2-associated complex sul1-type class 1 integrons were identified in Salmonella enterica serovar Choleraesuis, Pseudomonas aeruginosa, and Enterobacter cloacae, respectively. In addition, conjugation and Southern hybridization demonstrated that unusual class 1 integrons were located on plasmids or integrated into chromosomal DNA. Thus, an inverse PCR assay can be a valuable tool for the analysis of unusual structures of the 3' conserved region of class 1 integrons.

Molecular characterisation of the metallo-beta-lactamase genes in imipenem-resistant Gram-negative bacteria from a university hospital in southern Taiwan.

In this study, 260 non-replicate imipenem-resistant Gram-negative bacteria isolated between January 2002 and December 2006 were subjected to a screening test for detection of metallo-beta-lactamase (MBL) using the Etest containing imipenem and ethylene diamine tetra-acetic acid (EDTA). Among the 260 strains, 123 (47.3%) appeared to produce MBL. Of these 123 strains, 113 (91.9%) were found by polymerase chain reaction (PCR) to carry MBL genes of types blaVIM-2, blaVIM-3, blaVIM-11 (blaVIM-11a), blaIMP-8 and novel blaIMP-24. One strain of Serratia marcescens harboured two MBL genes (blaVIM-11 and blaIMP-8) simultaneously. Of the 123 strains, 116 strains (94.3%) carrying the intI1 gene and 21 strains carrying integron-associated blaVIM-3, blaVIM-11 and blaIMP-8 genes were identified among Acinetobacter baumannii, Pseudomonas aeruginosa, Acinetobacter haemolyticus and S. marcescens. Using pulsed-field gel electrophoresis (PFGE) and Southern hybridisation with the blaVIM gene probe for I-CeuI-digested genomic DNA, P. aeruginosa 9527 strain harboured two class 1 integron-associated MBL genes in the chromosome, including blaVIM-3-orf2-aacA4 and novel bla(VIM-3)-orf2-aacA4-aadB-aacA4. This is the first description of the blaVIM-11 gene spreading among P. aeruginosa and A. baumannii strains in southern Taiwan. This finding suggests that clinical spread of this blaVIM-11 gene is a matter of great concern for carbapenem resistance in southern Taiwan.

Diagnosis of fusariosis in urine cytology.

Fusarium is a filamentous fungus widely distributed in plants and in the soil. Most species are more common at tropical and subtropical areas. Besides being a common contaminant and a well-known plant pathogen, Fusarium sp may cause various infections in humans. However, it has not yet been reported as being the pathogen of urinary tract infection. A 67-year-old woman had extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy for renal stones 7 and 6 years ago, respectively. She had had fever, chillness, urinary urgency and frequency for 6 days. Routine testing of urine showed numerous leucocytes. She was admitted under the impression of urinary tract infection. On admission, many spindle-shaped structures were found in the urine smears. This shows that Fusarium was identified. Fusarium may be the pathogen of the urinary tract infection, particularly when urolithiasis is present.

Characterization of class 1 integrons and antimicrobial resistance in CTX-M-3-producing Serratia marcescens isolates from southern Taiwan.

The objective of this study was to characterize the gene cassettes of class 1 integrons and antimicrobial resistance among CTX-M-3-producing Serratia marcescens isolates from different specimens in southern Taiwan. One hundred and twenty-two isolates (70.5%) of 173 CTX-M-3-producing S. marcescens isolates were positive for class 1 integrons, including 53.3% of blood isolates, 94.1% of urine isolates, and 87.2% of sputum isolates. No class 2 or class 3 integrons were detected in this study. By PCR with primers 5'-CS and 3'-CS for the amplification of gene cassettes regions, amplicons ranging from 0.7 to 3.0 kb in length were found in 108 (88.5%) of the 122 class 1 integron-containing isolates of CTX-M-3-producing S. marcescens isolates. Ten different types by pattern of amplicons for class 1 integrons were obtained. The Type I amplicon (46.3%) harbors two different class 1 integrons containing the gene cassette of aadA2 and aadB-catB3, respectively, and was most prevalent in the gene cassette region-positive S. marcescens isolates, followed by the Type II amplicon, which harbors one class 1 integron containing the gene cassette dfrA12-orfF-aadA2 (28.7%). Most of the S. marcescens isolates (66.7%, 8/12) harboring three different class 1 integrons (Type IV amplicon) were found in blood isolates. Class 1 integrons were conjugally transferred to recipients in 92.0% of S. marcescens harboring two different class 1 integrons containing the gene cassettes aadA2 and aadB-catB3, respectively. The transfer rate of class 1 integron carrying dfrA12-orfF-aadA2 was detected in 77.4% of S. marcescens isolates. The results showed that all those isolates with conjugative transfer of integrons carried their class 1 integrons on the conjugative plasmids.

Prototheca algaemia: a rare but fatal opportunistic infection among immunocompromised individuals.

Infections due to Prototheca spp. are ubiquitous in nature, occurring in both immunocompetent and immunocompromised patients. The study cohort consisted of 14 cases of Prototheca algaemia reported over the past 5 decades and 2 recent cases from study hospitals. Prototheca wickerhamii was the most common species. The overall mortality rate was 62.5%. Prototheca algaemia, a healthcare-associated infection, was observed in immunocompromised patients and was associated with a poor prognosis.

Efficacy of combination oral antimicrobial agents against biofilm-embedded methicillin-resistant Staphylococcus aureus.

BACKGROUND: The combination of fusidic acid and rifampicin has a demonstrated synergistic effect against methicillin-resistant Staphylococcus aureus (MRSA), including planktonic and biofilm-related organisms. However, the in vitro efficacy of other combinations of oral anti-MRSA antibiotics in biofilm models has not been established. METHODS: The antibacterial activity of fusidic acid, linezolid, rifampicin, and minocycline against 33 biofilm-embedded MRSA isolates in low susceptibility and high resistance breakpoint concentrations was investigated using the 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide staining method. The compounds were further examined to determine their antibacterial efficacies in combination. The optical density ratio (ODr) was used to evaluate the antibacterial effects of these antibiotics, and the results indicate higher survival rates of MRSA on biofilm. A biofilm-positive phenotype (determined using the crystal violet stain) was defined as an optical density ≥ 0.17 at 492 nm, and strong biofilm formation was defined as an optical density ≥ 1.0. RESULTS: One-third of the MRSA isolates demonstrated weak biofilm formation, and two-thirds demonstrated strong biofilm formation. At low concentrations, linezolid alone lowered the ODr to 0.55 and was effective against biofilm-embedded MRSA (p < 0.001). The activity of minocycline was concentration-dependent and more effective against MRSA isolates that demonstrated weak biofilm formation. The effect of minocycline seems to be further enhanced when used in combination with either fusidic acid or linezolid at low concentrations, with the obtained results equal to those obtained with rifampicin-based regimens (p < 0.001). Rifampicin plus minocycline was also effective against MRSA in biofilm. CONCLUSION: In comparison with monotherapy, minocycline-based combinations exhibit highly effective bactericidal effects against biofilm-embedded MRSA.

Brevundimonas vesicularis bacteremia resistant to trimethoprim-sulfamethoxazole and ceftazidime in a tertiary hospital in southern Taiwan.

BACKGROUND: Over the past 20 years, Brevundimonas vesicularis has rarely been reported as a pathogen causing human infection. The clinical manifestations of B. vesicularis bacteremia and its susceptibility to antibiotics has not been characterized. METHODS: A retrospective study was conducted between 2006 and 2009 in a tertiary-care hospital in southern Taiwan. RESULTS: A total of 22 cases of B. vesicularis bacteremia were identified during the study with 86% being community-acquired primary bloodstream infections. Of the 22 patients, 15 (68%) presented with fever, fewer comorbidities, shorter hospital stays, lower mean creatinine levels (1.10 mg/dL vs. 1.74 mg/dL), lower aspartate aminotransferase levels (29.1 IU/L vs. 79.0 IU/L), and lower alanine aminotransferase levels (16.4 IU/L vs. 67.0 IU/L) when compared to afebrile patients. Among the bacterial isolates, 90.9% were susceptible to cefpirome, imipenem and piperacillin/tazobactam while 86.4% were susceptible to gentamicin, amikacin and ciprofloxacin. However, 63.6% of the bacterial isolates were susceptible to ceftazidime, and only 59.1% were susceptible to trimethoprim-sulfamethoxazole (TMP-SMX). The 30-day mortality rate from all causes was 4.5%. CONCLUSION: B. vesicularis is able to cause community-acquired and low-mortality primary bloodstream infections. The resistance of B. vesicularis to trimethoprim-sulfamethoxazole and ceftazidime limits the choice of available antibiotics for treatment.

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.