Repeated centrifuging and washing concentrates bacterial samples in peritoneal dialysis for optimal culture: an original article.

BACKGROUND: Bacterial cultures allow the identification of infectious disease pathogens. However, obtaining the results of conventional culture methods is time-consuming, taking at least two days. A more efficient alternative is the use of concentrated bacterial samples to accelerate culture growth. Our study focuses on the development of a high-yield sample concentrating technique. RESULTS: A total of 71 paired samples were obtained from patients on peritoneal dialysis (PD). The peritoneal dialysates were repeat-centrifuged and then washed with saline, namely the centrifuging and washing method (C&W method). The concentrated samples were Gram-stained and inoculated into culture plates. The equivalent unprocessed dialysates were cultured as the reference method. The times until culture results for the two methods were compared. The reference method yielded no positive Gram stain results, but the C&W method immediately gave positive Gram stain results for 28 samples (p < 0.001). The culture-negative rate was lower in the C&W method (5/71) than in the reference method (13/71) (p = 0.044). The average time for bacterial identification achieved with the C&W method (22.0 h) was shorter compared to using the reference method (72.5 h) (p < 0.001). CONCLUSIONS: The C&W method successfully concentrated bacterial samples and superseded blood culture bottles for developing adequate bacterial cultures. The C&W method may decrease the culture report time, thus improving the treatment of infectious diseases.

Diagnosis of Bacterial Pathogens in the Urine of Urinary-Tract-Infection Patients Using Surface-Enhanced Raman Spectroscopy.

(1) Background: surface-enhanced Raman spectroscopy (SERS) is a novel method for bacteria identification. However, reported applications of SERS in clinical diagnosis are limited. In this study, we used cylindrical SERS chips to detect urine pathogens in urinary tract infection (UTI) patients. (2) Methods: Urine samples were retrieved from 108 UTI patients. A 10 mL urine sample was sent to conventional bacterial culture as a reference. Another 10 mL urine sample was loaded on a SERS chip for bacteria identification and antibiotic susceptibility. We concentrated the urine specimen if the intensity of the Raman spectrum required enhancement. The resulting Raman spectrum was analyzed by a recognition software to compare with spectrum-form reference bacteria and was further confirmed by principal component analysis (PCA). (3) Results: There were 97 samples with single bacteria species identified by conventional urine culture and, among them, 93 can be successfully identified by using SERS without sample concentration. There were four samples that needed concentration for bacteria identification. Antibiotic susceptibility can also be found by SERS. There were seven mixed flora infections found by conventional culture, which can only be identified by the PCA method. (4) Conclusions: SERS can be used in the diagnosis of urinary tract infection with the aid of the recognition software and PCA.

Diagnosis of bacterial pathogens in the dialysate of peritoneal dialysis patients with peritonitis using surface-enhanced Raman spectroscopy.

BACKGROUND: Bacterial peritonitis is the most common cause of peritoneal dialysis (PD) therapy drop-out. A quick and accurate diagnosis of the bacterial pathogen can reduce the PD drop-out rate. Surface-enhanced Raman spectroscopy (SERS) can rapidly identify bacteria using chips coated with nano-sized metal particles. METHODS: Known bacteria were loaded in the SERS-chips and illuminated with laser light to establish a reference Raman spectra library. Dialysate from PD peritonitis patients was concentrated by centrifuge and examined with the same SERS, and the resulting Raman spectra were compared with library spectra for bacteria identification. Principal component analysis was used for further confirmation. The same batches of dialysate were sent to routine culture as a reference bacteria identification method. The results of the 2 identification methods were compared. RESULTS: A total of 43 paired-samples were sent for study. There were 37 samples with bacteria identified but 6 were culture-negative by the reference method. 31 bacteria were identified in paired-samples by SERS, among which, 29 bacteria were exactly the same as those identified by the reference method. Bacteria not included in the reference library spectra cannot be identified. CONCLUSIONS: SERS techniques can rapidly identify bacterial pathogens in the dialysate of PD peritonitis patients.

The medical diagnostic approaches with phylogenetic analysis for rare Brucella spp. diagnosis in Taiwan.

Brucellosis is a bacterial zoonotic disease which can be easy to misdiagnose in clinical microbiology laboratories. In the present study, we have tried to improve the current clinical method for detecting Brucella spp. and its antibiotic characteristics. Our method begins with detecting the clinical isolate through traditional biochemical methods and automatic identification systems. Then, we move on to editing the sequence for BLAST allows us to compare 16s rRNA sequences with sequences from other species, allowing the gene level to be determined. Next, the phylogenetic analysis of multiple genetic loci is able to determine the evolutionary relationships between our bacteria strain and those from other locations. Finally, an anti-microbial susceptibility test hones in on the level of antibacterial activity that the bacteria displays. Employing these four steps in concert is extremely effective in identifying rare bacteria. Thus, when attempting to determine the identity of rare bacteria such as Brucella, utilizing these four steps from our research should be highly effective and ultimately prevent further identification errors and misdiagnoses. The standards we have suggested to identify rare bacteria strains is applicable not only to Brucella, but also to other rarely encountered bacteria.

Comparison of genospecies and antimicrobial resistance profiles of isolates in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex from various clinical specimens.

This study was conducted to compare the prevalences of antimicrobial resistance profiles of clinical isolates in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex from sterile and nonsterile sites and to further study the relationship of antimicrobial resistance profiles and genospecies by amplified rRNA gene restriction analysis (ARDRA). A total of 1,381 isolates were tested with 12 different antibiotics to show their antimicrobial susceptibility profiles. A total of 205 clinical isolates were further analyzed by ARDRA of the intergenic spacer (ITS) region of the 16S-23S rRNA gene. It was found that the overall percentage of isolates from nonsterile sites (urine, sputum, pus, or catheter tip) that were resistant to the 12 antibiotics tested was significantly higher than that of isolates from sterile sites (cerebrospinal fluid [CSF], ascites fluid, and bloodstream) (46% versus 22%; P < 0.05). After ARDRA, it was found that 97% of the 62 isolates resistant to all antibiotics tested were the A. baumannii genospecies, which was identified in only 31% of the isolates susceptible to all antibiotics tested. More genospecies diversity was identified in the isolates susceptible to all antibiotics tested, including genospecies of 13TU (34%), genotype 3 (29%), and A. calcoaceticus (5%). Furthermore, as 91% (10/11) of the isolates from CSF were susceptible to all antibiotics tested, the A. calcoaceticus-A. baumannii complex isolates with multidrug resistance could be less invasive than the more susceptible isolates. This study also indicated current emergence of carbapenem-, fluoroquinolone-, aminoglycoside-, and cephalosporin-resistant A. calcoaceticus-A. baumannii complex isolates in Taiwan.

Multilocus sequence typing of invasive group B Streptococcus in central area of Taiwan.

BACKGROUND: Group B Streptococcus (GBS) (Streptococcus agalactiae) is an important pathogen in neonates, pregnant women, and adults with underlying disease. METHODS: Fifty clinical isolates were collected during the period 2001-2004 and analyzed by multilocus sequence typing and capsular serotyping. RESULTS: The six major sequence types (STs) identified by multilocus sequence typing were ST1, ST12, ST19, ST17, ST23, and ST10. Five major clonal complexes (CCs) and one single ST (ST61) from 11 different STs were found. CC1 (n=14) was the most common one, followed by CC12 (n=13), CC19 (n=9), CC17 (n=7), and CC23 (n=6). The most common serotypes were serotype III, followed by Ib, V, Ia, and IV. The most invasive strains in adults belonged to ST1 (CC1) and serotype V, and those in neonates belonged to ST17 (CC17) and serotype III. In addition, ST19 was distributed in adults and neonates. CONCLUSIONS: These results are similar to those of previous reports, but some geographic differences were found, suggesting that limited clonal lineages play important roles in GBS-associated diseases worldwide. Continued tracking of GBS in the population through clinical isolates is important for epidemiologic investigations and vaccine development.