Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Cholera Toxin Production in Vibrio cholerae O1 El Tor Biotype Strains in Single-Phase Culture.

Vibrio cholerae O1 serogroup strains have been classified into classical and El Tor biotypes. Cholera, a life-threatening diarrheal disease, can be caused by either biotype through the cholera toxin (CT) that they produce. To increase our knowledge of the pathogenicity of bacteria, we must understand the toxigenicity of bacteria. CT production by classical biotype strains in simple single-phase cell cultures has been established; however, special culture media and growth conditions that are not appropriate for mass production of CT are required to facilitate CT production in El Tor biotype strains. In this report, we produced CT in El Tor biotype strains using simple media and single-phase culture conditions. A single point mutation in ToxT, a transcriptional activator of toxin co-regulated pilus (TCP) and CT, enabled the El Tor biotype strains to produce CT in similar quantities as classical biotype strains in single-phase laboratory culture conditions. CT production capacity varied between El Tor biotype strains. Wave 2 and 3 atypical El Tor strains tended to produce more CT than prototype Wave 1 strains. Wave 2 and 3 strains lack neutral fermentation; however, the capacity for neutral fermentation was not associated with significant differences in CT production by El Tor biotype strains. The Wave 3 strain that caused the 2010 cholera outbreak in Haiti produced CT only when neutral fermentation was abolished. The disparity in CT production between the seventh cholera pandemic strains highlight the differences in virulence between strains and the cause of population changes in V. cholerae.

Alterations in glucose metabolism in Vibrio cholerae serogroup O1 El Tor biotype strains.

The 2 biotypes of Vibrio cholerae O1 serogroup strains-classical and El Tor-use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.