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Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J - Front Microbiol (2015)

Bottom Line: Klebsiella pneumoniae is a wide-spread nosocomial pathogen.The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR.In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus

Specificity of the LAMP reaction in detectingK. pneumoniae. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. Amplification was performed at 61°C for 60 min. 1, positive control (K. pneumoniae ATCC BAA-2146); 2, negative control (double-distilled water); 3, Klebsiella oxytoca ATCC 700324; 4, Klebsiella rhinoscleromatis CMCC 46111; 5, Citrobacter freundii CMCC 48001; 6, Enterobacter aerogenes ATCC 13048; 7, Enterobacter cloacae ATCC 13047; 8, Proteus mirabilis CMCC 49005; 9, Proteus vulgaris CMCC 49027; 10, Serratia marcescens ATCC 14756; 11, Morganella morganii ATCC 25830; 12, Streptococcus pneumoniae 112-07; 13, Mycobacterium tuberculosis 005; 14, Pseudomonas aeruginosa D104; 15, Haemophilus influenza ATCC 49247; 16, Yersinia enterocolitica 027; 17, Yersinia pestis 2638; 18, Bacillus tularense 3450; 19, Vibrio cholera 3802; 20, Salmonella aberdeen 9264; 21, Neisseria meningitides CMCC 29022; 22, Staphylococcus aureus 2740; 23, Pseudomonas pseudomallei 029; 24, Salmonella typhimurium 4030; 25, Corynebacterium diphtheriae CMCC 38001; 26, Bacillus megatherium 4623; 27, Stenotrophomonas maltophilia K279a; 28, Legionella pneumophila 9135; 29, Acinetobacter baumannii 12101; 30, enteroinvasive E. coli 44825; 31, enterotoxigenic E. coli 44824; 32, enteropathogenic E. coli 2348.
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Figure 3: Specificity of the LAMP reaction in detectingK. pneumoniae. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. Amplification was performed at 61°C for 60 min. 1, positive control (K. pneumoniae ATCC BAA-2146); 2, negative control (double-distilled water); 3, Klebsiella oxytoca ATCC 700324; 4, Klebsiella rhinoscleromatis CMCC 46111; 5, Citrobacter freundii CMCC 48001; 6, Enterobacter aerogenes ATCC 13048; 7, Enterobacter cloacae ATCC 13047; 8, Proteus mirabilis CMCC 49005; 9, Proteus vulgaris CMCC 49027; 10, Serratia marcescens ATCC 14756; 11, Morganella morganii ATCC 25830; 12, Streptococcus pneumoniae 112-07; 13, Mycobacterium tuberculosis 005; 14, Pseudomonas aeruginosa D104; 15, Haemophilus influenza ATCC 49247; 16, Yersinia enterocolitica 027; 17, Yersinia pestis 2638; 18, Bacillus tularense 3450; 19, Vibrio cholera 3802; 20, Salmonella aberdeen 9264; 21, Neisseria meningitides CMCC 29022; 22, Staphylococcus aureus 2740; 23, Pseudomonas pseudomallei 029; 24, Salmonella typhimurium 4030; 25, Corynebacterium diphtheriae CMCC 38001; 26, Bacillus megatherium 4623; 27, Stenotrophomonas maltophilia K279a; 28, Legionella pneumophila 9135; 29, Acinetobacter baumannii 12101; 30, enteroinvasive E. coli 44825; 31, enterotoxigenic E. coli 44824; 32, enteropathogenic E. coli 2348.

Mentions: K. pneumoniae ATCC BAA-2146 was used as the positive control and double-distilled water as the negative control when evaluating the specificity of LAMP for the detection of K. pneumoniae. Twenty-one other non- K. pneumoniae bacterial strains (Supplemental Materials) were also tested. As shown in Figure 3, both methods of analysis positively identified the K. pneumoniae. All other strains, including the blank control, tested negative, indicating that the LAMP assay was specific to K. pneumoniae.


Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China.

Dong D, Liu W, Li H, Wang Y, Li X, Zou D, Yang Z, Huang S, Zhou D, Huang L, Yuan J - Front Microbiol (2015)

Specificity of the LAMP reaction in detectingK. pneumoniae. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. Amplification was performed at 61°C for 60 min. 1, positive control (K. pneumoniae ATCC BAA-2146); 2, negative control (double-distilled water); 3, Klebsiella oxytoca ATCC 700324; 4, Klebsiella rhinoscleromatis CMCC 46111; 5, Citrobacter freundii CMCC 48001; 6, Enterobacter aerogenes ATCC 13048; 7, Enterobacter cloacae ATCC 13047; 8, Proteus mirabilis CMCC 49005; 9, Proteus vulgaris CMCC 49027; 10, Serratia marcescens ATCC 14756; 11, Morganella morganii ATCC 25830; 12, Streptococcus pneumoniae 112-07; 13, Mycobacterium tuberculosis 005; 14, Pseudomonas aeruginosa D104; 15, Haemophilus influenza ATCC 49247; 16, Yersinia enterocolitica 027; 17, Yersinia pestis 2638; 18, Bacillus tularense 3450; 19, Vibrio cholera 3802; 20, Salmonella aberdeen 9264; 21, Neisseria meningitides CMCC 29022; 22, Staphylococcus aureus 2740; 23, Pseudomonas pseudomallei 029; 24, Salmonella typhimurium 4030; 25, Corynebacterium diphtheriae CMCC 38001; 26, Bacillus megatherium 4623; 27, Stenotrophomonas maltophilia K279a; 28, Legionella pneumophila 9135; 29, Acinetobacter baumannii 12101; 30, enteroinvasive E. coli 44825; 31, enterotoxigenic E. coli 44824; 32, enteropathogenic E. coli 2348.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440914&req=5

Figure 3: Specificity of the LAMP reaction in detectingK. pneumoniae. (A) Turbidity was monitored every 6 s using a Loopamp Realtime Turbidimeter at 650 nm. (B) The results were visualized by the addition of 1 μl of fluorescent detection reagent to the 25 μl LAMP reaction mixture before the LAMP reaction. Amplification was performed at 61°C for 60 min. 1, positive control (K. pneumoniae ATCC BAA-2146); 2, negative control (double-distilled water); 3, Klebsiella oxytoca ATCC 700324; 4, Klebsiella rhinoscleromatis CMCC 46111; 5, Citrobacter freundii CMCC 48001; 6, Enterobacter aerogenes ATCC 13048; 7, Enterobacter cloacae ATCC 13047; 8, Proteus mirabilis CMCC 49005; 9, Proteus vulgaris CMCC 49027; 10, Serratia marcescens ATCC 14756; 11, Morganella morganii ATCC 25830; 12, Streptococcus pneumoniae 112-07; 13, Mycobacterium tuberculosis 005; 14, Pseudomonas aeruginosa D104; 15, Haemophilus influenza ATCC 49247; 16, Yersinia enterocolitica 027; 17, Yersinia pestis 2638; 18, Bacillus tularense 3450; 19, Vibrio cholera 3802; 20, Salmonella aberdeen 9264; 21, Neisseria meningitides CMCC 29022; 22, Staphylococcus aureus 2740; 23, Pseudomonas pseudomallei 029; 24, Salmonella typhimurium 4030; 25, Corynebacterium diphtheriae CMCC 38001; 26, Bacillus megatherium 4623; 27, Stenotrophomonas maltophilia K279a; 28, Legionella pneumophila 9135; 29, Acinetobacter baumannii 12101; 30, enteroinvasive E. coli 44825; 31, enterotoxigenic E. coli 44824; 32, enteropathogenic E. coli 2348.
Mentions: K. pneumoniae ATCC BAA-2146 was used as the positive control and double-distilled water as the negative control when evaluating the specificity of LAMP for the detection of K. pneumoniae. Twenty-one other non- K. pneumoniae bacterial strains (Supplemental Materials) were also tested. As shown in Figure 3, both methods of analysis positively identified the K. pneumoniae. All other strains, including the blank control, tested negative, indicating that the LAMP assay was specific to K. pneumoniae.

Bottom Line: Klebsiella pneumoniae is a wide-spread nosocomial pathogen.The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR.In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.

ABSTRACT
Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

No MeSH data available.


Related in: MedlinePlus