Efficacy of a membrane concentration method combined with real-time PCR for detection of Giardia and Cryptosporidium in drinking water.

The occurrence of Giardia and Cryptosporidium (oo)cysts in drinking source water poses a serious public health risk. Here, we established a method that combines membrane concentration and real-time polymerase chain reaction (PCR) to quantify Giardia and Cryptosporidium in drinking water. The water samples were filtered through a cellulose membrane to collect Giardia and Cryptosporidium, and then nucleic acids were extracted. Specific primers and probes were designed and synthesized according to the gph gene sequence of Giardia and 18S rRNA gene sequence of Cryptosporidium. The concentrations of the two targets were determined using real-time PCR technology. The sensitivity, specificity, and stability of the method were evaluated. Our findings revealed that the detection limits of real-time PCR method for detecting Giardia and Cryptosporidium were 0.926 and 0.65 copy/µL, respectively; the spiked recovery rates were above 60% and 38%, respectively, and relative standard deviations were under 0.95% and 2.26%, respectively. Therefore, this effective procedure based on the membrane concentration method and real-time PCR will be useful for detecting Giardia and Cryptosporidium in drinking water for purpose of continuous environmental monitoring.

Pressure response of carbapenems Klebsiella pneumoniae under antibiotic stress.

To analyze the drug-resistant phenotype and genetic characteristics of Carbapenem resistant Klebsiella pneumoniae (CRKP) in this region, and to study its different expression profiles in RNA level under the pressure of low levels of antibiotics. Trace dilution method and PCR method were used to detect the antibiotic resistance phenotype and antibiotic resistance gene carrying of CRKP strain, simulate the antibiotic stress process, and RNAseq was used to analyze the transcriptomic changes of CRKP strain. 37 CRKP strains, 27 Carbapenem sensitive Klebsiella pneumoniae (CSKP) CSKP strains and 42 sensitive strains were detected. The antibiotic resistance rate of CRKP strain was significantly higher than that of other drug-resistant strains, and there were many kinds of antibiotic resistance genes. Transcriptomic analysis showed that CRKP strain showed compensatory rise under meropenem stress at low concentration, and the expression of genes related to biofilm formation, pressure induction, pressure tolerance and transcriptional regulation was significantly changed. It was speculated that mrkAB, fimDH, phoHP and pspABCD clusters significantly altered their expression under the antibiotics stress response in CRKP strain. The detection rate of CRKP strain is high in this area. Under low levels of antibiotic stress, CRKP strain can not only survive by synthesizing antibiotic modified enzyme, but also respond by transcriptional regulation and biofilm changes, resulting in stress compensation. The discovery of this phenomenon explains the failure of treatment due to improper use of higher-order antibiotics from the perspective of genetic interaction.