Evaluation of the Zybio EXS3000 mass spectrometry in routine identification of Clinical isolates.

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.

The small RNA PrrH of Pseudomonas aeruginosa regulates hemolysis and oxidative resistance in bloodstream infection.

Small regulatory RNAs (sRNAs) regulate multiple physiological functions in bacteria, and sRNA PrrH can regulate iron homeostasis and virulence. However, the function of PrrH in Pseudomonas aeruginosa (P. aeruginosa) bloodstream infection (BSI) is largely unknown. The aim of this study was to investigate the role of PrrH in P. aeruginosa BSI model. First, P. aeruginosa PAO1 was co-cultured with peripheral blood cells for 6 h. qRT-PCR results showed a transient up-regulation of PrrH expression at 1 h. Simultaneously, the expression of iron uptake genes fpvA, pvdS and phuR were upregulated. In addition, the use of iron chelator 2,2'-dipyridyl to create low-iron conditions caused up-regulation of PrrH expression, a result similar to the BSI model. Furthermore, the addition of FeCl3 was found to decrease PrrH expression. These results support the hypothesis that the expression of PrrH is regulated by iron in BSI model. Then, to clarify the effect of PrrH on major cells in the blood, we used PrrH mutant, overexpressing and wild-type strains to act separately on erythrocytes and neutrophils. On one hand, the hemolysis assay revealed that PrrH contributes to the hemolytic activity of PAO1, and its effect was dependent on the T3SS system master regulator gene exsA, yet had no association with the hemolytic phospholipase C (plcH), pldA, and lasB elastase genes. On the other hand, PrrH mutant enhanced the oxidative resistance of PAO1 in the neutrophils co-culture assay, H2O2-treated growth curve and conventional plate spotting assays. Furthermore, the katA was predicted to be a target gene of PrrH by bioinformatics software, and then verified by qRT-PCR and GFP reporter system. In summary, dynamic changes in the expression of prrH are iron-regulated during PAO1 bloodstream infection. In addition, PrrH promotes the hemolytic activity of P. aeruginosa in an exsA-dependent manner and negatively regulates katA to reduce the oxidative tolerance of P. aeruginosa.

Quorum sensing regulates heteroresistance in Pseudomonas aeruginosa.

The prevalence and genetic mechanism of antibiotic heteroresistance (HR) have attracted significant research attention recently. However, non-genetic mechanism of HR has not been adequately explored. The present study aimed to evaluate the role of quorum sensing (QS), an important mechanism of behavioral coordination in different subpopulations and consequent heteroresistance. First, the prevalence of HR to 7 antibiotics was investigated in 170 clinical isolates of P. aeruginosa using population analysis profiles. The results showed that P. aeruginosa was significantly heteroresistant to meropenem (MEM), amikacin (AMK), ciprofloxacin (CIP), and ceftazidime (CAZ). The observed HR was correlated with down-regulation of QS associated genes lasI and rhlI. Further, loss-of-function analysis results showed that reduced expression of lasI and rhlI enhanced HR of P. aeruginosa to MEM, AMK, CIP, and CAZ. Conversely, overexpression of these genes or treatment with 3-oxo-C12-HSL/C4-HSL lowered HR of P. aeruginosa to the four antibiotics. Additionally, although downregulation of oprD and upregulation of efflux-associated genes was evident in heteroresistant subpopulations, their expression was not regulated by LasI and RhlI. Moreover, fitness cost measurements disclosed higher growth rates of PAO1ΔlasI and PAO1ΔrhlI in the presence of sub-MIC antibiotic as compared with that of wild-type PAO1. Our data suggest that under temporary antibiotic pressure, downregulation of QS might result in less fitness cost and promote HR of P. aeruginosa.

Evaluation of humoral pattern detection performance of Mindray BC-6000PLUS blood analyzer based on WS/T 662-2020.

BACKGROUND: Manual microscopic examination is the gold standard of humoral cell count test. However, it has some limitations and cannot fully meet clinical needs. Compared with the manual method, the automatic blood cell analyzer has the advantages of a high degree of automation, minimal error, high speed, high precision, and easy standardization. This study intends to verify the detection performance of the body fluid model of the Mindray BC-6000PLUS automatic hematology analyzer. METHODS: This study was performed in accordance with the International Committee for Standardization in Haematology (ICSH) Hematology Analyzer Evaluation Guide (version 2014) and the requirements of WS/T662-2020 "Clinical humoral examination technique". The humoral white blood cell-body fluid (WBC-BF), humoral red blood cell-body fluid (RBC-BF), monocyte (MN), polymorphonuclear (PMN) were measured to verify the performance indicators of the instrument, including background counting, intra-batch precision, accuracy, carrying contamination rate, and linear range. Referring to the WS/T514-2017 (Establishment and verification of detection capability for clinical laboratory measurement procedures), the limit of blank (LoB) and limit of detection (LoD) values of WBC-BF and RBC-BF in the humoral mode of the instrument were established. RESULTS: The blank count of WBC-BF and RBC-BF, and contamination of Mindray BC-6000PLUS analyzer were zero; the coefficient of variation (CV) of intra-batch precision at different levels of each item was less than 10%. There was a high correlation between instrument test results and manual microscopic examination results (r>0.95). The linear range of the instrument was wide, and the linear verification parameters was good (R2>0.999). The LoB value and LoD value of WBC-BF established by the instrument were 0×109/L and 0.004×109/L, respectively. The LoB value and LoD value of the RBC-BF established by the instrument were 0×1012/L and 0.004×1012/L, respectively. The lower detection limits of WBC-BF and RBC-BF were set as 0.004×109/L and 0.004×1012/L, respectively. CONCLUSIONS: All performance indicators of the Mindray BC-6000PLUS automatic blood analyzer met the requirements of the manufacturer's criteria. This instrument can fulfill the requirement of body fluid sample routine test in clinical practice.