The prevalence and molecular epidemiology of vancomycin-resistant Enterococcus (VRE) carriage in patients admitted to intensive care units in Beijing, China.

BACKGROUND: Vancomycin-resistant Enterococcus (VRE) can be carried in the gut for a long period and its carriage status is associated with subsequent infections. This study aimed to investigate the frequency of intestinal VRE carriage in intensive care patients in Beijing. METHODS: A multicenter, retrospective cross-sectional study was conducted at six hospitals in Beijing, China. All patients admitted to intensive care units (ICUs) between April 2 and May 1, 2017, were enrolled, and their clinical data were gathered by reviewing electronic medical records. Rectal swabs collected from patients were stored at -80 °C in the Institute of Clinical Pharmacology, Peking University First Hospital, and they were selectively cultured for VRE, then the identified strains were analyzed by polymerase chain reaction (PCR) to detect the glycopeptide resistance gene and were characterized by multilocus sequence typing (MLST). RESULTS: Of 148 patients recruited, 46 (31.1%) carried VRE, with the majority (n = 42) being Enterococcus faecium. In total, 78.3% of the VRE were vanA positive and 15.2% vanM positive, while 6.5% undetected glycopeptide resistance gene. The predominant ST was ST78 (47.6%) followed by ST192 (14.3%), ST555 (9.5%), and ST789 (9.5%). Multivariate analysis showed that factors associated VRE carriage were patients aged >65 years (odds ratio [OR], 3.786; 95% confidence interval [CI], 1.402-10.222) and recent third-generation cephalosporins use (OR, 6.360; 95% CI, 1.873-21.601). CONCLUSIONS: The overall proportion of VRE carriage in patients admitted to ICUs was markedly high in Beijing, China. The vanM gene has been spread widely but vanA gene was the dominant resistance determinant in VRE in Beijing.

Lactobacillus rhamnosus and Lactobacillus casei Affect Various Stages of Gardnerella Species Biofilm Formation.

Bacterial vaginosis (BV) and its recurrence are most commonly associated with the formation of Gardnerella species biofilm. Probiotics are typically used to treat BV; however, the optimal period of Lactobacillus probiotic application in BV treatment remains uncertain. The present study aimed to explore the effects of Lactobacillus rhamnosus and Lactobacillus casei on various stages of biofilm formation in Gardnerella species. The biofilm-forming ability of seven strains, including one Gardnerella vaginalis ATCC 14018 and six clinically isolated Gardnerella species, was determined via gentian violet staining assay. Moreover, the sensitivity of the planktonic and biofilm forms toward metronidazole and clindamycin was assessed via microdilution broth method. L. rhamnosus Xbb-LR-1 and L. casei Xbb-LC-1 were added during various stages of biofilm formation in Gardnerella species and were cocultured for 24 h. The biofilm thickness of each sample was determined via confocal laser scanning microscopy (CLSM). The absolute quantities of Gardnerella species in each sample was obtained via real time polymerase chain reaction method, and the pH value was obtained using a pH indicator paper. Biofilm formation by Gardnerella species in a medium with distinct pH values was observed via gentian violet staining, CLSM, and scanning electron microscopy (SEM). The biofilm increased the resistance of Gardnerella species toward metronidazole and clindamycin. L. rhamnosus added at the initial biofilm formation stage in Gardnerella species exhibited highest inhibitory effect, with a percentage inhibition of 38.17% ± 1.35%. When the pH value of the culture medium was <4.5 or >6.5, ATCC 14018 could hardly form a biofilm; however, at pH ≥4.5 and ≤6.5, it was able to form a stronger biofilm. The amount of biofilm attained maximum value at optical density of 3.29 ± 0.28 (595 nm), pH 5.5, and at 36 h. Biofilm formation increases the resistance of Gardnerella species toward antibiotics. Maintaining an acidic vaginal environment with pH <4.5 and a vaginal microbiota dominated by Lactobacillus remarkably prevents the formation of Gardnerella species biofilm at the initial stage, which further has a significant impact on the treatment and prevention of biofilm-related infections.

Evaluation of Quadruple Real-Time PCR Method to Detect Enterococci Carrying Vancomycin-Resistant Genes vanA, vanB, vanM in Rectal Swabs.

Purpose: To evaluate the sensitivity and accuracy of the quadruple real-time PCR method for the detection of enterococci carrying vancomycin-resistant genes vanA, vanB, and vanM in rectal swabs. Methods: Choosing PCR-sequenced DNA extracted directly from rectal swabs as the gold standard, the results of the quadruple real-time PCR method and the traditional method (screening culture combined PCR-sequencing method whose DNA extracted from single colony) were compared with the gold standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the quadruple real-time PCR method and the traditional method were obtained. The time required for the three methods was calculated. Results: The results between gold standard and the quadruple real-time PCR method were similar. Compared to the traditional method, the quadruple real-time PCR method had a much higher sensitivity, specificity, PPV, NPV, and consistency. Our study found that the quadruple real-time PCR method is beneficial for detection of enterococci carrying vanM with vancomycin heteroresistance. The traditional method had high specificity and NPV, but its sensitivity and PPV were not ideal. The time needed for gold standard is a minimum of 28 h; the quadruple real-time PCR method takes 2-3 h while the traditional method consumes a minimum of 72 h. Conclusion: The quadruple real-time PCR method can provide a rapid and reliable result for the diagnosis of patients with colonized vancomycin-resistant enterococci. This new method is beneficial for the active screening, timely clinical treatment measures, epidemiological research, and hospital monitoring of the enterococci carrying vancomycin-resistant gene, especially for the enterococci with vancomycin heteroresistance carrying vanM.

Antimicrobial Susceptibility Testing of Metronidazole and Clindamycin against Gardnerella vaginalis in Planktonic and Biofilm Formation.

BACKGROUND: Bacterial vaginosis (BV), one of the most common vaginal ecosystem-related microbiologic syndromes, is the most common disorder in women of reproductive age. Gardnerella (G.) vaginalis is the predominant species causing this infection. Our aim was to compare the antimicrobial susceptibilities of metronidazole and clindamycin against G. vaginalis at planktonic and biofilm levels. METHODS: From September 2019 to October 2019, we recruited a total of 10 patients with BV who underwent gynecological examinations at Beijing Obstetrics and Gynecology Hospital. G. vaginalis isolates were obtained from the vagina and identified using their characteristic colony morphology. Sequence data of clinical G. vaginalis isolates were confirmed by comparing 16S rDNA sequences. Subsequently, clinical isolates were evaluated for antimicrobial susceptibilities in vitro to metronidazole and clindamycin at planktonic and biofilm levels. The minimum inhibitory concentration (MIC) for metronidazole and clindamycin was evaluated by antimicrobial susceptibility testing. The minimum biofilm eradication concentration (MBEC) was evaluated by the biofilm inhibition assay. RESULTS: Planktonic clinical isolates showed a significantly higher susceptibility rate (76.67%) and lower resistance rate (23.33%) to clindamycin than to metronidazole (susceptibility rate: 38.24%; resistance rate: 58.82%; P < 0.05 for both). Furthermore, in comparison to planktonic isolates, the minimum inhibitory concentration (MIC) of metronidazole was significantly higher for biofilm-forming isolates (7.3 ± 2.6 µg/mL vs. 72.4 ± 18.3 µg/mL; P=0.005); the resistance rate was 27.3%, and the minimum biofilm eradication concentration (MBEC) was >128 µg/mL. Moreover, the MIC of clindamycin was higher too for biofilm-forming isolates (0.099 ± 0.041 µg/mL vs. 23.7 ± 9.49 µg/mL; P=0.034); the resistance rate was 27.3%, and the MBEC of clindamycin was 28.4 ± 6.50 µg/mL. CONCLUSION: Our results indicate that in comparison to metronidazole, clindamycin seems to be a better choice to tackle G. vaginalis as it exhibits a relatively higher susceptibility rate and lower resistance rate.

Evaluation of the Inhibitory Effects of Lactobacillus gasseri and Lactobacillus crispatus on the Adhesion of Seven Common Lower Genital Tract Infection-Causing Pathogens to Vaginal Epithelial Cells.

Background/Purpose: Lactobacillus colonization is important to maintain urogenital flora stability and prevent pathogenic infection. Different Lactobacillus species have distinct properties and effects on the urogenital flora. To select probiotics that colonize the vagina and provide protection against pathogenic infection, we evaluated the adhesion of five Lactobacillus strains and their inhibitory effects on the adhesion of pathogens to vaginal epithelial cells (VECs). Methods and Materials: (1) Lactobacillus adhesion experiments: VK2/E6E7 and primary VECs were used to evaluate the adhesion of two Lactobacillus gasseri and three Lactobacillus crispatus strains. The adhesion of these five Lactobacillus strains was compared. (2) Adhesion inhibition experiments: The inhibitory effects of the five Lactobacillus strains on the adhesion of pathogens (Gardnerella, Mobiluncus, Candida albicans, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis) were evaluated by adhesion exclusion, displacement, and competition experiments. Results: (1) Lactobacillus adhesion was stronger in the primary VECs than in the VK2/E6E7 VECs (P < 0.05). The adhesion of the three L. crispatus strains was stronger than that of the two L. gasseri strains (P < 0.05). L. crispatus 4# showed the strongest adhesion. (2) The exclusion, displacement, and competition experiments showed that all five Lactobacillus strains significantly inhibited the adhesion of the seven pathogenic strains to the VECs (P < 0.05). The displacement effect was stronger than the exclusion and competition effects of each Lactobacillus strain. (3) The results of the exclusion, displacement, and competition experiments indicated that L. gasseri 1# showed the strongest adhesion inhibition of C. albicans and S. agalactiae. L. crispatus 3# showed the strongest adhesion inhibition of S. aureus, whereas L. crispatus 4# showed the strongest adhesion inhibition of Gardnerella, Mobiluncus, E. coli, and E. faecalis. Conclusion: The source of the VECs might not affect the selection of the most adhesive Lactobacillus strain. L. crispatus showed stronger VEC adhesion than L. gasseri. The degree of antagonism of the Lactobacillus strains toward the different pathogens varied. This result provides incentives for personalized clinical treatment.

Real-time PCR for the rapid detection of vanA, vanB and vanM genes.

BACKGROUND/PURPOSE: To evaluate the ability of quadruple Taqman probe real-time PCR to the detection of vanA, vanB and vanM in enterococcal isolates. METHODS: A total of 343 strains, including 253 vancomycin-resistant enterococcus (VRE) strains and 90 non-VRE strains, were tested by both quadruple Taqman probe real-time PCR and gel-based PCR assay. RESULTS: When differentiating among three genotypes of vanA, vanB and vanM in VRE strains, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy and consistency of the quadruple Taqman probe real-time PCR were all 100%. Minimum. Inhibitory concentration (MIC) results showed that there was a wide MIC range of vancomycin and teicoplanin for the strains that harboring vanA/vanM gene respectively or harboring vanA and vanM genes simultaneously. However, the VRE strains with vanB genotype all were sensitive to teicoplanin. CONCLUSION: Considering the excellent PPV and low NPV, real-time PCR would be useful to monitor VRE-colonized or infected patients. However, further evaluation of the assay's performance in the clinical specimens is required, especially when considering that high level of PCR inhibitors present in these samples.