Antibiotic Resistance Profile of Enterococcus faecalis and Enterococcus faecium Isolates from Urine and Pleural Fluid in Two Hospitals of Cameroon

Enterococcus faecalis and Enterococcus faecium rank among the leading causes of nosocomial bacteremia and urinary tract infections. They often persist on hospital surfaces due to their ability to withstand adverse environmental conditions (low or high temperatures, high pH, and high salinity). The global Enterococcus faecalis-Enterococcus faecium ratio is currently shifting towards Enterococcus faecium. Enterococci present variable levels of resistance to certain families of antibiotics. This is the case for aminoglycosides, beta-lactams and cephalosporins. In 2017, WHO ranked Enterococci among priority pathogens for research and development of new antibiotics. The objective of our study was to determine the antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolates from urine and pleural fluid in two hospitals in Cameroon. This cross-sectional and analytic study was carried out between June to August 2023 on hospitalized and day patients in which a cytobacteriological test of urine and pleural fluid was done. The samples were inoculated on CLED Agar for urine and on Chocolate + polyvitex and blood agar (prepared from Columbia agar) for pleural fluid samples and incubated at 37℃ for 18 to 24 hours. Identification of isolates was carried out using the API 20 STREP micro gallery (Biomerieux, France) and tested for antimicrobial susceptibility. The data on socio-demographical and potential risk factors were recorded using self-administered questionnaires and data from laboratory analyses of the specimen were collected in a data capture sheet. Potential risk factors associated with the presence of Enterococci, were evaluated using the logistic regression in univariate and multivariate analysis. P value < 0.05 was considered as significant. A total of 511 patients were recruited who were predominantly females. Enterococcus spp were isolated in 27.79% of our samples with Enterococcus faecalis mostly encountered. Enterococcus spp showed a high level of resistance to penicilline (99.3% to Ampicilline), macrolides (66.2% to Erythromycin) and cyclines (85.2% to Doxycycline). Hospitalisation, access to health facilities, contact with urine specimen and hand hygiene practices were risk factors related to infection with Enterococcus spp while hospitalisation, health facility and hand hygiene were related to glycopeptide resistant Enterococcus. Strict compliance with hygiene rules and appropriate antibiotic consumption could help in the fight against these infections.

Pretreatment with probiotics Enterococcus faecium NCIMB 11181 attenuated Salmonella Typhimurium-induced gut injury through modulating intestinal microbiome and immune responses with barrier function in broiler chickens

Background:Preventing Salmonella infection and colonization in young birds is key to improving poultry gut health and reducing Salmonella contamination of poultry products and decreasing salmonellosis for human consumption(poultry meat and eggs).Probiotics can improve poultry health.The present study was conducted to investigate the impact of a probiotics,Enterococcus faecium NCIMB 11181(E.faecium NCIMB 11181)on the intestinal mucosal immune responses,microbiome and barrier function in the presence or absence of Salmonella Typhimurium(S.Typh-imurium,ST)infection.Methods:Two hundred and forty 1-day-old Salmonella-free male broiler chickens(Arbor Acres AA+)were randomly allocated to four groups with 6 replicate cages of 10 birds each.The four experimental groups were follows:(1)nega-tive control(NC),(2)S.Typhimurium,challenged positive control(PC),(3)the E.faecium NCIMB 11181-treated group(EF),(4)the E.faecium NCIMB 11181-treated and S.Typhimurium-challenged group(PEF).Results:Results indicated that,although continuous feeding E.faecium NCIMB 11181 did not obviously alleviate growth depression caused by S.Typhimurium challenge(P>0.05),E.faecium NCIMB 11181 addition significantly blocked Salmonella intestinal colonization and translocation(P<0.05).Moreover,supplemental E.faecium NCIMB 11181 to the infected chickens remarkably attenuated gut morphological structure damage and intestinal cell apoptosis induced by S.Typhimurium infection,as evidenced by increasing gut villous height and reducing intes-tinal TUNEL-positive cell numbers(P<0.05).Also,E.faecium NCIMB 11181 administration notably promoting the production of anti-Salmonella antibodies in intestinal mucosa and serum of the infected birds(P<0.05).Addition-ally,16S rRNA sequencing analysis revealed that E.faecium NCIMB 11181 supplementation ameliorated S.Typhimu-rium infection-induced gut microbial dysbiosis by enriching Lachnospiracease and Alistipes levels,and suppressing Barnesiella abundance.Predicted function analysis indicated that the functional genes of cecal microbiome involved in C5-branched dibasic acid metabolism;valine,leucine and isoleucine biosynthesis;glycerolipid metabolism and lysine biosynthesis were enriched in the infected chickens given E.faecium NCIMB 11181.While alanine,asparate and glutamate metabolism;MAPK signal pathway-yeast;ubiquine and other terpenoid-quinore biosynthesis,protein processing in endoplasmic reticulum;as well as glutathione metabolism were suppressed by E.faecium NCIMB 11181 addition.Conclusion:Collectively,our data suggested that dietary E.faecium NCIBM 11181 supplementation could ameliorate S.Typhimurium infection-induced gut injury in broiler chickens.Our findings also suggest that E.faecium NCIMB 11181 may serve as an effective non-antibiotic feed additive for improving gut health and controlling Salmonella infection in broiler chickens.

Effect of Bacillus subtilis,Enterococcus faecium,and Enterococcus faecalis supernatants on serotonin transporter expression in cells and tissues

BACKGROUND Bacillus subtilis(B.subtilis),Enterococcus faecium(E.faecium),and Enterococcus faecalis(E.faecalis)are probiotics that are widely used in the clinical treatment of irritable bowel syndrome(IBS).Whether the supernatants of these three probiotics can improve gastrointestinal sensation and movement by regulating the serotonin transporter(SERT)expression needs to be clarified.AIM To investigate whether B.subtilis,E.faecium,and E.faecalis supernatants can upregulate SERT expression in vitro and in vivo.METHODS Caco-2 and HT-29 cells were stimulated with probiotic culture supernatants for 12 and 24 h,respectively.A male Sprague-Dawley rat model of post-infectious irritable bowel syndrome(PI-IBS)was established and the rats were treated with phosphate-buffered saline(group A)and three probiotics culture supernatants(groups B,C,and D)for 4 wk.The levels of SERT were detected by quantitative PCR and western blotting.RESULTS The levels of SERT at post-treatment 12 and 24 h were significantly elevated in Caco-2 cells treated with B.subtilis supernatant compared with those in the control group(aP<0.05).Those levels were markedly upregulated in Caco-2 cells stimulated with E.faecium and E.faecalis supernatants at 24 h(aP<0.05).In addition,SERT expression in groups B,C,and D was significantly higher than that in group A in the 2nd wk(aP<0.05).Increased SERT expression was only found in group D in the 3rd wk(aP<0.05).However,there was no significant difference in SERT expression between the groups in the last week(P>0.05).CONCLUSION The supernatants of B.subtilis,E.faecium,and E.faecalis can upregulate SERT expression in intestinal epithelial cells and the intestinal tissues in the rat model of PI-IBS.

Isolation of Enterococcus faecium with Feeding Attractant Function from Pacific White Shrimp (Litopenaeus vannamei) Intestine

In the present study,we isolated the lactic acid bacterium strain SC-01 from Pacific white shrimp(Litopenaeus vannamei)intestine.Using conventional and molecular methods,we identified the bacterium as Enterococcus faecium,and found it had the function of feeding attractant and could inhibit the development of Vibrio parahaemolyticus(zone of inhibition:14 mm).The attractant effect of its fermentation broth is significantly better than that of the chemical attractant trimethylamine oxide(TMAO)(P<0.05),and is equivalent to that of dimethyl-beta-propiothetin(DMPT)based on the feeding behavior of shrimp.High performance liquid chromatography(HPLC)analysis suggested that inosine-5’-monophosphate(IMP)may be a component of the attractant.A biosecurity evaluation revealed a negative result in hemolytic assays,and no shrimp mortality was resulted from SC-01 fermentation broth challenge.Feeding trials(60 days)indicated that the SC-01 fermentation broth(viable counts:5.7×109 cfu mL−1)could improve feed intake,weight gain rate(WGR)and specific growth rate(SGR),and decrease the count of Vibrio sp.in the intestine of shrimp.

Identification of a New Peptide Deformylase Gene From Enterococcus faecium and Establishment of a New Screening Model Targeted on PDF for Novel Antibiotics

Objective To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. Methods A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E.coli Bl21(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. Result A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. Conclusion A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Bloodstream Infections Caused by Enterococcus spp：A 10-year Retrospective Analysis at a Tertiary Hospital in China

In order to discover the risk factors for 30-day mortality in bloodstream infections（BSI） caused by Enterococcus spp.strains,we explored the clinical and therapeutic profile of patients with Enterococcus spp.BSI and the characteristics of this condition.A total of 64 patients with BSI caused by Enterococcus spp.who were treated in our hospital between 2006 and 2015 were included in the study.The clinical features of patients,microbiology,and 30-day mortality were collected from the electronic medical records database and analyzed.The results showed that there were 38 patients infected by Enterococcus faecalis（E.faecalis）,24 by Enterococcus faecium（E.faecium）,1 by Enterococcus casseliflavus（E.casseliflavus）,and 1 by Enterococcus gallinarum（E.gallinarum）.A Charlson comorbidity score ≥5,corticosteroid treatment,placement of catheters or other prosthetic devices and history of antibiotic use were found more frequently in E.faecium BSI patients than in E.faecalis patients（P=0.017,P=0.027,P=0.008 and P=0.027,respectively）.Furthermore,the univariate and multivariate analysis showed that corticosteroid treatment（OR=17.385,P=0.008）,hospital acquisition（OR=16.328,P=0.038）,and vascular catheter infection（OR=14.788,P=0.025） were all independently associated with 30-day mortality.Our results indicate that E.faecalis and E.faecium are two different pathogens with unique microbiologic characteristics,which cause different clinical features in BSI,and the empiric antimicrobial treatments are paramount for patients with enterococcal BSI.

In vitro study on the transmission of multidrug-resistant bacteria from textiles to pig skin

BACKGROUND The survival of microorganisms on textiles and specifically on healthcare profes-sionals’(HCP)attire has been demonstrated in several studies.The ability of microorganisms to adhere and remain on textiles for up to hours or days raises questions as to their possible role in transmission from textile to skin via HCP to patients.AIM To evaluate the presence,survival and transmission of different multidrug-resistant bacteria(MDRB)from HCP attire onto skin.METHODS Three MDRB[methicillin-resistant Staphylococcus aureus(MRSA);vancomycin-resistant Enterococcus faecium(VRE);carbapenem-resistant Klebsiella pneumoniae,(CRKP)]were inoculated on textiles from scrubs(60%cotton-40%polyester)and white coat(100%cotton)at concentrations of 108 colony-forming units(CFU),105 CFU,and 103 CFU per mL.The inoculation of swatches was divided in time intervals of 1 min,5 min,15 min,30 min,1 h,2 h,3 h,4 h,5 h,and 6 h.At the end of each period,textiles were imprinted onto pig skins and each skin square was inverted onto three different selective chromogenic media.Growth from the pig skin squares was recorded for the 3 MDRB at the three above concentrations,for the whole length of the 6-h experiment.RESULTS MRSA was recovered from pig skins at all concentrations for the whole duration of the 6-h study.VRE was recovered from the concentration of 108 CFU/mL for 6 h and from 105 CFU/mL for up to 3 h,while showing no growth at 103 CFU/mL.CRKP was recovered from 108 CFU/mL for 6 h,up to 30 min from 105 CFU/mL and for 1 min from the concentration of 103 CFU/mL.CONCLUSION Evidence from the current study shows that MRSA can persist on textiles and transmit to skin for 6 h even at low concentrations.The fact that all MDRB can be sustained and transferred to skin even at lower concentrations,supports that textiles are implicated as vectors of bacterial spread.

Nosocomial spread of hospital-adapted CC17 vancomycin-resistant Enterococcus faecium in a tertiary-care hospital of Beijing, China

Background The incidence of vancomycin-resistant enterococci （VRE） appeared to be increasing in China, but very few nosocomial outbreaks have been reported. Our hospital had experienced an outbreak of VRE since March 2008 to March 2009. The objective of this study was to analyze the molecular features of the isolates and the control measures used to eradicate a VRE outbreak in a tertiary institution in China.Methods We characterized VRE isolates from 21 infected and 11 colonized inpatients from a single hospital by pulsed field gel electrophoresis （PFGE）, multilocus sequence typing （MLST）, the analysis of Tn 1546-like elements and virulence genes detection. Infection control measures, including more environmental disinfection, screening for VRE colonization,contact precautions, education and strict antibiotic restriction, were implemented to control the outbreak.Results During the outbreak, a total of 32 VRE strains were obtained. There were 21 strains found in Emergency Intensive Care Unit （EICU）, 9 isolates from Geriatric Ward, and two from other units. All the isolates harbored the vanA gene, however,four of them exhibited the VanB phenotype. Meanwhile, MLST analysis revealed that all isolates belonged to clonal complex （CC） 17. With the infection-control measures, the epidemic was constrained in two units （EICU and Geriatric Ward）. After March 2009, no further case infected with VRE was detected in the following one-year period.Conclusion The outbreak was controlled by continuous implementation of the infection control programme, and more rigorous infection control policy is needed.

First Complete Genome Sequence of a Probiotic Enterococcus faecium Strain T-110 and Its Comparative Genome Analysis with Pathogenic and Non-pathogenic Enterococcus faecium Genomes

Enterococci bacteria are important in environmental, food and clinical microbiology. Enterococcus faecium is a nosocomial pathogen that causes bacteremia, endocarditis and other infections. It is among the most prevalent organisms encountered in hospital-associated infections accounting for approximately 12% of nosocomial infections in the USA （Linden and Miller, 1999）. However, certain strains of E. faecium are not only non-pathogenic but also have beneficial effects on human health with probiotic potential. For example, E. faecium T-110 is a consortium member in several probiotic products including BIO-THREE~ which is widely prescribed for human, animal and aqua-cultural use. This strain was originally developed by TOA Pharmaceuticals in Japan, and later used in the probiotic products of several other companies.

Extensive contact tracing and screening to control the spread of vancomycin-resistant Enterococcus faecium ST414 in Hong Kong

Background Proactive infection control management is crucial in preventing the introduction of multiple drug resistant organisms in the healthcare setting. In Hong Kong, where vancomycin-resistant enterococci （VRE） endemicity is not yet established, contact tracing and screening, together with other infection control measures are essential in limiting intra- and inter-hospital transmission. The objective of this study was to illustrate the control measures used to eradicate a VRE outbreak in a hospital network in Hong Kong. Methods We described an outbreak of VRE in a healthcare region in Hong Kong, involving a University affiliated hospital and a convalescent hospital of 1600 and 550 beds respectively. Computer-assisted analysis was utilized to facilitate contact tracing, followed by VRE screening using chromogenic agar. Multi-locus sequence typing （MLST） was performed to assess the clonality of the VRE strains isolated. A case-control study was conducted to identify the risk factors for nosocomial acquisition of VRE. Results Between November 26 and December 17, 2011, 11 patients （1 exogenous case and 10 secondary cases） in two hospitals with VRE colonization were detected during our outbreak investigation and screening for 361 contact patients, resulting in a clinical attack rate of 2.8% （10/361）. There were 8 males and 3 females with a median age of 78 years （range, 40-87 years）. MLST confirmed sequence type ST414 in all isolates. Case-control analysis demonstrated that VRE positive cases had a significantly longer cumulative length of stay （P 〈0.001）, a higher proportion with chronic cerebral and cardiopulmonary conditions （P=0.001）, underlying malignancies （P 〈0.001）, and presence of urinary catheter （P 〈0.001）, wound or ulcer （P 〈0.001）, and a greater proportion of these patients were receiving β-lactam/ β-1actamase inhibitors （P=0.009）, carbapenem group （P 〈0.001）, fluoroquinolones （P=0.003）, or vancomycin （P=0.001） when compared with the controls. Conclusion Extensive contact tracing and screening with a ＂search-and-confine＂ strategy was a successful tool for outbreak control in our healthcare reqion.