vanA Genotype Research Articles

Molecular detection and occurrence of vancomycin resistance genes (van A, B, C1, C2/C3) among Enterococcus species isolated from farm ostriches.

Evaluating the prevalence of vancomycin resistance genes (van genes) in enterococcal isolates from food-producing animals is an important public health issue because of the possibility of resistance genes spread to human. The present study aimed to determine the occurrence of vancomycin resistance genes among Enterococcus species obtained from ostrich faecal samples. One hundred and twenty-five faecal samples of apparently healthy ostriches from five different farms were investigated. Genes encoding vancomycin resistance were studied by multiplex-PCR, and susceptibility to six antibiotics was evaluated by disk-diffusion method. In total, 107 Enterococcus spp. isolates were obtained and confirmed by biochemical and molecular tests. Enterococcus faecium was the prevailing species (56 isolates of 107; 52.3%), followed by E. hirae (24 isolates; 22.4%) and E. gallinarum (12 isolates; 11.2%). Of the 107 recovered isolates, 44% harboured at least a type of van genes. vanA, vanC2/3 and vanC1 were identified in 34 (31.7%), 13 isolates (12.1%) and 4 (3.7%) isolates respectively. Additionally, four isolates (E. gallinarum, E. rafinosus) co-harboured the the vanA and vanC1 or vanA and vanC2/3. Enterococcus faecium and Enterococcus hirae strains with the vanA genotype were the most frequent van-carrying enterococci from ostrich faecal samples. Among van-carrying enterococcal isolates, 23.4% were phenotypically resistant to vancomycin. This study revealed a relatively high prevalence (44%) of van-carrying enterococci in ostrich faecal samples. Results of the present study suggest that ostrich faeces could be considered as a reservoir of vancomycin resistance genes, especially vanA containing enterococci that could be potentially transferred to human through the food chain.

Isolation, Identification, Speciation, and Antibiogram of Enterococcus species by conventional methods and Assessment of the Prevalence of vana genotype among VRE

Enterococci are Gram-positive, ovoid cocci that can be found as diplococci or in short chains. These species are found in the typical microbiota of the intestine, oral cavity, vagina, and other areas. Despite being commensals with moderate virulence, these species have lately emerged as important nosocomial infections with increasing drug resistance particularly to vancomycin. This has resulted in their changing patterns of infection that are resistant to conventional antimicrobial therapy. The main objective of this study was to assess the frequency of the VanA genotype among Vancomycin Resistant Enterococci, as well as the isolation, identification, speciation, and antibiotic sensitivity pattern of Enterococci from various clinical specimens. In this six-month study, 500 different clinical specimens such as urine, blood, and pus were collected aseptically from patients suffering from urinary tract infection (UTI), septicemia, and pyogenic illnesses, and a total of 94 Enterococci strains were identified. These isolates were identified and speciated using traditional tests and biochemical processes. Following culture, the disc diffusion technique was used to determine their antibiotic susceptibility pattern, as suggested for common antibiotics. RCR was also used to evaluate the prevalence of the vanA gene among VRE isolates. 88.29% were E. faecalis, 7.88% were E. faecium, 2.12% were E. durans, 1.06 % was E. casseliflavus, and 1.06 % was E. gallinarum, out of a total of 94 isolates. Urine (70.21 %) yielded the most isolates, followed by blood (17.02 %) and pus (17.02 %). Female patients had a higher isolation rate of 73.40 % (69/94) than male patients, who had a rate of 26.59 % (25/94), and the majority of the Enterococcal isolates were from inpatients (62.77 %) rather than outpatients (37.23 %). 30 % of the isolates were hemolytic, whereas 70% were non-hemolytic. The isolates sensitivity patterns revealed that they were resistant to antibiotics such as ampicillin, ciprofloxacin, and gentamicin. The susceptibility test also included 30g discs of vancomycin. Vancomycin resistance is greatest in E. faecium (28 %) 6/83, followed by E. faecalis (8 %) 2\7. All other isolates were vancomycin-susceptible. Vancomycin-resistant isolates made up 8.51 % of the total. Barely urine isolates were tested for nitrofurantoin resistance, which was found to be only 5%. Linezolid sensitivity was found in all of the Enterococci. According to the PCR test for the vanA gene, among total VRE isolates 5 E. faecalis and 1 E. faecium isolates had vanA genotypes with bands of 473bp. A number of studies have revealed an increment in the infection rate and antimicrobial resistance of Enterococci. Elevated isolation rate of VRE has posed threat to therapeutic strategies as now only few antibiotics are susceptible to such isolates. It is thus imperative to execute measures that could stable the VRE augmentation lest a normal commensal would get the status of SARS-CoV-2

Antibiogram Pattern and Virulence Trait Characterization of Enterococcus Species Clinical Isolates in Eastern India: A Recent Analysis.

Objective We aimed to evaluate the current antimicrobial susceptibility pattern and characterize putative virulence traits among Enterococcus species isolates from various clinical specimens in view of their increased isolation rates in both community-related and serious nosocomial infections, as well as resistance to many antibiotics. Methods Study (April 2017–March 2018) included consecutive, nonrepeated, discrete, and clinically significant isolates of enterococci. Susceptibility testing included detection of high-level aminoglycoside-resistant (HLAR) and glycopeptide-resistant enterococci (GRE). All screen-positive GRE isolates were investigated by polymerase chain reaction for species confirmation and presence of vanA/vanB genes. Virulence genes ace , asa1 , cyt , efa , esp , gelE , and hyl were investigated by molecular methods. Hemolysin and biofilm production were studied using phenotypic methods. Results Of 111 isolates, 89 (80.1%), 16 (14.4%), and 6 (5.4%) were from urine, pus, and blood, respectively, consisting predominantly of E. faecalis (67, 60.4%) and E. faecium (32, 28.8%). E. hirae (5, 4.5%) was the predominant non- E. faecalis non- E. faecium isolate. Other species were E. durans (4, 3.6%), E. avium (2, 1.8%), and E. mundtii (1, 0.9%). Seven (6.3%) out of the 111 isolates were GRE, all vanA genotype. HLAR was observed in 70 (63.1%) isolates, significantly higher in E. faecium than E. faecalis (81.2 vs. 58.2%; p < 0.05). All were susceptible to daptomycin. Hemolysin activity and biofilm production were observed in 38 (34.2%) and 36 (32.4%) isolates. Most frequent virulence genes were efa (77, 69.4%), ace (71, 63.9%), asa1 (67, 60.3%), and gelE (66, 59.4%). There was a predominant association of esp and hyl genes with E. faecium and that of the other genes with E. faecalis . Conclusion The study will contribute to the existing limited data on virulence trait characterization of clinical E. spp. isolates in India. At the same time, it will help to serve as a guide in the choice of empirical therapy in enterococcal infections leading to favorable clinical outcomes by decreasing the clinical failure, microbiological persistence, and associated mortality, and will lead to future studies on controlling the spread of virulent and multiresistant isolates.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8-16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Epidemiological typing of ST80 vancomycin-resistant Enterococcus faecium: core genome multilocus sequence typing versus single nucleotide polymorphism-based typing

Vancomycin-resistant Enterococcus faecium (VRE) belonging to sequence type 80 (ST80) have been successfully disseminating in hospital settings. This study aimed to explore whole-genome sequencing (WGS)-based approaches in epidemiological typing of VRE by investigating a collection of ST80 strains. In total, 41 VRE ST80 isolates in three suspected transmission chains were subjected to WGS. The genetic relationship of the isolates was analysed by core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based typing. The published genome sequence of a predominant ST80 clone in Copenhagen was also included in the analyses. Group 1 isolates (n=10) were recovered in the same geographic area, including two isolates from an outbreak of vanA genotype. Group 2 isolates (n=4) were detected from another outbreak. Group 3 consisted of 3 non-outbreak isolates and 24 representative isolates from an outbreak of vanB genotype. Our findings indicated ≥24 allelic difference and >16 SNPs as the cut-off for excluding VRE strains from an outbreak. cgMLST identified >200 differing alleles between genomes of the Copenhagen clone and outbreak strains in this study. Our findings also demonstrated that SNP analysis was not suitable for typing samples from different groups, even with the same ST, owing to lack of an optimal reference genome. cgMLST and SNPs provided comparable epidemiological discrimination for isolates with a suspected transmission chain. For diverse isolates, SNP analysis could be suboptimal. An approach applying cgMLST as the first-line typing method and SNPs as a complementary tool is suggested.

Vancomycin-resistant Enterococcus faecium in Algeria: phenotypic and genotypic characterization of clinical isolates.

vancomycin-resistant Enterococcus faecium (VREfm) is a major public health problem worldwide. The aim of our study was to determine the microbiological, epidemiological and molecular characteristics of VREfm isolated in north-central, eastern and western Algeria. a collection of 48 VREfm isolated from September 2010 to April 2017 in several Algerian hospitals were studied. Minimum inhibitory concentrations (MICs) were determined by E-test method according to CLSI guidelines. the detection of van genotype of all strains was performed by PCR. Clonal relationship of five VREfm targeted by region were characterized using multilocus sequence typing (MLST). All isolates have multidrug-resistance (MDR) and were resistant to at least five classes of antibiotics; however, all were susceptible to tigecycline and daptomycin with MIC50 at 0.094 µg/mL and 2 µg/mL respectively. All strains belonged to vanA genotype and have high level of resistance to vancomycin and teicoplanin. MLST revealed two sequence types (STs): ST80 (from the four regions of Algeria) and ST789, both belonging to the former hospital-adapted clonal complex CC17. the alarming dissemination of MDR E. faecium vanA and the ST80 in several regions of Algeria suggest a clonal spread of VREfm strains, which urgently require implementation of adequate infection control measures.

Thirty years of VRE in Germany - "expect the unexpected": The view from the National Reference Centre for Staphylococci and Enterococci.

Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

Therapeutic Options for Infections due to vanB Genotype Vancomycin-Resistant Enterococci.

Enterococci are ubiquitous, facultative, anaerobic Gram-positive bacteria that mainly reside, as part of the normal microbiota, in the gastrointestinal tracts of several animal species, including humans. These bacteria have the capability to turn from a normal gut commensal organism to an invasive pathogen in patients debilitated by prolonged hospitalization, concurrent illnesses, and/or exposed to broad-spectrum antibiotics. The majority of vancomycin-resistant enterococcus (VRE) infections are linked to the vanA genotype; however, outbreaks caused by vanB-type VREs have been increasingly reported, representing a new challenge for effective antimicrobial treatment. Teicoplanin, daptomycin, fosfomycin, and linezolid are useful antimicrobials for infections due to vanB enterococci. In addition, new drugs have been developed (e.g., dalbavancin, telavancin, and tedizolid), new molecules will soon be available (e.g., eravacycline, omadacycline, and oritavancin), and new treatment strategies are progressively being used in clinical practice (e.g., combination therapies and bacteriophages). The aim of this article is to discuss the pathogenesis of infections due to enterococci harboring the vanB operon (vanBVRE) and their therapeutic, state-of-the-art, and future treatment options and provide a comprehensive and easy to use review for clinical purposes.

Increase of vancomycin-resistant Enterococcus faecium strain type ST117 CT71 at Charit\xe9 - Universit\xe4tsmedizin Berlin, 2008 to 2018

BackgroundIn addition to an overall rise in vancomycin-resistant Enterococcus faecium (VREfm), an increase in certain strain types marked by sequence type (ST) and cluster type (CT) has been reported in Germany over the past few years. Outbreak analyses at Charité - Universitätsmedizin Berlin revealed the frequent occurrence of VREfm ST117 CT71 isolates in 2017 and 2018. To investigate whether ST117 CT71 have emerged in recent years or whether these strains have been circulating for a longer time, we retrospectively analyzed non-outbreak strains that occurred between 2008 and 2018 to identify frequent STs and CTs.MethodsIn total, 120 VREfm isolates obtained from clinical and surveillance cultures from the years 2008, 2013, 2015, and 2018 were analyzed. Thirty isolates per year comprising the first 7–8 non-outbreak isolates of each quarter of the respective year were sequenced using whole genome sequencing. MLST and cgMLST were determined as well as resistance genes and virulence factors. Risk factors for VREfm ST117 were analyzed in a multivariable analysis with patient characteristics as possible confounders.ResultsThe percentage of VREfm of type ST117 increased from 17% in 2008 to 57% in 2018 (p = 0.012). In 2008, vanA genotype accounted for 80% of all ST117 isolates compared to 6% in 2018. VanB CT71 first appeared in 2018 and predominated over all other ST117 at 43% (p < 0.0001).The set of resistance genes (msrC, efmA, erm(B), dfrG, aac(6′)-Ii, gyrA, parC and pbp5) and virulence factors (acm, esp, hylEfm, ecbA and sgrA) in CT71 was also found in other ST117 non-CT71 strains, mainly in CT36. The study population did not differ among the different calendar years analyzed in terms of age, gender, length of stay, or ward type (each p > 0.2).ConclusionThis study revealed an increase in ST117 strains from 2008 to 2018, accompanied by a shift toward CT71 strains with the vanB genotype in 2018. We did not detect resistance or virulence traits in CT71 that could confer survival advantage compared to other CTs among ST117 strains. To date, it is not clear why ST117 and in particular strain type ST117 CT71 predominates over other strains.

2478. Surveillance of antibacterial resistance among clinical isolates from hospitals in Shanghai: results of 2018

BackgroundTo investigate the current state of antibacterial resistance of clinical isolates from hospitals in Shanghai, China.MethodsAntimicrobial susceptibility testing (AST) was carried out for the clinical isolates from 50 hospitals (including 30 grade A tertiary hospitals and 20 grade B tertiary hospitals/grade A secondary hospitals, and there were 3 children hospitals among them) according to a unified protocol using Kirby–Bauer(KB) method or automated AST systems. Results were analyzed according to CLSI 2018 breakpoints.ResultsOf the 144373 clinical isolates, Gram-positive cocci and Gram-negative bacilli accounted for 29.6% and 70.4%, respectively. The overall prevalence of MRSA in Staphylococcus aureus was 45.9% and 78.4% for MRCNS in coagulase-negative Staphylococcus. No strains were found resistant to vancomycin in Staphylococcus spp. 84.1% of the 1204 strains of non-meningitis S. pneumoniae isolated from children were penicillin-susceptible (PSSP), 15.9% were penicillin-nonsusceptible, including penicillin-intermediate (PISP, 10.5%) and penicillin-resistant (PRSP, 5.4%) strains. Of the 361 strains isolated from adults, 94.5%, 3.0% and 2.5% were PSSP, PISP, and PRSP, respectively. Vacomycin-resistance E. feacium was 0.7% and no vacomycin-resistant E. feacalis were identified. According to PCR results, most of these resistant strains were vanA genotype. The prevalence linezolid-nonsusceptible E. faecalis was about 1.6%, few E. feacium was resistant to Linezolid. The overall prevalence of ESBL-producing strains was 54.0% in E. coli, 35.0% in Klebsiella pneumoniae and 47.1% in Proteus mirabilis. Enterobacteriaceae isolates were still mainly susceptible to carbapenems. Overall, 11.7% and 11.2% of the Enterobacteriaceae isolates were resistant to imipenem and meropenem, respectively. The predominant organism of CRE isolates was K. pneumoniae. The prevalence of CRAB and CRPA were 62.5% and 28.7%, respectively.ConclusionAntimicrobial resistance remains to be a problematic issue in healthcare settings, especially in Gram-negative bacilli, effective infection-control measures should be promoted to tackle this critical threat.Disclosures All authors: No reported disclosures.

First detection of vanB phenotype-vanA genotype vancomycin-resistant enterococci in Egypt.

Enterococci have emerged in last two decades as serious hospital acquired pathogens particularly vancomycin resistant strains (VRE). The study aimed to identify the prevalence of enterococcal isolation from hospital infections and colonization as well as determine vancomycin resistance phenotypes and genotypes. Sixty enterococcus isolates were isolated from patients, health care workers and hospital environment, identified and tested for antimicrobial susceptibility. Enterococcus species were identified by Real-time PCR and vancomycin resistance was assessed by agar dilution method and Real-time PCR. out of 300 samples (20%) were enterococci (53.3% were E. faecium, 31.7% E. faecalis and 10% other enterococci). Among of them 40/60 (66, 6%) were isolated from infections and 33.3% were isolated from colonization. multiple drug resistance was reported in (100%) of isolates, while (95%) and (45%) of isolates were resistant to vancomycin and ticoplanin respectively. VanA phenotype, vanA genotype was identified in (47.4%) of isolates, while vanB phenotype, vanA genotype was identified in (33.3%) of vancomycin resistant isolates. VanB phenotype-vanA genotype was identified in (33.3%) of vancomycin resistant enterococcal isolates. To our knowledge it is the first identified incidence of such strains in Egypt and Africa.

Distribution and transmission characteristics of thirty-five dual genotype vancomycin-resistant Enterococcus faecium carrying both vanA and vanM in an intensive care unit

Objective To investigate the distribution and transmission characteristics of vancomycin-resistant Enterococcus faecium (VREF) carrying both vanA and vanM in the intensive care unit. Methods VREF strains were isolated from patients in the intensive care unit of Jinshan Hospital, Fudan University in Shanghai from 2013 to 2017. Antimicrobial susceptibilities of the VREF strains to nine antibiotics, including vancomycin, teicoplanin, linezolid and chloromycetin, were tested by broth microdilution method. Multiple polymerase chain reaction (PCR) was used for van genotyping and pulsed field gel electrophoresis (PFGE) was used for homology analysis. Results Thirty-five strains were mainly isolated from urine (16 strains), blood (11 strains), feces (five strains), bile (two strains) and pleural effusion (one strain). All the strains (100.00%) were resistant to vancomycin, ampicillin and levofloxacin, but only 40.00% were resistant to teicoplanin. All the strains were sensitive to linezolid. The results of van genotyping showed that 33 (94.3%) strains belonged to vanA and vanM dual genotype VREF, and the other two were vanA type VREF. PFGE results showed that 35 strains could be divided into 14 PFGE patterns, and seven out of 10 strains isolated in 2014 were identical and the other three belonged to three different PFGE patterns. Conclusions A dual genotype VREF carrying both vanA and vanM has been emerging and spreading in the intensive care unit of Jinshan Hospital, Fudan University in Shanghai. Key words: Enterococcus faecium; Vancomycin; Drug resistance; Genes; Genotype

Horizontal transfer of vanA between probiotic Enterococcus faecium and Enterococcus faecalis in fermented soybean meal and in digestive tract of growing pigs

BackgroundThe aim of this study was to investigate the intergeneric transfer of vancomycin resistance gene vanA between probiotic enterococci in the fermentation progress of soybean meal and in the digestive tract of growing pigs. One vanA genotype vancomycin resistant E. faecium strain, Efm4, and one chloramphenicol-resistant E. faecalis strain, Efs2, were isolated from twenty-nine probiotic basis feed material / additive samples. For in vitro conjugation, Efm4 and Efs2 were used as starter to ferment soybean meal. For in vivo conjugation, thirty growing pigs were randomly assigned to five groups (n = 6), treated with a basic diet, or supplemented with 10% fermented soybean meal, 1% Efm4, 5% Efs2 or a combination of 1% Efm4 + 5% Efs2 for 7 d, respectively. Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of Efm4, Efs2 and transconjugants. The sequence types (STs) of Efm4, Efs2 and transconjugants were analyzed by multilocus sequence typing (MLST). The vanA harboring plasmid in Efm4 and transconjugants was analyzed by S1-pulsed field gel electrophoresis (PFGE) and further verified by multiple alignments.ResultsThe results showed that, in FSBM, transconjugants were detected 1 h after the fermentation, with a conjugation frequency of ~ 10− 3 transconjugants / recipient. Transconjugants proliferated with Efm4 and Efs2 in the first 8 h and maintained steadily for 10 d till the end of the experiment. Additionally, in vivo experiment showed that transcojugants were recovered in one of six pigs in both FSBM and Efm4 + Efs2 groups, with conjugation frequency of ~ 10− 5 and ~ 10− 4, respectively. MLST revealed the ST of Efm4, Efs2 and transconjugants was ST1014, ST69 and ST69, respectively. S1-PFGE confirmed the existence of the vanA-harboring, 142,988-bp plasmid, which was also a multi-drug resistant plasmid containing Tn1546-like transposon.ConclusionsThe findings revealed the potential safety hazard existing in the commercial probiotic enterococci in China, because the horizontal transfer from farm to fork could potentially pose a safety risk to the public.

Emerging resistance of van genotype in enterococci: A potential menace for therapeutic failure.

Objectives:Emerging cases of vancomycin-resistant enterococci (VRE) are detrimental for the patients. The current study aimed to ascertain the occurrence of VRE, their antibiogram and the van genotype responsible for vancomycin resistance.Methods:A total number of 2,958 clinical specimens were processed at Microbiology Department of the Alrazi Health Care, Lahore during the one year (2016-2017) using microbiological culture media, biochemical and serology. Antibiogram of enterococcal strains was performed using disc diffusion and E-test. ATCC Enterococcus faecalis 29212 was used as a quality control strain. The detection of van genotypes was accomplished by multiplex PCR assay.Results:Out of the 147 enterococci, 139 (94.6%) were E. faecalis, and 8 (5.4%) were E. faecium. Statistically significant associations of urine (p < 0.001), pus (p < 0.001) and wound swabs (p = 0.001) were observed with E. faecalis. A significant correlation of enterococcal infections (p = 0.05) was seen with female patients. Four (2.9%) strains of E. faecalis found to be VRE with vanB (75%) and vanA (25%) genotypes.Conclusion:The emerging strains of VRE (vanB and vanA genotype) in the current study are a potential menace for therapeutic failure, which left the physicians with only linezolid as a therapeutic option.

Impact on Public Health of the Spread of High-Level Resistance to Gentamicin and Vancomycin in Enterococci.

Antibiotic resistance has turned into a global public health issue. Enterococci are intrinsically resistant to many antimicrobials groups. These bacteria colonize dairy and meat products and integrate the autochthonous microbiota of mammal’s gastrointestinal tract. Over the last decades, detection of vanA genotype in Enterococcus faecium from animals and from food of animal origin has been reported. Vancomycin-resistant E. faecium has become a prevalent nosocomial pathogen. Hospitalized patients are frequently treated with broad-spectrum antimicrobials and this leads to an increase in the presence of VanA or VanB vancomycin-resistant enterococci in patients’ gastrointestinal tract and the risk of invasive infections. In humans, E. faecium is the main reservoir of VanA and VanB phenotypes. Acquisition of high-level aminoglycoside resistance is a significant therapeutic problem for patients with severe infections because it negates the synergistic effect between aminoglycosides and a cell-wall-active agent. The aac(6′)-Ie-aph (2″)-Ia gene is widely spread in E. faecalis and has been detected in strains of human origin and in the food of animal origin. Enzyme AAC(6′)-Ie-APH(2″)-Ia confers resistance to available aminoglycosides, except to streptomycin. Due to the fast dissemination of this genetic determinant, the impact of its horizontal transferability among enterococcal species from different origin has been considered. The extensive use of antibiotics in food-producing animals contributes to an increase in drug-resistant animal bacteria that can be transmitted to humans. Innovation is needed for the development of new antibacterial drugs and for the design of combination therapies with conventional antibiotics. Nowadays, semi-purified bacteriocins and probiotics are becoming an attractive alternative to the antibiotic in animal production. Therefore, a better understanding of a complex and relevant issue for Public Health such as high-level vancomycin and gentamicin resistance in enterococci and their impact is needed. Hence, it is necessary to consider the spread of vanA E. faecium and high-level gentamicin resistant E. faecalis strains of different origin in the environment, and also highlight the potential horizontal transferability of these resistance determinants to other bacteria.

Co-occurrence of antimicrobial resistance and virulence determinants in enterococci isolated from traditionally fermented fish products.

Fermented foods frequently consumed in Northeast India can act as a reservoir for disseminating pathogenic organisms. Enterococci are often responsible for contamination of food products. This study investigated the antimicrobial resistance and co-existing virulence determinants of enterococci found in traditionally processed foods in India. A total of 38 enterococci isolates identified as Enterococcus faecalis isolated from fermented fish samples from retail markets of Northeast India were selected for screening of pathogenic traits. Of the 38 isolates, 8 (21%) were able to hydrolyse gelatin and 13 (34%) showed protease activity. Screening for haemolytic activity of the isolates showed no positive test on sheep blood. The presence of virulence genes (gelE, agg, esp, cpd, efaAfs and cylA) was investigated by PCR. gelE, agg and esp were present in 17, 13 and 4 isolates, respectively. cpd and efaAfs were found in all isolates, whereas cylA was not detected. High resistance percentages to various antibiotics included kanamycin (63%), vancomycin and gentamicin (58%), tetracycline (53%) and rifampicin (50%). The vanA genotype was confirmed in 15 multidrug- and vancomycin-resistant strains. The simultaneous occurrence of virulence determinants and antimicrobial resistance in enterococci prevalent in the fermented fish products studied poses a potential threat of transmission to humans through the food chain. This study highlights the importance of E. faecalis as a reservoir of antimicrobial resistance and virulence factors and their potential transfer to humans. The findings reopen the issue of food safety regarding enterococci prevalent in traditionally processed fish products in the region.

The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study.

Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm. A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid. vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission. Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

Presence of the esp gene in Enterococcus faecium derived from oropharyngeal microbiota of haematology patients

ObjectivesAntibiotic use and immunocompromised status in haematology patients have been shown to determine the constituents of commensal microbiota with highly increased resistance, including vancomycin resistant enterococci. We compared the carriage of virulence factor genes and the capacity for biofilm formation in vancomycin resistant enterococci (VRE) originating from the oropharyngeal and stool cultures of haematology patients. DesignPCR tests were used to investigate the presence of genes encoding pathogenicity factors (esp and hyl) in VRE isolates. The genotype of vancomycin resistance was investigated by multiplex PCR tests for vanA and vanB genes. PFGE typing was conducted to exclude the duplicate isolates. ResultsOf 3310 pharyngeal swabs taken from inpatients at a clinic for haematology, Enterococcus species were recovered in 6.46%. All VRE investigated were identified as Enterococcus faecium and were highly vancomycin resistant. VanA genotype was confirmed in all. In the group of oropharyngeal carriers (n = 8 patients), 15 strains were recovered from oropharyngeal specimens and PFGE typing revealed 5 types and 3 subtypes. Identical types of VRE in the oropharynx and stool cultures were found in three patients from this group. In the group of stool carriers (n = 24 patients) VRE were obtained from stools only and placed in 21 macro-restriction patterns. The esp gene was more common in VRE isolated from the oropharynx than in isolates from stools (p = 0.014). Results were not significant when we compared the presence of hyl genes in oropharyngeal isolates with those from stool cultures (p = 0.66) or when we investigated the association between esp and hyl gene carriage and capability of biofilm formation in non-repeated VRE. ConclusionsIn the present study, isolation of VRE from the oropharynx in haematology patients was associated with esp gene carriage. Further research is needed to investigate the clinical and long-term effects of this finding.

Vancomycin-resistant enterococci with vanA and vanB genes in Australian gulls.

This study is revealing the possible dissemination of vancomycin-resistant enterococci (VRE) from humans into the wildlife. We studied silver gulls (Chroicocephalus novaehollandiae) in Australia as potential carriers and reservoirs of VRE with acquired vancomycin resistance. In New South Wales (Australia), we have found two multi-resistant isolates belonging to Enterococcus faecium (sequence type 341, vanB genotype) and Enterococcus dispar (vanA genotype). Based on our knowledge, this is the first report of VRE in Australian wildlife.

Rapid detection of high-level vancomycin-resistant genes, vanA, vanB, vanD and vanM, in enterococci

Objective To develop a multiple polymerase chain reaction (PCR) technique based assay for rapid detection of vanA, vanB, vanD and vanM in high-level vancomycin-resistant enterococci. Methods After analyzing the uncleotide sequence divergence among D-Ala∶D-Lac ligase genes, an multiplex PCR assay for vanA, vanB, vanD and vanM genes in high-level vancomycin-resistant enterococci were designed. By using recombination plasmids containing vanA, vanB, vanD and vanM genes as positive control, and non-vancomycin resistant enterococci (non-VRE) common pathogenic bacterial DNA as negative control, the sensitivity and specificity of the assay were evaluated. Fifty vancomycin-resistant enterococci (VRE) isolates were detected by the assay. Fifty clinical strains of VRE were isolated from 9 hospitals in Shanghai from January 2006 to December 2014. The results were compared with the conventional PCR and sequencing methods. Results The identity of the D-Ala∶D-Lac ligase genes were 60.8%—71.3% of vanA, vanB, vanD and vanM genes. The multiplex PCR assay could identify the genotypes of the positive control samples accurately. No false positive results were found in negative control samples. Among fifty VRE strains detected by the assay, 18 were vanA genotype and 32 were vanM genotype. Comparison of the multiplex PCR assay and sequencing methods revealed sensitivity and specificity of 100%. The detection limit of the assay was 2×10 copies/PCR reaction. The experiment could be done within 3.5 h. Conclusions A multiplex PCR assay is developed to rapid identify the genotype of the high-level vancomycin-resistant enterococci, which can be used for the molecular epidemiology research and detection of VRE. Key words: Enterococcus; Vancomycin resistance; Genes; Genotype