Resistance Genes vanA Research Articles

Genomic analysis of enterococci carrying optrA, poxtA, and vanA resistance genes from wild boars, Italy.

To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars. Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings.

Role of membrane vesicles in the transmission of vancomycin resistance in Enterococcus faecium

Clonal transmission and horizontal gene transfer (HGT) contribute to the spread of vancomycin-resistant enterococci (VRE) in global healthcare. Our study investigated vesiduction, a HGT mechanism via membrane vesicles (MVs), for vanA and vanB genes that determine vancomycin resistance. We isolated MVs for VRE of different sequence types (STs) and analysed them by nanoparticle tracking analysis. Selected MV samples were subjected to DNA sequence analysis. In resistance transfer experiments, vancomycin-susceptible enterococci were exposed to MVs and bacterial supernatants of VRE. Compared to bacteria grown in lysogeny broth (MVs/LB), cultivation under vancomycin stress (MVs/VAN) resulted in increased particle concentrations of up to 139-fold (ST80). As a key finding, we could show that VRE isolates of ST80 and ST117 produced remarkably more vesicles at subinhibitory antibiotic concentrations (approx. 9.2 × 1011 particles/ml for ST80 and 2.4 × 1011 particles/ml for ST117) than enterococci of other STs (range between 1.8 × 1010 and 5.3 × 1010 particles/ml). In those MV samples, the respective resistance genes vanA and vanB were completely verifiable using sequence analysis. Nevertheless, no vancomycin resistance transfer via MVs to vancomycin-susceptible Enterococcus faecium was phenotypically detectable. However, our results outline the potential of future research on ST-specific MV properties, promising new insights into VRE mechanisms.

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system

ObjectiveThe purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.MethodsTwo specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.ResultsIn the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.ConclusionIn conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Rapid Detection of vanA Resistance Gene from E. faecalis Clinical Isolates Using Duplex Loop-Mediated Isothermal Amplification and Triplex PCR Assay.

Today, the spread of vancomycin-resistant strains isolated from Enterococcus faecalis (E. faecalis) has become a major health concern worldwide. Therefore, it is essential to provide a rapid and sensitive assay for identifying vanA gene for timely and appropriate antimicrobial control of resistant enterococcal infections. For this purpose, a cross-sectional study was performed on different clinical specimens of enterococci from Imam Reza hospital, Kermanshah, Iran. The antimicrobial susceptibility testing was determined by disk diffusion and MIC methods. Triplex-PCR and duplex-LAMP assays were also used to identify vanA E. faecalis resistance gene isolates. The results of this study shown that out of 108 Enterococcus isolates, 86, 18, 2, 1, and one isolates of E. faecalis, E. faecium, E. avium, E. psudoavium, and E. raffinosus were identified, respectively. On the other hand, E. faecalis was confirmed in 87 and 88 isolates using duplex-LAMP and triplex PCR, respectively. The LAMP primer set designed in this study can reliably identify seven distinct regions of the vanA gene, and finally the sensitivity, specificity, and the positive and negative predictive values of LAMP assay were shown to be 94.19%, 72.73%, 76.19%, and 93.10%, respectively. In general, sample processing, isothermal reaction and result reporting were completed using the LAMP assay in 75 minutes. Our findings suggest that LAMP assay has been approved as an alternative to the vancomycin resistance Enterococcus genotype (vanA and vanB) compared to other methods and has the advantage of being rapid, time-consuming, and easy for diagnosis.

The Role of Emergency Departments in Cross-Contamination: The Case of VRE

Colonized surfaces, equipment, individuals, and infected patients can be sources for the hospital spread of vancomycin-resistant enterococci (VRE), which is one of the important nosocomial pathogens. The basic epidemiological tools for the prevention and control of hospital-acquired infections are the typing methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminating method used frequently to detect clonal associations in epidemics and hospital-acquired VRE infections. This study aimed to investigate the presence of cross-contamination, which service or services come to the forefront in case of possible cross-contamination and clonal relationship between VRE strains isolated from rectal swab, clinical and environmental swab samples taken in two different periods in various clinics of Istanbul University Istanbul Medical Faculty Hospital by PFGE method. A total of 125 VREs isolated in two different periods were included in the study. Rectal and environmental swab samples were inoculated on Enterococcosel agar and sodium azide broth, urine samples were inoculated on chromogenic agar, and other clinical samples were inoculated on 5% sheep blood agar and incubated for 18-24 hours. VITEK 2 Compact automation system GP panel (bioMerieux, MarcyL'Etoile, France) was used for the species identification of catalase-negative, gram-positive colonies and disc diffusion and minimum inhibitory concentration (MIC) gradient tests were used to determine vancomycin susceptibility. Multiplex polymerase chain reaction was used to search for vanA and vanB resistance genes in isolates identified as VRE, and PFGE was used to determine clonal association. All isolates were identified as Enterococcus faecium with the vanA resistance gene. It was shown that PFGE clones were divided into six types with 65% similarity (A-F), and in this polyclonal spread, the major type was type A, which contained 108 isolates in 37 subtypes existed in the hospital for years. In patients from whom similar isolates were obtained, the high rate of hospitalizations in the same emergency room or in different emergency services in the same building drew attention. Our results showed that the presence of VRE was established in our hospital, new isolates were added from time to time, and there was a cross-contamination. It was observed that emergency services, where infection control measures were neglected due to working conditions, were among the high-risk areas for VRE contamination. In order to better understand the importance of emergency services in cross-contamination, it would be useful to conduct surveillance studies among patients hospitalized in emergency services and monitor the rate of VRE in the services where positive patients were transferred.

Clinically Relevant Enterococcus Species and PCR Screening of Antibiotic Resistance and Virulence Factor Coding Genes

Enterococci are Gram positive cocci which are common residence of gastro intestinal tracts of humans and animals. But they are capable of causing severe infections, most often in hospitalised patients. Enterococci are important nosocomial pathogens and their intrinsic property of antibiotic resistance makes treatment difficult. Against this clinical significance, a study was conducted on the prevalence of vancomycin resistant enterococcal infections and their antibiotic sensitivity patterns at Sunrise Institute of Medical Sciences, a tertiary care hospital in Kochi, Kerala state. Various clinical specimens like blood, urine, abscess, vaginal swab etc. were microbiologically screened for the presence of antibiotic resistant Enterococci. The common phenotyping methods and genotyping protocols were used to identify the species for the study. The vancomycin resistant strains were typed genotypically by using 16S rDNA sequencing and the isolates were speciated to be E. faecium. A molecular screening of the isolates was also done for the presence of various virulence factor coding genes and for the genes which confer antibiotic resistance. The findings of the study revealed that 89.1 % isolated strains were multidrug resistant and a total of 4 antibiotic resistance genes were detected. Among the isolates the vancomycin resistance genes vanA, vanB; tetracycline resistance genes tetA and macrolide resistance gene ermA were screened. The presence of genes coding for various virulence factors were also detected among the isolates. However, we present an overview of antibiotic resistance pattern and virulence factors present in enterococci and we strongly believe that these results will surely help to design a new disinfection regime and antibiotic stewardship to control the incidence of infection.

Faecal microbiota transplantation reduces amounts of antibiotic resistance genes in patients with multidrug-resistant organisms

BackgroundMultidrug-resistant organisms (MDROs) such as vancomycin-resistant enterococci (VRE) and carbapenemase-producing Enterobacteriaceae (CPE) are associated with prolonged hospitalisation, increased medical costs, and severe infections. Faecal microbiota transplantation (FMT) has emerged as an important strategy for decolonisation. This study aimed to evaluate the genetic response of MDROs to FMT.MethodsA single-centre prospective study was conducted on patients infected with VRE, CPE, or VRE/CPE who underwent FMT between May 2018 and April 2019. Genetic response was assessed as the change in the expression of the resistance genes VanA, blaKPC, blaNDM, and blaOXA on days 1, 7, 14, and 28 by real-time reverse-transcription polymerase chain reaction.ResultsTwenty-nine patients received FMT, of which 26 (59.3%) were infected with VRE, 5 (11.1%) with CPE, and 8 (29.6%) with VRE/CPE. The mean duration of MDRO carriage before FMT was 71 days. Seventeen patients (63.0%) used antibiotics within a week of FMT. In a culture-dependent method, the expression of VanA and overall genes significantly decreased (p = 0.011 and p = 0.003 respectively). In a culture-independent method, VanA, blaNDM, and overall gene expression significantly decreased over time after FMT (p = 0.047, p = 0.048, p = 0.002, respectively). Similar results were confirmed following comparison between each time point in both the culture-dependent and -independent methods. Regression analysis did not reveal important factors underlying the genetic response after FMT. No adverse events were observed.ConclusionFMT in patients infected with MDROs downregulates the expression of resistance genes, especially VanA, and facilitates MDRO decolonisation.

Isolation and antimicrobial resistance of vancomycin resistant Enterococcus spp. (VRE) and methicillin-resistant S. aureus (MRSA) on beef and chicken meat, and workers hands from slaughterhouses and retail shops in Turkey

The objectives of this study were to determine the presence and antimicrobial resistance of Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococci (VRE) on beef and chicken carcasses and meat, and workers hands’ at processing time from a cattle and a poultry slaughterhouse, and beef and chicken meat at retail level. Disk diffusion method was used to determine the antimicrobial resistance profile of the Enterococcus spp. and S. aureus isolates. Minimum Inhibitory Concentration (MIC) values were determined for vancomycin and oxacillin resistance. Finally, conventional PCR was performed to determine the presence of the mecA and vanA resistance genes in isolates classified resistant to oxacillin and vancomycin according to MIC values. S. aureus and Enterococcus faecium isolated from 17 (17%) and eight (8%) samples, respectively. E. faecalis was not detected in any sample. The highest resistance rates were to ampicillin (3/5, 60 %) and penicillin G (5/5, 100 %) in MRSA and tetracycline (4/5, 80 %) in VRE isolates. While the mecA gene was detected in all MRSA isolates, vanA gene was not detected in any of the phenotypically vancomycin resistant E. faecium isolates. The present study provides data for multiple antimicrobial resistance and presence of VRE and MRSA isolated from an ongoing surveillance in humans, livestock and poultry in Turkey.

Resistant Genes and Multidrug-Resistant Bacteria in Wastewater: A Study of Their Transfer to the Water Reservoir in the Czech Republic.

Wastewater is considered the most serious source of the spread of antibiotic resistance in the environment. This work, therefore, focuses on the fate and spread of antibiotic resistance genes (ARGs) in wastewater and the monitoring of multidrug-resistant strains. ARGs were monitored in the nitrification and sedimentation tanks of the wastewater treatment plant (WWTP) and in the dam into which this WWTP flows, at various times. The highest relative abundance was found for the blaTEM > tetW > blaNDM-1 > vanA resistance genes, respectively. An increased concentration of tetracycline (up to 96.00 ng/L) and ampicillin (up to 19.00 ng/L) was found in water samples compared to other antibiotics detected. The increased incidence of seven ARGs and four antibiotics was observed in the November and December sampling times. Isolated ampicillin-resistant strains showed a high degree of resistance to ampicillin (61.2% of the total isolates had a minimum inhibitory concentration (MIC) ≥ 20 mg/mL). In 87.8% of isolates, out of the total number, the occurrence of two or more ARGs was confirmed. These multidrug-resistant strains were most often identified as Aeromonas sp. This strain could represent a significant role in the spread of multidrug resistance through wastewater in the environment.

Inactivation of antibiotic resistant bacteria and their resistance genes in sewage by applying pulsed electric fields

We evaluated the suitability of pulsed electric field (PEF) technology as a new disinfection option in the sewage treatment plants (STPs) that can inactivate antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). It was shown that PEF applied disinfection could inactivate not only vancomycin-resistant enterococci (VRE), but also vanA resistance gene. Cultivable VRE could be effectively inactivated by PEF applied disinfection, and were reduced to below the detection limit (log reduction value of VRE > 5 log). Although the vanA also showed a reduction of more than 4 log, it remained in the order of 105 copies/mL, suggesting that ARGs are more difficult to be inactivated than ARB in PEF applied disinfection. Among parameters in each applying condition verified in this study, the initial voltage was found to be the most important for inactivation of ARB and ARGs. Furthermore, frequency was a parameter that affects the increase or decrease of the duration time, and it was suggested that the treatment time could be shortened by increasing the frequency. Our results strongly suggested that PEF applied disinfection may be a new disinfection technology option for STPs that contributes to the control of ARB and ARGs contamination in the aquatic environments.

Analysis of Vancomycin-Resistant Enterococci in Hemato-Oncological Patients.

Enterococci are important bacterial pathogens, and their significance is even greater in the case of vancomycin-resistant enterococci (VRE). The study analyzed the presence of VRE in the gastrointestinal tract (GIT) of hemato-oncological patients. Active screening using selective agars yielded VRE for phenotypic and genotypic analyses. Isolated strains were identified with MALDI-TOF MS, (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) their susceptibility to antibiotics was tested, and resistance genes (vanA, vanB, vanC-1, vanC2-C3) and genes encoding virulence factors (asa1, gelE, cylA, esp, hyl) were detected. Pulsed-field gel electrophoresis was used to assess the relationship of the isolated strains. Over a period of three years, 103 VanA-type VRE were identified in 1405 hemato-oncological patients. The most frequently detected virulence factor was extracellular surface protein (84%), followed by hyaluronidase (40%). Unique restriction profiles were observed in 33% of strains; clonality was detected in 67% of isolates. The study found that 7% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was Enterococcus faecium. No clone persisted for the entire 3-year study period. However, genetically different clusters were observed for shorter periods of time, no longer than eight months, with identical VRE spreading among patients.

Investigation of the presence of slime production, VanA gene and antiseptic resistance genes in Staphylococci isolated from bovine mastitis in Algeria

Staphylococcus strains are frequently as- sociated with clinical and subclinical bovine intra-mammary infection. The virulence factors of staphylococcus have not been widely studied in Algeria. The objective of this study was to determine the frequency of slime production, VanA gene and antiseptic resistance genes in staphylococci strains isolated from bovine mas- titis in Algeria. The study examined 35 Staphy- lococci strains obtained from the inflammatory secretion of mammary glands of cows with mastitis. Slime production was determined by detecting the icaA and icaD genes using the polymerase chain reaction (PCR) method and Congo red agar (CRA) method. The presence of qacAB and qac C antiseptic resistance genes and the VanA resistance gene in these isolates was investigated by PCR. The results of the current study revealed that of the 35 Staphylo- cocci isolates, 42.85% (15/35) and 17.14% (6/35) of the isolates harboured the slime production gene by analysing icaA and icaD genes, respec- tively and 71.42% (25/35) by the CRA method. However, VanA and antiseptic resistance genes (qacAB and qac C) were not detected in any of the isolates. Therefore, the majority of Staphylo- coccus strains were capable of producing slime, and the CRA detection rate was higher than the PCR method for the biofilm-producing capac- ity of Staphylococcus strains. Thus, the presence of the ica genes in Staphylococcus strains con- firms its role as a virulence factor in the patho- genesis of bovine mastitis.

Emergence of vanA-Type Vancomycin-Resistant Enterococcus faecium ST 78 Strain with a rep2-Type Plasmid Carrying a Tn1546-Like Element Isolated from a Urinary Tract Infection in China.

PurposeThe emergence of vancomycin-resistant enterococci (VRE) dramatically narrows therapeutic options. Although the prevalence of VRE in China has maintained a low level, VRE outbreaks have been reported in some tertiary hospitals in the developed areas of China. The clonal background of vanA-positive Enterococcus faecium strains has not been well characterized in China. Here, we report the whole-genome sequence of a vanA-type vancomycin-resistant E. faecium belonging to sequence type (ST) 78 isolated from a urinary tract infection in China.Patients and MethodsA vancomycin-resistant E. faecium was isolated from a 66-year-old male patient diagnosed with brainstem hemorrhage. Antibiotic susceptibility assays were performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Complete genome sequencing was performed using both the HiSeqTM 4000 platform and the MinION platform. Plasmid, genomic and phylogenetic relationship analysis were further performed.ResultsE. faecium VRE1 was resistant to all antimicrobials tested except for tetracyclines and oxazolidinones. The whole genome of E. faecium VRE1 was composed of one chromosomal DNA and four plasmids. Two virulence genes and five antimicrobial resistance genes were identified. In silico multilocus sequence typing (MLST) showed that it belonged to ST78 (clonal complex CC17), a well-known epidemic clone that is widespread in Europe and the United States. Three antimicrobial resistance genes, including aminoglycoside resistance genes ant(6)-Ia and aph(3ʹ)-III; and glycopeptide resistance gene vanA, were located on a rep2-type plasmid carrying a Tn1546-like element that has not been reported. The most closely related strain harboring a similar plasmid backbone was recovered from fodder sample in China that differed by 178 cgMLST loci.ConclusionOur study characterizes the genomic feature of a vancomycin-resistant E. faecium ST78 strain harboring a vanA-carrying plasmid in China. The ST78 clonal group possessed the potential to emerge as a successful vanA-carrying epidemic lineage in China.

CRISPR elements and their association with antimicrobial resistance and virulence genes among vancomycin-resistant and vancomycin-susceptible enterococci recovered from human and food sources

We aimed to investigate the occurrence of CRISPR elements in the genomes of vancomycin-resistant (VRE) and vancomycin-susceptible (VSE) enterococci and their association with the presence of antimicrobial resistance and virulence genes. We analyzed 180 isolates, including 91 VRE and 89 VSE. Isolates were identified by PCR or MALDI-TOF. Antimicrobial susceptibility and MICs for vancomycin were determined by the disk-diffusion method and E-test®, respectively. The presence of resistance and virulence genes, as well as CRISPR elements, was investigated by PCR. We identified 95 (53%) E. faecalis, 78 (43%) E. faecium, five (2.8%) E. gallinarum, and one (0.6% each) E. casseliflavus and E. durans. The highest and the lowest non-susceptibility frequencies were observed for erythromycin (n = 152; 84.4%) and fosfomycin (n = 5; 2.8%), respectively. Most erythromycin-resistant isolates had the erm(B) gene (106/152; 69.7%). Of 118 (65.6%) isolates with high-level resistance to aminoglycoside, 69 (58.5%) had at least one aminoglycoside resistance gene, mostly ant(6)-Ia and aac(6′)-Ie + aph(2″)-Ia. We found at least one virulence gene among 135 (75%) isolates, mostly gelE (79/180; 43.9%). Ninety-two (51.1%) isolates had at least one CRISPR element, especially CRISPR3 (62/92; 67.4%). CRISPR elements were more common among E. faecalis, in which we observed a relationship between the absence of CRISPR and the presence of the vanA resistance gene, and the hyl and esp virulence genes. Among VRE. faecium, a relationship was found between the absence of CRISPR and the hyl gene. In conclusion, we found evident associations between the lack of CRISPR elements with species, multidrug resistance, and major resistance- and virulence-associated genes.

Determination of clonal relationships and virulence genes of vancomycin-resistant enterococcus spp. isolated from colonized and infected patients

Purpose: In this study, we investigated the possible relationship between virulence factors and clonal relationship between Vancomycin-resistant colonization and infection isolates. Materials and Methods: A total of 156 Vancomycin-resistant Enterococcus spp. (VRE) were collected and grouped as infection and colonization isolates. A multiplex polymerase chain reaction analysis was performed to screen specific virulence genes (esp, hyl, asa1, cylA and gelE) and vancomycin resistance genes (vanA, vanB). The clonal relationship among isolates was investigated by the Pulsed Field Gel Electrophoresis (PFGE) method. Results: The vanA gene was determined in 86 infection and 64 colonization isolates. esp was the most common virulence gene for both groups (55.8% and 56.25%), followed by hyl (51.1% and 35.9%) that statistically differed between the two groups. The 150 E. faecium isolates carrying the vanA resistance gene were divided into 24 main clusters (A-Y) in PFGE analysis. Conclusion: When the distribution of virulence genes or genes combinations of the infection and colonization groups was examined, there was no statistical significance of cluster distribution in the two groups. However, hyl gene was found to be more common in infection isolates.

Molecular analysis and epidemiological typing of Vancomycin-resistant Enterococcus outbreak strains

Outbreaks of multidrug resistant bacteria including vancomycin-resistant enterococci (VRE) in healthcare institutions are increasing in Norway, despite a low level of resistance compared to other European countries. In this study, we describe epidemiological relatedness of vancomycin-resistant Enterococcus faecium isolated during an outbreak at a Norwegian hospital in 2012–2013. During the outbreak, 9454 fecal samples were screened for VRE by culture and/or PCR. Isolates from 86 patients carrying the vanA resistance gene were characterized using pulsed-field gel electrophoresis (PFGE), MALDI-TOF mass spectrometry and single nucleotide polymorphism typing. PFGE revealed two main clusters, the first comprised 56 isolates related to an initial outbreak strain, and the second comprised 21 isolates originating from a later introduced strain, together causing two partly overlapping outbreaks. Nine isolates, including the index case were not related to the two outbreak clusters. In conclusion, the epidemiological analyses show that the outbreak was discovered by coincidence, and that infection control measures were successful. All typing methods identified the two outbreak clusters, and the experiment congruence between the MALDI-TOF and the PFGE clustering was 63.2%, with a strong correlation (r = 72.4%). Despite lower resolution compared to PFGE, MALDI-TOF may provide an efficient mean for real-time monitoring spread of infection.

Molecular characterization of vancomycin resistant enterococci from various clinical samples in a tertiary care hospital

Aim: The aim of this present study was to investigate the molecular characterization of vancomycin resistant enterococci isolated from various clinical samples. Enterococci are aerobic and anaerobic gram positive cocci found in gastrointestinal tract of humans and other animals. Materials and Methods: Enterococcal isolates were isolated from various clinical samples according to standard protocol and sample size of the study was 52. Vancomycin resistance genes VanA and VanB were detected using conventional PCR. Results: The six isolates resistant to Vancomycin confirmed phenotypically were confirmed using Polymerase Chain Reaction. Conclusion: in the present study conclude that, Vancomycin resistant enterococci is a major health problem in the coming years and hence it is necessary to take all adequate measures to identify the resistant strains. Keywords: Vancomycin resistance, Enterococci and polymerase chain reaction.

<p>Genetic characterization of two vancomycin-resistant <em>Staphylococcus aureus</em> isolates in Kerman, Iran</p>

AimThe aim of this study was the genetic characterization of two clinical vancomycin-resistant Staphylococcus aureus (VRSA) isolates.Materials and methodsResistance to vancomycin was determined by phenotypic method. PCR was used for detection of mecA, vanA, ermA, ermB, ermC, msrA/B, aph(2”)-Ic, aph(3ʹ)-IIIa, pvl, Immune Evasion Cluster [sea, sep, chip, sak and scn] genes and biofilm operon icaABCD. On the other hand, multilocus sequence typing and agr typing methods were performed for the determination of clonal relationship and van operon was detected and sequenced.ResultsVancomycin-resistant Staphylococcus aureus strain 1 (VRSA-1) was positive for vanA, ermA, ermC, aph(2”)-Ic, aph(3ʹ)-IIIa, sea, sep, icaD genes, belonging to agr type I; SCCmec type III; spa type t030; and ST239. However, the genetic characterization of Vancomycin-resistant Staphylococcus aureus strain 2 (VRSA-2) revealed the presence of various types of resistance genes vanA, ermA, ermC, aph(2”)-Ic, aph(3ʹ)-IIIa, sea, icaD, relating to agr type I; SCCmec type III; spa type t459; and ST239. The presence of transposon Tn1546 was determined by PCR sequencing.The Basic Local Alignment Search Tool analysis of van operon in the VRSA isolates showed 99.6% sequence homology to Tn1546 in vancomycin-resistant enterococci, indicating the vanA operon has an enterococcal origin.ConclusionIn conclusion, the ST239 is one of the most common clones of MRSA isolates which involved the hospital-associated infections, therefore, the emergence of VRSA isolates with ST239 increased the spread of resistance to vancomycin in the hospital settings.

Highly variable vancomycin-resistant enterococci in the north-eastern part of the Czech Republic.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens of increasing medical importance. This study involved 121 VRE selectively obtained from a representative set of 1464 samples collected from various sources in the north-eastern part of the Czech Republic. In total, 119 VRE belonged to Enterococcus faecium and two to Enterococcus faecalis. All isolates of E. faecium were resistant to at least three antibiotic classes. The resistance genes vanA, erm(B), tet(M), tet(L), aac(3)-IIIa and aac(6')-aph(2'') were detected. We assigned the E. faecium to sequence types ST5, ST18, ST38, ST64, ST92, ST273, ST549 and ST640. In E. faecium isolates, we identified the presence of replicases rep20p LG 1 , rep2p RE 25 , rep17p RUM , rep21p VEF 1/2 and rep14p RI 1 , as well as relaxases relp EF 1 , relp LG 1 , relp CIZ 2 , relp RE 25 and relp RUM . The presence of the toxin-antitoxin system axe-txe was detected mainly among isolates of hospital origin. The A and D types of transposon Tn1546 were those occurring most frequently. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first extensive study of vancomycin-resistant enterococci of diverse origin in a single well-defined area of the Czech Republic. The isolates were investigated for their antibiotic resistance, epidemiological characteristics and plasmid characteristics. Based on the results obtained, we can make assumptions as to the ways that vancomycin resistance is disseminated throughout the environment including humans and animals.

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.