Acquisition Of Vancomycin Resistance Research Articles

Reversible vancomycin susceptibility within emerging ST1421 Enterococcus faecium strains is associated with rearranged vanA-gene clusters and increased vanA plasmid copy number

Vancomycin variable enterococci (VVE) are van-positive enterococci with a vancomycin-susceptible phenotype (VVE-S) that can convert to a resistant phenotype (VVE-R) and be selected for during vancomycin exposure. VVE-R outbreaks have been reported in Canada and Scandinavian countries. The aim of this study was to examine the presence of VVE in whole genome sequenced (WGS) Australian bacteremia Enterococcus faecium (Efm) isolates collected through the Australian Group on Antimicrobial resistance (AGAR) network. Eight potential VVEAus isolates, all identified as Efm ST1421, were selected based on the presence of vanA and a vancomycin-susceptible phenotype. During vancomycin selection, two potential VVE-S harboring intact vanHAX genes, but lacking the prototypic vanRS and vanZ genes, reverted to a resistant phenotype (VVEAus-R). Spontaneous VVEAus-R reversion occurred at a frequency of 4-6 × 10−8 resistant colonies per parent cell in vitro after 48 h and led to high-level vancomycin and teicoplanin resistance. The S to R reversion was associated with a 44-bp deletion in the vanHAX promoter region and an increased vanA plasmid copy number. The deletion in the vanHAX promoter region enables an alternative constitutive promoter for the expression of vanHAX. Acquisition of vancomycin resistance was associated with a low fitness cost compared with the corresponding VVEAus-S isolate. The relative proportion of VVEAus-R vs. VVEAus-S decreased over time in serial passages without vancomycin selection. Efm ST1421 is one of the predominant VanA-Efm multilocus sequence types found across most regions of Australia, and has also been associated with a major prolonged VVE outbreak in Danish hospitals.

VanA-Enterococcus faecalis in Poland: hospital population clonal structure and vanA mobilome

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1pVEF1, rep1pIP501, rep2pRE25, rep9pAD1/pTEF2/pCF10 and rep6pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18ap200B and repUS1pVEF1 genes and located on ~ 20 kb and 150–200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals.

First Report of the Local Spread of Vancomycin-Resistant Enterococci Ascribed to the Interspecies Transmission of a vanA Gene Cluster-Carrying Linear Plasmid.

Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid.IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.

Prevalence of vancomycin resistance phenotypes among Enterococcus species isolated from clinical samples in a Romanian hospital

Vancomycin resistant Enterococcus (VRE) has been increasingly reported since the 1980s. Although normally regarded as harmless commensals, enterococci can become the etiological agents of nosocomial infections causing urinary tract infections, peritonitis, bacteriemia, wound infections and endocarditis. The acquisition of vancomycin resistance has seriously affected the treatment and infection control of these bacteria. VRE are frequently resistant to most antibiotics that are effective against vancomycin susceptible enterococci, the therapeutic options of VRE infections being thus very limited. The aim of this study was to assess the genetic support of vancomycin resistance among VRE strains isolated from patients admitted to intensive care units in one hospital unit from Bucharest, Romania Out of 84 enterococcal strains isolated from urine prelevated from catheterized and non-catheterized patients with urinary tract infections, wound secretions, pus, blood, ascite liquid and stool, 28 strains had glycopeptide resistance. Out of these, in the 12 vancomycin-resistant Enterococcus faecalis strains, the VanA phenotype was detected in 9 strains and VanB in 3 strains. From the 16 E. faecium strains resistant to vancomycin, 10 strains showed the VanA phenotype and 6 strains had the VanB phenotype. According to the results of this study, the VanA phenotype, conferring high-level resistance to vancomycin and teicoplanin, was more prevalent than VanB in the Enterococcus sp. strains isolated from hospitalized patients.

VraR Binding to the Promoter Region of agr Inhibits Its Function in Vancomycin-Intermediate Staphylococcus aureus (VISA) and Heterogeneous VISA.

Acquisition of vancomycin resistance in Staphylococcus aureus is often accompanied by a reduction in virulence, but the mechanisms underlying this change remain unclear. The present study was undertaken to investigate this process in a clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain, 10827; an hVISA reference strain, Mu3; and a VISA reference strain, Mu50, along with their respective series of vancomycin-induced resistant strains. In these strains, increasing MICs of vancomycin were associated with increased expression of the vancomycin resistance-associated regulator gene (vraR) and decreased expression of virulence genes (hla, hlb, and coa) and virulence-regulated genes (RNAIII, agrA, and saeR). These results suggested that VraR might have a direct or indirect effect on virulence in S. aureus In electrophoretic mobility shift assays, VraR did not bind to promoter sequences of hla, hlb, and coa genes, but it did bind to the agr promoter region. In DNase I footprinting assays, VraR protected a 15-nucleotide (nt) sequence in the intergenic region between the agr P2 and P3 promoters. These results indicated that when S. aureus is subject to induction by vancomycin, expression of vraR is upregulated, and VraR binding inhibits the function of the Agr quorum-sensing system, causing reductions in the virulence of VISA/hVISA strains. Our results suggested that VraR in S. aureus is involved not only in the regulation of vancomycin resistance but also in the regulation of virulence.

Dispersion of the Vancomycin Resistance Genes vanA and vanC of Enterococcus Isolated from Nile Tilapia on Retail Sale: A Public Health Hazard.

Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The acquisition of vancomycin resistance by enterococci (VRE) has seriously affected the treatment and infection control of these organisms. VRE are frequently resistant to all antibiotics that are effective treatment for vancomycin-susceptible enterococci, which leaves clinicians treating VRE infections with limited therapeutic options. With VRE emerging as a global threat to public health, we aimed to isolate, identify enterococci species from tilapia and their resistance to van-mediated glycopeptide (vanA and vanC) as well as the presence of enterococcal surface protein (esp) using conventional and molecular methods. The cultural, biochemical (Vitek 2 system) and polymerase chain reaction results revealed eight Enterococcus isolates from the 80 fish samples (10%) to be further identified as E. faecalis (6/8, 75%) and E gallinarum (2/8, 25%). Intraperitoneal injection of healthy Nile tilapia with the eight Enterococcus isolates caused significant morbidity (70%) within 3 days and 100% mortality at 6 days post-injection with general signs of septicemia. All of the eight Enterococcus isolates were found to be resistant to tetracycline. The 6/6 E. faecalis isolates were susceptible for penicillin, nitrofurantoin, gentamicin, and streptomycin. On the other hand 5/6 were susceptible for ampicillin, vancomycin, chloramphenicol, and ciprofloxacin. The two isolates of E. gallinarum were sensitive to rifampicin and ciprofloxacin and resistant to vancomycin, chloramphenicol, and erythromycin. Molecular characterization proved that they all presented the prototypic vanC element. On the whole, one of the two vancomycin resistance gene was present in 3/8 of the enterococci isolates, while the esp virulence gene was present in 1/8 of the enterococci isolates. The results in this study emphasize the potential role that aquatic environments are correlated to proximity to anthropogenic activities in determining the antimicrobial resistance patterns of Enterococcus spp. recovered from fish in the river Nile in Giza, Elmounib, Egypt as a continuation of our larger study on the reservoirs of antibiotic resistance in the environment.

Prevalence of Virulence Factors and Drug Resistance in Clinical Isolates of Enterococci: A Study from North India.

Along with emergence of multidrug resistance, presence of several virulence factors in enterococci is an emerging concept. This study was undertaken to determine the prevalence of various virulence factors phenotypically and genotypically in enterococci and study their association with multidrug resistance. A total of 310 enterococcal isolates were studied, comprising 155 E. faecium and 155 E. faecalis. Antimicrobial susceptibility testing was done by disc diffusion and agar dilution method. Hemolysin, gelatinase, biofilm production, and haemagglutination were detected phenotypically and presence of virulence genes, namely, asa1, gelE, cylA, esp, and hyl, was detected by multiplex PCR. Of the total, 47.41% isolates were high level gentamicin resistant (HLGRE) and 7.09% were vancomycin resistant (VRE). All the virulence traits studied were found in varying proportions, with majority in E. faecalis (p > 0.05). Strong biofilm producers possessed either asa1 or gelE gene. gelE silent gene was detected in 41.37% (12/29). However, increase in resistance was associated with significant decrease in expression or acquisition of virulence genes. Further, acquisition of vancomycin resistance was the significant factor responsible for the loss of virulence traits. Though it is presumed that increased drug resistance correlates with increased virulence, acquisition of vancomycin resistance might be responsible for reduced expression of virulence traits to meet the “biological cost” relating to VRE.

Could a Phosphotransferase System Provide the Means to Control Outbreaks of Enterococcus faecium Infection?

The epidemiology of enterococcal infections has changed dramatically over the past 100 years [1]. Until relatively recently, the vast majority were caused by Enterococcus faecalis and were acquired in the community; then, during the 1970s– 1980s, the percentage of nosocomial infections due to E. faecalis tripled [2,3],an increase temporally associated with increasing use of third-generation cephalosporins. In the United States, an increase in the proportion of nosocomial infections caused by Enterococcus faecium was observed in the 1980s [4, 5], and by 2006–2007, a Centers for Disease Control survey of healthcare-associated infections found that approximately 38% of clinical enterococcal isolates identified to the species level were E. faecium [6]. This increase was blamed on the acquisition of vancomycin resistance by E. faecium (which seldom occurred in E. faecalis) and the increasing use of vancomycin in hospitalized patients. However, the epidemiology of vancomycin-resistant E. faecium (VREfm) presented a quandary: at the time, VREfm was alreadyacommon cause of US healthcare-associated infections, such organisms were not found in the community [7].Yet, concurrently, VREfm was rare as a cause of infection in the European Union but were commonly found in feces of food animals in Europe, in products derived from these animals, in feces of individuals handling them, and even in feces of

Global Spread of the hyl Efm Colonization-Virulence Gene in Megaplasmids of the Enterococcus faecium CC17 Polyclonal Subcluster

Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumptively associated with the expansion of a human-associated Enterococcus faecium polyclonal subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin and vancomycin) and virulence (esp(Efm), hyl(Efm), and fms) traits. We analyzed the presence and the location of a putative glycoside hydrolase hyl(Efm) gene among E. faecium strains obtained from hospitalized patients (255 patients; outbreak, bacteremic, and/or disseminated isolates from 23 countries and five continents; 1986 to 2009) and from nonclinical origins (isolates obtained from healthy humans [25 isolates], poultry [30], swine [90], and the environment [55]; 1999 to 2007). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid analysis included determination of content and size (S1-PFGE), transferability (filter mating), screening of Rep initiator proteins (PCR), and location of vanA, vanB, ermB, and hyl(Efm) genes (S1/I-CeuI hybridization). Most E. faecium isolates contained large plasmids (>150 kb) and showed variable contents of van, hyl(Efm), or esp(Efm). The hyl(Efm) gene was associated with megaplasmids (170 to 375 kb) of worldwide spread (ST16, ST17, and ST18) or locally predominant (ST192, ST203, ST280, and ST412) ampicillin-resistant CC17 clones collected in the five continents since the early 1990s. All but one hyl(Efm)-positive isolate belonged to the CC17 polyclonal subcluster. The presence of hyl(Efm) megaplasmids among CC17 from Europe, Australia, Asia, and Africa since at least the mid-1990s was documented. This study further demonstrates the pandemic expansion of particular CC17 clones before acquisition of vancomycin resistance and putative virulence traits and describes the presence of megaplasmids in most of the contemporary E. faecium isolates with different origins.

Characterization of Vancomycin-Resistant Enterococci Isolated from Stools and Their Acquisition of Vancomycin Resistance

We have isolated 6 vancomycin resistant (VR) Enterococcus faecium and 5 VR-E. gallinarum. Vancomycin resistant enterococcus (VRE) isolates were resistant to multi-drugs, but susceptible to linezolid and quinupristin/dalfopristin. VRE isolates showed 10 VanA phenotypes and 1 VanB phenotype (E. gallinarum). However, all of them showed vanA genotype. vanA gene was detected on both genomic and plasmid DNA from all VRE isolates. Almost of VR-E. faecium had IS1216V which is worldwide type and almost of VR-E. gallinarum had IS1542 which is European type. IS1216V and IS1542 genes were not related with antibiotic types of VRE. Copy numbers of vanA were decreased in VRE with IS1216V or IS1542 but not in VRE with both ISs in broth without vancomycin. The copy numbers of vanA were significantly decreased in VanB phenotype of VRE with IS1542 in broth without vancomycin. Copy numbers of vanA were recovered in the presence of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis when cultured in the broth containing vancomycin. Reference strains cultured in the broth containing vancomycin showed intermediate resistance or resistance to antibiotics without acquisition of van genes. Naturally, multidrugresistant E. faecium might be fast adapted in the presence of vancomycin compared to E. faecalis. Taken together, VanA phenotype E. gallinarum as well as E. feacium have been increasing in nosocomial infection and showed acquired inducible resistance. E. faecium and E. faecalis showed intermediate resistance in long exposure of vancomycin without acquisition of vanA.

Enterococci and streptococci

Besides Staphylococcus aureus, other Gram-positive bacteria have become multidrug-resistant and cause therapeutic problems, particularly amongst hospitalised patients. The acquisition of vancomycin resistance by strains of Enterococcus faecium and Enterococcus faecalis is of particular concern and has resulted in treatment failures. Some of the infections caused by these bacteria do respond to treatment with new antibiotics that have been released in the last few years, however more options are required as not all enterococci are inherently susceptible and resistance is beginning to emerge amongst those that were susceptible. Resistance to commonly used antibiotics is also emerging in Streptococcus spp., particularly to the tetracyclines and macrolides. In both genera, multiresistant strains spread between patients and between hospitals. In the laboratory, these bacteria show considerable susceptibility to tigecycline, with little propensity to develop resistance, indicating that tigecycline could assume an important role in controlling infections caused by these Gram-positive bacteria.

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP–area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with β-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8–16 μg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 μg/mL to >16 μg/mL, with a PAP–AUC ratio of >1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and β-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.

Biofilm formation and esp gene carriage in enterococci

Aims: To investigate biofilm production and esp carriage in enterococci. Methods: Biofilm production in vancomycin resistant enterococci (VRE) and vancomycin susceptible enterococci (VSE) was tested on a microtitre plate method,...

Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus.

To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype.