Ellen C. Brouwer

Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium

ABSTRACT Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism (SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.

Methicillin Resistance Transfer from Staphylocccus epidermidis to Methicillin-Susceptible Staphylococcus aureus in a Patient during Antibiotic Therapy

Background The mecA gene, encoding methicillin resistance in staphylococci, is located on a mobile genetic element called Staphylococcal Cassette Chromosome mec (SCCmec). Horizontal, interspecies transfer of this element could be an important factor in the dissemination of methicillin-resistant S. aureus (MRSA). Previously, we reported the isolation of a closely related methicillin-susceptible Staphylococcus aureus (MSSA), MRSA and potential SCCmec donor Staphylococcus epidermidis isolate from the same patient. Based on fingerprint techniques we hypothesized that the S. epidermidis had transferred SCCmec to the MSSA to become MRSA. The aim of this study was to show that these isolates form an isogenic pair and that interspecies horizontal SCCmec transfer occurred. Methodology/Results Whole genome sequencing of both isolates was performed and for the MSSA gaps were closed by conventional sequencing. The SCCmec of the S. epidermidis was also sequenced by conventional methods. The results show no difference in nucleotide sequence between the two isolates except for the presence of SCCmec in the MRSA. The SCCmec of the S. epidermidis and the MRSA are identical except for a single nucleotide in the ccrB gene, which results in a valine to alanine substitution. The main difference with the closely related EMRSA-16 is the presence of SaPI2 encoding toxic shock syndrome toxin and exfoliative toxin A in the MSSA-MRSA pair. No transfer of SCCmec from the S. epidermidis to the MSSA could be demonstrated in vitro. Conclusion The MSSA and MRSA form an isogenic pair except for SCCmec. This strongly supports our hypothesis that the MRSA was derived from the MSSA by interspecies horizontal transfer of SCCmec from S. epidermidis O7.1.

Characterization of Dutch Staphylococcus aureus from bovine mastitis using a Multiple Locus Variable Number Tandem Repeat Analysis.

Current typing methods for Staphylococcus aureus have important drawbacks. We evaluated a Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) scheme with 6 loci which lacks most drawbacks on 85 bovine mastitis isolates from The Netherlands. For each locus the number of repeat units (RU) was calculated. Each combination of repeat units was assigned a MLVA-type (MT). We compared the MLVA typing result with Multi Locus Sequence Typing (MLST), spa-typing and Pulsed-Field Gel Electrophoresis (PFGE). MLVA typing resulted in 18 MTs, although 3 loci could not always be amplified. Spa-typing distinguished 10 spa-types including 3 dominant and 2 new types. PFGE showed 5 dominant profiles with 15 related profiles and 6 unique profiles. MLST showed 4 dominant STs. Some types appeared to be bovine specific. The Simpsons Indices of diversity for PFGE, MLST, spa-typing and MLVA were 0.887, 0.831, 0.69 and 0.781, respectively, indicating that discriminatory power of MLVA was between MLST and spa-typing, whereas PFGE displayed the highest discriminatory power. However, MLVA is fast and cheap when compared to the other methods. The Adjusted Rand index and Wallaces coefficient indicated that MLVA was highly predictive for spa-type, but not vice versa. Analysis of the region neighboring SIRU05 showed a difference in the genetic element bordering the repeats of SIRU05 that explained the negative SIRU05 PCRs. PFGE, MLST, and MLVA are adequate typing methods for bovine-associated S. aureus.

Virulence factors of Escherichia coli isolated from urine of diabetic women with asymptomatic bacteriuria: correlation with clinical characteristics

Since Escherichia coli isolated from compromised patients with symptomatic urinary tract infections (UTIs) express fewer virulence factors than those isolated from healthy controls, the question arises whether this is also the case for diabetic patients with asymptomatic bacteriuria (ASB). Polymerase chain reaction (PCR) assays were conducted on 111E. coli strains, isolated from the urine of diabetic women with ASB, using primers for the major subunit A and the G-adhesin (I, II, and III) of P fimbriae, type 1 fimbriae, S fimbriae, afimbrial adhesin, cytotoxic necrotizing factor (CNF), and aerobactin. Phenotypically, hemolysis, mannose-sensitive hemagglutination, mannose-resistant hemagglutination and O:K:H-serotypes were determined. Furthermore, we investigated the associations between virulence factors and patient characteristics (including deterioration of renal function). Type 1 fimbriae were the most prevalent virulence factor (86% by genotyping and 59% phenotypically). Except for a lower prevalence of known uropathogenic O-serotypes, we found the same number of virulence factors in our compromised patient group as listed in the literature in noncompromised patients with ASB. Certain virulence factors (type 1 and S fimbriae and CNF) of the causative E. colicorrelated with the risk of a decline in renal function. In conclusion, the number of virulence factors in E. coli isolated from the urine of diabetic women with ASB are comparable with the results found in other (noncompromised) patients with ASB. Furthermore, certain virulence factors of E. colimight contribute to a decline in renal function.

Chicken cathelicidins display antimicrobial activity against multiresistant bacteria without inducing strong resistance.

The increased prevalence of multidrug-resistant (MDR) bacteria in combination with the relatively limited development of new antibiotics presents a serious threat to public health. In chicken, especially Extended-Spectrum ß-Lactamase (ESBL) carrying Enterobacteriaceae are often asymptomatically present but can infect humans. Due to their broad range antimicrobial activity cathelicidins and other host defence peptides, are considered to be an attractive alternative to conventional antibiotics. In this study, the antimicrobial activity of three chicken cathelicidins against a broad array of multidrug resistant bacteria was determined. All three peptides showed high antibacterial activity independent of the presence of MDR characteristics. Induction experiments using S. aureus and K. pneumoniae showed that although an increase in resistance was initially observed, susceptibility towards chicken cathelicidins remained high and no major resistance was developed. The combined results underline the potential of chicken cathelicidins as a new alternative to antibiotics.

Is a Second Urine Specimen Necessary for the Diagnosis of Asymptomatic Bacteriuria

By use of pulse-field gel electrophoresis, we evaluated the molecular identity of 32 Escherichia coli isolates obtained in 2 consecutive urine cultures from 16 patients as part of a large study of asymptomatic bacteriuria in diabetic women and found different E. coli isolates in 7 of 16 patients, meaning that nearly half (44%) of the patients who had been previously classified as having asymptomatic bacteriuria were reinfected with a different strain.

Lipoteichoic acid synthesis inhibition in combination with antibiotics abrogates growth of multidrug-resistant Enterococcus faecium

Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections.

The two component system ChtRS contributes to chlorhexidine tolerance in Enterococcus faecium

ABSTRACT Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium. Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR. Transmission electron microscopy showed that upon challenge with CHX, the ΔchtR and ΔchtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans. Morphological abnormalities were also observed upon exposure of the ΔchtR and ΔchtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials.

Whole Genome Analysis of Epidemiologically Closely Related Staphylococcus aureus Isolates

The change of the bacteria from colonizers to pathogens is accompanied by a drastic change in expression profiles. These changes may be due to environmental signals or to mutational changes. We therefore compared the whole genome sequences of four sets of S. aureus isolates. Three sets were from the same patients. The isolates of each pair (S1800/S1805, S2396/S2395, S2398/S2397, an isolate from colonization and an isolate from infection, respectively) were obtained within <30 days of each other and the isolate from infection caused skin infections. The isolates were then compared for differences in gene content and SNPs. In addition, a set of isolates from a colonized pig and a farmer from the same farm at the same time (S0462 and S0460) were analyzed. The isolates pair S1800/S1805 showed a difference in a prophage, but these are easily lost or acquired. However, S1805 contained an integrative conjugative element not present in S1800. In addition, 92 SNPs were present in a variety of genes and the isolates S1800 and S1805 were not considered a pair. Between S2395/S2396 two SNPs were present: one was in an intergenic region and one was a synonymous mutation in a putative membrane protein. Between S2397/S2398 only one synonymous mutation in a putative lipoprotein was found. The two farm isolates were very similar and showed 12 SNPs in genes that belong to a number of different functional categories. However, we cannot pinpoint any gene that explains the change from carrier status to infection. The data indicate that differences between the isolate from infection and the colonizing isolate for S2395/S2396 and S2397/S2398 exist as well as between isolates from different hosts, but S1800/S1805 are not clonal.

Virulence Factors of Eescherichia coli Isolated from Urine of Diabetic Women with Asymptomatic Bacteriuria

1. Type 1 fimbriae is the most prevalent virulence factor in E. coli, isolated from urine of diabetic women with ASB. 2. E. coli, isolated from urine of diabetic women with ASB, express comparable numbers of virulence factors as E. coli isolated from urine of noncompromised patients with ASB.