Sarcomere Disassembly and Transfection Efficiency in Proliferating Human iPSC-Derived Cardiomyocytes.

Contractility of the adult heart relates to the architectural degree of sarcomeres in individual cardiomyocytes (CMs) and appears to be inversely correlated with the ability to regenerate. In this study we utilized multiple imaging techniques to follow the sequence of sarcomere disassembly during mitosis resulting in cellular or nuclear division in a source of proliferating human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We observed that both mono- and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with the CHIR99021 small molecule, we found that Wnt and Hippo signaling was more present when compared to metabolic matured non-proliferative hiPSC-CMs and adult human heart tissue. Furthermore, we found that CHIR99021 increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after the chromosomal segregation (M phase). This study provides a tool for gene manipulation studies in hiPSC-CMs and engineered cardiac tissue. Moreover, our data illustrate that there is a complex biology behind the cellular and nuclear division of mono- and binuclear CMs, with a shared-phenomenon of sarcomere disassembly during mitosis.

Identification of a Novel Genomic Island Associated with vanD-Type Vancomycin Resistance in Six Dutch Vancomycin-Resistant Enterococcus faecium Isolates.

Genomic comparison of the first six Dutch vanD-type vancomycin-resistant Enterococcus faecium (VRE) isolates with four vanD gene clusters from other enterococcal species and anaerobic gut commensals revealed that the vanD gene cluster was located on a genomic island of variable size. Phylogenetic inferences revealed that the Dutch VRE isolates were genetically not closely related and that genetic variation of the vanD-containing genomic island was not species specific, suggesting that this island is transferred horizontally between enterococci and anaerobic gut commensals.

The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium.

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.

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.

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.

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.

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.

Association between Escherichia coli bacteriuria and renal function in women: long-term follow-up.

BACKGROUND: We sought to investigate whether Escherichia coli bacteriuria is associated with a decline in renal function or with the development of end-stage renal failure after long-term follow-up. METHODS: We performed a full cohort analysis for women who participated in 2 population-based studies. The baseline cohort consisted of women who collected morning midstream urine samples that were stored. In the cohort study, the presence of E coli bacteriuria was subsequently determined by real-time polymerase chain reaction. After a mean +/- SD follow-up of 11.5 +/- 1.7 years, blood samples were drawn from 490 women. In the nested case-control study, cases comprised all women who underwent kidney therapy (hemodialysis or renal transplantation) between participation in the baseline cohort study and a mean +/- SD of 13.8 +/- 7.4 years later. RESULTS: The mean +/- SD age at baseline was 45.0 +/- 3.2 years, and 48 women (10%) had E coli bacteriuria. After 11.5 years, the mean +/- SD creatinine clearance (Cockroft-Gault formula) was similar between the 2 groups (87 +/- 21 mL/min [1.5 +/- 0.4 mL/s] and 85 +/- 18 mL/min [1.4 +/- 0.3 mL/s] for women who had and those who did not have bacteriuria, respectively). In the nested case-control study, the prevalence of E coli bacteriuria was 14% among cases and control subjects. The odds ratio corrected for age for the development of end-stage renal failure in the presence of E coli bacteriuria at baseline was 1.1 (95% confidence interval, 0.4-2.8; P = .86). CONCLUSION: Escherichia coli bacteriuria is not associated with a decline in renal function or with the development of end-stage renal failure in a population of generally healthy women during 12 to 14 years of follow-up.

Fimch antiserum inhibits the adherence of Escherichia coli to cells collected by voided urine specimens of diabetic women.

PURPOSE: With the increasing problem of resistance in pathogenic microorganisms the development of nonantimicrobial therapies is important. Diabetes mellitus (DM) is associated with an increased incidence of urinary tract infections. The majority of Escherichia coli strains, which is the most prevalent uropathogen, have type 1 fimbriae that bind to uroplakin in the bladder, as mediated by the adhesin FimH. A vaccine is being developed based on FimH adhesion. MATERIALS AND METHODS: The sequence of FimH adhesion of 29 E. coli strains isolated from women with DM was determined. For adherence experiments we used E. coli isolated from women with DM and a T24 bladder cell line as well as the 2 well-defined type 1 fimbriated E. coli strains Ctrl 39 and NU14, and uroepithelial cells from women with DM. RESULTS: The fimH sequence of E. coli strains isolated from women with DM was highly homologous to the known fimH sequence of E. coli from patients without DM. Adherence assays in a T24 bladder cell line showed that adherence of these E. coli strains from women with DM could be inhibited by pre-incubation with antiserum raised against the chaperone-adhesin complex FimC-FimH. AntiFimCH antiserum also inhibited the adherence of the 2 well-defined E. coli strains expressing type 1 fimbriae, NU14 and Ctrl 39, but not of the FimH mutant strain NU14 H-, to uroepithelial cells from women with DM. CONCLUSIONS: These findings suggest that a vaccine based on FimH adhesin of type 1 fimbriated E. coli is a potential method of preventing urinary tract infection in women with DM.

Adherence of type 1-fimbriated Escherichia coli to uroepithelial cells: more in diabetic women than in control subjects.

OBJECTIVE: Women with diabetes have bacteriuria more often than women without diabetes. Because Escherichia coli adhere better to vaginal cells of nondiabetic patients with recurrent urinary tract infections (UTIs) than to those obtained from healthy control subjects, it was hypothesized that E. coli adhere more to the uroepithelial cells of diabetic women, either because of substances excreted in the urine (e.g., albumin, glucose, and Tamm Horsfall protein) or because of a difference in the uroepithelial cells. RESEARCH DESIGN AND METHODS: A T24 bladder cell line and uroepithelial cells of 25 diabetic women and 19 control subjects were incubated with three different E. coli strains. RESULTS: The mean numbers of type 1-fimbriated E. coli that adhered to diabetic and control cells were 12.9 and 6.1 (P = 0.001), respectively, whereas those of P-fimbriated E. coli were 8.8 and 8.1 (P = 0.8), and those of nonfimbriated E. coli were 2.7 and 3.4 (P = 0.4). The addition of various substances did not influence the adherence of E. coli to a T24 bladder cell line. CONCLUSIONS: Type 1-fimbriated E. coli adhere more to diabetic than to control uroepithelial cells.

Effect of glucose and pH on uropathogenic and non-uropathogenic Escherichia coli: studies with urine from diabetic and non-diabetic individuals.

It is generally assumed that one of the reasons why diabetics are more susceptible to urinary tract infections than non-diabetics is their 'sweet urine'. However, very little information is available on this subject. Therefore, the growth rates of different Escherichia coli strains were studied in human urine with and without added glucose and with and without a constant pH, and compared with their growth rates in Mueller-Hinton broth (MHB). Eight isolates were used (three from blood cultures from urosepsis patients, two urinary isolates, two faecal isolates and one laboratory strain K12). All isolates grew better in MHB than in urine, but with the exception of the laboratory strain, they had the same growth rate in urine. No significant difference was found between the growth rate in urine from diabetics without glucosuria and that in urine from non-diabetics. The addition of glucose (up to a concentration of 1000 mg/dl) to urine and MHB enhanced the growth rate of all isolates. However, very high concentrations of glucose (up to 10000 mg/dl) in urine and MHB caused a decrease in bacterial growth rate when the urinary pH was not kept constant. The stationary phase was reached later and the final bacterial yield was greater when the urine was made less acidic. As the uropathogenic strains did not grow better in urine than the other isolates, it may be concluded that better growth in urine is not one of the causes of the greater virulence of these strains.