Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. IMPORTANCE: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Evolution of a small phage protein overcomes antiphage defense in Enterococcus faecalis.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of new antibiotics needed to combat these infections remains stagnant. MDR enterococci, which are a common cause of hospital-acquired infections, are emerging as one of the major contributors to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which entails the use of lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of phage resistance and the mechanisms underlying this resistance are unknown. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from vancomycin-resistant Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) and we show that this enzyme is sufficient to restrict the replication of the lytic phage in E. faecalis. We further find that phages can evolve to overcome restriction by acquiring a missense mutation in a novel TIV-RE inhibitor protein encoded by many enterococcal phages. We show that this inhibitor, which we have named anti-restriction-factor A (arfA), directly binds to and inactivates diverse TIV-REs. Overall, our findings significantly advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages can evolve to overcome antiphage defense systems.

Molecular insights into effector binding by DgoR, a GntR/FadR family transcriptional repressor of D-galactonate metabolism in Escherichia coli.

Several GntR/FadR transcriptional regulators govern sugar acid metabolism in bacteria. Although effectors have been identified for a few sugar acid regulators, the mode of effector binding is unknown. Even in the overall FadR subfamily, there are limited details on effector-regulator interactions. Here, we identified the effector-binding cavity in Escherichia coli DgoR, a FadR subfamily transcriptional repressor of D-galactonate metabolism that employs D-galactonate as its effector. Using a genetic screen, we isolated several dgoR superrepressor alleles. Blind docking suggested eight amino acids corresponding to these alleles to form a part of the effector-binding cavity. In vivo and in vitro assays showed that these mutations compromise the inducibility of DgoR without affecting its oligomeric status or affinity for target DNA. Taking Bacillus subtilis GntR as a representative, we demonstrated that the effector-binding cavity is similar among FadR subfamily sugar acid regulators. Finally, a comparison of sugar acid regulators with other FadR members suggested conserved features of effector-regulator recognition within the FadR subfamily. Sugar acid metabolism is widely implicated in bacterial colonization and virulence. The present study sets the basis to investigate the influence of natural genetic variations in FadR subfamily regulators on their sensitivity to sugar acids and ultimately on host-bacterial interactions.

Molecular and Functional Insights into the Regulation of d-Galactonate Metabolism by the Transcriptional Regulator DgoR in Escherichia coli.

d-Galactonate, an aldonic sugar acid, is used as a carbon source by Escherichia coli, and the structural dgo genes involved in its metabolism have previously been investigated. Here, using genetic, biochemical and bioinformatics approaches, we present the first detailed molecular and functional insights into the regulation of d-galactonate metabolism in E. coli K-12 by the transcriptional regulator DgoR. We found that dgoR deletion accelerates the growth of E. coli in d-galactonate concomitant with the strong constitutive expression of dgo genes. In the dgo locus, sequence upstream of dgoR alone harbors the d-galactonate-inducible promoter that likely drives the expression of all dgo genes. DgoR exerts repression on the dgo operon by binding two inverted repeats overlapping the dgo promoter. Binding of d-galactonate induces a conformational change in DgoR to derepress the dgo operon. The findings from our work firmly place DgoR in the GntR family of transcriptional regulators: DgoR binds an operator sequence [5'-TTGTA(G/C)TACA(A/T)-3'] matching the signature of GntR family members that recognize inverted repeats [5'-(N) yGT(N) xAC(N) y -3', where x and y indicate the number of nucleotides, which varies], and it shares critical protein-DNA contacts. We also identified features in DgoR that are otherwise less conserved in the GntR family. Recently, missense mutations in dgoR were recovered in a natural E. coli isolate adapted to the mammalian gut. Our results show these mutants to be DNA binding defective, emphasizing that mutations in the dgo-regulatory elements are selected in the host to allow simultaneous induction of dgo genes. The present study sets the basis to explore the regulation of dgo genes in additional enterobacterial strains where they have been implicated in host-bacterium interactions.IMPORTANCE d-Galactonate is a widely prevalent aldonic sugar acid. Despite the proposed significance of the d-galactonate metabolic pathway in the interaction of enteric bacteria with their hosts, there are no details on its regulation even in Escherichia coli, which has been known to utilize d-galactonate since the 1970s. Here, using multiple methodologies, we identified the promoter, operator, and effector of DgoR, the transcriptional repressor of d-galactonate metabolism in E. coli We establish DgoR as a GntR family transcriptional regulator. Recently, a human urinary tract isolate of E. coli introduced in the mouse gut was found to accumulate missense mutations in dgoR Our results show these mutants to be DNA binding defective, hence emphasizing the role of the d-galactonate metabolic pathway in bacterial colonization of the mammalian gut.

Immunohistochemical phospho tensin tumor suppressor gene staining patterns in endometrial hyperplasias: a 2-year study.

BACKGROUND: Endometrial carcinoma is a common neoplasm associated with the female genital tract with considerable morbidity. Endometrial hyperplasias have been widely regarded as precursor lesions. It is of importance to the pathologist to identify the subset of hyperplasias or the associated factor which could be a possible forerunner of malignancy. Phospho tensin gene (PTEN) has gained importance as one of the factors responsible. AIM: To determine the variability in PTEN expression patterns in different types of endometrial hyperplasias. SETTINGS AND DESIGN: The study was undertaken on samples received at the Department of Pathology from 2005 to 2007. MATERIALS AND METHODS: One hundred samples with 76 showing hyperplasias of different types formed the core "study group" with simple hyperplasia without atypia predominating. The rest belonged to the control groups. PTEN intensity and percentage positivity, variability in patterns of glandular and stromal expression, the number and type of PTEN null glands in different types of hyperplasia were evaluated. Statistical analyses used were Fisher's exact test based on Monte Carlo test and chi-square test. RESULTS: Complex hyperplasia was associated with a reduction in number of strongly PTEN positive glands, with an increase in null glands, seen in clusters. Co-existing atypia was associated with the weakest staining and in fewer glands. CONCLUSIONS: PTEN expression in endometrial hyperplasias can be used as an early warning of heightened cancer risk and a potential target for preventive treatment. However, extensive research is needed along this line to conclusively establish its effectiveness.