Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

Virological and immunological responses to efavirenz or boosted lopinavir as first-line therapy for patients with HIV

BACKGROUND: Efavirenz and lopinavir boosted with ritonavir are both recommended as first-line therapies for patients with HIV when combined with two nucleoside reverse transcriptase inhibitors. It is uncertain which therapy is more effective for patients starting therapy with an advanced infection. METHODS: We estimated the relative effect of these two therapies on rates of virological and immunological failure within the Swiss HIV Cohort Study and considered whether estimates depended on the CD4(+) T-cell count when starting therapy. We defined virological failure as either an incomplete virological response or viral rebound after viral suppression and immunological failure as failure to achieve an expected CD4(+) T-cell increase calculated from EuroSIDA statistics. RESULTS: Patients starting efavirenz (n=660) and lopinavir (n=541) were followed for a median of 4.5 and 3.1 years, respectively. Virological failure was less likely for patients on efavirenz, with the adjusted hazard ratio (95% confidence interval) of 0.63 (0.50-0.78) then multiplied by a factor of 1.00 (0.90-1.12) for each 100 cells/mm(3) decrease in CD4(+) T-cell count below the mean when starting therapy. Immunological failure was also less likely for patients on efavirenz, with the adjusted hazard ratio of 0.68 (0.51-0.91) then multiplied by a factor of 1.29 (1.14-1.46) for each 100 cells/mm(3) decrease in CD4(+) T-cell count below the mean when starting therapy. CONCLUSIONS: Virological failure is less likely with efavirenz regardless of the CD4(+) T-cell count when starting therapy. Immunological failure is also less likely with efavirenz; however, this advantage disappears if patients start therapy with a low CD4(+) T-cell count.

Long-term trends of HIV type 1 drug resistance prevalence among antiretroviral treatment-experienced patients in Switzerland

BACKGROUND:Accurate quantification of the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance in patients who are receiving antiretroviral therapy (ART) is difficult, and results from previous studies vary. We attempted to assess the prevalence and dynamics of resistance in a highly representative patient cohort from Switzerland. METHODS:On the basis of genotypic resistance test results and clinical data, we grouped patients according to their risk of harboring resistant viruses. Estimates of resistance prevalence were calculated on the basis of either the proportion of individuals with a virologic failure or confirmed drug resistance (lower estimate) or the frequency-weighted average of risk group-specific probabilities for the presence of drug resistance mutations (upper estimate). RESULTS:Lower and upper estimates of drug resistance prevalence in 8064 ART-exposed patients were 50% and 57% in 1999 and 37% and 45% in 2007, respectively. This decrease was driven by 2 mechanisms: loss to follow-up or death of high-risk patients exposed to mono- or dual-nucleoside reverse-transcriptase inhibitor therapy (lower estimates range from 72% to 75%) and continued enrollment of low-risk patients who were taking combination ART containing boosted protease inhibitors or nonnucleoside reverse-transcriptase inhibitors as first-line therapy (lower estimates range from 7% to 12%). A subset of 4184 participants (52%) had >or= 1 study visit per year during 2002-2007. In this subset, lower and upper estimates increased from 45% to 49% and from 52% to 55%, respectively. Yearly increases in prevalence were becoming smaller in later years. CONCLUSIONS:Contrary to earlier predictions, in situations of free access to drugs, close monitoring, and rapid introduction of new potent therapies, the emergence of drug-resistant viruses can be minimized at the population level. Moreover, this study demonstrates the necessity of interpreting time trends in the context of evolving cohort populations.

Molecular Surface Features in Modeling the HIV-1 RT Inhibitory Activity of 2-(2,6-Disubstituted phenyl)-3-(substituted pyrimidin-2-yl)-thiazolidin-4-ones

The human immunodeficiency virus-1 reverse transcriptase inhibitory activity of 2-(2,6-disubstituted phenyl)-3-(substituted pyrimidin-2-yl)-thiazolidin-4-ones have been analyzed using combinatorial protocol in multiple linear regression (CP-MLR) with several electronic and molecular surface area features of the compounds obtained from Molecular Operating Environment (MOE) software. The study has indicated the role of different charged molecular surface areas in modeling the inhibitory activity of the compounds. The derived models collectively suggested that the compounds should be compact without bulky substitutions on its peripheries for better HIV-1 RT inhibitory activity. It also emphasized the necessity of hydrophobicity and compact structural features for their activity. The scope of the descriptors identified for these analogues have been verified by extending the dataset with different 2-(disubstituted phenyl)-3-(substituted pyridin-2-yl)-thiazolidin-4-ones. The joint analysis of extended dataset highlighted the information content of identified descriptors in modeling the HIV-1 RT inhibitory activity of the compounds.

Zidovudine impairs immunological recovery on first-line antiretroviral therapy: collaborative analysis of cohort studies in southern Africa

OBJECTIVES Zidovudine (ZDV) is recommended for first-line antiretroviral therapy (ART) in resource-limited settings. ZDV may, however, lead to anemia and impaired immunological response. We compared CD4+ cell counts over 5 years between patients starting ART with and without ZDV in southern Africa. DESIGN Cohort study. METHODS Patients aged at least 16 years who started first-line ART in South Africa, Botswana, Zambia, or Lesotho were included. We used linear mixed-effect models to compare CD4+ cell count trajectories between patients on ZDV-containing regimens and patients on other regimens, censoring follow-up at first treatment change. Impaired immunological recovery, defined as a CD4+ cell count below 100 cells/μl at 1 year, was assessed in logistic regression. Analyses were adjusted for baseline CD4+ cell count and hemoglobin level, age, sex, type of regimen, viral load monitoring, and calendar year. RESULTS A total of 72,597 patients starting ART, including 19,758 (27.2%) on ZDV, were analyzed. Patients on ZDV had higher CD4+ cell counts (150 vs.128 cells/μl) and hemoglobin level (12.0 vs. 11.0 g/dl) at baseline, and were less likely to be women than those on other regimens. Adjusted differences in CD4+ cell counts between regimens containing and not containing ZDV were -16 cells/μl [95% confidence interval (CI) -18 to -14] at 1 year and -56 cells/μl (95% CI -59 to -52) at 5 years. Impaired immunological recovery was more likely with ZDV compared to other regimens (odds ratio 1.40, 95% CI 1.22-1.61). CONCLUSION In southern Africa, ZDV is associated with inferior immunological recovery compared to other backbones. Replacing ZDV with another nucleoside reverse transcriptase inhibitor could avoid unnecessary switches to second-line ART.

Impact of recommendation updates in well-controlled patients on nonrecommended antiretroviral therapies: the Swiss HIV cohort study

BACKGROUND HIV treatment recommendations are updated as clinical trials are published. Whether recommendations drive clinicians to change antiretroviral therapy in well-controlled patients is unexplored. METHODS We selected patients with undetectable viral loads (VLs) on nonrecommended regimens containing double-boosted protease inhibitors (DBPIs), triple-nucleoside reverse transcriptase inhibitors (NRTIs), or didanosine (ddI) plus stavudine (d4T) at publication of the 2006 International AIDS Society recommendations. We compared demographic and clinical characteristics with those of control patients with undetectable VL not on these regimens and examined clinical outcome and reasons for treatment modification. RESULTS At inclusion, 104 patients were in the DBPI group, 436 in the triple-NRTI group, and 19 in the ddI/d4T group. By 2010, 28 (29%), 204 (52%), and 1 (5%) patient were still on DBPIs, triple-NRTIs, and ddI plus d4T, respectively. 'Physician decision,' excluding toxicity/virological failure, drove 30% of treatment changes. Predictors of recommendation nonobservance included female sex [adjusted odds ratio (aOR) 2.69, 95% confidence interval (CI) 1 to 7.26; P = 0.01] for DPBIs, and undetectable VL (aOR 3.53, 95% CI 1.6 to 7.8; P = 0.002) and lack of cardiovascular events (aOR 2.93, 95% CI 1.23 to 6.97; P = 0.02) for triple-NRTIs. All patients on DBPIs with documented diabetes or a cardiovascular event changed treatment. Recommendation observance resulted in lower cholesterol values in the DBPI group (P = 0.06), and more patients having undetectable VL (P = 0.02) in the triple-NRTI group. CONCLUSION The physician's decision is the main factor driving change from nonrecommended to recommended regimens, whereas virological suppression is associated with not switching. Positive clinical outcomes observed postswitch underline the importance of observing recommendations, even in well-controlled patients.

Higher CNS Penetration-Effectiveness of Long-term Combination Antiretroviral Therapy Is Associated With Better HIV-1 Viral Suppression in Cerebrospinal Fluid

Objectives: To determine HIV-1 RNA in cerebrospinal fluid (CSF) of successfully treated patients and to evaluate if combination antiretroviral treatments with higher central nervous system penetration-effectiveness (CPE) achieve better CSF viral suppression. Methods: Viral loads (VLs) and drug concentrations of lopinavir, atazanavir, and efavirenz were measured in plasma and CSF. The CPE was calculated using 2 different methods. Results: The authors analyzed 87 CSF samples of 60 patients. In 4 CSF samples, HIV-1 RNA was detectable with 43–82 copies per milliliter. Median CPE in patients with detectable CSF VL was significantly lower compared with individuals with undetectable VL: CPE of 1.0 (range, 1.0–1.5) versus 2.3 (range, 1.0–3.5) using the method of 2008 (P = 0.011) and CPE of 6 (range, 6–8) versus 8 (range, 5–12) using the method of 2010 (P = 0.022). The extrapolated CSF trough levels for atazanavir (n = 12) were clearly above the 50% inhibitory concentration (IC50) in only 25% of samples; both patients on atazanavir/ritonavir with detectable CSF HIV-1 RNA had trough levels in the range of the presumed IC50. The extrapolated CSF trough level for lopinavir (n = 42) and efavirenz (n = 18) were above the IC50 in 98% and 78%, respectively, of samples, including the patients with detectable CSF HIV-1 RNA. Conclusions: This study suggests that treatment regimens with high intracerebral efficacy reflected by a high CPE score are essential to achieve CSF HIV-1 RNA suppression. The CPE score including all drug components was a better predictor for treatment failure in the CSF than the sole concentrations of protease inhibitor or nonnucleoside reverse transcriptase inhibitor in plasma or CSF.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Persistent Productive Epstein‐Barr Virus Replication in Normal Epithelial Cells In Vivo

Productive Epstein‐Barr virus (EBV) replication characterizes hairy leukoplakia, an oral epithelial lesion typically occurring in individuals infected with human immunodeficiency virus (HIV). Serial tongue biopsy specimens were obtained from HIV‐infected subjects before, during, and after valacyclovir treatment. EBV replication was detected by Southern hybridization to linear terminal EBV genome fragments, reverse‐transcriptase polymerase chain reaction amplification of EBV replicative gene transcripts, immunohistochemical detection of EBV replicative protein, and in situ hybridization to EBV DNA. EBV replication was detected in both hairy leukoplakia and normal tongue tissues. Valacyclovir treatment completely abrogated EBV replication in vivo, resulting in resolution of hairy leukoplakia when it was present. EBV replication returned in normal tongue epithelial cells after valacyclovir treatment. These data suggest that normal oral epithelium supports persistent EBV infection in individuals infected with HIV and that productive EBV replication is necessary but not sufficient for the pathogenesis of oral hairy leukoplakia.

CREB mediates prostaglandin F2alpha-induced MUC5AC overexpression.

Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are PGE(2) and PGF(2alpha). PGE(2)-induced mucin production has been well studied, but the effect of PGF(2alpha) on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF(2alpha) on MUC5AC production, we investigated the signal transduction of PGF(2alpha) associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF(2alpha) induces MUC5AC overproduction via a signaling cascade involving protein kinase C, ERK, p90 ribosomal S6 protein kinase, and CREB. The regulation of PGF(2alpha)-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF(2alpha) receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE(2) and other inflammatory mediators, our findings have important clinical implications for the management of airway mucus hypersecretion.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Sp1 up-regulates cAMP-response-element-binding protein expression during retinoic acid-induced mucous differentiation of normal human bronchial epithelial cells.

CREB [CRE (cAMP-response element)-binding protein] is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA (retinoic acid) rapidly activates CREB without using RARs (RA receptors) or RXRs (retinoid X receptors) in NHTBE cells (normal human tracheobronchial epithelial cells). However, little is known about the role of RA in the physiological regulation of CREB expression in the early mucous differentiation of NHTBE cells. In the present study, we report that RA up-regulates CREB gene expression and that, using 5'-serial deletion promoter analysis and mutagenesis analyses, two Sp1 (specificity protein 1)-binding sites located at nt -217 and -150, which flank the transcription initiation site, are essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nt -119 and -98 contributed to basal promoter activity. Interestingly, RA also up-regulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using siRNA (small interfering RNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA up-regulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in up-regulating human CREB gene expression. This result implies that co-operation of these two transcription factors plays a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells.

Direct intrathecal implantation of mesenchymal stromal cells leads to enhanced neuroprotection via an NFkappaB-mediated increase in interleukin-6 production.

Mesenchymal stromal cell (MSC) therapy has shown promise for the treatment of traumatic brain injury (TBI). Although the mechanism(s) by which MSCs offer protection is unclear, initial in vivo work has suggested that modulation of the locoregional inflammatory response could explain the observed benefit. We hypothesize that the direct implantation of MSCs into the injured brain activates resident neuronal stem cell (NSC) niches altering the intracerebral milieu. To test our hypothesis, we conducted initial in vivo studies, followed by a sequence of in vitro studies. In vivo: Sprague-Dawley rats received a controlled cortical impact (CCI) injury with implantation of 1 million MSCs 6 h after injury. Brain tissue supernatant was harvested for analysis of the proinflammatory cytokine profile. In vitro: NSCs were transfected with a firefly luciferase reporter for NFkappaB and placed in contact culture and transwell culture. Additionally, multiplex, quantitative PCR, caspase 3, and EDU assays were completed to evaluate NSC cytokine production, apoptosis, and proliferation, respectively. In vivo: Brain supernatant analysis showed an increase in the proinflammatory cytokines IL-1alpha, IL-1beta, and IL-6. In vitro: NSC NFkappaB activity increased only when in contact culture with MSCs. When in contact with MSCs, NSCs show an increase in IL-6 production as well as a decrease in apoptosis. Direct implantation of MSCs enhances neuroprotection via activation of resident NSC NFkappaB activity (independent of PI3 kinase/AKT pathway) leading to an increase in IL-6 production and decrease in apoptosis. In addition, the observed NFkappaB activity depends on direct cell contact.

The hylEfm gene in pHylEfm of Enterococcus faecium is not required in pathogenesis of murine peritonitis.

BACKGROUND: Plasmids containing hylEfm (pHylEfm) were previously shown to increase gastrointestinal colonization and lethality of Enterococcus faecium in experimental peritonitis. The hylEfm gene, predicting a glycosyl hydrolase, has been considered as a virulence determinant of hospital-associated E. faecium, although its direct contribution to virulence has not been investigated. Here, we constructed mutants of the hylEfm-region and we evaluated their effect on virulence using a murine peritonitis model. RESULTS: Five mutants of the hylEfm-region of pHylEfmTX16 from the sequenced endocarditis strain (TX16 [DO]) were obtained using an adaptation of the PheS* system and were evaluated in a commensal strain TX1330RF to which pHylEfmTX16 was transferred by mating; these include i) deletion of hylEfm only; ii) deletion of the gene downstream of hylEfm (down) of unknown function; iii) deletion of hylEfm plus down; iv) deletion of hylEfm-down and two adjacent genes; and v) a 7,534 bp deletion including these four genes plus partial deletion of two others, with replacement by cat. The 7,534 bp deletion did not affect virulence of TX16 in peritonitis but, when pHylEfmTX16Δ7,534 was transferred to the TX1330RF background, the transconjugant was affected in in vitro growth versus TX1330RF(pHylEfmTX16) and was attenuated in virulence; however, neither hylEfm nor hylEfm-down restored wild type function. We did not observe any in vivo effect on virulence of the other deletions of the hylEfm-region CONCLUSIONS: The four genes of the hylEfm region (including hylEfm) do not mediate the increased virulence conferred by pHylEfmTX16 in murine peritonitis. The use of the markerless counterselection system PheS* should facilitate the genetic manipulation of E. faecium in the future.