Patients exposed to vancomycin-resistant enterococci during in-hospital outbreaks in a low endemic setting: a proposal for risk-based screening.

BACKGROUND: The optimal extent of screening of contact patients (CoPat) after exposure to patients infected or colonized with vancomycin-resistant enterococci (VRE) remains controversial. METHODS: We retrospectively developed a new risk stratification for screening patients exposed to VRE, based on data from three outbreaks-two with Enterococcus faecium vanB and one with Enterococcus faecium vanA involving 1096 CoPat-in a low endemic setting. We classified them into four risk groups: three on environmental exposure, one by healthcare exposure: high (sharing the same room/bathroom with a VRE-colonized patient), medium (hospitalization in the same room after a VRE-colonized patient's discharge until terminal disinfection including ultraviolet C (UVc)-disinfection), low (hospitalized in the same room within three weeks before the VRE-colonized patient), and "staff" (screening of patients having the same medical care team). RESULTS: VRE-transmission occurred in 7.9% in the high-risk group compared to 0.6% and 0% in the medium and low risk groups. There was a significant trend to higher rates of transmission by risk level of exposure (p < 0.001). In the "staff" group, VRE transmission rate was 2.3%. CONCLUSION: Based on this stratification, we recommend to focus screening of exposed CoPat on the high-risk and "staff" group, saving resources and costs, but larger studies will allow to further improve the yield of VRE screening in the outbreak setting.

Multicenter Prevalence Study Comparing Molecular and Toxin Assays for Clostridioides difficile Surveillance, Switzerland.

Public health authorities in the United States and Europe recommend surveillance for Clostridioides difficile infections among hospitalized patients, but differing diagnostic algorithms can hamper comparisons between institutions and countries. We compared surveillance based on detection of C. difficile by PCR or enzyme immunoassay (EIA) in a nationwide C. difficile prevalence study in Switzerland. We included all routinely collected stool samples from hospitalized patients with diarrhea in 76 hospitals in Switzerland on 2 days, 1 in winter and 1 in summer, in 2015. EIA C. difficile detection rates were 6.4 cases/10,000 patient bed-days in winter and 5.7 cases/10,000 patient bed-days in summer. PCR detection rates were 11.4 cases/10,000 patient bed-days in winter and 7.1 cases/10,000 patient bed-days in summer. We found PCR used alone increased reported C. difficile prevalence rates by <80% compared with a 2-stage EIA-based algorithm.

Moraxella catarrhalis AcrAB-OprM efflux pump contributes to antimicrobial resistance and is enhanced during cold shock response.

Moraxella catarrhalis is a common pathogen of the human respiratory tract. Multidrug efflux pumps play a major role in antibiotic resistance and virulence in many Gram-negative organisms. In the present study, the role of the AcrAB-OprM efflux pump in antibiotic resistance was investigated by constructing mutants that lack the acrA, acrB, and oprM genes in M. catarrhalis strain O35E. We observed a moderate (1.5-fold) decrease in the MICs of amoxicillin and cefotaxime and a marked (4.7-fold) decrease in the MICs of clarithromycin for acrA, acrB, and oprM mutants in comparison with the wild-type O35E strain. Exposure of the M. catarrhalis strains O35E and 300 to amoxicillin triggered an increased transcription of all AcrAB-OprM pump genes, and exposure of strains O35E, 300, and 415 to clarithromycin enhanced the expression of acrA and oprM mRNA. Inactivation of the AcrAB-OprM efflux pump genes demonstrated a decreased ability to invade epithelial cells compared to the parental strain, suggesting that acrA, acrB, and oprM are required for efficient invasion of human pharyngeal epithelial cells. Cold shock increases the expression of AcrAB-OprM efflux pump genes in all three M. catarrhalis strains tested. Increased expression of AcrAB-OprM pump genes after cold shock leads to a lower accumulation of Hoechst 33342 (H33342), a substrate of AcrAB-OprM efflux pumps, indicating that cold shock results in increased efflux activity. In conclusion, the AcrAB-OprM efflux pump appears to play a role in the antibiotic resistance and virulence of M. catarrhalis and is involved in the cold shock response.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression.

BACKGROUND: Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS: In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION: Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

Molecular pathogenesis of infections caused by Moraxella catarrhalis in children.

Moraxella catarrhalis (M. catarrhalis) is a human-restricted commensal of the normal bacterial flora in the upper respiratory tract of children, and - during the previous two decades - has been recognised as a true human pathogen. M. catarrhalis is the third most common pathogen causing acute otitis media in children, which is the most common reason to visit a paediatrician during childhood. Acute otitis media thus causes a high clinical and economical burden. With the introduction of the conjugate pneumococcal vaccines the microbiomic pattern in the nasopharyngeal flora of children has changed, and the frequency of isolation of M. catarrhalis has increased. Compared to adults, children are more often colonised with M. catarrhalis. Over the last three decades there has been a dramatic increase in the acquisition of ß-lactam resistance in M. catarrhalis. Today 95-100% of clinically isolated M. catarrhalis produce ß-lactamase. It is thus desirable to reduce the burden of M. catarrhalis disease by developing a vaccine. There are several potential vaccine antigen candidates in different stages of development, but none of them has entered clinical trials at the present time.

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion.

BACKGROUND: Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence. RESULTS: A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis. CONCLUSION: Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.

Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?

PURPOSE: Curcumin exerts its anti-inflammatory activity via inhibition of nuclear factor κB. Oropharyngeal epithelia and residing bacteria closely interact in inflammation and infection. This in vitro model investigated the effects of curcumin on bacterial survival, adherence to, and invasion of upper respiratory tract epithelia, and studied its anti-inflammatory effect. We aimed to establish a model, which could offer insights into the host-pathogen interaction in cancer therapy induced mucositis. METHODS: Moraxella catarrhalis (Mcat) and the oropharyngeal epithelial cell line Detroit 562 were used. Time-kill curves assessed the inhibition of bacterial growth and adherence assays and gentamicin protection assays determined the effect of curcumin-preincubated cells on bacterial adherence and invasion. Curcumin-mediated inhibition of pro-inflammatory activation by Mcat was determined via interleukin-8 concentrations in the supernatants. The synergistic role of secretory IgA (sIgA) on adherence was investigated. RESULTS: Curcumin was bactericidal at concentrations >50 µM. Preincubation of Detroit cells for 60 min demonstrated that concentrations >100 µM inhibited bacterial adherence. Together with sIgA, curcumin inhibited adherence at concentrations ≥50 µM. Both 100 and 200 µM curcumin significantly inhibited Mcat cell invasion. Finally, curcumin inhibited Mcat-induced pro-inflammatory activation by strongly suppressing IL-8 release. At a concentration of 200 µM, 10 min of curcumin exposure inhibited IL-8 release significantly, and complete suppression required a pre-exposure time of ≥45 min. CONCLUSION: Curcumin, in clinically relevant concentrations for topical use, displayed strong antibacterial effect against a facultative upper respiratory tract pathogen by inhibiting bacterial growth, adherence, invasion, and pro-inflammatory activation of upper respiratory tract epithelial cells in vitro.

Down-regulation of porin M35 in Moraxella catarrhalis by aminopenicillins and environmental factors and its potential contribution to the mechanism of resistance to aminopenicillins.

OBJECTIVES: The outer membrane protein M35 of Moraxella catarrhalis is an antigenically conserved porin. Knocking out M35 significantly increases the MICs of aminopenicillins. The aim of this study was to determine the biological mechanism of this potentially new antimicrobial resistance mechanism of M. catarrhalis and the behaviour of M35 in general stress situations. METHODS: PCR using m35-specific primers was used to detect the m35 gene in clinical isolates. The m35 mRNA expression of strains 300, O35E and 415 after exposure to amoxicillin and different stress conditions was measured by real-time PCR and normalized in relation to their 16S rRNA expression. The expression of M35 protein was analysed by SDS-PAGE and western blotting. RESULTS: Screening of 52 middle ear isolates resulted in positive PCR products for all tested strains. The analysis of m35 mRNA expression after amoxicillin treatment showed 24%-85% down-regulation compared with the respective amoxicillin-free controls in all three strains tested. Also, analysis of protein concentrations revealed lower M35 expression after growth with amoxicillin. Investigation of M35 during general stress responses showed down-regulation of the porin with growth at 26°C and 42°C, under hyperosmolar stress and under iron restriction. CONCLUSIONS: The reduced expression of M35 after aminopenicillin exposure indicates a novel resistance mechanism against aminopenicillins in M. catarrhalis, which may be relevant in vivo. The differences in expression after different stress treatments demonstrate that M35 is involved in general stress responses.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells.

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins.

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis.

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue.

BACKGROUND: Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. METHODS: Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). RESULTS: Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P<.001). Histologic analysis identified M. catarrhalis in crypts, intraepithelially, subepithelially, and (using CLSM) intracellularly. M. catarrhalis colocalized with macrophages and B cells in lymphoid follicles. CONCLUSIONS: Colonization by M. catarrhalis is more frequent than is determined by surface culture, because the organism resides both within and beneath the epithelium and invades host cells.

Indinavir inhibits sterol-regulatory element-binding protein-1c-dependent lipoprotein lipase and fatty acid synthase gene activations.

BACKGROUND: A syndrome characterized by hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia, and lipodystrophy has been found to be associated with highly active antiretroviral treatment (HAART) including protease inhibitors. A marker predicting this syndrome has been previously identified in the gene encoding the sterol-regulatory element-binding protein (SREBP)-1c, a regulator of triglycerides, cholesterol, insulin, and adipocytes. OBJECTIVE: A possible inhibition of SREBP-1c-dependent genes by the protease inhibitor indinavir and its possible reversal by the lipid-lowering drug simvastatin were studied. METHODS: The effects of indinavir and simvastatin on the inhibition/activation of SREBP-1c-dependent genes were compared with the effects of indinavir and simvastatin on the inhibition/activation of SREBP-1c-independent genes. RESULTS: Indinavir inhibited the SREBP-1c-dependent genes encoding the lipoprotein lipase (103 nmol/l resulted in an inhibition of 12.4%; P = 0.0051) and the fatty acid synthase (103 nmol/l resulted in an inhibition of 30.3%; P = 0.036) in a dose-dependent fashion but not the SREBP-1c-independent gene encoding the low-density lipoprotein receptor. Simvastatin antagonized the indinavir-induced SREBP-1c-inhibition. CONCLUSIONS: Indinavir inhibits important effector genes of the SREBP-1c pathway, explaining major HAART-related adverse effects.