Evaluation of novel disinfection methods for the remediation of heavily contaminated thermostatic mixing valves and water systems with Pseudomonas aeruginosa biofilm: considerations for new and existing healthcare water systems.

BACKGROUND: Pseudomonas aeruginosa is a leading cause of nosocomial Gram-negative bacteraemia. Water systems are a well-documented source of P. aeruginosa and established biofilms are difficult to remove. AIM: To evaluate the efficacy of regular flushing, peracetic acid disinfection, in-tap thermal disinfection, and in-line thermal disinfection to eradicate P. aeruginosa biofilm in a colonized tap model. METHODS: A simulated tap system was constructed and inoculated with a reference and an environmental strain of P. aeruginosa to form biofilm. Water samples were collected from the taps and P. aeruginosa levels enumerated following disinfection methods. To simulate regular flushing, taps were flushed for 5 min, five times per day with water tested daily. Peracetic acid (4000 ppm) was manually injected into the system and flushed through the system with a pump. Thermal flushing at 60 °C was performed in-line and with an in-tap bypass valve. Tests were conducted with cross-linked polyethylene (PEX) piping and repeated with copper piping. FINDINGS: Regular flushing and peracetic acid applied with a pump did not reduce P. aeruginosa levels. A limited reduction was observed when manually injecting peracetic acid. In-tap thermal flushing eradicated P. aeruginosa in copper piping but not PEX. In-line thermal flushing was the most effective at reducing P. aeruginosa levels; however, it did not eradicate the biofilm. CONCLUSION: In-line thermal flushing was the most effective method to remove P. aeruginosa biofilm. Results vary significantly with the strain of bacteria and the composition of the plumbing. Several methods used in combination may be necessary to remove established biofilm.

Concordance between IDEXX Legiolert® (liquid culture assay) and plate culture (ISO 11731:2017) for the detection and quantification of Legionella pneumophila in water samples.

BACKGROUND: Legionella pneumophila is a water-borne bacterium that can cause Legionnaires' disease. Legiolert® (IDEXX, USA) is a low-labour liquid culture assay for the detection and enumeration of L. pneumophila (SG1-15) from water. AIM: To analyse concordance between Legiolert and ISO 11731:2017 plate culture method (membrane filtration and culture on selective agars) using hospital water samples (N = 100). METHODS: Incubation was at 39 °C and 36 °C, respectively, for seven days, followed by most-probable enumeration for Legiolert and subculturing and serogrouping of suspected Legionella colonies, with plate culture. FINDINGS: L. pneumophila (SG1-15) was isolated from 25 out of 100 samples when using Legiolert or plate culture. Fourteen additional Legiolert samples tested positive for L. pneumophila; analysis of the same samples by plate culture was negative (12 out of 14) or yielded only Legionella rubrilucens (two out of 14; confirmed via matrix-assisted ionization/desorption time-of-flight mass spectrometry). L. pneumophila was not captured from Quanti-Tray/Legiolert pouch wells of these positive samples after subculture of puncture aliquots on buffered charcoal yeast-extract agar. Both methods in concordance did not detect L. pneumophila in 61 out of 100 samples. CONCLUSION: Legiolert and plate culture are both satisfactory methods to detect L. pneumophila from water samples, and both to detect isolated L. pneumophila in 25% of the sample population. Legiolert provides a faster time to result, and is less resource-demanding and labour-intensive; however, there may be a low risk of cross-reactivity with other organisms. Both methods are suitable for the analysis of water in healthcare settings, where the monitoring of L. pneumophila is imperative in preventing cases of Legionnaires' disease.

Platensimycin is a selective FabF inhibitor with potent antibiotic properties.

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Thermal disinfection at suboptimal temperature of Pseudomonas aeruginosa biofilm on copper pipe and shower hose materials.

BACKGROUND: Hospital-acquired infections caused by Pseudomonas aeruginosa have been linked to contaminated shower systems in health care. Thermal disinfection, whereby colonized outlets are flushed with existing hot water supplies, is a commonly used method to disinfect contaminated systems. Temperatures of 60°C are recommended for inactivation of P. aeruginosa; however, this is often not achievable at outlets. AIM: To investigate whether thermal disinfection at a suboptimal temperature (58°C) can effectively eradicate planktonic P. aeruginosa and biofilm adherent on copper piping and shower hoses. Exposure times of up to 60 min and efficacy of repeated cycles were evaluated. METHODS: A type culture and an environmental strain of P. aeruginosa isolated from a hospital shower were tested. Planktonic bacteria and biofilm adhered to sections of copper pipe and shower hoses were exposed to water at 58°C for up to 60 min. Biofilms were tested with static water, flushing water and repeated cycles of disinfection. Remaining viable bacteria after disinfection were enumerated. FINDINGS: Planktonic P. aeruginosa remained viable after up to 60 min of thermal disinfection. With static water, biofilm was removed from copper piping after 15 min, but remained viable in shower hoses for up to 60 min. With thermal flushing, biofilm was fully eradicated from copper piping after 2 min, but remained viable on shower hoses. Repeated cycles did not shorten thermal disinfection exposure times. CONCLUSION: Thermal disinfection at 58°C was effective at eliminating biofilm on copper; however, biofilm on shower hoses remained viable after 60 min of exposure.

Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist.

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2'-oxo-1,1',2',3-tetrahydrospiro[indene-2,3'-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (K(i) = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

Evaluation of droplet production by a new design of clinical handwash basin for the healthcare environment.

Splashing from handwash basins may be a source of bacteria in the healthcare environment. A novel splash-reducing basin was assessed for its ability to reduce droplet formation during simulated handwashing. The basin was compared to two conventional basins commonly used in healthcare. Basins were mounted in a test system and tap flushed for 30-s with and without handwashing. Droplets were visualized with fluorescent dye. With conventional basins, >1000 droplets were formed during 30-s flushes and found to spread further than 2-m. The novel basin significantly reduced the number of droplets formed during handwashing and reduced the distance spread.

Ultraviolet-C decontamination of hand-held tablet devices in the healthcare environment using the Codonics D6000™ disinfection system.

Mobile phones and tablet computers may be contaminated with micro-organisms and become a potential reservoir for cross-transmission of pathogens between healthcare workers and patients. There is no generally accepted guidance on how to reduce contamination on mobile devices in healthcare settings. Our aim was to determine the efficacy of the Codonics D6000™ UV-C disinfection device. Daily disinfection reduced contamination on screens and on protective cases (test) significantly, but not all cases (control) could be decontaminated. The median aerobic colony count on the control and the test cases was 52 cfu/25 cm2 (interquartile range: 33-89) and 22 cfu/25 cm2 (10.5-41), respectively, before disinfection.

Comparison of two whole-room ultraviolet irradiation systems for enhanced disinfection of contaminated hospital patient rooms.

BACKGROUND: Ultraviolet (UV) light decontamination systems are being used increasingly to supplement terminal disinfection of patient rooms. However, efficacy may not be consistent in the presence of soil, especially against Clostridium difficile spores. AIM: To demonstrate in-use efficacy of two whole-room UV decontamination systems against three hospital pathogens with and without soil. METHODS: For each system, six patient rooms were decontaminated with UV irradiation (enhanced disinfection) following manual terminal cleaning. Total aerobic colony counts of surface contamination were determined by spot-sampling 15 environmental sites before and after terminal disinfection and after UV irradiation. Efficacy against biological indicator coupons (stainless-steel discs) was performed for each system using test bacteria (106 cfu EMRSA-15 variant A, carbapenemase-producing Klebsiella pneumoniae) or spores (105 cfu C. difficile 027), incorporating low soiling [0.03% bovine serum albumin (BSA)], heavy soiling (10% BSA) or synthetic faeces (C. difficile only) placed at five locations in the room. FINDINGS: UV disinfection eliminated contamination after terminal cleaning in 8/14 (57%) and 11/14 (79%) sites. Both systems demonstrated 4-5 log10 reductions in meticillin-resistant Staphylococcus aureus and K. pneumoniae at low soiling. Lower and more variable log10 reductions were achieved when heavy soiling was present. Between 0.1 and 4.8 log10 reductions in C. difficile spores were achieved with low but not heavy soil challenge. CONCLUSION: Terminal disinfection should be performed on all surfaces prior to UV decontamination. In-house validation studies should be considered to ensure optimal positioning in each room layout and sufficient cycle duration to eliminate target pathogens.

Complex molecular genetic abnormalities involving three or more genetic mutations are important prognostic factors for acute myeloid leukemia.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P<0.0001). In conclusion, CMGAs in AML were found to be strong independent adverse prognostic factors and simplified co-mutation analysis to have clinical usefulness and applicability.

Neutral lipid accumulation in macrophages during lipid-induced macrophage growth.

We previously reported that lipids such as cholesterol esters, triglycerides, and some phospholipids that are components of cell membranes or serum lipoproteins induced the growth of mouse peritoneal macrophages in vitro. Here, we report that macrophage growth was directly induced by lipids without any soluble factors and that the continuous presence of lipids was required for their growth during culture. When macrophages were cultured with several phospholipids that stimulated macrophage growth, their triglyceride content was dramatically increased during culture, whereas their contents of phospholipids, cholesterol, and esterified cholesterols were scarcely changed. On the other hand, phosphatidylcholine and sphingomyelin, which did not stimulate macrophage growth, caused less accumulation of triglycerides in the cells. Macrophages in which triglycerides accumulated resembled foam cells, since they contained many particles that stained with oil red O. Macrophages that cultured with cholesteryl linoleate also contained many oil red O-stainable particles. These data suggest that accumulation of lipid droplets is correlated with macrophage growth induced by lipids and that foam cells in pathological states such as those in atherosclerotic or xanthoma lesions might proliferate in situ.

Relationship of ability of phospholipids to stimulate growth and bind to macrophages.

Among the phospholipids normally present in mammalian cell membranes, the negatively charged phospholipids, phosphatidylserine (PS), phosphatidylglycerol, and cardiolipin, and the neutral phospholipid phosphatidylethanolamine--but not phosphatidylcholine (PC) or sphingomyelin--were found to induce growth of peripheral macrophages. By use of liposomes prepared from PS, which stimulated growth, and PC, which did not, the relation between growth-stimulating activity and binding of the phospholipids to macrophages was studied. The growth-stimulating activity of PS/PC liposomes decreased with increase in their relative content of PC. The amount of PS bound to macrophages also decreased with increase in the proportion of PC in PS/PC liposomes. These decreases in growth-stimulating and binding activities were both partly recovered by additional incorporation into the PS/PC liposomes of cholesterol, which is also a cell membrane component. Phospholipids that stimulated macrophage growth showed high binding ability to macrophages, whereas those that did not stimulate growth scarcely bound to macrophages. Thus the macrophage growth-stimulating activities of phospholipids correlated well with their ability to bind to macrophages. These liposome models should be useful in elucidating the early mechanism of induction of macrophage growth by lipid materials such as cell debris and lipoproteins.

Induction of macrophage growth by negatively charged phospholipids.

The effects on macrophage growth of seven phospholipids that are normally present in cell membranes were examined. Phosphatidylcholine and sphingomyelin which are the main phospholipid constituents of mammalian cell membranes, had no effect on growth of macrophages. Phosphatidylinositol also had little effect. On the other hand, the relatively minor components phosphatidylserine, phosphatidylglycerol, cardiolipin, and phosphatidic acid, which are negatively charged phospholipids, significantly enhanced macrophage growth. These findings suggest that some acidic phospholipids may increase survival or growth of tissue macrophages when they are ingested by the cells in materials containing phospholipids, such as effete autologous cells or bacteria.

Induction of macrophage growth by effete cells.

When mouse peritoneal cells in early phase-inflammatory exudates were cultured in vitro, the number of polymorphonuclear leukocytes (PMNs) rapidly decreased, whereas the number of macrophages gradually increased during culture. Macrophage growth was also induced by addition of effete PMNs or their debris to macrophage monolayers. This phenomenon was not restricted to senescent PMNs, because effete spleen cells or effete tumor cells also induced macrophage growth. The activity of these cell debris was stable on heating at 100 degrees C and was partially recovered in the lipid fraction. These data suggest that tissue macrophages may proliferate when they scavenge effete PMNs or other host cells and that these cell debris may be important mediators in inducing growth of peripheral macrophages in inflammation and tumors or the normal steady state.

Purification and characterization of the cytotoxic factor in rat peritoneal exudate cells: its identification as the calcium binding protein complex, calprotectin.

We previously reported the existence of a growth inhibitory factor for mitogen-stimulated lymphocytes and murine tumor cell lines, MM46 and L-929, in inflammatory polymorphonuclear leukocytes. In this study, by using mouse MM46 mammary carcinoma as target, we purified the inhibitor from lysate of rat inflammatory peritoneal exudate cells by ammonium sulfate precipitation, gel filtration, isoelectrofocusing, and anion exchange chromatography. Although the in vitro inhibitory activity for MM46 growth was partitioned into three peaks in the final step, it was found that these inhibitory samples all consist of 8- and 13-kDa peptides. Analysis of amino acid sequences revealed that the partial sequences of the 8- and 13-kDa peptides completely agree with the smaller and larger components of rat calprotectin, which are predicted from cDNA, respectively, suggesting the cell growth inhibitory factor is calprotectin. In addition to MM46, the partially purified calprotectin inhibited the growth of a rat, three mice, and a human tumor cell line in similar dose-response relationships in vitro. Moreover, it exerted a cytolytic effect against all examined tumor cells. It was confirmed that the purified calprotectin induces growth inhibition and the lysis of MM46 cells and that the minimum effective concentration is between 50 and 100 micrograms/ml. The factor also inhibited the growth of bone marrow cells and macrophages. These results suggest that calprotectin is a negative regulatory factor for the growth and/or survival states of normal and tumor cells.

Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells.

We have previously shown that the calcium-binding protein complex, calprotectin, purified from rat inflammatory peritoneal cells exerts marked cytotoxic activity against rat, mouse, and human tumor cells. We studied here whether the cytotoxicity is caused by induction of apoptosis, using mouse EL-4 lymphoma and human MOLT-4 leukemia lines as targets. The rat calprotectin sample inhibited [3H]thymidine incorporation into these cells by partially 24 h and almost completely in 48 h of culture at concentrations of 100-200 micrograms/ml. Morphological changes, that is, loss of cell volume and nuclear condensation and/or fragmentation, appeared in both cell types cultured with calprotectin from 20 h, and such apoptotic cells subsequently increased in number to compose the great majority of the cells at 40 h. Cell death, measured by stainability with trypan blue, lagged behind the emergence of the apoptotic morphology by about 2 and 10 h in EL-4 and MOLT-4 cells, respectively. DNA fragmentation was observed in EL-4 cells cultured with calprotectin, whereas it was not observed in MOLT-4 cells, consistent with results of flow cytometry showing that loss of cell DNA content caused by the factor was greater in EL-4 cells. The data indicate that calprotectin induces the apoptosis of certain tumor cells but that the occurrence of DNA fragmentation is dependent on cell type. Finally, the apoptosis-inducing activity of the calprotectin sample was abrogated by the presence of 10 microM zinc, whereas it was not affected by 5 mM calcium or magnesium.

Induction of macrophage growth by the lipid moiety of lipoprotein and its augmentation by denaturation of the lipoproteins.

Lipoprotein (d less than 1.21) isolated from mouse tumor ascitic fluid or mouse-serum induced growth of peritoneal macrophages in vitro. Lipoprotein fractions that stimulated macrophage growth were the chylomicron, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL), whereas the high-density lipoprotein (HDL) fraction did not. Lipids extracted from total lipoprotein also showed significant macrophage-growth-stimulating activity and lost this activity when hydrolyzed. The macrophage-growth-stimulating activity of the lipoprotein (d less than 1.21) was increased about ten times by heat treatment of the lipoprotein (100 degrees C, 30 min). The HDL fraction that had no activity in the native form also showed activity after heat treatment. Lipoprotein-depleted ascitic fluid and simple proteins such as bovine serum albumin (BSA) had no activity even after heat treatment. These results show that the lipid moiety of lipoproteins caused proliferation of macrophages and that denatured lipoproteins were more effective than native ones.

Growth-inhibitory and apoptosis-inducing activities of calprotectin derived from inflammatory exudate cells on normal fibroblasts: regulation by metal ions.

We have shown previously that a calcium-binding protein complex, calprotectin, derived from polymorphonuclear leukocytes exerts a cytostatic and cytolytic effect against a very broad range of tumor cell lines. We also described that calprotectin is an apoptosis-inducing factor for certain tumor cells and that zinc ion attenuates the calprotectin activities. The titers of the factor in body fluids are known to increase greatly in various types of inflammation. In this study, to learn the role of calprotectin in inflammation, the growth-inhibitory and the apoptosis-inducing activities of the factor against normal fibroblasts were examined because fibroblasts are a cell type constituting a local inflammatory site. Rat calprotectin inhibited the growth of murine embryonic as well as human dermal fibroblasts. Although calprotectin induced apoptotic morphology in both fibroblasts, the reaction was slower and less efficient than cases using tumor cells as targets. The activities were significantly abrogated by 10-50 microM Zn2+, Cu2+, Mn2+, or Fe2+, respectively, whereas the trivalent cations Al3+ and Fe3+ had no effect. The dose-response curves of the calprotectin effects were shifted to about 10-fold lower concentration ranges in the divalent metal ion-depleted medium. These results suggest that calprotectin extracellularly affects the inflammatory processes by modulating the growth and survival states of normal fibroblasts, and that the effects are physiologically controlled by several metal ions.

Phase II study of a novel oral formation of 5-fluorouracil in combination with low-dose cisplatin as preoperative chemotherapy of oral squamous cell carcinoma.

TS-1 is a novel oral 5-fluorouracil containing tegaful (prodrug of 5-FU) and two biochemical modulators. These modulators feature effect-enhancing and adverse reaction-reducing activity. We investigated the histological response and toxicities of combination chemotherapy with TS- 1 and low-dose cisplatin and evaluated its usefulness as preoperative chemotherapy Forty-four newly diagnosed patients with stage Il-IV oral squamous cell carcinoma were enrolled in this study from February 2002 to April 2004. Patients were administered TS-1 80 mg/m2/day (days 1-14) and cisplatin 5 mg/m2/day (days 1-5 and 8-12) followed by radical surgery within 2 weeks. The histopathological effect of chemotherapy, which was a surrogate endpoint of this trial, was evaluated with surgical or biopsy specimens. The rate of histological antitumor effect was as follows: complete response (CR) 36.4%, partial response (PR) 25.0%, minor response (MR) 18.1% and no change (NC) 20.5%. The rate of histological response (CR + PR) was 61.4%. The CR rate of effective cases was 59.3%. The main toxicities occurred in bone marrow and the digestive tract. The incidence of severe toxicity such as grade 3 or 4 was 4.5% in anemia, 9% in leukocytopenia, 11.4% in neutropenia, 4.5% in thrombocytopenia and 2.3% in anorexia, diarrhea and urticaria. Most patients showed no toxicity or mild toxicities. TS- 1 with low-dose cisplatin has highly effective antitumor activity and mild toxicities. In particular, the CR rate was very high. It is suggested that this regimen is suitable for neoadjuvant chemotherapy. We expect that this chemotherapy will contribute to avoidance of surgery for small tumors (stages I and II) and will enable function-preserving surgery for advanced tumors.

A new reaction useful for chemical cross-linking between nucleic acids and proteins.

Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.