Efficient removal of antibiotics from water by highly crosslinked metal-alginate particles: Preparation, isotherms, kinetics, and microbiological assay.

Traces of antibiotics reaching aquatic environment lead to the emergence of antimicrobial resistance (AMR). The efficient removal of antibiotics (ATBs) traces from wastewater is essential to tackle the AMR. In this study, a novel solid-state crosslinking method of alginate (ALG) was developed and applied to specifically remove ATBs from water. A wide range of crosslinkers (Ca2+, Zn2+, Cu2+, Ni2+, Fe3+ and Al3+) was used and the crosslinking nature, density, and distribution were evidenced by FTIR, ICP-MS, and SEM-EDS. Compared with ionotropic gelation, the novel solid-state crosslinking method proved superior in term of ease of production, high crosslinking degree, and ATBs removal capacity. Fe-ALG and Zn-ALG showed high removal capacity of ciprofloxacin (356.5 mg/g and 928.6 mg/g) and doxycycline (90 mg/g and 690 mg/g), however, they were less effective toward amoxicillin (11.5 mg/g and 6 mg/g). Removal isotherms and kinetics followed type I and pseudo-second order suggesting a chemisorption removal mechanism. Fe-ALG was successfully regenerated with no loss in ATB removal capacity. The microbiological assay showed significant reductions of antibacterial activities after ATBs removal from water. Overall, metal-ALG systems obtained by solid-state crosslinking are promising for ATBs removal from wastewater giving the ease of production, high efficiency, regenerability, and scalability potential.

Visualisation and biovolume quantification in the characterisation of biofilm formation in Mycoplasma fermentans.

The ability of mycoplasmas to persist on surfaces has been widely acknowledged, despite their fastidious nature. However, the organism's capability to form a recognisable biofilm structure has been identified more recently. In the current study Mycoplasma fermentans was found to adhere to the glass surface forming highly differentiated biofilm structures. The volumes of biofilm microcolonies were quantified and observed to be greater at late growth stage than those at early growth stage. The channel diameters within biofilms were measured with Scanning Electron Microscopy images and found to be consistent with the size observed in Confocal Laser Scanning Microscope images. The combination of imaging methods with 3D visualisation provides key findings that aid understanding of the mycoplasma biofilm formation and true biofilm architecture. The observations reported here provide better understanding of the persistence of these minimalist pathogens in nature and clinical settings.

Investigating Potential Chromosomal Rearrangements during Laboratory Culture of Neisseria gonorrhoeae.

Comparisons of genome sequence data between different strains and isolates of Neisseria spp., such as Neisseria gonorrhoeae, reveal that over the evolutionary history of these organisms, large scale chromosomal rearrangements have occurred. Factors within the genomes, such as repetitive sequences and prophage, are believed to have contributed to these observations. However, the timescale in which rearrangements occur is not clear, nor whether it might be expected for them to happen in the laboratory. In this study, N. gonorrhoeae was repeatedly passaged in the laboratory and assessed for large scale chromosomal rearrangements. Using gonococcal strain NCCP11945, for which there is a complete genome sequence, cultures were passaged for eight weeks in the laboratory. The resulting genomic DNA was assessed using Pulsed Field Gel Electrophoresis, comparing the results to the predicted results from the genome sequence data. Three cultures generated Pulsed Field Gel Electrophoresis patterns that varied from the genomic data and were further investigated for potential chromosomal rearrangements.

The effect of copper(II), iron(II) sulphate, and vitamin C combinations on the weak antimicrobial activity of (+)-catechin against Staphylococcus aureus and other microbes.

Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H(2)O(2)via the action of added metal(II) ions. H(2)O(2) generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin-iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.

Enhancement of antimicrobial activities of whole and sub-fractionated white tea by addition of copper (II) sulphate and vitamin C against Staphylococcus aureus; a mechanistic approach.

BACKGROUND: Enhancement of antimicrobial plant products e.g. pomegranate extract by copper (II) sulphate is known. Such combinations have applications in various settings, including the identification of novel compositions to study, treat and control infection. METHODS: A combination of white tea (WT) (made allowing 10 minutes infusion time at 100°C) was combined with 4.8 mM copper (II) sulphate and tested for antimicrobial effect on the viability of Staphylococcus aureus NCTC 06571. Comparisons were made with green (GT) and black (BT) teas. A WT sub-fraction (WTF < 1000 Da) was tested with copper (II) sulphate and 4.8 mM vitamin C. pH measurements of samples were taken for controls and to observe any changes due to tea/agent interaction. Catalase was used to investigate hydrogen peroxide release. UV-vis. was used to compare WT and WTF. RESULTS: A 30 minute incubation at room temperature of copper (II) sulphate alone and combined with WT reduced the viability of S. aureus NCTC 06571 by c.a 1 log10 cfu mL-1. GT and BT with copper (II) sulphate negated activity to buffer values. Combined with copper (II) sulphate, vitamin C, WTF and, vitamin C plus WTF all reduced the viability of S. aureus NCTC 06571 by c.a. 3.5 log10 cfu mL-1. Independent experiments showed the results were not due to pH effects. Adding WT or WTF to copper (II) sulphate resulted in increased acidity. Copper (II) sulphate alone and combined with WT required c.a 300 µg mL-1 (final concentration) catalase to restore S. aureus viability, WTF with copper (II) sulphate and added vitamin C required c.a 600 µg mL-1. WT and WTF UV-visible spectra were similar. CONCLUSIONS: WT showed no efficacy in the combinations tested. WTF was enhanced with copper (II) sulphate and further with vitamin C. WT and WTF increased acidity of copper (II) sulphate possibly via the formation of chemical complexes. The difference in WT/WTF absorbance possibly represented substances less concentrated or absent in WTF. Investigations to establish which WTF component/s and in what proportions additives are most effective against target organisms are warranted.

The need for continued monitoring of antibiotic resistance patterns in clinical isolates of Staphylococcus aureus from London and Malta.

BACKGROUND: Antibiotic resistance is an increasing problem in isolates of Staphylococcus aureus (S. aureus) worldwide. In 2001 The National Health Service in the UK introduced a mandatory bacteraemia surveillance scheme for the reporting of S. aureus and methicillin-resistant S. aureus (MRSA). This surveillance initiative reports on the percentage of isolates that are methicillin resistant. However, resistance to other antibiotics is not currently reported and therefore the scale of emerging resistance is currently unclear in the UK. In this study, multiple antibiotic resistance (MAR) profiles against fourteen antimicrobial drugs were investigated for 705 isolates of S. aureus collected from two European study sites in the UK (London) and Malta. RESULTS: All isolates were susceptible to linezolid, teicoplanin and vancomycin. Multiple antibiotic resistance profiles from both countries were determined, a total of forty-two and forty-five profiles were seen in the UK cohort (MRSA and MSSA respectively) and comparatively, sixty-two and fifty-two profiles were shown in the Maltese group. The largest MAR profile contained six antibiotics (penicillin G, methicillin, erythromycin, ciprofloxacin, clindamycin and clarithromycin) and was observed in the MRSA isolates in both the UK and Maltese cohorts. CONCLUSION: The data presented here suggests that the monitoring of changing resistance profiles locally in maintaining treatment efficacy to resistant pathogens.

Anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of S. aureus, MRSA and PVL positive CA-MSSA.

BACKGROUND: Recently, natural products have been evaluated as sources of antimicrobial agents with efficacies against a variety of micro-organisms. METHODS: This report describes the antimicrobial activities of pomegranate rind extract (PRE) singularly and in combination with cupric sulphate against methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA respectively), and Panton-Valentine Leukocidin positive community acquired MSSA (PVL positive CA-MSSA). RESULTS: PRE alone showed limited efficacy against MRSA and MSSA strains. Exposure to copper (II) ions alone for 2 hours resulted in moderate activity of between 102 to 103 log10 cfu mL-1 reduction in growth. This was enhanced by the addition of PRE to 104 log10 cfu mL-1 reduction in growth being observed in 80% of the isolates. However, the PVL positive CA-MSSA strains were more sensitive to copper (II) ions which exhibited moderate activities of between 103 log10 cfu mL-1 reduction in growth for 60% of the isolates. CONCLUSION: PRE, in combination with Cu(II) ions, was seen to exhibit moderate antimicrobial effects against clinical isolates of MSSA, MRSA and PVL positive CA-MSSA isolates. The results of this study indicate that further investigation into the active ingredients of natural products, their mode of action and potential synergism with other antimicrobial agents is warranted. This is the first report of the efficacy of pomegranate against clinical PVL positive CA-MSSA isolates.

The evaluation of novel chromogenic substrates for the detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups.

Eight novel chromogenic substrates were evaluated for their efficacy in detecting lipase activity in clinical isolates of Staphylococcus aureus from the United Kingdom and Malta. All isolates metabolized the chromogenic lipase substrates 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothia-zolidin-4-one-3-ethanoic acid (SRA)-propionate, SRA-butyrate, SRA-octanoate and 2-[2-(4-hydroxy-3,5-dimethoxyphenyl)-vinyl]-3-methy-benzothiazolium salt (SB(Z)TM)-acetate. Over 90% of the isolates metabolized the lipase substrates SRA-decanoate and SRA-laurate. However, only 0.6% of UK isolates and 2% of Maltese isolates metabolized the lipase substrate SRA-myristate; none of the isolates tested metabolized SB(Z)TM-butyrate. Traditional Tween 80 assays showed that over 73% of the UK methicillin-resistant Staphylococcus aureus (MRSA) isolates and 83% of the UK methicillin-sensitive Staphylococcus aureus (MSSA) isolates demonstrated lipolytic activity. In contrast, Maltese isolates showed lipase activity in 94% and 88% of the MRSA and MSSA strains, respectively. Lipases in MRSA and MSSA demonstrated substrate specificity whose activity appeared dependent upon hydrocarbon chain length of the chromogen. These novel chromogens can be used for lipase enzyme detection and have application for full characterization of numerous S. aureus lipases.

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C.

BACKGROUND: Punica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C. METHODS: PRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. RESULTS: The screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus. CONCLUSION: This is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations.

UK epidemic strains of meticillin-resistant Staphylococcus aureus in clinical samples from Malta.

Since 1999, the European Antimicrobial Resistance Surveillance System (EARSS) has monitored the rise in infection due to a number of organisms, including meticillin-resistant Staphylococcus aureus (MRSA). The EARSS reported that MRSA infections within intensive care units account for 25-50 % of infections in many central and southern European countries, these included France, Spain, Great Britain, Malta, Greece and Italy. Each country has defined epidemic MRSA (EMRSA) strains; however, the method of spread of these strains from one country to another is unknown. In this current study, DNA profiles of 473 isolates of MRSA collected from the UK and Malta were determined by PFGE. Analysis of the data showed that two countries separated by a large geographical distance had a similar DNA profile pattern. Additionally it was demonstrated that strains of EMRSA normally found in the UK were also found in the Maltese cohort (EMRSA 15 and 16). A distinct DNA profile was found in the Maltese cohort, which may be a local EMRSA, and accounted for 14.4 % of all Maltese isolates. The appearance of the same MRSA and EMRSA profiles in two separate countries suggests that MRSA can be transferred out of their country of origin and potentially establish in a new locality or country.

The withdrawal of antimicrobial treatment as a mechanism for defeating resistant microorganisms.

Antimicrobial resistance is a major concern in health care and farming settings throughout the world. The level of antimicrobial resistance continues to increase and the requirement for a novel and possibly dramatic change in therapy choices is required. One possible mechanism for overcoming resistance is the actual removal of antimicrobial treatment from the therapeutic armoury. This review examines the potential for success of a policy advocating the reduction of antimicrobial use and additionally the withdrawal of such treatments. Evidence from agriculture suggests that the removal of certain drugs from animal husbandry can result in concomitant falls in certain drug resistances in human patients.