- Volume 65, Issue 1, 2016

Discoveries associated with antibacterial activity of hydrated clays necessitate assessments of in vivo efficacy, practical use and safety. Surface properties of clays can lead to variations in the composition and abundance of bound compounds or ions, thus affecting antibacterial activity. Since exchangeable metal ions released from the clay surface are responsible for in vitro antibacterial activity, we evaluated the in vivo antibacterial efficacy of four natural clays (one illite clay, two montmorillonite clays and one kaolinite clay) and three ion-exchanged, antibacterial clays against superficial, cutaneous meticillin-resistant Staphylococcus aureus (MRSA) infections in mice. Superficial, cutaneous wounds on the back of SKH1-Elite mice were generated and subsequently infected with MRSA. Following twice daily applications of a hydrated clay poultice to infected wounds for 7 days, we observed significant differences in the in vivo antibacterial efficacy between different types of clays. The natural and ion-exchanged illite clays performed best, as measured by bacterial load, inflammatory response and gross wound morphology with significant decreases in bacterial viability and dermatitis. Topical application of kaolinite clay was the least effective, resulting in the lowest decrease in bacterial load and exhibiting severe dermatitis. These data suggest that specific types of clays may offer a complementary and integrative strategy for topically treating MRSA and other cutaneous infections. However, since natural clays exhibit in vitro antibacterial variability and vary vastly in surface chemistries, adsorptive/absorptive characteristics and structural composition, the properties and characteristics of illite clays could aid in the development of standardized and customized aluminosilicates for topical infections.

Listeria monocytogenes, Salmonella enterica and verocytotoxigenic Escherichia coli (VTEC) are amongst the most important agents responsible for food outbreaks occurring worldwide. In this work, two Lactobacillus spp. strains (LABs), Lactobacillus plantarum (LB95) and Lactobacillus paraplantarum (LB13), previously isolated from spontaneously fermenting olive brines, and two reference probiotic strains, Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, were investigated for their ability to attenuate the virulence of the aforementioned pathogens using animal cell culture assays. In competitive exclusion assays, the relative percentages of adhesion and invasion of S. enterica subsp. enterica serovar Enteritidis were significantly reduced when the human HT-29 cell line was previously exposed to LB95. The relative percentage of invasion by Listeria monocytogenes was significantly reduced when HT-29 cells were previously exposed to LB95. In the cytotoxicity assays, the cell-free supernatant of the co-culture (CFSC) of VTEC with LB95 accounted for the lowest value obtained amongst the co-cultures of VTEC with LABs, and was significantly lower than the value obtained with the co-culture of VTEC with the two probiotic reference strains. The cytotoxicity of CFSC of VTEC with both LB95 and LB13 exhibited values not significantly different from the cell-free supernatant of the non-pathogenic E. coli B strain. Our results suggested that LB95 may be able to attenuate the virulence of Gram-positive and Gram-negative food-borne pathogens; together with other reported features of these strains, our data reveal their possible use in probiotic foods due to their interesting potential in preventing enteric infections in humans.

It is often difficult to obtain a bacteriological diagnosis in patients with cellulitis. We examined the utility of molecular techniques and skin and throat cultures, as well as serology, in providing evidence of either Staphylococcus aureus or group A Streptococcus (GAS) presence in patients with cellulitis. Samples were collected from patients with a clinical diagnosis of cellulitis who were recruited into a prospective placebo-controlled clinical trial (C4C study, EudraCT 2013-001218-14). Specific PCR, paired serology and culture for both organisms were carried out on a variety of samples where appropriate. Despite utilizing a range of diagnostic methods, a bacteriological diagnosis was only achieved in 43 % of patients with a clinical diagnosis of cellulitis. Seventeen per cent of patients tested positive for GAS by any method but only 4 % were positive by PCR, whilst S. aureus was detected in 34% of samples. Bacterial diagnosis in cases of cellulitis remains challenging. This is probably due to a very low bacterial burden with toxin production resulting in inflammation mediating skin damage. Further consideration for the need for long courses of antimicrobial therapy for cellulitis therefore appears merited.

The efficient medical treatment of infections requires detailed information about the pathogens involved and potential antibiotic-resistance mechanisms. The dramatically increasing incidence of multidrug-resistant bacteria especially highlights the importance of sophisticated diagnostic tests enabling a fast patient-customized therapy. However, the current molecular detection methods are limited to either the detection of species or only a few antibiotic-resistance genes. In this work, we present a human pathogen characterization assay using a rRNA gene microarray identifying 75 species comprising bacteria and fungi. A statistical classifier was developed to facilitate the automated species identification. Additionally, the clinically most important β-lactamases were identified simultaneously in a 100-plex reaction using padlock probes and the same microarray. The specificity and sensitivity of the combined assay was determined using clinical isolates. The detection limit was 105 c.f.u. ml− 1, recovering 89 % of the detectable β-lactamase-encoding genes specifically. The total assay time was less than 7 h and the modular character of the antibiotic-resistance detection allows the easy integration of further genetic targets. In summary, we present a fast, highly specific and sensitive multiplex pathogen characterization assay.

Rapid identification of bacteria isolated from blood cultures by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now in widespread use in major centres but is not yet feasible in smaller hospital laboratories. A FilmArray multiplex PCR panel for blood culture isolate identification (BCID) provides an alternative approach to near point-of-care microbial identification in regional hospitals. We assessed the accuracy and time to identification of the BCID FilmArray in a consecutive series of 149 blood cultures from 143 patients in a teaching hospital and smaller regional hospitals, currently identified by direct MALDI-TOF and proprietary molecular methods. The BCID FilmArray contained 18 of 34 species and 20 of 23 species isolated from teaching and regional hospital, respectively. Overall, 85 % of the teaching hospital and 100 % of the regional hospital monomicrobial blood cultures were identified, compared with 60 and 68 %, respectively, for direct MALDI-TOF on the same cultures. There were no incorrect results from blood cultures containing Staphylococcus aureus, streptococci, Pseudomonas aeruginosa or Enterobacteriaceae. The three discrepant results were all in mixed cultures. The mean reduction in time to identification of blood culture isolates was 53 h, which did not include the time required to transport cultures from regional centres to a central laboratory. The overall performance of the BCID FilmArray is stronger in blood cultures from smaller regional hospitals that encounter a narrower range of bacterial species dominated by the commonest species. This approach is more suited to smaller clinical laboratories than the MALDI-TOF direct method.

This study demonstrates a novel detection assay able to identify and subtype strains of Clostridium difficile. Primers carefully designed for melting curve analysis amplify DNA from three C. difficile genes, tcdB, tcdC and cdtB, during quantitative (q)PCR. The tcdB gene allows for confirmation of organism presence, whilst the tcdC and cdtB genes allow for differentiation of virulence status, as deletions in the tcdC gene and the concurrent presence of the cdtB gene, which produces binary toxin, are associated with hypervirulence. Following qPCR, subtyping is then achieved by automated, inline melting curve analysis using only a single intercalating dye and verified by microchip electrophoresis. This assay represents a novel means of distinguishing between toxigenic and hypervirulent C. difficile strains NAP1/027/BI and 078 ribotype, which are highly prevalent hypervirulent strains in humans. This methodology can help rapidly detect and identify C. difficile strains that impose a significant health and economic burden in hospitals and other healthcare settings.

Extra-intestinal pathogenic Escherichia coli (ExPEC) are the predominant cause of Gram-negative bloodstream infections. In this study, 20 E. coli isolates that were the causative agents of bacteraemia and subsequent mortality were characterized. Whole-genome sequencing was used to define the predominant sequence types (ST) among the isolates and to identify virulence factors associated with pathogenicity of ExPEC. The ability of the isolates to resist killing by both serum and polymorphonuclear leukocytes (PMNLs) was also assessed. In line with global trends, ST131 occurred most frequently among the bloodstream isolates and all isolates of this sequence type were multidrug resistant. Other common STs included ST73 and ST69. All isolates encoded multiple virulence factors across a range of categories, including factors involved in adhesion, immune evasion, iron acquisition and synthesis of toxins. None of these factors could be associated with serum and neutrophil resistance. The majority of isolates were resistant to the bactericidal action of serum and PMNLs, and most of those that were sensitive were isolated from patients with compromised immunity.