Antimicrobial effects of Melaleuca alternifolia (tea tree) essential oil against biofilm-forming multidrug-resistant cystic fibrosis-associated Pseudomonas aeruginosa as a single agent and in combination with commonly nebulized antibiotics.

Broth microdilution assays were used to determine minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indices (FICIs) of tea tree oil (TTO), tobramycin, colistin and aztreonam (ATM) against clinical cystic fibrosis-associated Pseudomonas aeruginosa (CFPA) isolates (n = 20). The minimum biofilm eradication concentration (MBEC) and fractional biofilm eradication concentration index (FBECI) were also determined using a similar microbroth dilution checkerboard assay, with biofilms formed using the MBEC device® . TTO was effective at lower concentrations against multidrug-resistant (MDR) CFPA isolates (n = 3) in a biofilm compared to in a planktonic state (MBEC 18·7-fold lower than MIC). CFPA within biofilm was less susceptible to ATM, colistin and tobramycin compared to planktonic cells (MBEC 6·3-fold, 9·3-fold, and 2·1-fold higher than MIC respectively). All combinations of essential oil and antibiotic showed indifferent relationships (FICI 0·52-1·72) when tested against planktonic MDR CFPA isolates (n = 5). Against CFPA isolates (n = 3) in biofilm, combinations of TTO/aztreonam and TTO/colistin showed indifferent relationships (mean FBECI 0·85 and 0·60 respectively), whereas TTO/tobramycin showed a synergistic relationship (mean FBECI 0·42). The antibiofilm properties of TTO and the synergistic relationship seen between TTO and tobramycin against CFPA in vitro make inhaled TTO a promising candidate as a potential therapeutic agent.

Clostridium difficile infection diagnosis in a paediatric population: comparison of methodologies.

The increasing incidence of Clostridium difficile infection (CDI) in paediatric hospitalised populations, combined with the emergence of hypervirulent strains, community-acquired CDI and the need for prompt treatment and infection control, makes the rapid, accurate diagnosis of CDI crucial. We validated commonly used C. difficile diagnostic tests in a paediatric hospital population. From October 2011 to January 2012, 150 consecutive stools were collected from 75 patients at a tertiary paediatric hospital in Perth, Western Australia. Stools were tested using: C. Diff Quik Chek Complete, Illumigene C. difficile, GeneOhm Cdiff, cycloserine cefoxitin fructose agar (CCFA) culture, and cell culture cytotoxin neutralisation assay (CCNA). The reference standard was growth on CCFA or Cdiff Chromagar and PCR on isolates to detect tcdA, tcdB, cdtA, and cdtB. Isolates were PCR ribotyped. The prevalence of CDI was high (43 % of patients). Quik Chek Complete glutamate dehydrogenase (GDH) demonstrated a low negative predictive value (NPV) (93 %). Both CCNA and Quik Chek Complete toxin A/B had poor sensitivity (33 % and 29 % respectively). Molecular methods both had 89 % sensitivity. Algorithms using GDH + Illumigene or GeneOhm reduced the sensitivity to 85 % and 83 % respectively. Ribotype UK014/20 predominated. GDH NPV and GeneOhm and Illumigene sensitivities were reduced compared with adult studies. Quik Chek Complete and CCNA cannot reliably detect toxigenic CDI. A GDH first algorithm showed reduced sensitivity. In a high prevalence paediatric population, molecular methods alone are recommended over the use of GDH algorithm or culture and CCNA, as they demonstrate the best test performance characteristics.

Clostridioides difficile colonization and infection in a cohort of Australian adults with cystic fibrosis.

BACKGROUND: Little is known about Clostridioides difficile infection (CDI) in patients with cystic fibrosis (CF). The aim of this study was to investigate the prevalence, molecular epidemiology and risk factors for CDI in asymptomatic and symptomatic adults with CF in Western Australia. METHODS: Faecal samples from symptomatic and asymptomatic patients were prospectively collected and tested for the presence of C. difficile by toxigenic culture. Ribotyping was performed by established protocols. Logistic regression analysis was performed to analyse the risk factors for C. difficile colonization and infection. Extensive environmental sampling was performed within the CF clinic in Perth. RESULTS: The prevalence rates of asymptomatic toxigenic and non-toxigenic C. difficile colonization were 30% (14/46 patients) and 24% (11/46 patients), respectively. Fifteen ribotypes (RTs) of C. difficile were identified, of which non-toxigenic RT 039 was the most common. Among the symptomatic patients, the prevalence of toxigenic CDI was 33% (11/33 patients). Impaired glucose tolerance/diabetes mellitus and duration of intravenous antibiotic use in the past 12 months were significantly associated with increased risk of asymptomatic toxigenic C. difficile carriage and CDI. A trend towards higher CF transmembrane conductance regulator modulator treatment was observed in the CDI group. Extensive environmental sampling showed no evidence of toxigenic C. difficile contamination within the CF clinic. CONCLUSIONS: A high prevalence of asymptomatic carriage of toxigenic C. difficile was observed in adults with CF, comparable with that observed in the symptomatic CF population. There was no evidence of direct person-to-person transmission.

Prevalence and molecular epidemiology of Clostridium difficile infection in Thailand.

Little is known about Clostridium difficile infection (CDI) in Asia generally, and specifically in Thailand. Given the high prevalence of inappropriate antibiotic usage in this region, CDI is likely to be common. This study investigated the prevalence and molecular epidemiology of CDI in Thailand. Stool specimens collected from inpatients with diarrhoea at Siriraj hospital in Bangkok (n = 422) were cultured on ChromID Cdiff agar and any presumptive C. difficile colonies were identified, PCR ribotyped and toxin profiled. As part of the routine C. difficile testing at Siriraj Hospital, 370 specimens also underwent testing with the BD MAX Cdiff assay to detect the presence of tcdB. With direct culture, 105 different isolates of C. difficile were recovered from 23.7% (100/422) of the stool specimens. The prevalence of toxigenic and nontoxigenic isolates was 9.2% (39/422) and 15.6% (66/422), respectively. Of the toxigenic isolates, 69.2% (27/39) and 30.8% (12/39) were tcdA and tcdB positive (A+B+), and A-B+, respectively; none contained binary toxin genes. The five most prevalent ribotypes (RTs) were 014/020 group (17/105), 010 (12/105), 017 (12/105), 039 (9/105) and 009 (6/105). Using toxigenic culture as the reference standard, the sensitivity, specificity, positive predictive value and negative predictive value of the BD MAX Cdiff assay were 68.6, 95.1, 63.2 and 96.1%, respectively. The high proportion of A-B+, RT 017 strains emphasises the need for diagnostic tests that detect either both toxins or just tcdB. Continued surveillance that involves stool culturing will allow molecular tracking and assist in elucidating the epidemiology of CDI in Thailand.

Contamination of Australian newborn calf carcasses at slaughter with Clostridium difficile.

In North America and Europe, reports of a genetic overlap between toxigenic strains of Clostridium difficile isolated from humans, livestock and retail meat suggest that food-borne transmission may be occurring. We investigated the prevalence, concentration and genetic diversity of C. difficile on the carcasses (n = 300) and in the faeces (n = 30) of neonatal veal calves at three abattoirs in Australia in 2013. Selective culture (both direct and enrichment) was performed, and all isolates were characterized by PCR for the toxin genes tcdA, tcdB and cdtA/B and by PCR ribotyping. Prevalence of C. difficile was 25.3% (76/300) on carcasses and 60.0% (18/30) in faeces. Multiple PCR ribotypes (RT) were detected, with four binary toxin-positive RTs accounting for 70.3% (71/101) of isolates; 127 (A(+), B(+), CDT(+), 32.7%), 288 (A(-), B(-), CDT(+), 28.7%), 033 (A(-), B(-), CDT(+), 6.9%) and 126 (A(+), B(+), CDT(+), 2.0%). Viable counts of a subset of samples revealed detectable numbers of C. difficile in 66.7% (10/15) of faecal samples (range 2.0 × 10(3) to 2.3 × 10(6) CFU/mL, median count 2.5 × 10(4) CFU/mL) and in 16.7% (25/150) of carcase samples (range 3 to 33 CFU/cm(2), median count 7 CFU/cm(2)). These data further confirm that Australian neonatal veal calf carcasses are contaminated with potentially significant strains of C. difficile at slaughter.