- Volume 63, Issue 9, 2014

Leptospirosis, a worldwide zoonotic infection, is an important human and veterinary health problem. We have previously identified a leptospiral multipurpose adhesin, Lsa66, capable of binding extracellular matrix (ECM) components and plasminogen (PLG). In this work, we report the cloning, expression, purification and characterization of three fragments derived from the full-length Lsa66: N-terminal, intermediate and C-terminal regions. We employed Escherichia coli BL21-SI as expression cells. The recombinant fragments tagged with N-terminal His6 were purified by metal-charged chromatography to major protein bands that were recognized by anti-His-tag mAbs. The recombinant fragments were evaluated for their capacity to attach to ECM components and to PLG. The intermediate region bound to laminin, plasma fibronectin and PLG. Laminin also bound to the C-terminal region. Antibodies in leptospirosis-positive serum samples recognized Lsa66, being the immune epitopes located at the N-terminal and intermediate fragments. The data confirm that Lsa66 is expressed during infection and that this protein might have a role in bacterial infection.

Influenza virus infections in humans remain a healthcare concern, and the need for vaccines, therapeutics and prophylactics remains a high priority. Understanding the molecular events associated with influenza-virus-induced pathology may lead to the identification of clinical disease biomarkers and novel antiviral targets. MicroRNAs (miRNAs) are well-conserved endogenous non-coding RNAs known to regulate post-transcriptional gene expression as well as play a major role in many biological processes and pathways. Animal studies have demonstrated that miRNAs are involved in viral disease and controlling inflammation. In this study, we examined the differences in the miRNA expression profiles associated with the lung in mice infected with influenza viruses that varied in virulence and pathogenicity. A statistical model was employed that utilized changes in miRNA expression to identify the virus that was used to infect the mice. This study identified a unique fingerprint of viral pathogenicity associated with seasonal H1N1, swine H1N1 and highly pathogenic H5N1 in the mouse model, and may lead to the identification of novel therapeutic and prophylactic targets.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the ‘Streptococcus anginosus group’ (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide ‘gold standard’ identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.

Biofilms are commonly involved in medical device-related infections. The purpose of this study was to determine the antimicrobial and anti-biofilm activity of polyethyleneimine (PEI) and PEI-based nanoparticles (nanoPEI) against Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii and Candida albicans (clinical and ATCC strains), and to evaluate their effect upon biofilm formation on polyurethane (PUR)-like catheters. MICs and minimal lethal concentrations of PEI and nanoPEI were determined according to CLSI microdilution reference protocols. For PEI, the MIC value was 195.31 mg l−1 for all the bacteria and 48.83 mg l−1 for the yeast strains. For nanoPEI, the MIC value was 1250 mg l−1 for all the strains except A. baumannii, for which it was 2500 mg l−1. Biofilm formation was assessed with PUR-like catheter segments and biofilm metabolic activity was quantified by colorimetry with a tetrazolium reduction assay. Plasma membrane integrity and membrane potential were assessed by flow cytometry after staining microbial cells with a membrane-impermeable dye, propidium iodide, and a membrane-potential marker, DiBAC4(3). PEI inhibited growth of all microbial species; higher concentrations of nanoPEI were needed to inhibit growth of all species. Biofilm formation in the presence of anti-bacterial PEI activity was dose-dependent (except for S. epidermidis) and species-related. NanoPEI at 0.5×MIC and MIC significantly reduced the metabolic activity of biofilms of S. aureus, S. epidermidis and A. baumannii, whereas 2×MIC was required in order to inhibit biofilm metabolic activity.

The implementation of direct testing of clinical faecal specimens for gastrointestinal (GI) pathogens by PCR offers a sensitive and comprehensive approach for the detection of Shiga toxin-producing Escherichia coli (STEC). The introduction of a commercial PCR assay, known as GI PCR, for the detection of GI pathogens at three frontline hospital laboratories in England between December 2012 and December 2013 led to a significant increase in detection of STEC other than serogroup O157 (non-O157 STEC). In 2013, 47 isolates were detected in England, compared with 57 in the preceding 4 years (2009–2012). The most common non-O157 STEC serogroup detected was O26 (23.2 %). A total of 47 (47.5 %) STEC isolates had stx2 only, 28 (28.3 %) carried stx1 and stx2, and the remaining 24 (24.2 %) had stx1 only. Stx2a (64.0 %) was the most frequently detected Stx2 subtype. The eae (intimin) gene was detected in 52 (52.5 %) non-O157 STEC isolates. Six strains of STEC O104 had aggR, but this gene was not detected in any other STEC serogroups in this study. Haemolytic ureamic syndrome was significantly associated with STEC strains possessing eae [odds ratio (OR) 5.845, P = 0.0235] and/or stx2a (OR 9.56, P = 0.0034) subtypes. A matched case–control analysis indicated an association between non-O157 STEC cases and contact with farm animals. Widespread implementation of the PCR approach in England will determine the true incidence of non-O157 STEC infection, highlight the burden in terms of morbidity and mortality, and facilitate the examination of risk factors to indicate whether there are niche risk exposures for particular strains.

Multidrug-resistant (MDR) Pseudomonas aeruginosa isolated from cystic fibrosis (CF) sputum was shown to be more tolerant to the most commonly used chlorine-based disinfecting agent in the UK, with approximately 7 out of 10 isolates surviving a residual free chlorine (RFC) concentration of 500 p.p.m., when compared with antibiotic-sensitive invasive P. aeruginosa from a non-CF blood culture source, where 8 out of 10 isolates were killed at a RFC concentration of 100 p.p.m. All CF isolates were killed at 1000 p.p.m. chlorine. Additional studies were performed to examine factors that influenced the concentration of RFC from chlorine-based (sodium dichloroisocyanurate) disinfecting agents in contact with CF sputum and their components (bacterial cells, glycocalyx) to assess the reduction of the bactericidal activity of such disinfecting agents. Pseudomonas glycocalyx had a greater inhibitory effect of chlorine deactivation than bacterial cells. Calibration curves demonstrated the relative deactivating capacity on RFC from clinical soils, in the order pus>CF sputum>wound discharge fluid/synovial fluid>ascites fluid>bile, where quantitatively each 1 % (w/v) CF sputum reduced the RFC by 43 p.p.m. Sublethal stressing of P. aeruginosa with chlorine resulted in lowered susceptibility to colistin (P = 0.0326) but not to meropenem, tobramycin or ciprofloxacin. In conclusion, heavy contamination of healthcare fomites with CF sputum containing MDR P. aeruginosa may result in exhaustion of RFC, and this, combined with an increased resistance to chlorine with such strains, may lead to their survival and increased antibiotic resistance in such environments. CF infection prevention strategies in such scenarios should therefore target interventions with increased concentrations of chlorine to ensure the eradication of MDR P. aeruginosa from the CF healthcare environment.

Meticillin-resistant Staphylococcus pseudintermedius (MRSP) has recently emerged as a worldwide cause of canine pyoderma. In this study, we characterized 22 S. pseudintermedius isolates cultured from 19 dogs with pyoderma that attended a veterinary dermatology referral clinic in Australia in 2011 and 2012. Twelve isolates were identified as MRSP by mecA real-time PCR and phenotypic resistance to oxacillin. In addition to β-lactam resistance, MRSP isolates were resistant to erythromycin (91.6 %), gentamicin (83.3 %), ciprofloxacin (83.3 %), chloramphenicol (75 %), clindamycin (66 %), oxytetracycline (66 %) and tetracycline (50 %), as shown by disc-diffusion susceptibility testing. Meticillin-susceptible S. pseudintermedius isolates only showed resistance to penicillin/ampicillin (90 %) and tetracycline (10 %). PFGE using the SmaI restriction enzyme was unable to type nine of the 12 MRSP isolates. However the nine isolates provided the same PFGE pulsotype using the Cfr91 restriction enzyme. Application of the mec-associated direct repeat unit (dru) typing method identified the nine SmaI PFGE-untypable isolates as dt11cb, a dru type that has only previously been associated with MRSP sequence type (ST)45 isolates that possess a unique SCCmec element. The dt11cb isolates shared a similar multidrug-resistant antibiogram phenotype profile, whereas the other MRSP isolates, dt11a, dt11af (dt11a-associated) and dt10h, were resistant to fewer antibiotic classes and had distinct PFGE profiles. This is the first report of MRSP causing pyoderma in dogs from Australia. The rapid intercontinental emergence and spread of multidrug-resistant MRSP strains confirms the urgent need for new treatment modalities for recurrent canine pyoderma in veterinary practice.

A total of 15 adult ewes from one flock known to be seroreactive for leptospirosis was studied. Urine and vaginal fluid were collected from each animal to test for the presence of leptospires using bacterial culture and conventional PCR methods. One pure culture of Leptospira sp. was obtained from the vaginal fluid sample of a non-pregnant ewe. The isolate was characterized by DNA sequencing of the rrs and secY genes, variable-number of tandem-repeats (VNTR) analysis and serogrouping, and the isolate was typed as Leptospira interrogans serogroup Sejroe serovar Hardjo type Hardjoprajitno. This report indicates the presence of viable Leptospira in the vaginal fluid of a ewe, suggesting the potential for venereal transmission of leptospires in sheep.