Melioidosis in Birds and Burkholderia pseudomallei Dispersal, Australia

To the Editor: Melioidosis is an emerging infectious disease of humans and animals caused by the gram-negative bacterium Burkholderia pseudomallei, which inhabits soil and surface water in the disease-endemic regions of Southeast Asia and northern Australia (1). The aim of this study was to assess the potential for birds to spread B. pseudomallei. Birds are known carriers of various human pathogens, including influenza viruses, West Nile virus, Campylobacter jejuni, and antimicrobial drug–resistant Escherichia coli (2)

Comparison of TaqMan PCR assays for detection of the melioidosis agent Burkholderia pseudomallei in clinical specimens

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin

Comparison of TaqMan PCR assays for detection of the melioidosis agent Burkholderia pseudomallei in clinical specimens

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin

High-Resolution Melting Genotyping of Enterococcus faecium Based on Multilocus Sequence Typing Derived Single Nucleotide Polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 “melting types” (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure

Characterization of Ceftazidime Resistance Mechanisms in Clinical Isolates of Burkholderia pseudomallei from Australia

Burkholderia pseudomallei is a Gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen

Diversity of emm sequence types in group A beta-haemolytic streptococci in two remote Northern Territory Indigenous communities: implications for vaccine development

There is a high burden of disease due to group A streptococcus (GAS) in remote Northern Territory (NT) Indigenous communities. A proposed 26-valent GAS M-type vaccine covers 80–90% of pharyngeal and invasive isolates in the US. We examined the diversity and distribution of emm types in two remote Indigenous communities in the NT Top End over a 17-year period and compared them to the proposed vaccine types. Eighty emm types were identified between 1991 and 2007. Diversity in both communities was high (overall Simpson's index 0.976), but varied between communities. Prior to 2004, 71 emm types were identified and an additional 9 emm types were identified during a period of active surveillance in 2004–2005. The proposed 26-valent vaccine would be expected to cover only 20% of emm types recovered in this study. Of the 80 emm types, 16 (20%) were new sequence types identified since the last assignment of M types in 2002. The diversity of streptococcal isolates was higher than that reported from most industrialized countries, and similar to that described in several developing countries. A vaccine based on such a variable antigen is unlikely to provide effective protection in the highest risk populations

Active surveillance for multidrug-resistant Gram-negative bacteria in the intensive care unit

Summary: A short-term program of performing serial active screening cultures (ASC) in the intensive care unit was instituted to establish a method for the detection of antibiotic-resistant Gram-negative bacteria (GNB) and the local rates of colonisation. Of all submitted ASC, 25.9% (30/116 collected swabs) isolated an antibiotic-resistant GNB. ChromID ESBL agar (bioMérieux, France) identified the majority of these organisms, with the additional antibiotic-impregnated media [MacConkey agar (MCA) with ciprofloxacin, MCA with gentamicin and MCA with ceftazidime] adding limited benefit. Compared to swabs performed on admission, 37.8% (14/37) of patients cultured a new antibiotic-resistant isolate on discharge. Serial screening in intensive care has the ability to identify patients with unrecognised colonisation with antibiotic-resistant GNB; however, the increase in the laboratory workload and logistical challenges in the collection of the surveillance swabs may limit this program's expansion

Surveillance snapshot of Clostridium difficile infection in hospitals across Queensland detects binary toxin producing ribotype UK 244

In North America and Europe, the binary toxin positive Clostridium difficile strains of the ribotypes 027 and 078 have been associated with death, toxic megacolon and other adverse outcomes. Following an increase in C. difficile infections (CDIs) in Queensland, a prevalence study involving 175 hospitals was undertaken in early 2012, identifying 168 cases of CDI over a 2 month period. Patient demographics and clinical characteristics were recorded, and C. difficile isolates were ribotyped and tested for the presence of binary toxin genes. Most patients (106/168, 63.1%) were aged over 60 years. Overall, 98 (58.3%) developed symptoms after hospitalisation; 89 cases (53.0%) developed symptoms more than 48 hours after admission. Furthermore, 27 of the 62 (67.7%) patients who developed symptoms in the community ad been hospitalised within the last 3 months. Thirteen of the 168 (7.7%) cases identified had severe disease, resulting in admission to the Intensive Care Unit or death within 30 days of the onset of symptoms. The 3 most common ribotypes isolated were UK 002 (22.9%), UK 014 (13.3%) and the binary toxin-positive ribotype UK 244 (8.4%). The only other binary toxin positive ribotype isolated was UK 078 (n = 1). Of concern was the detection of the binary toxin positive ribotype UK 244, which has recently been described in other parts of Australia and New Zealand. No isolates were of the international epidemic clone of ribotype UK 027, although ribotype UK 244 is genetically related to this clone. Further studies are required to track the epidemiology of ribotype UK 244 in Australia and New Zealand. Commun Dis Intell 2014;38(4):E279–E284