A systematic approach to microbial forensics.

The coronavirus disease 2019 pandemic accelerated developments in biotechnology that underpin infection science. These advances present an opportunity to refresh the microbial forensic toolkit. Integration of novel analytical techniques with established forensic methods will speed up acquisition of evidence and better support lines of enquiry. A critical part of any such investigation is demonstration of a robust causal relationship and attribution of responsibility for an incident. In the wider context of a formal investigation into agency, motivation and intent, the quick and efficient assembly of microbiological evidence sets the tone and tempo of the entire investigation. Integration of established and novel analytical techniques from infection science into a systematic approach to microbial forensics will therefore ensure that major perspectives are correctly used to frame and shape the evidence into a clear narrative, while recognizing that forensic hypothesis generation, testing and refinement comprise an iterative process. Development of multidisciplinary training exercises that use this approach will enable translation into practice and efficient implementation when the need arises.

Author Correction: Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae.

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Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae.

The expanding global distribution of multi-resistant Klebsiella pneumoniae demands faster antimicrobial susceptibility testing (AST) to guide antibiotic treatment. Current ASTs rely on time-consuming differentiation of resistance and susceptibility after initial isolation of bacteria from a clinical specimen. Here we describe a flow cytometry workflow to determine carbapenem susceptibility from bacterial cell characteristics in an international K. pneumoniae isolate collection (n = 48), with a range of carbapenemases. Our flow cytometry-assisted susceptibility test (FAST) method combines rapid qualitative susceptible/non-susceptible classification and quantitative MIC measurement in a single process completed shortly after receipt of a primary isolate (54 and 158 minutes respectively). The qualitative FAST results and FAST-derived MIC (MICFAST) correspond closely with broth microdilution MIC (MICBMD, Matthew's correlation coefficient 0.887), align with the international AST standard (ISO 200776-1; 2006) and could be used for rapid determination of antimicrobial susceptibility in a wider range of Gram negative and Gram positive bacteria.

First bacteraemic human infection with Escherichia albertii.

The facultative anaerobic Gram-negative species Escherichia albertii has been isolated from human faeces in gastrointestinal infection and from a range of wild bird species. Here we report the first case of a febrile infection associated with E. albertii bacteraemia in a 76-year-old woman with gastric dysplasia.

First case of Chlorella wound infection in a human in Australia.

A 30-year-old man developed an infected knee wound 2 days after jumping his bicycle into a freshwater dam. He required repeated debridement and tissue grew bright green colonies typical of the alga Chlorella plus Aeromonas hydrophila. This, and one previously reported case, responded to surgical debridement and careful wound management.

Distribution of 13 virulence genes among clinical and environmental Aeromonas spp. in Western Australia.

We evaluated the pathogenic potential of 98 clinical and 31 environmental Aeromonas isolates by detecting the presence of 13 virulence genes using a polymerase chain reaction (PCR)-based method. The majority (96 %) of the strains contained at least one of the virulence genes. The overall distribution was aerA/haem (77 %), alt (53 %), lafA (51 %), ast (39 %), flaA (32 %), aspA (29 %), vasH (26 %), ascV (16 %) and aexT (13 %). No amplification products were detected for the genes encoding a bundle-forming pilus (BfpA and BfpG) or a Shiga-like toxin (stx-1 and stx-2). Five or more virulence genes were detected in 42 % of environmental and 24 % of clinical isolates. Among the major species, 48 % of A. hydrophila and 42 % of A. dhakensis isolates harboured five or more virulence genes compared with 19 % in A. veronii bv. sobria and none in A. caviae isolates. Our results suggest that, in Western Australia, strains of A. dhakensis and A. hydrophila are potentially more virulent than those of A. veronii bv. sobria and A. caviae, although the pathogenic potential of Aeromonas spp. is probably strain- rather than species-dependent.

Canibacter oris gen. nov., sp. nov., isolated from an infected human wound.

A facultatively anaerobic, Gram-reaction-positive, catalase- and oxidase-negative, rod-shaped bacterium isolated from an infected human wound caused by a dog bite was characterized by phenotypic and molecular genetic methods. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain IMMIB Q2029717T was a member of the order Micrococcales of the class Actinobacteria, displaying 91.6% to 96% sequence similarity with members of the family Microbacteriaceae. Phylogentic trees generated by different algorithms indicated that the strain forms an independent phylogenetic line of descent that consistently clustered proximal to the base of the genus Leucobacter. Chemical studies revealed the presence of a cell-wall murein based on L-lysine (type B1α), major menaquinone (MK-10) and a DNA G+C content of 56.9 mol%. The distinct phylogenetic position, ribotyping and matrix-assisted laser desorption/ionization time-of-flight MS profiles and the significant phenotypic differences clearly separate strain IMMIB Q2029717T from its nearest phylogenetic neighbour and support its classification as a representative of a novel genus and species, with the suggested name Canibacter oris gen. nov., sp. nov. The type strain is IMMIB Q2029717T (=DSM 27064T=CCUG 64069T).

Cruoricaptor ignavus gen. nov., sp. nov., a novel bacterium of the family Flavobacteriaceae isolated from blood culture of a man with bacteraemia.

A Gram-reaction-negative bacterium, strain IMMIB L-12475(T), was isolated from blood cultures of a human with septicaemia. The yellowish orange pigmented strain contained flexirubin pigment. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain IMMIB L-12475(T) belonged to the family Flavobacteriaceae, forming a distinct phyletic line that is distantly related (79.1-89.4% sequence similarity) to described genera of this family. Membership to the family was confirmed by a fatty acid profile consisting of branched-chain and 3-hydroxy fatty acids with major amounts of iso-C(17:0) 3-OH and iso-C(15:0), by the presence of menaquinone MK-6 as the only respiratory quinone and a polyamine pattern that contained sym-homospermidine as major component. The phospholipids consisted of phosphatidylethanolamine and an unknown phospholipid. The genomic DNA mol% G+C content was 45.6%. The distant phylogenetic position as compared to other representative of the family and the significant phenotypic properties such as pigment composition, morphology, and physiology support the proposal of a novel genus and species Cruoricaptor ignavus gen. nov., sp. nov. The type strain is IMMIB L-12475(T) (=DSM 25479(T)=CCUG 62025(T)).

Antimicrobial susceptibility testing of cystic fibrosis and non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa: a comparison of three methods.

Pseudomonas aeruginosa is an important pathogen in humans, particularly in the context of nosocomial infection and infections of the cystic fibrosis (CF) lung. In order to provide clinicians with information about the likely effectiveness of specific antimicrobial treatment for P. aeruginosa infections, clinical laboratories employ in vitro antimicrobial susceptibility testing. Two commonly employed methods are the CLSI disc-diffusion and Etest methods. The purpose of this study is to compare the accuracy of susceptibility results generated by these two methods against agar dilution as the reference method. Susceptible or nonsusceptible (resistant and intermediate) results of the Etest and CLSI disc-diffusion methods are compared with CLSI agar dilution results for a large cohort of clinical cystic fibrosis (n = 71) and non-cystic fibrosis (n = 83) isolates using CLSI interpretive criteria. An unacceptable number of major and very major errors were observed for various antimicrobials tested against both CF and non-CF isolates when using the Etest and CLSI disc-diffusion methods. The potential for error in standard laboratory antimicrobial susceptibility testing should be considered by clinicians when being guided by the results of such tests in the prescription of antimicrobial agents for P. aeruginosa infection.

Emergence of multi-resistant Pseudomonas aeruginosa in a Western Australian hospital.

Multi-resistant Pseudomonas aeruginosa (MRPa) has been isolated from patients in a Western Australian teaching hospital with increasing frequency since first encountered in 2006. Between 2006 and 2008 the number of patients with MRPa increased from three to nine per annum, and their location shifted from intensive care to a high dependency unit. A novel water-saving device (aerator) in a staff hand basin was identified as a likely disseminator, with MRPa being isolated from biofilm in the basin's plumbing. The disposal of patient waste, surplus intravenous antibiotic infusions and solid items via hand basins were possible contributory factors. Genotyping of MRPa from patients in other hospitals showed distinct genotypic lineages. The third seasonal cluster persisted for longer, indicating adaption to environment. More effective environmental control of P. aeruginosa is urgently needed.

Australian multicentre comparison of subtyping methods for the investigation of Campylobacter infection.

In order to identify subtyping methods able to contribute to the surveillance or investigation of Australian Campylobacter infection, six genotypic and three phenotypic subtyping methods were evaluated on a collection of 84 clinical isolates collected over a 30-month period from one region in Australia. The aim was to compare the logistics of various subtyping methods and examine their ability to assist in finding outbreaks or common sources of sporadic infection. The genotypic subtyping methods used were sequencing of the short variable region of the flaA gene, two methods using restriction fragment length polymorphism (RFLP) of the flaA gene using either DdeI or EcoRI with PstI, automated ribotyping, pulsed field gel electrophoresis and multilocus sequence typing. The phenotypic methods employed included Laboratory of Enteric Pathogens serotyping, Lior biotyping and antibiotic resistotyping. The level of agreement between subtyping results was determined. Phenotypic methods showed little agreement whereas genotypic typing methods showed a high level of agreement. Using the premise that five of the six genotypic typing methods were in agreement 15 genotypic groupings were identified. Sequencing of the short variable region of the flaA gene, RFLP of the flaA gene or automated ribotyping in conjunction with multilocus sequence typing best identified genotypic groupings. An alternative combination of RFLP of the flaA gene followed by ribotyping was equally satisfactory. RFLP of the flaA gene appeared to be suitable as a preliminary typing method based on ease of operation, equipment availability and cost.

Preliminary report on the northern Australian melioidosis environmental surveillance project.

An environmental surveillance programme was developed to determine whether water supplies could be a source of Burkholderia pseudomallei as noted during previous melioidosis outbreak investigations. Water supplies to communities in the three northern Australian jurisdictions (Western Australia, Northern Territory and Queensland) were sampled periodically during 2001 and 2002. Water and soil samples were collected from communities known to have had recent culture-positive melioidosis cases and nearby communities where no cases had been diagnosed. Clinical isolates of B. pseudomallei obtained from northern Australian patients during 2001 and 2002 were compared with the environmental B. pseudomallei isolates by ribotyping and pulsed-field gel electrophoresis. B. pseudomallei was isolated from 11 distinct locations, all in the Northern Territory, seven of which were associated with culture-positive melioidosis cases (>1 case at three locations). Water was implicated as a possible environmental source of melioidosis in six locations. A variety of free-living amoebae including Acanthamoeba and Hartmannella spp. that are potential hosts to B. pseudomallei were recovered from environmental specimens. Culturable B. pseudomallei was not found to be widely dispersed in the environments sampled.

Comparison of the susceptibilities of Burkholderia pseudomallei to meropenem and ceftazidime by conventional and intracellular methods.

The effect of the two antibiotics ceftazidime and meropenem on a collection of 46 Burkholderia pseudomallei isolates representing clinical and environmental sources across northern Australia was investigated by using a series of in vitro test methods. The susceptibility testing methods used included Kirby-Bauer disk diffusion, Etest MIC, broth microdilution MIC, and a modification of the microdilution method in which Acanthamoeba cells were added to simulate the effect of a professional phagocytic cell on test outcome. In a semiquantitative validation coculture series, the majority of bacteria were intracellular up to a multiplicity of infection of 10 bacteria to one ameba. The optical density and bacterial count (log10 CFU/ml) correlated across the range tested (r2 = 0.77; P < 0.0001). Susceptibility test results were compared against clinical outcomes. The MICs of ceftazidime were consistently higher than those of meropenem by all three methods. The MICs of both agents were significantly higher when Acanthamoeba trophozoites were added to the broth microdilution method. Conventional and intracellular MIC results were consistent for clinical isolates from the Western Australian outbreak cluster despite the wide variety of clinical outcomes. Further development of the intracellular MIC method is expected to help assess the efficacy of antimicrobial agents on this bacterial species in an intracellular setting.

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak.

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Novel selective medium for isolation of Burkholderia pseudomallei.

Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA). We designed a new selective agar (Burkholderia pseudomallei selective agar [BPSA]) to improve recovery of the more easily inhibited strains of B. pseudomallei. B. pseudomallei, Burkholderia cepacia, and Pseudomonas aeruginosa were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to P. aeruginosa and B. cepacia and should make recognition of Burkholderia species easier due to distinctive colony morphology. BPSA also inhibited Enterococcus, Escherichia, Staphylococcus, and Streptococcus: These results indicate that BPSA is a potential replacement for ASA.

Case report: recovery from persistent septicemic melioidosis.

Septicemic melioidosis is often fatal despite treatment with antibiotics such as ceftazidime to which Burkholderia pseudomallei, the causal pathogen, is sensitive in vitro. We report a near-fatal case of septicemic melioidosis with persistent B. pseudomallei bacteremia despite intravenous ceftazidime in which combination therapy with meropenem and ciprofloxacin, splenectomy and correction of metabolic acidosis allowed for hospital discharge. The choice of antibiotic agents was supported by intracellular minimum inhibitory concentration analysis using B. pseudomallei co-culture in Acanthamoeba trophozoites. The patient's B. pseudomallei isolates were indistinguishable by pulsed-field gel electrophoresis from clinical and environmental isolates previously analyzed during investigation of a Western Australian melioidosis outbreak. A combination of antibiotics known to possess intracellular activity against B. pseudomallei, surgery and supportive critical care may provide a means of improving the probability of survival in persistent septicemic melioidosis.

Molecular epidemiology of Scedosporium apiospermum infection determined by PCR amplification of ribosomal intergenic spacer sequences in patients with chronic lung disease.

Respiratory tract colonization with Scedosporium apiospermum in patients with chronic suppurative lung disease is a significant concern for lung transplantation candidates, since Scedosporium infections occurring posttransplantation are usually untreatable. Up to 10% of patients with cystic fibrosis attending our respiratory medicine unit have had Scedosporium organisms isolated from sputum samples. We therefore developed a molecular typing method to examine these isolates. Typing by PCR amplification of ribosomal intergenic spacer sequences demonstrated 20 different types from 52 isolates collected from the respiratory medicine unit and elsewhere in Australia. A single common type was isolated from 11 respiratory medicine unit inpatients. Two other types were isolated from more than one source: one from two respiratory medicine unit inpatients and one from two epidemiologically linked nonhuman sources. Multiple isolates were obtained from nine patients. This method demonstrated persistent carriage of isolates of the same type in one patient for 7 months. Two patients showed carriage of isolates with multiple typing patterns within a 3-month period. The high rate of isolation and the predominance of isolates with a single typing pattern from respiratory medicine unit patients may suggest transmission to patients from a source in the unit. There was no epidemiological evidence of direct patient-to-patient spread, and Scedosporium organisms were not isolated from dust, soil, or air samples from the unit. The source and route of transmission have yet to be determined.

Burkholderia pseudomallei traced to water treatment plant in Australia.

Burkholderia pseudomallei was isolated from environmental specimens 1 year after an outbreak of acute melioidosis in a remote coastal community in northwestern Australia. B. pseudomallei was isolated from a water storage tank and from spray formed in a pH-raising aerator unit. Pulsed-field gel electrophoresis confirmed the aerator and storage tank isolates were identical to the outbreak strain, WKo97.