Barbara J. Chang

Interaction between Burkholderia pseudomallei and Acanthamoeba Species Results in Coiling Phagocytosis, Endamebic Bacterial Survival, and Escape

ABSTRACT Burkholderia pseudomallei causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial pathogens, B. pseudomallei is capable of survival in human phagocytic cells, but unlike mycobacteria, Listeria monocytogenes, andSalmonella serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae. We investigated the consequences of exposing Acanthamoeba astronyxis, A. castellani, and A. polyphaga to B. pseudomallei NCTC 10276 in a series of coculture experiments. Bacterial endocytosis was observed in all three Acanthamoeba species. A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with A. astronyxis in timed coculture experiments. Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture. Confocal microscopy was used to confirm the intracellular location of endamebic B. pseudomallei cells. Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 × 102 CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture. Demonstration of an interaction between B. pseudomallei and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival ofB. pseudomallei and subsequent human exposure. Endamebic passage of B. pseudomallei warrants further investigation as a potential in vitro model of intracellular B. pseudomallei infection.

Invasion of Spores of the Arbuscular Mycorrhizal Fungus Gigaspora decipiens by Burkholderia spp.

ABSTRACT Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 × 106 CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 × 105 CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.

Diversity and Evolution in the Genome of Clostridium difficile

SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen.

New types of toxin A-negative, toxin B-positive strains among clinical isolates of Clostridium difficile in Australia

A total of 817 human clinical isolates of Clostridium difficile from all Australian states were screened for A(-)B(+) strains by toxin gene PCR assays. Nine (1.1 %) strains were confirmed to be A(-)B(+) by enzyme immunoassay for toxin production. Of these, six (66.7 %) were binary toxin-positive by PCR. Using PCR ribotyping and toxinotyping, the A(-)B(+) strains could be grouped into seven ribotypes and three toxinotypes. Only one of the ribotypes had been reported previously (017). The prevalence of ribotype 017 was low in this study with only two strains detected. Two new A(-)B(+) toxinotypes were also defined (XXX, XXXI). Toxinotype XXX had a toxin B gene similar to that of toxinotype IV (A(+)B(+)) but with a novel cytopathic region. Toxinotype XXXI was similar to other A(-)B(+) types (X, XVII), but had a larger deletion to the toxin A gene than in either of those types. The types of A(-)B(+) strains identified in this study differed markedly from those described in other regions.

Antimicrobial Susceptibilities of Aeromonas Strains Isolated from Clinical and Environmental Sources to 26 Antimicrobial Agents

ABSTRACT We determined the susceptibilities of 144 clinical and 49 environmental Aeromonas strains representing 10 different species to 26 antimicrobial agents by the agar dilution method. No single species had a predominantly nonsusceptible phenotype. A multidrug nonsusceptible pattern was observed in three (2.1%) clinical strains and two (4.0%) strains recovered from diseased fish. Common clinical strains were more resistant than the corresponding environmental isolates, suggesting that resistance mechanisms may be acquired by environmental strains from clinical strains.

Isolation and Characterization of Temperate Bacteriophages of Clostridium difficile

ABSTRACT The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages φC2, φC5, φC6, and φC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of φC2 showed nucleotide homology to the sequenced C. difficile strain CD630.

Role of the MexAB-OprM Efflux Pump of Pseudomonas aeruginosa in Tolerance to Tea Tree (Melaleuca alternifolia) Oil and Its Monoterpene Components Terpinen-4-ol, 1,8-Cineole, and α-Terpineol

ABSTRACT Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organisms tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and α-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.

Flagellum-Mediated Adhesion by Burkholderia pseudomallei Precedes Invasion of Acanthamoeba astronyxis

ABSTRACT In this study we investigated the role of the bacterial flagellum in Burkholderia pseudomallei entry to Acanthamoeba astronyxis trophozoites. B. pseudomallei cells were tethered to the external amoebic surface via their flagella. MM35, the flagellum-lacking fliC knockout derivative of B. pseudomallei NCTC 1026b did not demonstrate flagellum-mediated endocytosis in timed coculture, confirming that an intact flagellar apparatus assists B. pseudomallei entry into A. astronyxis.

Extended epidemic of nosocomial urinary tract infections caused by Serratia marcescens.

ABSTRACT In recent years a significant increase in the incidence of Serratia marcescens infections was noted at the Chang Gung Memorial Hospital, Taoyuan, Taiwan. A review of laboratory (1991 to 2002) and infection control (1995 to 2002) records showed the possibility of an extended epidemic of nosocomial urinary tract infections (UTIs) caused by S. marcescens. Therefore, in 1998 and 1999, 87 isolates were collected from patients with such infections and examined and another 51 isolates were collected in 2001 and 2002. The patients were mostly elderly or the infections were associated with the use of several invasive devices. S. marcescens was usually the only pathogen found in urine cultures in our study. Neither prior infections nor disseminated infections with the organism were observed in these patients. Resistance to most antibiotics except imipenem was noted. Two genotyping methods, pulsed-field gel electrophoresis and infrequent-restriction-site PCR, were used to examine the isolates. A total of 12 genotypes were identified, and 2 predominant genotypes were found in 72 (82.8%) of the 87 isolates derived from all over the hospital. However, 63.9% of the isolates of the two genotypes were from neurology wards. A subsequent intervention by infection control personnel reduced the infection rate greatly. The number and proportion of the two predominant genotypes were significantly reduced among the 51 isolates collected in 2001 and 2002. Thus, a chronic and long-lasting epidemic of nosocomial UTIs caused by S. marcescens was identified and a successful intervention was carried out. Both a cautious review of laboratory and infection control data and an efficient genotyping system are necessary to identify such a cryptic epidemic and further contribute to the quality of patient care.

Aeromonas aquariorum is widely distributed in clinical and environmental specimens and can be misidentified as Aeromonas hydrophila

ABSTRACT Genotypic characterization of 215 Aeromonas strains (143 clinical, 52 environmental, and 20 reference strains) showed that Aeromonas aquariorum (60 strains, 30.4%) was the most frequently isolated species in clinical and water samples and could be misidentified as Aeromonas hydrophila by phenotypic methods.