Mark A. Fisher

Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system.

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.

Optimization of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis for Bacterial Identification

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.

A Novel Human-Infection-Derived Bacterium Provides Insights into the Evolutionary Origins of Mutualistic Insect-Bacterial Symbioses

Despite extensive study, little is known about the origins of the mutualistic bacterial endosymbionts that inhabit approximately 10% of the worlds insects. In this study, we characterized a novel opportunistic human pathogen, designated “strain HS,” and found that it is a close relative of the insect endosymbiont Sodalis glossinidius. Our results indicate that ancestral relatives of strain HS have served as progenitors for the independent descent of Sodalis-allied endosymbionts found in several insect hosts. Comparative analyses indicate that the gene inventories of the insect endosymbionts were independently derived from a common ancestral template through a combination of irreversible degenerative changes. Our results provide compelling support for the notion that mutualists evolve from pathogenic progenitors. They also elucidate the role of degenerative evolutionary processes in shaping the gene inventories of symbiotic bacteria at a very early stage in these mutualistic associations.

Novel Approach for Differentiating Shigella Species and Escherichia coli by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Shigella species are so closely related to Escherichia coli that routine matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, “inactive” isolates of E. coli are biochemically very similar to Shigella species and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate between Shigella species and E. coli. Sixty-six Shigella spp. and 72 E. coli isolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% of E. coli and Shigella clinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification of E. coli and Shigella species.

Susceptibility Profiles of Nocardia Isolates Based on Current Taxonomy

ABSTRACT The genus Nocardia has undergone rapid taxonomic expansion in recent years, and an increasing number of species are recognized as human pathogens. Many established species have predictable antimicrobial susceptibility profiles, but sufficient information is often not available for recently described organisms. Additionally, the effectiveness of sulfonamides as first-line drugs for Nocardia has recently been questioned. This led us to review antimicrobial susceptibility patterns for a large number of molecularly identified clinical isolates. Susceptibility results were available for 1,299 isolates representing 39 different species or complexes, including 11 that were newly described, during a 6-year study period. All tested isolates were susceptible to linezolid. Resistance to trimethoprim-sulfamethoxazole (TMP-SMX) was rare (2%) except among Nocardia pseudobrasiliensis (31%) strains and strains of the N. transvalensis complex (19%). Imipenem susceptibility varied for N. cyriacigeorgica and N. farcinica, as did ceftriaxone susceptibility of the N. nova complex. Resistance to more than one of the most commonly used drugs (amikacin, ceftriaxone, TMP-SMX, and imipenem) was highest for N. pseudobrasiliensis (100%), N. transvalensis complex (83%), N. farcinica (68%), N. puris (57%), N. brasiliensis (51%), N. aobensis (50%), and N. amikacinitolerans (43%). Thus, while antimicrobial resistance can often be predicted, susceptibility testing should still be considered when combination therapy is warranted, for less well characterized species or those with variable susceptibility profiles, and for patients with TMP-SMX intolerance.

Identification of HACEK clinical isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.

A Systematic Approach for Discovering Novel, Clinically Relevant Bacteria

We identified 95 isolates from novel taxa that may have clinical relevance.

Leptotrichia Bacteremia in Patients Receiving High-Dose Chemotherapy

ABSTRACT Leptotrichia spp. are anaerobic, pencil-shaped, Gram-negative rods that are part of the normal oral and intestinal human flora. Although not typically considered pathogenic, invasive Leptotrichia infections have been reported in immunosuppressed patients. A perceived rise in the identification of Leptotrichia spp. at our institution prompted a retrospective evaluation of these infections. Laboratory and clinical records were reviewed to identify Leptotrichia culture-positive patients. Over a 5-year period, 68 Leptotrichia-positive specimens were identified. Of these, 21% (14/68) were identified in original samples submitted from 13 different patients at our institution, and the remainder (79% [54/68]) were unknown isolates referred from outside hospitals for molecular identification. All in-house Leptotrichia were identified from blood cultures. Only 64% (9/14) of these grew on solid media, and 5 were a part of polymicrobial bacteremias containing other enteric pathogens. All local patients were receiving chemotherapy and a majority received hematopoietic stem cell transplant (HSCT) (11/13). All had neutropenic fever with symptoms of mucositis and/or enteritis. Most of the HSCT patients (73% [8/11]) were autologous recipients hospitalized after recent high-dose chemotherapy for multiple myeloma. L. hongkongensis, a novel species, was found in the majority of myeloma cases (63% [5/8]). In conclusion, we suggest that Leptotrichia spp. may be an underappreciated cause of bacteremia, particularly in multiple myeloma patients receiving cytotoxic chemotherapy for autologous HSCT. In our cohort, these infections were associated with neutropenic fever from an enteric source, and most isolates remained sensitive to standard antibiotics.

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples

A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7-100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA-DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332(T)  = DSM 25276(T)  = LMG 26725(T)) is proposed.

Performance of the Phoenix bacterial identification system compared with disc diffusion methods for identifying extended-spectrum β-lactamase, AmpC and KPC producers

Phenotypic identification of AmpC, KPC and extended-spectrum beta-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam +/- clavulanic acid (CA) and cefpodoxime +/- CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC(+), but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC(+) isolates, all of which tested as ESBL(+) or ESBL(+) AmpC(+) by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.