Publications by Year: 2017

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae generally cannot be treated with penicillins and cephalosporins. However, some later-generation cephalosporins, including cefepime, are poorly hydrolyzed by specific ESBL enzymes, and certain strains demonstrate in vitro susceptibility to these agents, potentially affording additional treatment opportunities. Moreover, the ability to adjust both the dose and dosing interval of beta-lactam agents allows the treatment of strains with elevated MICs that were formerly classified in the intermediate range. The ability to treat strains with elevated cefepime MICs is codified in new susceptible dose-dependent (SDD) breakpoints promulgated by the Clinical and Laboratory Standards Institute. In the interest of validating and implementing new cefepime SDD criteria, we evaluated the performances of Vitek 2, disk diffusion, and a MicroScan panel compared to that of reference broth microdilution (BMD) during the testing of 64 strains enriched for presumptive ESBL phenotype (based on nonsusceptibility to ceftriaxone). Surprisingly, categorical agreement with BMD was only 47.6%, 57.1%, and 44.6% for the three methods, respectively. Given these findings, we tested the performance of the HP D300 inkjet-assisted broth microdilution digital dispensing method (DDM), which was previously described by our group as an at-will testing alternative. In contrast to commercial methods, DDM results correlated well with the reference method, with 86% categorical agreement, 91.1% evaluable essential agreement, and no major or very major errors. The reproducibility and accuracy of MIC determinations were statistically equivalent to BMD. Our results provide support for the use of the DDM as a BMD equivalent methodology that will enable hospital-based clinical laboratories to support cefepime MIC-based dosing strategies.

Candida guilliermondii was isolated from sterile specimens with increasing frequency over a several-month period despite a paucity of clinical evidence suggesting true Candida infections. However, a health care-associated outbreak was strongly considered due to growth patterns in the microbiology laboratory that were more consistent with true infection than environmental contamination. Therefore, an extensive investigation was performed to identify its cause. With the exception of one case, patient clinical courses were not consistent with true invasive fungal infections. Furthermore, no epidemiologic link between patients was identified. Rather, extensive environmental sampling revealed C. guilliermondii in an anaerobic holding jar in the clinical microbiology laboratory, where anaerobic plates were prereduced and held before inoculating specimens. C. guilliermondii grows poorly under anaerobic conditions. Thus, we postulate that anaerobic plates became intermittently contaminated. Passaging from intermittently contaminated anaerobic plates to primary quadrants of aerobic media during specimen planting yielded a colonial growth pattern typical for true specimen infection, thus obscuring laboratory contamination. A molecular evaluation of the C. guilliermondii isolates confirmed a common source for pseudo-outbreak cases but not for the one true infection. In line with Reason's model of organizational accidents, active and latent errors coincided to contribute to the pseudo-outbreak. These included organism factors (lack of growth in anaerobic conditions obscuring plate contamination), human factors (lack of strict adherence to plating order, leading to only intermittent observation of aerobic plate positivity), and laboratory factors (novel equipment). All of these variables should be considered when evaluating possible laboratory-based pseudo-outbreaks.

We established a new Brucella neotomaein vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus, B. melitensis, and B. suis, B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila, we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitroBrucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection.

Bacteria frequently carry mobile genetic elements capable of being passed to other bacterial cells. An example of this is the transfer of plasmids (small, circular DNA molecules) that often contain antibiotic resistance genes from one bacterium to another. Plasmids have evolved mechanisms to ensure their survival through generations by employing plasmids segregation and replication machinery and plasmid addiction systems. Plasmid addiction systems utilize a post-segregational killing of cells that have not received a plasmid. In this review, the types of plasmid addiction systems are described as well as their prevalence in antibiotic resistant bacteria. Lastly, the possibility of targeting these plasmid addiction systems for the treatment of antibiotic resistant bacterial infections is explored.

The in vitro activity of apramycin was compared to that of amikacin, gentamicin, and tobramycin against multidrug-resistant, extensively drug-resistant, and pandrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. Apramycin demonstrated an MIC50/MIC90 of 8/32μg/ml for A. baumannii and 16/32μg/ml for P. aeruginosa. Only 2% of A. baumannii and P. aeruginosa had an MIC greater than an epidemiological cutoff value of 64μg/ml. In contrast, the MIC50/MIC90 for amikacin, gentamicin, and tobramycin were ≥64/>256μg/ml for A. baumannii with 57%, 95%, and 74% of isolates demonstrating resistance, respectively, and the MIC50/MIC90 were ≥8/256μg/ml for P. aeruginosa with 27%, 50%, and 57% of strains demonstrating resistance, respectively. Apramycin appears to offer promising in vitro activity against highly resistant pathogens. It therefore may warrant further pre-clinical study to assess potential for repurposing as a human therapeutic and relevance as a scaffold for further medicinal chemistry exploration.

Antimicrobial susceptibility testing (AST) is a fundamental mission of the clinical microbiology laboratory. Reference AST methods are based on bacterial growth in antibiotic doubling dilution series, which means that any error in the reference method inherently represents at least a 2-fold difference. We describe the origins of current AST reference methodology, highlight the sources of AST variability, and propose ideas for improving AST predictive power.

BACKGROUND: Synergistic combination antimicrobial therapy may provide new options for treatment of MDR infections. However, comprehensive in vitro synergy data are limited and facile methods to perform synergy testing in a clinically actionable time frame are unavailable.

OBJECTIVES: To systematically investigate a broad range of antibiotic combinations for evidence of synergistic activity against a collection of carbapenem-resistant Enterobacteriaceae (CRE) isolates.

METHODS: We made use of an automated method for chequerboard array synergy testing based on inkjet printer technology in the HP D300 digital dispenser to test 56 pairwise antimicrobial combinations of meropenem, aztreonam, cefepime, colistin, gentamicin, levofloxacin, chloramphenicol, fosfomycin, trimethoprim/sulfamethoxazole, minocycline and rifampicin, as well as the double carbapenem combination of meropenem and ertapenem.

RESULTS: In a screening procedure, we tested these combinations against four CRE strains and identified nine antibiotic combinations that showed potential clinically relevant synergy. In confirmatory testing using 10 CRE strains, six combinations demonstrated clinically relevant synergy with both antimicrobials at the minimum fractional inhibitory concentration (FICI-MIN) in the susceptible or intermediate range in at least one trial. These included two novel combinations: minocycline plus colistin and minocycline plus meropenem. In 80% of strains at least one combination demonstrated clinically relevant synergy, but the combinations that demonstrated synergy varied from strain to strain.

CONCLUSIONS: This work establishes the foundation for future systematic, broad-range investigations into antibiotic synergy for CRE, emphasizes the need for individualized synergy testing and demonstrates the utility of inkjet printer-based technology for the performance of automated antimicrobial synergy assays.