A hybridization target enrichment approach for pathogen genomics.

IMPORTANCE: Emerging infectious diseases require continuous pathogen monitoring. Rapid clinical diagnosis by nucleic acid amplification is limited to a small number of targets and may miss target detection due to new mutations in clinical isolates. Whole-genome sequencing (WGS) identifies genome-wide variations that may be used to determine a pathogen's drug resistance patterns and phylogenetically characterize isolates to track disease origin and transmission. WGS is typically performed using DNA isolated from cultured clinical isolates. Culturing clinical specimens increases turn-around time and may not be possible for fastidious bacteria. To overcome some of these limitations, direct sequencing of clinical specimens has been attempted using expensive capture probes to enrich the entire genomes of target pathogens. We present a method to produce a cost-effective, time-efficient, and large-scale synthesis of probes for whole-genome enrichment. We envision that our method can be used for direct clinical sequencing of a wide range of microbial pathogens for genomic epidemiology.

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory.

Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior.

Commonly upregulated in human cancers, the scaffolding protein NEDD9/HEF1 is a known regulator of mesenchymal migration and cancer cell plasticity. However, the functional role of NEDD9 as a regulator of different migration/invasion modes in the context of breast cancer metastasis is currently unknown. Here, it is reported that NEDD9 is necessary for both mesenchymal and amoeboid individual cell migration/invasion in triple-negative breast cancer (TNBC). NEDD9 deficiency results in acquisition of the amoeboid morphology, but severely limits all types of cell motility. Mechanistically, NEDD9 promotes mesenchymal migration via VAV2-dependent Rac1 activation, and depletion of VAV2 impairs the ability of NEDD9 to activate Rac1. In addition, NEDD9 supports a mesenchymal phenotype through stimulating polymerization of actin via promoting CTTN phosphorylation in an AURKA-dependent manner. Interestingly, an increase in RhoA activity in NEDD9-depleted cells does not facilitate a switch to functional amoeboid motility, indicating a role of NEDD9 in the regulation of downstream RhoA signaling effectors. Simultaneous depletion of NEDD9 or inhibition of AURKA in combination with inhibition of the amoeboid driver ROCK results in an additional decrease in cancer cell migration/invasion. Finally, we confirmed that a dual targeting strategy is a viable and efficient therapeutic approach to hinder the metastasis of breast cancer in xenograft models, showcasing the important need for further clinical evaluation of this regimen to impede the spread of disease and improve patient survival.Implications: This study provides new insight into the therapeutic benefit of combining NEDD9 depletion with ROCK inhibition to reduce tumor cell dissemination and discovers a new regulatory role of NEDD9 in the modulation of VAV2-dependent activation of Rac1 and actin polymerization. Mol Cancer Res; 15(6); 670-82. ©2017 AACR.

Recent Outbreaks of Shigellosis in California Caused by Two Distinct Populations of Shigella sonnei with either Increased Virulence or Fluoroquinolone Resistance.

Shigella sonnei has caused unusually large outbreaks of shigellosis in California in 2014 and 2015. Preliminary data indicated the involvement of two distinct bacterial populations, one from San Diego and San Joaquin (SDi/SJo) and one from the San Francisco (SFr) Bay area. Whole-genome analysis and antibiotic susceptibility testing of 68 outbreak and archival isolates of S. sonnei were performed to investigate the microbiological factors related to these outbreaks. Both SDi/SJo and SFr populations, as well as almost all of the archival S. sonnei isolates belonged to sequence type 152 (ST152). Genome-wide single nucleotide polymorphism (SNP) analysis clustered the majority of California (CA) isolates to an earlier described lineage III. Isolates in the SDi/SJo population had a novel lambdoid bacteriophage carrying genes encoding Shiga toxin (STX) that were most closely related to that found in Escherichia coli O104:H4. However, the STX genes (stx1A and stx1B) from this novel phage had sequences most similar to the phages from Shigella flexneri and S. dysenteriae. The isolates in the SFr population were resistant to ciprofloxacin due to point mutations in gyrA and parC genes and were related to the fluoroquinolone-resistant S. sonnei clade within lineage III that originated in South Asia. The emergence of a highly virulent S. sonnei strain and introduction of a fluoroquinolone-resistant strain reflect the changing traits of this pathogen in California. An enhanced monitoring is advocated for early detection of future outbreaks caused by such strains. IMPORTANCE Shigellosis is an acute diarrheal disease causing nearly half a million infections, 6,000 hospitalizations, and 70 deaths annually in the United States. S. sonnei caused two unusually large outbreaks in 2014 and 2015 in California. We used whole-genome sequencing to understand the pathogenic potential of bacteria involved in these outbreaks. Our results suggest the persistence of a local S. sonnei SDi/SJo clone in California since at least 2008. Recently, a derivative of the original clone acquired the ability to produce Shiga toxin (STX) via exchanges of bacteriophages with other bacteria. STX production is connected with more severe disease, including bloody diarrhea. A second population of S. sonnei that caused an outbreak in the San Francisco area was resistant to fluoroquinolones and showed evidence of connection to a fluoroquinolone-resistant lineage from South Asia. These emerging trends in S. sonnei populations in California must be monitored for future risks of the spread of increasingly virulent and resistant clones.

Laboratory Investigation of Salmonella enterica serovar Poona Outbreak in California: Comparison of Pulsed-Field Gel Electrophoresis (PFGE) and Whole Genome Sequencing (WGS) Results.

INTRODUCTION: Recently, Salmonella enterica serovar Poona caused a multistate outbreak, with 245 out of 907 cases occurring in California. We report a comparison of pulsed-field gel electrophoresis (PFGE) results with whole genome sequencing (WGS) for genotyping of Salmonella Poona isolates. METHODS: CA Salmonella Poona isolates, collected from July to August 2015, were genotyped by PFGE using XbaI restriction enzyme. WGS was done using Nextera XT library kit with 2x300 bp or 2x250 bp sequencing chemistry on the Illumina MiSeq Sequencer.  Reads were mapped to the de novo assembled serovar Poona draft genome (48 contigs, N50= 223,917) from the outbreak using CLCbio GW 8.0.2. The phylogenetic tree was generated based on hqSNPs calling. Genomes were annotated with CGE and PHAST online tools. In silico MLST was performed using the CGE online tool. RESULTS: Human (14) and cucumber (2) Salmonella Poona isolates exhibited 3 possibly related PFGE patterns (JL6X01.0018 [predominant], JL6X01.0375, JL6X01.0778).  All isolates that were related by PFGE also clustered together according to the WGS. One isolate with a divergent PFGE pattern (JL6X01.0776) served as an outlier in the phylogenetic analysis and substantially differed from the outbreak clade by WGS. All outbreak isolates were assigned to MLST sequence type 447. The majority of the outbreak-related isolates possessed the same set of Salmonella Pathogenicity Islands with few variations. One outbreak isolate was sequenced and analyzed independently by CDC and CDPH laboratories; there was 0 SNP difference in results. Additional two isolates were sequenced by CDC and the raw data was processed through CDPH and CDC analysis pipelines. Both data analysis pipelines also generated concordant results.  Discussion: PFGE and WGS results for the recent CA Salmonella enterica serovar Poona outbreak provided concordant assignment of the isolates to the outbreak cluster. WGS allowed more robust determination of genetic relatedness, provided information regarding MLST-type, pathogenicity genes, and bacteriophage content. WGS data obtained independently at two laboratories showed complete agreement.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809-22. ©2016 AACR.

Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1-Dependent Trafficking of Integrins.

UNLABELLED: The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand-integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity. IMPLICATIONS: This study provides valuable new insight into the potential therapeutic benefit of NEDD9 depletion to reduce dissemination of tumor cells and discovers a new regulatory role of NEDD9 in promoting migration through modulation of CAV1-dependent trafficking of integrins.

Long-term dissemination of CTX-M-5-producing hypermutable Salmonella enterica serovar typhimurium sequence type 328 strains in Russia, Belarus, and Kazakhstan.

In this paper, we present evidence of long-term circulation of cefotaxime-resistant clonally related Salmonella enterica serovar Typhimurium strains over a broad geographic area. The genetic relatedness of 88 isolates collected from multiple outbreaks and sporadic cases of nosocomial salmonellosis in various parts of Russia, Belarus, and Kazakhstan from 1996 to 2009 was established by multilocus tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). The isolates belong to sequence type 328 (ST328) and produce CTX-M-5 ß-lactamase, whose gene is carried by highly related non-self-conjugative but mobilizable plasmids. Resistance to nalidixic acid and low-level resistance to ciprofloxacin is present in 37 (42%) of the isolates and in all cases is determined by various single point mutations in the gyrA gene quinolone resistance-determining region (QRDR). Isolates of the described clonal group exhibit a hypermutable phenotype that probably facilitates independent acquisition of quinolone resistance mutations.

NEDD9 regulates actin dynamics through cortactin deacetylation in an AURKA/HDAC6-dependent manner.

UNLABELLED: The prometastatic protein NEDD9 (neural precursor cell expressed, developmentally downregulated 9) is highly expressed in many cancers and is required for mesenchymal individual cell migration and progression to the invasive stage. Nevertheless, the molecular mechanisms of NEDD9-driven migration and the downstream targets effecting metastasis are not well defined. In the current study, knockdown of NEDD9 in highly metastatic tumor cells drastically reduces their migratory capacity due to disruption of actin dynamics at the leading edge. Specifically, NEDD9 deficiency leads to a decrease in the persistence and stability of lamellipodial protrusions similar to knockdown of cortactin (CTTN). Mechanistically, it was shown that NEDD9 binds to and regulates acetylation of CTTN in an Aurora A kinase (AURKA)/HDAC6-dependent manner. The knockdown of NEDD9 or AURKA results in an increase in the amount of acetylated CTTN and a decrease in the binding of CTTN to F-actin. Overexpression of the deacetylation mimicking (9KR) mutant of CTTN is sufficient to restore actin dynamics at the leading edge and migration proficiency of the tumor cells. Inhibition of AURKA and HDAC6 activity by alisertib and Tubastatin A in xenograft models of breast cancer leads to a decrease in the number of pulmonary metastases. Collectively, these findings identify CTTN as the key downstream component of NEDD9-driven migration and metastatic phenotypes. IMPLICATIONS: This study provides a mechanistic platform for therapeutic interventions based on AURKA and HDAC6 inhibition for patients with metastatic breast cancer to prevent and/or eradicate metastases.

NEDD9 depletion leads to MMP14 inactivation by TIMP2 and prevents invasion and metastasis.

UNLABELLED: The scaffolding protein NEDD9 is an established prometastatic marker in several cancers. Nevertheless, the molecular mechanisms of NEDD9-driven metastasis in cancers remain ill-defined. Here, using a comprehensive breast cancer tissue microarray, it was shown that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly, it was shown that NEDD9 overexpression is a hallmark of highly invasive breast cancer cells. Moreover, NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo, leading to decreased circulating tumor cells and lung metastases in xenograft models. Mechanistically, NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Reexpression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of breast cancer cells in vitro and in vivo. Collectively, these findings uncover critical steps in NEDD9-dependent invasion of breast cancer cells. IMPLICATIONS: This study provides a mechanistic basis for potential therapeutic interventions to prevent metastasis.

Bacterial resistance studies using in vitro dynamic models: the predictive power of the mutant prevention and minimum inhibitory antibiotic concentrations.

In light of the concept of the mutant selection window, i.e., the range between the MIC and the mutant prevention concentration (MPC), MPC-related pharmacokinetic indices should be more predictive of bacterial resistance than the respective MIC-related indices. However, experimental evidence of this hypothesis remains limited and contradictory. To examine the predictive power of the ratios of the area under the curve (AUC24) to the MPC and the MIC, the selection of ciprofloxacin-resistant mutants of four Escherichia coli strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant E. coli was intensively enriched at AUC24/MPCs from 1 to 10 h (AUC24/MIC from 60 to 360 h) but not at the lower or higher AUC24/MPC and AUC24/MIC ratios. AUC24/MPC and AUC24/MIC relationships of the areas under the time courses of ciprofloxacin-resistant E. coli (AUBCM) were bell-shaped. A Gaussian-like function fits the AUBCM-AUC24/MPC and AUBCM-AUC24/MIC data combined for all organisms (r(2) = 0.69 and 0.86, respectively). The predicted anti-mutant AUC24/MPC ratio was 58 ± 35 h, and the respective AUC24/MIC ratio was 1,080 ± 416 h. Although AUC24/MPC was less predictive of strain-independent E. coli resistance than AUC24/MIC, the established anti-mutant AUC24/MPC ratio was closer to values reported for Staphylococcus aureus (60 to 69 h) than the respective AUC24/MIC ratio (1,080 versus 200 to 240 h). This implies that AUC24/MPC might be a better interspecies predictor of bacterial resistance than AUC24/MIC.

Convergent in vivo and in vitro selection of ceftazidime resistance mutations at position 167 of CTX-M-3 beta-lactamase in hypermutable Escherichia coli strains.

We report on a novel CTX-M extended-spectrum beta-lactamase (ESBL), designated CTX-M-42, with enhanced activity toward ceftazidime. CTX-M-42 was identified in a hypermutable Escherichia coli nosocomial isolate (isolate Irk2320) and is a Pro167Thr amino acid substitution variant of CTX-M-3. By molecular typing of ESBL-producing E. coli strains previously isolated in the same hospital ward, we were able to identify a putative progenitor (strain Irk1224) of Irk2320, which had a mutator phenotype and harbored the CTX-M-3 beta-lactamase. To reproduce the natural evolution of CTX-M-3, we selected for ceftazidime resistance mutations in bla CTX-M-3 gene in vitro both in clinical isolate Irk1224 and in laboratory-derived hypermutable (mutD5) strain GM2995. These experiments yielded CTX-M-3 Pro167Ser and CTX-M-3 Asn136Lys mutants which conferred higher levels of resistance to ceftazidime than to cefotaxime. CTX-M-3 Asn136Lys had a level of low activity toward ampicillin, which may explain its absence from clinical isolates. We conclude that the selection of CTX-M-42 could have occurred in vivo following treatment with ceftazidime and was likely facilitated by the hypermutable background.