Distinct Origins and Transmission Pathways of blaKPC Enterobacterales across Three U.S. States.

Carbapenem-resistant Enterobacterales (CRE) are among the most concerning antibiotic resistance threats due to high rates of multidrug resistance, transmissibility in health care settings, and high mortality rates. We evaluated the potential for regional genomic surveillance to track the spread of blaKPC-carrying CRE (KPC-CRE) by using isolate collections from health care facilities in three U.S. states. Clinical isolates were collected from Connecticut (2017 to 2018), Minnesota (2012 to 2018), and Tennessee (2016 to 2017) through the U.S. Centers for Disease Control and Prevention's Multi-site Gram-negative Surveillance Initiative (MuGSI) and additional surveillance. KPC-CRE isolates were whole-genome sequenced, yielding 255 isolates from 214 patients across 96 facilities. Case report data on patient comorbidities, facility exposures, and interfacility patient transfer were extracted. We observed that in Connecticut, most KPC-CRE isolates showed evidence of importation from outside the state, with limited local transmission. In Minnesota, cases were mainly from sporadic importation and transmission of blaKPC-carrying Klebsiella pneumoniae ST258, and clonal expansion of blaKPC-carrying Enterobacter hormaechei ST171, primarily at a single focal facility and its satellite facilities. In Tennessee, we observed transmission of diverse strains of blaKPC-carrying Enterobacter and Klesbiella, with evidence that most derived from the local acquisition of blaKPC plasmids circulating in an interconnected regional health care network. Thus, the underlying processes driving KPC-CRE burden can differ substantially across regions and can be discerned through regional genomic surveillance. This study provides proof of concept that integrating genomic data with information on interfacility patient transfers can provide insights into locations and drivers of regional KPC-CRE burden that can enable targeted interventions.

Comparing shear wave elastography and fine needle aspiration in the diagnosis of solid thyroid nodules.

This descriptive study was performed on individuals who were referred to Imam Reza Hospital for fine needle aspiration biopsy (FNAB) based on the results of gray scale ultrasound and the decision of the referring physician. In addition to determining the gray scale characteristics of the nodules, shear wave elastography (SWE) was also performed and the results were recorded. These were also taken from the patients FNAB results. Finally, the findings of SWE and FNAB methods were compared and analyzed using SPSS software version 16. Based on the results presented herein, a significant relationship was observed between the results of SWE and FNA in the diagnosis of malignancy in solid thyroid nodules. This agreement was found to be higher in men (K = 0.866) than women (K = 0.849). Taken together, our data suggest that shear wave elastography can replace FNA in the diagnosis of malignancy in solid thyroid nodules.

Threshold-free genomic cluster detection to track transmission pathways in health-care settings: a genomic epidemiology analysis.

BACKGROUND: A crucial barrier to the routine application of whole-genome sequencing (WGS) for infection prevention is the insufficient criteria for determining whether a genomic linkage is consistent with transmission within the facility. We evaluated the use of single-nucleotide variant (SNV) thresholds, as well as a novel threshold-free approach, for inferring transmission linkages in a high-transmission setting. METHODS: We did a retrospective genomic epidemiology analysis of samples previously collected in the context of an intervention study at a long-term acute care hospital in the USA. We performed WGS on 435 isolates of Klebsiella pneumoniae harbouring the blaKPC carbapenemase (KPC-Kp) collected from 256 patients through admission and surveillance culturing (once every 2 weeks) of almost every patient who was admitted to hospital over a 1-year period. FINDINGS: Our analysis showed that the standard approach of using an SNV threshold to define transmission would lead to false-positive and false-negative inferences. False-positive inferences were driven by the frequent importation of closely related strains, which were presumably linked via transmission at connected health-care facilities. False-negative inferences stemmed from the diversity of colonising populations that were spread among patients, with multiple examples of hypermutator strain emergence within patients and, as a result, putative transmission links separated by large genetic distances. Motivated by limitations of an SNV threshold, we implemented a novel threshold-free transmission cluster inference approach, in which each of the acquired KPC-Kp isolates were linked back to the imported KPC-Kp isolate with which it shared the most variants. This approach yielded clusters that varied in levels of genetic diversity but where 105 (81%) of 129 unique strain acquisition events were associated with epidemiological links in the hospital. Of 100 patients who acquired KPC-Kp isolates that were included in a cluster, 47 could be linked to a single patient who was positive for KPC-Kp at admission, compared with 31 and 25 using 10 SNV and 20 SNV thresholds, respectively. Holistic examination of clusters highlighted extensive variation in the magnitude of onward transmission stemming from more than 100 importation events and revealed patterns in cluster propagation that could inform improvements to infection prevention strategies. INTERPRETATION: Our results show how the integration of culture surveillance data into genomic analyses can overcome limitations of cluster detection based on SNV-thresholds and improve the ability to track pathways of pathogen transmission in health-care settings. FUNDING: US Center for Disease Control and Prevention and University of Michigan.

Strain Variation in Clostridioides difficile Cytotoxicity Associated with Genomic Variation at Both Pathogenic and Nonpathogenic Loci.

Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes located within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the cytotoxicity of C. difficile may be modulated by genomic variants outside the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between cytotoxicity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model cytotoxicity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci and changes in cytotoxicity. Combined, these data support a model of C. difficile disease wherein cytotoxicity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections, including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Finally, we highlight the intersection of loci identified by the GWAS for different phenotypes and clinical severity. This strategy to identify overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes. IMPORTANCE Clostridioides difficile has two major disease-mediating toxins, A and B, encoded within the pathogenicity locus (PaLoc). In this study, we demonstrate via multiple approaches that genomic variants outside the PaLoc are associated with changes in cytotoxicity. These genomic variants may provide new avenues of exploration in the hunt for novel disease-modifying interventions. Additionally, we provide insight into the evolution of several additional phenotypes also critical for clinical infection, such as sporulation, germination, and growth rate. These in vitro phenotypes display a range of responses to evolutionary pressures and, as such, vary in their appropriateness for certain bacterial genome-wide association study approaches. We used a convergence-based association method to identify the genomic variants most correlated with both changes in these phenotypes and disease severity. These overlapping loci may be important for both bacterial function and human clinical disease.

Ferric Citrate Uptake Is a Virulence Factor in Uropathogenic Escherichia coli.

More than half of women will experience a urinary tract infection (UTI), with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if HM7 possesses unidentified iron acquisition systems, we performed RNA sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is enriched in UPEC isolates compared to fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔfecA/ΔentB) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

Phenotypic and Genomic Diversification in Complex Carbohydrate-Degrading Human Gut Bacteria.

Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multilocus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors. IMPORTANCE Nonharmful bacteria are the primary microbial symbionts that inhabit the human gastrointestinal tract. These bacteria play many beneficial roles and in some cases can modify disease states, making it important to understand which nutrients sustain specific lineages. This knowledge will in turn lead to strategies to intentionally manipulate the gut microbial ecosystem. We designed a scalable, high-throughput platform for measuring the ability of gut bacteria to utilize polysaccharides, of which many are derived from dietary fiber sources that can be manipulated easily. Our results provide paths to expand phenotypic surveys of more diverse gut bacteria to understand their functions and also to leverage dietary fibers to alter the physiology of the gut microbial community.

Replication Dynamics for Six Gram-Negative Bacterial Species during Bloodstream Infection.

Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated <60-min generation times. The relatively short generation times observed in the spleen were in contrast to an overall decrease in bacterial burden for some species, suggesting that the rate of immune-mediated clearance exceeded replication. Furthermore, bacterial generation times measured in the murine spleen approximated those measured during growth in human serum cultures. Together, these findings provide insight into the infection kinetics of six medically important species during bacteremia. IMPORTANCE Bloodstream infections are a global public health problem. The goal of this work was to determine the replication characteristics of Gram-negative bacterial species in the host following bloodstream infection. The number of bacteria in major organs is likely determined by a balance between replication rates and the ability of the host to clear bacteria. We selected a cohort of six species from three families that represent common causative agents of bloodstream infections in humans and determined their replication rates in a murine bacteremia model. We found that the bacteria grow rapidly in the spleen, demonstrating that they can obtain the necessary nutrients for growth in this environment. However, the overall number of bacteria decreased in most cases, suggesting that killing of bacteria outpaces their growth. Through a better understanding of how bacteria replicate during bloodstream infections, we aim to gain insight into future means of combating these infections.

Regional Spread of blaNDM-1-Containing Klebsiella pneumoniae ST147 in Post-Acute Care Facilities.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) harboring blaKPC have been endemic in Chicago-area healthcare networks for more than a decade. During 2016-2019, a series of regional point-prevalence surveys identified increasing prevalence of blaNDM-containing CRE in multiple long-term acute care hospitals (LTACHs) and ventilator-capable skilled nursing facilities (vSNFs). We performed a genomic epidemiology investigation of blaNDM-producing CRE to understand their regional emergence and spread. METHODS: We performed whole-genome sequencing on New Delhi metallo-beta-lactamase (NDM)+ CRE isolates from 4 point-prevalence surveys across 35 facilities (LTACHs, vSNFs, and acute care hospital medical intensive care units) in the Chicago area and investigated the genomic relatedness and transmission dynamics of these isolates over time. RESULTS: Genomic analyses revealed that the rise of NDM+ CRE was due to the clonal dissemination of an sequence type (ST) 147 Klebsiella pneumoniae strain harboring blaNDM-1 on an IncF plasmid. Dated phylogenetic reconstructions indicated that ST147 was introduced into the region around 2013 and likely acquired NDM around 2015. Analyzing the relatedness of strains within and between facilities supported initial increases in prevalence due to intrafacility transmission in certain vSNFs, with evidence of subsequent interfacility spread among LTACHs and vSNFs connected by patient transfer. CONCLUSIONS: We identified a regional outbreak of blaNDM-1 ST147 that began in and disseminated across Chicago area post-acute care facilities. Our findings highlight the importance of performing genomic surveillance at post-acute care facilities to identify emerging threats.

A Predictive Model for Respiratory Failure and Determining the Risk Factors of Prolonged Mechanical Ventilation in Children with Guillain-Barre Syndrome.

OBJECTIVE: Determining the predictors of respiratory failure and duration of intubation in children with Guillain-Barre syndrome (GBS). MATERIALS & METHODS: Children diagnosed with GBS at Tabriz Children's Hospital were studied. Factors associated with and influencing respiratory failure as well as the duration of intubation were determined using both univariate and multiple analyses. RESULTS: Overall, 324 children were enrolled in the study, 54.0% of whom were males. Thirty-one (9.6%) patients underwent mechanical ventilation, the patients under 5 years old were more prone to the requirement of mechanical ventilation (11.3% vs. 6.9%). Cases hospitalized in winter were more likely to need ventilation compared to those hospitalized in spring (OR =7.00; 95% CI:1.51-32.53). Also, autonomic involvement (OR=8.88, 95% CI:4.03-19.58; p<0.001) and cranial nerves involvement (OR=9.88, 95% CI:3.68 - 26.52; p<0.001) emerged as risk factors for mechanical ventilation requirement. Overall, 16.1% of patients with axonal electrophysiologic pattern required mechanical ventilation compared to 7.4% of those with demyelinating type (OR:2.15, 95% CI: 1.01-4.69). In univariate analysis, the only variable that showed a correlation with the duration of intubation was axonal electrophysiologic pattern (p= 0.028). CONCLUSION: Approximately, 10% of the patients required mechanical ventilation. Season, cranial nerve involvement, autonomic dysfunction and electrophysiologic pattern were the most important variables in predicting respiratory failure and duration of mechanical ventilation.

prewas: data pre-processing for more informative bacterial GWAS.

While variant identification pipelines are becoming increasingly standardized, less attention has been paid to the pre-processing of variants prior to their use in bacterial genome-wide association studies (bGWAS). Three nuances of variant pre-processing that impact downstream identification of genetic associations include the separation of variants at multiallelic sites, separation of variants in overlapping genes, and referencing of variants relative to ancestral alleles. Here we demonstrate the importance of these variant pre-processing steps on diverse bacterial genomic datasets and present prewas, an R package, that standardizes the pre-processing of multiallelic sites, overlapping genes, and reference alleles before bGWAS. This package facilitates improved reproducibility and interpretability of bGWAS results. prewas enables users to extract maximal information from bGWAS by implementing multi-line representation for multiallelic sites and variants in overlapping genes. prewas outputs a binary SNP matrix that can be used for SNP-based bGWAS and will prevent the masking of minor alleles during bGWAS analysis. The optional binary gene matrix output can be used for gene-based bGWAS, which will enable users to maximize the power and evolutionary interpretability of their bGWAS studies. prewas is available for download from GitHub.

Application of Combined Genomic and Transfer Analyses to Identify Factors Mediating Regional Spread of Antibiotic-resistant Bacterial Lineages.

BACKGROUND: Patients entering nursing facilities (NFs) are frequently colonized with antibiotic-resistant organisms (AROs). To understand the determinants of ARO colonization on NF admission, we applied whole-genome sequencing to track the spread of 4 ARO species across regional NFs and evaluated patient-level characteristics and transfer acute care hospitals (ACHs) as risk factors for colonization. METHODS: Patients from 6 NFs (n = 584) were surveyed for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis/faecium (VREfc/VREfm), and ciprofloxacin-resistant Escherichia coli (CipREc) colonization. Genomic analysis was performed to quantify ARO spread between NFs and compared to patient-transfer networks. The association between admission colonization and patient-level variables and recent ACH exposures was examined. RESULTS: The majority of ARO isolates belonged to major healthcare-associated lineages: MRSA (sequence type [ST] 5); VREfc (ST6); CipREc (ST131), and VREfm (clade A). While the genomic similarity of strains between NF pairs was positively associated with overlap in their feeder ACHs (P < .05 for MRSA, VREfc, and CipREc), limited phylogenetic clustering by either ACH or NF supported regional endemicity. Significant predictors for ARO colonization on NF admission included lower functional status and recent exposure to glycopeptides (adjusted odds ratio [aOR], > 2 for MRSA and VREfc/VREfm) or third-/fourth-generation cephalosporins (aOR, > 2 for MRSA and VREfm). Transfer from specific ACHs was an independent risk factor for only 1 ARO/ACH pair (VREfm/ACH19: aOR, 2.48). CONCLUSIONS: In this region, healthcare-associated ARO lineages are endemic among connected NFs and ACHs, making patient characteristics more informative of NF admission colonization risk than exposure to specific ACHs.

Relative Effectiveness of Electroacupuncture and Biofeedback in the Treatment of Neck and Upper Back Myofascial Pain: A Randomized Clinical Trial.

OBJECTIVE: To determine the differences between clinical effects of electroacupuncture and biofeedback therapy in addition to conventional treatment in patients with cervical myofascial pain syndrome (MPS). DESIGN: Randomized clinical trial. SETTING: Physical medicine and rehabilitation clinic of a university hospital. PARTICIPANTS: Fifty patients (N=50) aged 25-55 years of both sexes with chronic neck pain diagnosed with MPS (characterized by trigger points within taut bands) were randomly assigned to 2 equal groups of 25 individuals. INTERVENTIONS: The patients in electroacupuncture group were treated with standard acupuncture and concomitant electrical stimulation; those in biofeedback group received visual electromyography biofeedback therapy for muscle activity and relaxation. Both groups received the intervention 2 times a week for a total of 6 sessions. Basic exercise training and medicines were administered for all the patients. MAIN OUTCOME MEASURES: Pain severity based on the visual analog scale (VAS), functional status using Neck Disability Index (NDI), cervical range of motion (ROM) using and inclinometer, and pressure pain threshold (PPT) using an algometer were evaluated before and at 3 and 12 weeks after the treatment. Primary outcome was defined as 20% reduction in the 3-month neck pain and dysfunction compared to baseline, assessed through the NDI. RESULTS: Fifty patients (39 women, 11 men) with a mean age (years) ± SD of 39.0±5.5 and neck pain duration (weeks) of 6.0±2.2 were analyzed. All parameters, except for PPT of the lower trapezius and paravertebral muscles were improved significantly in both groups, while baseline values were controlled. The primary outcome was achieved more significantly in the acupuncture group than in the biofeedback group: 20 (80.0%) vs 10 (40.0%); rate ratio=2 with 95% confidence interval (CI), 1.19-3.36; number needed to treat (NNT)=2.5 with 95% CI, 1.54-6.58. Advantages of acupuncture over biofeedback were observed according to values obtained from the NDI, VAS, extension and left lateral-bending ROM, and PPT on the left upper trapezius after the last session of intervention until 3 months (P<.05). CONCLUSIONS: Both electroacupuncture and biofeedback therapies were found to be effective in management of MPS when integrated with conventional treatment. However, intergroup differences showed priority of acupuncture in some parameters vs biofeedback. Thus, electroacupuncture seems to be a better complementary modality for treatment of MPS in the neck and upper back area.

High throughput genotyping of structural variations in a complex plant genome using an original Affymetrix® axiom® array.

BACKGROUND: Insertions/deletions (InDels) and more specifically presence/absence variations (PAVs) are pervasive in several species and have strong functional and phenotypic effect by removing or drastically modifying genes. Genotyping of such variants on large panels remains poorly addressed, while necessary for approaches such as association mapping or genomic selection. RESULTS: We have developed, as a proof of concept, a new high-throughput and affordable approach to genotype InDels. We first identified 141,000 InDels by aligning reads from the B73 line against the genome of three temperate maize inbred lines (F2, PH207, and C103) and reciprocally. Next, we designed an Affymetrix® Axiom® array to target these InDels, with a combination of probes selected at breakpoint sites (13%) or within the InDel sequence, either at polymorphic (25%) or non-polymorphic sites (63%) sites. The final array design is composed of 662,772 probes and targets 105,927 InDels, including PAVs ranging from 35 bp to 129kbp. After Affymetrix® quality control, we successfully genotyped 86,648 polymorphic InDels (82% of all InDels interrogated by the array) on 445 maize DNA samples with 422,369 probes. Genotyping InDels using this approach produced a highly reliable dataset, with low genotyping error (~ 3%), high call rate (~ 98%), and high reproducibility (> 95%). This reliability can be further increased by combining genotyping of several probes calling the same InDels (< 0.1% error rate and > 99.9% of call rate for 5 probes). This "proof of concept" tool was used to estimate the kinship matrix between 362 maize lines with 57,824 polymorphic InDels. This InDels kinship matrix was highly correlated with kinship estimated using SNPs from Illumina 50 K SNP arrays. CONCLUSIONS: We efficiently genotyped thousands of small to large InDels on a sizeable number of individuals using a new Affymetrix® Axiom® array. This powerful approach opens the way to studying the contribution of InDels to trait variation and heterosis in maize. The approach is easily extendable to other species and should contribute to decipher the biological impact of InDels at a larger scale.

Genetically diverse uropathogenic Escherichia coli adopt a common transcriptional program in patients with UTIs.

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTIs). A common virulence genotype of UPEC strains responsible for UTIs is yet to be defined, due to the large variation of virulence factors observed in UPEC strains. We hypothesized that studying UPEC functional responses in patients might reveal universal UPEC features that enable pathogenesis. Here we identify a transcriptional program shared by genetically diverse UPEC strains isolated from 14 patients during uncomplicated UTIs. Strikingly, this in vivo gene expression program is marked by upregulation of translational machinery, providing a mechanism for the rapid growth within the host. Our analysis indicates that switching to a more specialized catabolism and scavenging lifestyle in the host allows for the increased translational output. Our study identifies a common transcriptional program underlying UTIs and illuminates the molecular underpinnings that likely facilitate the fast growth rate of UPEC in infected patients.

Replication Study: Intestinal inflammation targets cancer-inducing activity of the microbiota.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Eaton et al., 2015) that described how we intended to replicate selected experiments from the paper "Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota" (Arthur et al., 2012). Here we report the results. We observed no impact on bacterial growth or colonization capacity when the polyketide synthase (pks) genotoxic island was deleted from E. coli NC101, similar to the original study (Supplementary Figure 7; Arthur et al., 2012). However, for the experiment that compared inflammation, invasion, and neoplasia in azoxymethane (AOM)-treated interleukin-10-deficient mice mono-associated with NC101 or NC101[Formula: see text] pks the experimental timing of the replication attempt was longer than that of the original study. This difference was because in the original study the methodology was not clearly stated and likely led to the increased mortality and severity of inflammation observed in this replication attempt. Additionally, early death occurred during AOM treatment with higher mortality observed in NC101[Formula: see text] pks mono-associated mice compared to NC101, which was in the same direction, but more severe than the original study (Suppleme1ntal Figure 10; Arthur et al., 2012). A meta-analysis suggests that mice mono-associated with NC101[Formula: see text] pks have higher mortality compared to NC101. While these data were unable to address whether, under the conditions of the original study, NC101 and NC101[Formula: see text] pks differ in inflammation, invasion, and neoplasia this replication attempt demonstrates that clear description of experimental methods is essential to ensure accurate reproduction of experimental studies.

Identification of Pathogenicity-Associated Loci in Klebsiella pneumoniae from Hospitalized Patients.

Despite insights gained through experimental models, the set of bacterial genes important for human infection is unclear for many of our most threatening pathogens. Klebsiella pneumoniae is a leading cause of health care-associated infections (HAIs) and commonly colonizes hospitalized patients, but the factors that determine whether a particular isolate causes disease or remains a colonizer are poorly understood. To identify bacterial genes associated with K. pneumoniae infection, a case-control study was performed comparing infected and asymptomatic colonized patients. Comparative bacterial genomics was combined with a conditional logit model that identified patient factors differentiating cases from controls. This method identified five gene loci associated with infection after adjustment for patient factors, including a psicose sugar utilization locus that was validated as a fitness factor during mouse lung infection. These results indicate that bacterial genome-wide association studies of patients can identify loci associated with HAIs and important in infection models. IMPORTANCE Klebsiella pneumoniae is a common cause of infections in the health care setting. This work supports a paradigm for K. pneumoniae pathogenesis where the accessory genome, composed of genes present in some but not all isolates, influences whether a strain causes infection or asymptomatic colonization, after accounting for patient-level factors. Identification of patients at high risk of infection could allow interventions to prevent or rapidly treat K. pneumoniae infections.

Rapid Growth of Uropathogenic Escherichia coli during Human Urinary Tract Infection.

Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo, matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization.

Integrated genomic and interfacility patient-transfer data reveal the transmission pathways of multidrug-resistant Klebsiella pneumoniae in a regional outbreak.

Development of effective strategies to limit the proliferation of multidrug-resistant organisms requires a thorough understanding of how such organisms spread among health care facilities. We sought to uncover the chains of transmission underlying a 2008 U.S. regional outbreak of carbapenem-resistant Klebsiella pneumoniae by performing an integrated analysis of genomic and interfacility patient-transfer data. Genomic analysis yielded a high-resolution transmission network that assigned directionality to regional transmission events and discriminated between intra- and interfacility transmission when epidemiologic data were ambiguous or misleading. Examining the genomic transmission network in the context of interfacility patient transfers (patient-sharing networks) supported the role of patient transfers in driving the outbreak, with genomic analysis revealing that a small subset of patient-transfer events was sufficient to explain regional spread. Further integration of the genomic and patient-sharing networks identified one nursing home as an important bridge facility early in the outbreak-a role that was not apparent from analysis of genomic or patient-transfer data alone. Last, we found that when simulating a real-time regional outbreak, our methodology was able to accurately infer the facility at which patients acquired their infections. This approach has the potential to identify facilities with high rates of intra- or interfacility transmission, data that will be useful for triggering targeted interventions to prevent further spread of multidrug-resistant organisms.

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

BACKGROUND: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. METHODS: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. RESULTS: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. CONCLUSION: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.

Genomic and Epidemiological Evidence for Community Origins of Hospital-Onset Methicillin-Resistant Staphylococcus aureus Bloodstream Infections.

Background: We examined whether disparities existed in hospital-onset (HO) Staphylococcus aureus bloodstream infections (BSIs) and used whole-genome sequencing (WGS) to identify factors associated with USA300 transmission networks. Methods: We evaluated HO methicillin-susceptible S. aureus (MSSA) and HO methicillin-resistant S. aureus (MRSA) BSIs for 2009-2013 at 2 hospitals and used an adjusted incidence for modeling. WGS and phylogenetic analyses were performed on a sample of USA300 BSI isolates. Epidemiologic data were analyzed in the context of phylogenetic reconstructions. Results: On multivariate analysis, male sex, African-American race, and non-Hispanic white race/ethnicity were significantly associated with HO-MRSA BSIs whereas Hispanic ethnicity was negatively associated (rate ratio, 0.41; P = .002). Intermixing of community-onset and HO-USA300 strains on the phylogenetic tree indicates that these strains derive from a common pool. African-American race was the only factor associated with genomic clustering of isolates. Conclusions: In a multicenter assessment of HO-S. aureus BSIs, African-American race was significantly associated with HO-MRSA but not MSSA BSIs. There appears to be a nexus of USA300 community and hospital transmission networks, with a community factor being the primary driver. Our data suggest that HO-USA300 BSIs likely are due to colonizing strains acquired in the community before hospitalization. Therefore, prevention efforts may need to extend to the community for maximal benefit.