Pairs Of Isolates Research Articles

Strain tracking with uncertainty quantification.

The ability to detect and quantify microbiota over time has a plethora of clinical, basic science, and public health applications. One of the primary means of tracking microbiota is through sequencing technologies. When the microorganism of interest is well characterized or known a priori , targeted sequencing is often used. In many applications, however, untargeted bulk (shotgun) sequencing is more appropriate; for instance, the tracking of infection transmission events and nucleotide variants across multiple genomic loci, or studying the role of multiple genes in a particular phenotype. Given these applications, and the observation that pathogens (e.g. Clostridioides difficile, Escherichia coli, Salmonella enterica ) and other taxa of interest can reside at low relative abundance in the gastrointestinal tract, there is a critical need for algorithms that accurately track low-abundance taxa with strain level resolution. Here we present a sequence quality- and time-aware model, ChronoStrain , that introduces uncertainty quantification to gauge low-abundance species and significantly outperforms the current state-of-the-art on both real and synthetic data. ChronoStrain leverages sequences' quality scores and the samples' temporal information to produce a probability distribution over abundance trajectories for each strain tracked in the model. We demonstrate Chronostrain's improved performance in capturing post-antibiotic Escherichia coli strain blooms among women with recurrent urinary tract infections (UTIs) from the UTI Microbiome (UMB) Project. Other strain tracking models on the same data either show inconsistent temporal colonization or can only track consistently using very coarse groupings. In contrast, our probabilistic outputs can reveal the relationship between low-confidence strains present in the sample that cannot be reliably assigned a single reference label (either due to poor coverage or novelty) while simultaneously calling high-confidence strains that can be unambiguously assigned a label. We also analyze samples from the Early Life Microbiota Colonisation (ELMC) Study demonstrating the algorithm's ability to correctly identify Enterococcus faecalis strains using paired sample isolates as validation.

CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

Gut microbial genomes with paired isolates from China illustrate probiotic and cardiometabolic effects

The gut microbiome displays genetic differences among populations, and characterization of the genomic landscape of the gut microbiome in China remains limited. Here, we present the Chinese Gut Microbial Reference (CGMR) set, comprising 101,060 high-quality metagenomic assembled genomes (MAGs) of 3,707 nonredundant species from 3,234 fecal samples across primarily rural Chinese locations, 1,376 live isolates mainly from lactic acid bacteria, and 987 novel species relative to worldwide databases. We observed region-specific coexisting MAGs and MAGs with probiotic and cardiometabolic functionalities. Preliminary mouse experiments suggest a probiotic effect of two Faecalibacillus intestinalis isolates in alleviating constipation, cardiometabolic influences of three Bacteroides fragilis_A isolates in obesity, and isolates from the genera Parabacteroides and Lactobacillus in host lipid metabolism. Our study expands the current microbial genomes with paired isolates and demonstrates potential host effects, contributing to the mechanistic understanding of host-microbe interactions.

Microbial co-occurrences on catheters from long-term catheterized patients

Catheter-associated urinary tract infections (CAUTIs), a common cause of healthcare-associated infections, are caused by a diverse array of pathogens that are increasingly becoming antibiotic resistant. We analyze the microbial occurrences in catheter and urine samples from 55 human long-term catheterized patients collected over one year. Although most of these patients were prescribed antibiotics over several collection periods, their catheter samples remain colonized by one or more bacterial species. Examination of a total of 366 catheter and urine samples identify 13 positive and 13 negative genus co-occurrences over 12 collection periods, representing associations that occur more or less frequently than expected by chance. We find that for many patients, the microbial species composition between collection periods is similar. In a subset of patients, we find that the most frequently sampled bacteria, Escherichia coli and Enterococcus faecalis, co-localize on catheter samples. Further, co-culture of paired isolates recovered from the same patients reveals that E. coli significantly augments E. faecalis growth in an artificial urine medium, where E. faecalis monoculture grows poorly. These findings suggest novel strategies to collapse polymicrobial CAUTI in long-term catheterized patients by targeting mechanisms that promote positive co-associations.

Discordance Between Phenotypic and WGS-Based Drug Susceptibility Testing Results for Some Anti-Tuberculosis Drugs: A Snapshot Study of Paired Mycobacterium tuberculosis Isolates with Small Genetic Distance.

Current tuberculosis treatment regimens primarily rely on phenotypic drug susceptibility testing and rapid molecular assays. Although whole-genome sequencing (WGS) offers a promising alternative, disagreements between phenotypic and molecular testing methods remain. In this retrospective study, we compared the phenotypic and WGS-predicted drug resistance profiles of paired Mycobacterium tuberculosis isolates with small genetic distances (≤10 single nucleotide variants) obtained from patients with longitudinal single-episode or recurrent tuberculosis. Additionally, we investigated the distribution of drug-resistance-conferring variants among the identified M. tuberculosis genotypes. Paired M. tuberculosis isolates from 46 patients with pulmonary tuberculosis (2002-2019) were analyzed. Spoligotyping was performed for all the isolates. WGS data were processed using TB-Profiler software to genotype the strains and detect variants in M. tuberculosis genes associated with drug resistance. The significance of these variants was evaluated using the M. tuberculosis variant catalog developed by the World Health Organization. Phenotypic drug susceptibility test results were obtained from patients' medical records. Among the 46 isolate pairs, 25 (54.3%) harbored drug-resistance-associated variants, with 20 demonstrating identical WGS-predicted drug resistance profiles. Drug-resistant isolate pairs belonged to Lineages 2 and 4, with the most common sub-lineages being 2.2.1 (SIT1 and SIT190 spoligotypes), and 4.3.3 (SIT42). Agreement between phenotypic and WGS-based drug susceptibility testing was highest (>90%) for rifampicin, isoniazid, ethambutol, fluoroquinolones, streptomycin, and amikacin when calculated for M. tuberculosis isolates or isolate pairs. In most discordant cases, isolate pairs harbored variants that could cause low- or moderate-level resistance or were previously associated with variable minimum inhibitory concentrations. Notably, such discrepancies mostly occurred in one isolate from the pair. In addition, differences in resistance-related variant distributions among M. tuberculosis genotypes were observed for most of the analyzed drugs. The simultaneous performance of phenotypic and WGS-based drug susceptibility testing creates the most accurate drug resistance profile for M. tuberculosis isolates and eliminates important limitations of each method.

Vertical mother-to-infant transmission of herpes simplex virus 2 is correlated with tropism due to mutations in viral UL13.

Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission.

Synergistic Effect of Trichoderma reesei and Fusarium solani on growth and yield of Barley plant

Chemical fertilizers are known to improve plant yield but on the cost of environment, soil health as well as human health. Thus, alternatives of chemical fertilizers are the demand of time. Plant growth-promoting fungi (PGPF) are using nowadays as an alternative of chemical fertilizers to improve the growth, yield, and plant nutrient uptake. They are also involved in the improvement of rhizosphere fertility, without compromising environment and human health. A total of seven fungal isolates were obtained from rhizospheric soil of two crop plants viz., turnip and wheat. Fungal strains were examined for their In vitro plant growth promotional traits. A total of two strains found positive for 4 or more plant growth promoting traits. These potential fungal strains were characterized by amplification and sequencing of ITS region and were identified as Trichoderma reesei, and another was identified as Fusarium solani. Our current study is aimed to screen the effect of single inoculation as well as paired fungal inoculations and to provide evidence for the synergistic effects of paired isolates having high impact on growth and development of barley plant.

Sink-traps are a major source for carbapenemase-producing Enterobacteriaceae transmission.

We studied the extent of carbapenemase-producing Enterobacteriaceae (CPE) sink contamination and transmission to patients in a nonoutbreak setting. During 2017-2019, 592 patient-room sinks were sampled in 34 departments. Patient weekly rectal swab CPE surveillance was universally performed. Repeated sink sampling was conducted in 9 departments. Isolates from patients and sinks were characterized using pulsed-field gel electrophoresis (PFGE), and pairs of high resemblance were sequenced by Oxford Nanopore and Illumina. Hybrid assembly was used to fully assemble plasmids, which are shared between paired isolates. In total, 144 (24%) of 592 CPE-contaminated sinks were detected in 25 of 34 departments. Repeated sampling (n = 7,123) revealed that 52%-100% were contaminated at least once during the sampling period. Persistent contamination for >1 year by a dominant strain was common. During the study period, 318 patients acquired CPE. The most common species were Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. In 127 (40%) patients, a contaminated sink was the suspected source of CPE acquisition. For 20 cases with an identical sink-patient strain, temporal relation suggested sink-to-patient transmission. Hybrid assembly of specific sink-patient isolates revealed that shared plasmids were structurally identical, and SNP differences between shared pairs, along with signatures for potential recombination events, suggests recent sharing of the plasmids. CPE-contaminated sinks are an important source of transmission to patients. Although traditionally person-to-person transmission has been considered the main route of CPE transmission, these data suggest a change in paradigm that may influence strategies of preventing CPE dissemination.

The intra-host evolutionary landscape and pathoadaptation of persistent Staphylococcus aureus in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic sinonasal mucosal inflammation associated with Staphylococcus aureus biofilm and relapsing infections. This study aimed to determine rates of S. aureus persistence and pathoadaptation in CRS patients by investigating the genomic relatedness and antibiotic resistance/tolerance in longitudinally collected S. aureus clinical isolates. A total of 68 S. aureus paired isolates (34 pairs) were sourced from 34 CRS patients at least 6 months apart. Isolates were grown into 48 h biofilms and tested for tolerance to antibiotics. A hybrid sequencing strategy was used to obtain high-quality reference-grade assemblies of all isolates. Single nucleotide variants (SNV) divergence in the core genome and sequence type clustering were used to analyse the relatedness of the isolate pairs. Single nucleotide and structural genome variations, plasmid similarity, and plasmid copy numbers between pairs were examined. Our analysis revealed that 41 % (14/34 pairs) of S. aureus isolates were persistent, while 59 % (20/34 pairs) were non-persistent. Persistent isolates showed episode-specific mutational changes over time with a bias towards events in genes involved in adhesion to the host and mobile genetic elements such as plasmids, prophages, and insertion sequences. Furthermore, a significant increase in the copy number of conserved plasmids of persistent strains was observed. This was accompanied by a significant increase in biofilm tolerance against all tested antibiotics, which was linked to a significant increase in biofilm biomass over time, indicating a potential biofilm pathoadaptive process in persistent isolates. In conclusion, our study provides important insights into the mutational changes during S. aureus persistence in CRS patients highlighting potential pathoadaptive mechanisms in S. aureus persistent isolates culminating in increased biofilm biomass.

Combined mutations of the penA with ftsX genes contribute to ceftriaxone resistance in Neisseria gonorrhoeae and peptide nucleic acids targeting these genes reverse ceftriaxone resistance

To investigate the gene mutations associated with ceftriaxone (CRO) resistance among gonococcal isolates, and to determine the effects of the mutated genes on CRO minimum inhibitory concentrations (MICs) with transformation assays and antisense peptide nucleic acids (asPNAs). Ceftriaxone-resistant (CROR) and ceftriaxone-susceptible (CROS) isolates were identified using EUCAST and paired according to similarity in their MICs to other antimicrobials. The two groups of gonococci were sequenced and analysed. Mutated genes that showed a statistical difference between the two groups were transformed into gonococcal reference strains to determine their functions. AsPNAs were designed and transformed into the former transformant to further confirm the effects of the mutated genes. Twenty-two paired CROR and CROS isolates were obtained. The incidence of the penA-A501T and penA-G542S mutations individually, as well as combined mutations (penA-A501T and ftsX-R251H, penA-G542S and ftsX R251H), was statistically different between the two groups. The MIC of ATCC43069 (A43) increased 2 times following transformation with penA-A501T, and the MICs of A43 and ATCC49226 (A49) increased 32 times and 2 times following transformation with penA-A501T and ftsX-R251H, respectively. Antisense PNA-P3 reduced the MIC of the A43 transformant most significantly when transformed individually. PNA-P3 and PNA-F1 (asPNAs of the penA and ftsX) restored CRO susceptibility. PenA-A501T and penA-G542S mutations are important in CRO resistance among gonococci isolates. The ftsX-R251H mutation is also related to CRO resistance, and combined mutations of ftsX-R251H and penA-A501T comediate a significant reduction in CRO susceptibility. The combined application of PNA-P3 and PNA-F1 could effectively reverse the resistance to CRO in N. gonorrhoeae.

Comparative Genomic Analysis of ST131 Subclade C2 of ESBL-Producing E. coli Isolates from Patients with Recurrent and Sporadic Urinary Tract Infections.

The global emergence of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli), mainly causing urinary tract infections (UTI), is a major threat to human health. ESBL-E. coli sequence type (ST) 131 is the dominating clone worldwide, especially its subclade C2. Patients developing recurrent UTI (RUTI) due to ST131 subclade C2 appear to have an increased risk of recurrent infections. We have thus compared the whole genome of ST131 subclade C2 isolates from 14 patients with RUTI to those from 14 patients with sporadic UTI (SUTI). We aimed to elucidate if isolates causing RUTI can be associated with specific genomic features. Paired isolates from patients with RUTI were identical, presenting 2-18 single nucleotide polymorphism (SNP) differences for all six patients investigated. Comparative genomic analyses, including virulence factors, antibiotic resistance, pangenome and SNP analyses did not find any pattern associated with isolates causing RUTI. Despite extensive whole genome analyses, an increased risk of recurrences seen in patients with UTI due to ST131 subclade C2 isolates could not be explained by bacterial genetic differences in the two groups of isolates. Hence, additional factors that could aid in identifying bacterial properties contributing to the increased risk of RUTI due to ESBL-E. coli ST131 subclade C2 remains to be explored.

Lung SPLUNC1 Peptide Derivatives in the Lipid Membrane Headgroup Kill Gram-Negative Planktonic and Biofilm Bacteria.

SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in human respiratory tract with antimicrobial properties. In this work, we compare the biological activities of four SPLUNC1 antimicrobial peptide (AMP) derivatives using paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from 11 patients with/without colistin resistance. Secondary structural studies were carried out to study interactions between the AMPs and lipid model membranes (LMMs) utilizing circular dichroism (CD). Two peptides were further characterized using X-ray diffuse scattering (XDS) and neutron reflectivity (NR). A4-153 displayed superior antibacterial activity in both G(-) planktonic cultures and biofilms. NR and XDS revealed that A4-153 (highest activity) is located primarily in membrane headgroups, while A4-198 (lowest activity) is located in hydrophobic interior. CD revealed that A4-153 is helical, while A4-198 has little helical character, demonstrating that helicity and efficacy are correlated in these SPLUNC1 AMPs.

Surveillance for feline herpesvirus type 1 mutation and development of resistance in cats treated with antiviral medications.

Feline herpesvirus type 1 (FHV-1) commonly causes ocular surface disease in cats and is treated with antiviral medications targeting viral DNA polymerase (UL30/42). Herein, we describe a method to assess the FHV-1 genome for mutation development and to assess the functional impact of mutations, if present. Fourteen shelter-housed domestic cats with FHV-1 ocular surface disease were assigned to one of four treatment groups: placebo (n = 3), cidofovir 0.5% ophthalmic solution (n = 3), famciclovir oral solution (n = 5), or ganciclovir 0.15% ophthalmic solution (n = 3). Swabs were collected before (day 1) and after (day 8) 1 week of twice-daily treatments to isolate viable FHV-1. Viral DNA was extracted for sequencing using Illumina MiSeq with subsequent genomic variant detection between paired day 1 and day 8 isolates. Plaque reduction assay was performed on paired isolates demonstrating non-synonymous variants. A total of 171 synonymous and 3 non-synonymous variants were identified in day 8 isolates. No variants were detected in viral UL23, UL30, or UL42 genes. Variant totals were not statistically different in animals receiving antiviral or placebo (p = 0.4997). A day 8 isolate from each antiviral treatment group contained a single non-synonymous variant in ICP4 (transcriptional regulator). These 3 isolates demonstrated no evidence of functional antiviral resistance when IC50 was assessed. Most (10/14 pairs) day 1 and 8 viral isolate pairs from the same host animal were near-identical. While functional variants were not detected in this small sample, these techniques can be replicated to assess FHV-1 isolates suspected of having developed resistance to antiviral medications.

Detection of maternal transmission of resistant Gram-negative bacteria in a Cambodian hospital setting.

Infection with Extended-spectrum beta-lactamase -producing Enterobacterales (ESBL-E) is common in infants and leads to increased intensive care unit admission and mortality, but the role of maternal transmission in colonization of infants is unclear. Using paired isolates from 50 pairs of mothers and neonates admitted to a Cambodian hospital, we investigated antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae using whole genome sequencing. We detected a wide variety of ESBL-E genes present in this population along with high levels of multidrug resistance. From 21 pairs where the same organism was present in both mother and neonate, we identified eight pairs with identical or near-identical isolates from both individuals suggestive of transmission at or around birth, including a pair with transmission of multiple strains. We found no evidence for transmission of plasmids only from mother to infant. This suggests vertical transmission outside hospitals as a common cause of ESBL-E colonization in neonates.

Association between Intestinal Colonization and Extraintestinal Infection with Carbapenem-Resistant Klebsiella pneumoniae in Children.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public health threat. However, the association between intestinal colonization and parenteral infection among pediatric patients has not been elucidated. We collected 8 fecal CRKP strains and 10 corresponding CRKP strains responsible for extraintestinal infection from eight patients who did not manifest infection upon admission to the hospital. Paired isolates showed identical resistance to antimicrobials and identical virulence in vitro and in vivo. wzi capsule typing, multilocus sequence typing, and whole-genome sequencing (WGS) indicated high similarity between paired colonizing and infecting isolates. Mutations between colonizing and infecting isolate pairs found by WGS had a distinctive molecular signature of a high proportion of complex structural variants. The mutated genes were involved in pathways associated with infection-related physiological and pathogenic functions, including antibiotic resistance, virulence, and response to the extracellular environment. The latter is important for bacterial infection of environmental niches. Various mutations related to antibiotic resistance, virulence, and colonization that were not associated with any particular mutational hot spot correlated with an increased risk of extraintestinal infection. Notably, novel subclone carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) KL19-ST15 exhibited hypervirulence in experimental assays that reflected the severe clinical symptoms of two patients infected with the clonal strains. Taken together, our findings indicate the association between CRKP intestinal colonization and extraintestinal infection, suggesting that active screening for colonization on admission could decrease infection risk in children. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes an increasing number of nosocomial infections, which can be life-threatening, as carbapenems are last-resort antibiotics. K. pneumoniae is part of the healthy human microbiome, and this provides a potential advantage for infection. This study demonstrated that CRKP intestinal colonization is strongly linked to extraintestinal infection, based on the evidence given by whole-genome sequencing data and phenotypic assays of antimicrobial resistance and virulence. Apart from these findings, our in-depth analysis of point mutations and chromosome structural variants in patient-specific infecting isolates compared with colonizing isolates may contribute insights into bacterial adaptation underlying CRKP infection. In addition, a novel subclone of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) was observed in the study. This finding highlights the importance of CRKP active surveillance among children, targeting in particular the novel high-risk CR-hvKP clone.

Early initiation of three-drug combinations for the treatment of carbapenem-resistant A. baumannii among COVID-19 patients.

We evaluated the clinical characteristics and outcomes of patients with COVID-19 who received three-drug combination regimens for treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) infections during a single-centre outbreak. Our objective was to describe the clinical outcomes and molecular characteristics and in vitro synergy of antibiotics against CRAB isolates. Patients with severe COVID-19 admitted between April and July 2020 with CRAB infections were retrospectively evaluated. Clinical success was defined as resolution of signs/symptoms of infection without need for additional antibiotics. Representative isolates underwent whole-genome sequencing (WGS) and in vitro synergy of two- or three-drug combinations was assessed by checkerboard and time-kill assays, respectively. Eighteen patients with CRAB pneumonia or bacteraemia were included. Treatment regimens included high-dose ampicillin-sulbactam, meropenem, plus polymyxin B (SUL/MEM/PMB; 72%), SUL/PMB plus minocycline (MIN; 17%) or other combinations (12%). Clinical resolution was achieved in 50% of patients and 30-day mortality was 22% (4/18). Seven patients had recurrent infections, during which further antimicrobial resistance to SUL or PMB was not evident. PMB/SUL was the most active two-drug combination by checkerboard. Paired isolates collected before and after treatment with SUL/MEM/PMB did not demonstrate new gene mutations or differences in the activity of two- or three-drug combinations. Use of three-drug regimens for severe CRAB infections among COVID-19 resulted in high rates of clinical response and low mortality relative to previous studies. The emergence of further antibiotic resistance was not detected phenotypically or through WGS analysis. Additional studies are needed to elucidate preferred antibiotic combinations linked to the molecular characteristics of infecting strains.

Endogenous relapse and exogenous reinfection in recurrent pulmonary tuberculosis: A retrospective study revealed by whole genome sequencing.

Tuberculosis may reoccur due to reinfection or relapse after initially successful treatment. Distinguishing the cause of TB recurrence is crucial to guide TB control and treatment. This study aimed to investigate the source of TB recurrence and risk factors related to relapse in Hunan province, a high TB burden region in southern China. A population-based retrospective study was conducted on all culture-positive TB cases in Hunan province, China from 2013 to 2020. Phenotypic drug susceptibility testing and whole-genome sequencing were used to detect drug resistance and distinguish between relapse and reinfection. Pearson chi-square test and Fisher exact test were applied to compare differences in categorical variables between relapse and reinfection. The Kaplan-Meier curve was generated in R studio (4.0.4) to describe and compare the time to recurrence between different groups. p < 0.05 was considered statistically significant. Of 36 recurrent events, 27 (75.0%, 27/36) paired isolates were caused by relapse, and reinfection accounted for 25.0% (9/36) of recurrent cases. No significant difference in characteristics was observed between relapse and reinfection (all p > 0.05). In addition, TB relapse occurs earlier in patients of Tu ethnicity compared to patients of Han ethnicity (p < 0.0001), whereas no significant differences in the time interval to relapse were noted in other groups. Moreover, 83.3% (30/36) of TB recurrence occurred within 3 years. Overall, these recurrent TB isolates were predominantly pan-susceptible strains (71.0%, 49/69), followed by DR-TB (17.4%, 12/69) and MDR-TB (11.6%, 8/69), with mutations mainly in codon 450 of the rpoB gene and codon 315 of the katG gene. 11.1% (3/27) of relapse cases had acquired new resistance during treatment, with fluoroquinolone resistance occurring most frequently (7.4%, 2/27), both with mutations in codon 94 of gyrA. Endogenous relapse is the main mechanism leading to TB recurrences in Hunan province. Given that TB recurrences can occur more than 4 years after treatment completion, it is necessary to extend the post-treatment follow-up period to achieve better management of TB patients. Moreover, the relatively high frequency of fluoroquinolone resistance in the second episode of relapse suggests that fluoroquinolones should be used with caution when treating TB cases with relapse, preferably guided by DST results.

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

Unitary p-wave interactions between fermions in an optical lattice.

Exchange-antisymmetric pair wavefunctions in fermionic systems can give rise to unconventional superconductors and superfluids1-3. The realization of these states in controllable quantum systems, such as ultracold gases, could enable new types of quantum simulations4-8, topological quantum gates9-11 and exotic few-body states12-15. However, p-wave and other antisymmetric interactions are weak in naturally occurring systems16,17, and their enhancement via Feshbach resonances in ultracold systems has been limited by three-body loss18-24. Here we create isolated pairs of spin-polarized fermionic atoms in a multiorbital three-dimensional optical lattice. We spectroscopically measure elastic p-wave interaction energies of strongly interacting pairs of atoms near a magnetic Feshbach resonance. The interaction strengths are widely tunable by the magnetic field and confinement strength, and yet collapse onto a universal curve when rescaled by the harmonic energy and length scales of a single lattice site. The absence of three-body processes enables the observation of elastic unitary p-wave interactions, as well as coherent oscillations between free-atom and interacting-pair states. All observations are compared both to an exact solution using a p-wave pseudopotential and to numerical solutions using an ab initio interaction potential. The understanding and control of on-site p-wave interactions provides a necessary component for the assembly of multiorbital lattice models25,26 and a starting point for investigations of how to protect such systems from three-body recombination in the presence of tunnelling, for instance using Pauli blocking and lattice engineering27,28.

152. Molecular Epidemiological Analysis of Relapsed Gram-negative Bloodstream Infection Isolates

Abstract Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience multiple episodes of GNB-BSI for unclear reasons. This study examines the genetic differences between initial and subsequent isolates of recurrent GNB-BSI. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with &amp;gt;1 episode of GNB-BSI due to the same bacterial species. We performed pulsed field gel electrophoresis (PFGE) on paired isolates to determine if the events were Reinfection (paired isolates different) or Relapse (paired isolates genetically identical). We then used whole genome sequencing (WGS) to verify the PFGE findings and explore the genetic similarity between initial and relapsed GNB-BSI isolates. Results Among 1,423 unique patients with GNB-BSI, 60 (4%) experienced recurrent GNB-BSI with the same bacterial species. We performed genotyping (PFGE, followed by WGS) on the paired bacterial isolates from the index and recurrent bloodstream infections for the four most common species in our study population (Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Serratia marcescens) (n=48 pairs). We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. PFGE correctly identified relapse versus reinfection in 98% (47/48) of cases. The WGS data illustrated the microevolution of relapsed GNB-BSI isolates within patients. When comparing initial to relapsed isolates, we calculated a median of 5 single-nucleotide polymorphisms (SNPs) in relapsed E. coli isolates and 2 SNPs in Klebsiella spp. isolates. Of these SNPs, 37% (E. coli) and 12% (Klebsiella) were non-synonymous changes in coding regions, most commonly genes associated with antimicrobial resistance (AMR), energy metabolism and stress response. Alterations in the AMR profile was common, with 20/30 relapsed isolates demonstrating either loss or acquisition of AMR. Figure 1:Time from initial to recurrent episode of gram-negative bloodstream infection. Relapse (i.e., same bacterial strain noted by green circle) versus reinfection (i.e., different bacterial strain noted by red circle) was determined by pulse field gel electrophoresis followed by WGS. Whole genome sequencing was performed on the initial and recurrent isolate from patients with relapsed GNB-BSI. We identified single nucleotide polymorphisms (SNPs) present in the recurrent isolate, which were not present in the initial isolate. We identified the function of genes containing SNPs in E. coli (n=28 SNPs) and Klebsiella spp. (n=13 SNPs). Synonymous mutations, insertions, deletions, and non-synonymous mutations in hypothetical proteins were excluded. Conclusion Most recurrent GNB-BSI with same species are due to relapse. PFGE has comparable accuracy to WGS to determine reinfection versus relapse. Multiple mutations in genes involved in stress response, AMR and metabolism were identified in relapsed isolates. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds.