Clonal Relationship Research Articles

Molecular Characterization of Enterococcus faecalis and Enterococcus faecium Isolated from a Meat Source in Shahrekord Local Markets, Iran.

Enterococcus faecalis (E. faecalis) and E. faecium (E. faecium) are commensals of the gastrointestinal biota of humans and animals and are considered opportunistic pathogens. This study aimed to improve the knowledge about E. faecalis and E. faecium isolated from meat. To this end, 104 meat samples were collected from sheep, goats, cattle, and calves from Shahrekord local markets in Iran. Presumptive colonies of E. faecalis and E. faecium were isolated from the samples and subjected to identification tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was performed to detect some virulence genes. Furthermore, randomly amplified polymorphic DNA typing and repetitive element sequence-based PCR fingerprinting were used to show the clonal relatedness of the isolates. The results revealed that enterococci were present in 90 out of 104 samples. Specifically, E. faecalis and E. faecium were the commonly isolated species, with the predominance of E. faecalis, which exhibited high resistance to streptomycin (95%) but was susceptible to vancomycin (85.6%). Virulence genes detection showed that ccf and cpd genes were the most prevalent genes in both species. In addition, the molecular typing method indicated that the isolates belonged to separate subgroups. This study shows the contamination of meat products by potential pathogens and resistant enterococci. There is a need to implement regular surveillance to monitor the emergence of antimicrobial-resistant E. faecalis and E. faeciumin food, particularly in meat production.

Evaluation of Antibiotic Resistance and adeABC, adeR, adeS Efflux Pump Genes among Foodborne and Clinical Acinetobacter spp. in Türkiye.

The adeABC efflux pump has a crucial role in the resistance of Acinetobacter baumannii strains to antimicrobial agents; it is encoded by adeABC, adeR, adeS genes. We evaluated antibiotic resistance, efflux pump genes, clonal relationships, and analyzed a probable correlation that can exist between antibiotic resistance and the aforementioned genes. We conducted this study on 27 food-originated and 50 human clinical Acinetobacter spp. in Southern Türkiye. MALDI-TOF system and disc diffusion/agar dilution (colistin) methods were used for the identification and antibiotic susceptibility. The efflux pump genes and genetic relatedness of the two groups were investigated by (PCR) and (PFGE) methods. Foodborne A. dijkshoorniae strain was multidrug- resistant (MDR), and none of them resistant to colistin. Most of the clinical isolates (92%) were Extensive-Drug Resistant (XDR); highest resistant to ceftazidime, piperacillin-tazobactam, and imipenem (47, 94%), and were lowest to colistin (7, 14%), respectively. adeABC, and adeR, adeS genes were (23, 85.2%), (9, 33.3%), (27, 100%) and (10, 37.3%), (18, 66.7%) in foodborne strains respectively. These rates were (43, 86%), (48, 96%), (50, 100%), and (34, 68%), (48, 96.7%) in clinical strains respectively. A positive correlation existed between adeA gene positivity and piperacillintazobactam, ceftazidime, gentamycin, imipenem (P=0.048), amikacin (P=0.007) and trimethoprimsulfamethoxazole (P=0.029) resistance in clinical strains. A positive correlation of trimethoprimsulfamethoxazole resistance and adeS gene positivity was seen in foodborne strains (P=0.018). Multiple-efflux pump genes rise in parallel to multidrug-resistance in clinical isolates, while susceptible to diverse antibiotics; food may be a potential provenance for the dissemination of adeABC, adeR and adeS genes.

High Prevalence of blaOXA-48 and blaNDM-Producing Carbapenem-Resistant Klebsiella pneumoniae Isolated from Clinical Samples in Shahid Rajaei Hospital in Tehran, Iran

Background: Due to the increasing antibiotic resistance, treating infections caused by Klebsiella pneumoniae has become more challenging. Objectives: The present study aimed to investigate the prevalence of blaOXA-48 and blaNDM producing carbapenem-resistant K. pneumoniae isolated from clinical samples in Shahid Rajaei hospital in Tehran, Iran. Methods: Various clinical samples were collected from 1,186 patients admitted with open heart surgery in two wards (ICU and surgery) in Shahid Rajaei Heart Hospital in Tehran, Iran. Klebsiella pneumoniae isolates were identified by standard microbiologic tests. Antimicrobial susceptibility of isolates were determined by disk diffusion and E-test methods. A modified carbapenem inactivation method (mCIM) was performed to detect the presence of carbapenemase. Antibiotic resistance genes were detected using conventional polymerase chain reaction (PCR) by primers targeting blaOXA-48, blaSPM, blaIMP, blaVIM, and blaNDM genes. Results: A total of 131 clinical isolates of K. pneumoniae were isolated and 45.8% (60/131) of them were resistant to carbapenem. Klebsiella pneumoniae isolates showed the highest resistance rate (100%) to ceftriaxone, ceftazidime, cefazolin, and cefepime and the maximum sensitivity to tigecycline (96.7%). The carbapenemase-encoding blaOXA-48 and blaNDM-1 genes were detected in 96.7% and 66.7% of isolates, respectively. Eight different clusters of the isolates, considering a ≥ 80% homology cut-off, were shown with the same rep-PCR pattern. Clusters A, B, C, D, E, F, G, and H included 20, 11, 7, 6, 6, 3, 2, and 2 members, respectively. Conclusions: The RAPD-PCR method reveals the clonal relationship between isolates and may help improve infection control procedures.

Multidrug-resistant and clonal dispersion of enterotoxigenic Escherichia coli from ready-to-eat meat products in Duhok province, Iraq

This research evaluated the effluent proportion of E. coli and ETEC in RTE meat products, characterized the isolated strains' clonal relatedness, and determined their antibiotic resistance. 130 RTE products were gathered from various restaurants and street fast food vendors in Duhok and Zakho Province. The Isolates of E. coli identified by culture methods were confirmed as ETEC by multiplex PCR of the identified virulence genes. ERIC-PCR was applied to establish the clonal relationships between strains. The disk diffusion method performed the susceptibility of antibiotics on the isolated ETEC. Out of 130 examined samples, 39 (30%) isolates of E. coli and 16 (12.3%) ETEC were detected. Pan-fried burgers were revealed to be the most frequent contaminated sample type, with both E. coli and ETEC 50% and 23.3%, respectively (P≤0.05). A high clonal dispersion (12 genotypes) was observed among the isolated ETEC strains. A strong genetic linkage was discovered between a few isolates retrieved from the same sample type and within the strains from the same geographic source area. A high antibiotic resistance rate was observed with total resistance to Amoxicillin/clavulanate, Clarithromycin, Doxycycline, Erythromycin, and Clindamycin. Isolates from burger samples showed a higher resistance rate when compared with the other sample types (P≤0.05). Multi-drug resistance was noticed in all ETEC isolates. RTE meat products sold in our area have a high rate of clonally heterogeneous carrying multi-drug resistant ETEC and may constitute a significant public health risk.

Genotypic characterization and clonal relatednessof metallo-β-lactamase-producingnon-fermentative gram negative bacteria in the first 5years of their circulation in Paraguay(2011-2015).

Pseudomonas aeruginosa and species of Acinetobacter calcoaceticus-baumanii complex are multiresistant intrahospital opportunistic pathogens, able to acquire carbapenemases and produce outbreaks with high morbidity and mortality. Pseudomonas putida has also emerged with similar characteristics. The aim of this research was to characterize the Metallo-β-lactamases (MBLs) detected by surveillance in Paraguay in the first 5years of their circulation in hospitals. The coexistence of KPC and OXA-type carbapenemases was also investigated. 70 MBL-producing strains from inpatients were detected from clinical samples and rectal swab from 11 hospitals. The strains were identified by manual, automated, and molecular methods. Antimicrobial susceptibility was studied by Kirby-Bauer and automated methods, while colistin susceptibility was determined by broth macrodilution. MBLs were investigated by synergy with EDTA against carbapenems and PCR, and their variants by sequencing. KPC and OXA-carbapenemases were investigated by PCR. Clonality was studied by pulsed-field gel electrophoresis (PFGE). The results demonstrated the circulation of blaVIM-2 (60%), blaNDM-1 (36%), and blaIMP-18 (4%). The MBL-producing species were P. putida (45.7%), P. aeruginosa (17.2%), A. baumannii (24.3%), A. pittii (5.7%), A. nosocomialis, (4.3%) A. haemolyticus (1.4%), and A. bereziniae (1.4%). PFGE analysis showed one dominant clone for A. baumannii, a predominant clone for half of the strains of P. aeruginosa, and a polyclonal spread for P. putida. In the first 5years of circulation in Paraguay, MBLs were disseminated as unique variants per genotype, appeared only in Pseudomonas spp. and Acinetobacter spp., probably through horizontal transmission between species and vertical by some successful clones.

Lineage Reconstruction of In Vitro Identified Antigen-Specific Autoreactive B Cells from Adaptive Immune Receptor Repertoires.

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis.

Next-generation sequencing proves clonal relationship between two distinguished lung and liver carcinomas by standard histopathology approach

Introduction: Two tumors having different histopathologies at anatomically distinct sites giving the picture of dual primary malignancies. Here we presented a case of two possible primary tumors and one secondary mass. Case Report: A 74-year-old female, active smoker, without personal or family cancer history presented with early satiety and weakness for two months. Systems review was positive for a “raw” feeling in stomach, alleviated with antacids. Vital signs were stable with a negative abdominal exam. Lab showed leukocytosis 24.8 K/uL (3.5–10.8 K/uL) with left shift, microcytic anemia with hemoglobin 6.1 g/dL (12.0–16.0 g/dL), and reactive thrombocytosis 477 K/uL (130–400 K/uL). Contrast-enhanced computed tomography (CT) showed right upper lobe necrotizing cavitating lesion with reactive mediastinal and right hilar lymphadenopathy, two irregular hypodense lesions in pancreatic head and tail without ductal dilation with two irregular hypodense liver lesions. Immunohistochemistry of lung and pancreatic lesions were biopsied through endoscopic ultrasound (EUS), consistent with poorly differentiated squamous cell carcinoma (SCC) with extensive necrosis, which indicates pancreatic masses are likely metastases from the lung. Liver lesion biopsy exhibited high-grade neuroendocrine tumor (NET) with focal necrosis. Next gene sequencing was pursued. Given poor functional status, palliative immunotherapy was offered; however, the patient succumbed to respiratory failure. Conclusion: Given the morphology and immunoprofile, differential diagnosis includes dual primary cancers with one metastasis, or primary SCC with metastasis with neuroendocrine differentiation. Despite having different histopathology and immunophenotype, both lung and liver tumors harbor the same molecular profile even at the variants of unknown significance that show identical mutations. As a result, they are directly related. TP53, RB1, MYCL1, and MEK1 mutations are more prevalent in SCC than NET. Tumor mutation burden values may vary as the tumor clonal structure varies between primary and metastatic sites, with higher rates of monoclonal structure recorded in metastases due to clonal selection, leading to a reduction in overall genetic diversity (“bottlenecking”). This raises the suspicion that the liver tumor is a SCC with neuroendocrine differentiation. The paucity of the specimen and rapid clinical course limited further investigation. Germline testing would have been useful to determine whether these findings are somatic or germline.

One health clones of multidrug-resistant Escherichia coli carried by synanthropic animals in Brazil

WHO priority pathogens have disseminated beyond hospital settings and are now being detected in urban and wild animals worldwide. In this regard, synanthropic animals such as urban pigeons (Columba livia) and rodents (Rattus rattus, Rattus norvegicus and Mus musculus) are of interest to public health due to their role as reservoirs of pathogens that can cause severe diseases. These animals usually live in highly contaminated environments and have frequent interactions with humans, domestic animals, and food chain, becoming sentinels of anthropogenic activities. In this study, we report genomic data of Escherichia coli strains selected for ceftriaxone and ciprofloxacin resistance, isolated from pigeons and black rats. Genomic analysis revealed the occurrence of international clones belonging to ST10, ST155, ST224 and ST457, carrying a broad resistome to beta-lactams, aminoglycosides, trimethoprim/sulfamethoxazole, fluoroquinolones, tetracyclines and/or phenicols. SNP-based phylogenomic investigation confirmed clonal relatedness with high-risk lineages circulating at the human-animal-environmental interface globally. Our results confirm the dissemination of WHO priority CTX-M-positive E. coli in urban rodents and pigeons in Brazil, highlighting potential of these animals as infection sources and hotspot for dissemination of clinically relevant pathogens and their resistance genes, which is a critical issue within a One Health perspective.

1230. Identification of Subclinical Healthcare-Associated Clusters of Staphylococcus epidermidis in an Orthopedic Patient Population.

Abstract Background Prosthetic joint infections (PJIs) cause increased morbidity and mortality for patients. Staphylococcus epidermidis (S. epidermidis) can readily form biofilm on implanted medical devices, making this typically commensal species a common cause of PJIs. This study investigates genetic, epidemiologic, and environmental factors contributing to positive S. epidermidis joint cultures. Methods We identified S. epidermidis isolates from hip or knee cultures obtained between 2017-20 in patients with one or more prior corresponding intraarticular procedure at our hospital. Whole genome sequencing and single nucleotide polymorphism (SNP) based clonality analysis was performed using the epiXact® service at Day Zero Diagnostics, including species identification, in silico multi-locus sequence typing (MLST), phylogenomic analysis, along with genotypic assessment of the prevalence of specific antibiotic resistance and virulence genes. Additional epidemiologic review was performed to compare cluster and noncluster cases. Results Sixty phenotypically distinct S. epidermidis joint isolates were identified from 44 patients. After removal of duplicates and impure samples, 46 samples were used for genomic sequencing, and 45 were used for MLST. Four S. epidermidis strains showed phenotypic susceptibility to oxacillin yet harbored mecA, and two showed phenotypic resistance despite not having mecA (Figure 1). Notably, smr was found in all isolates. MupA positivity was not observed. We also identified six clonal clusters, all with SNP distances < 10 (Figure 2). Each cluster consisted of between 2-4 patients. Cluster isolates accounted for 14/45 (31.1%) of S. epidermidis isolates sequenced. Further epidemiologic investigation showed ties to common aspirations or operative procedures although no specific common source was identified. These subclinical clusters appear to be self-limited, suggesting benefit to common infection control practices. Phylogenetic tree of S. epidermidis population and heatmap showing the presence/absence of specific genomic features. Circle color on node tip corresponds to sequence type (MLST). Color strips (right panel) showing genomic feature (gene or marker) presence/absence inferred from WGS de novo assemblies. Green cells correspond to a full match found, and gray cells to no match found. Empty rows correspond to external reference genomes used as controls. *AMR: antimicrobial resistance; ST: sequence type; SNP distance values for all pairs of samples of matching multi-locus sequence types. Only samples belonging to matching multi-locus sequence types (MLSTs) with more than one sample are shown. Blue cells correspond to pairwise distance of < 25 SNPs indicating clonality. 13 isolates with unique MLSTs are not shown. 10 isolates were not included due to having multi-strain or multi-species contamination. *ST: sequence type; SNP: single-nucleotide polymorphism; Univariate analysis of clonal isolates versus non-clonal isolates. Samples not included in the multi-locus sequence type analysis due to contamination were excluded from the univariate analysis. *PJI: prosthetic joint infection; MSIS: Musculoskeletal Infection Society; Conclusion The majority of S. epidermidis isolated from clinical joint samples are diverse in origin, but we identified an important subset of 31% that belonged to subclinical healthcare-associated clusters. Clusters appeared to resolve spontaneously over time, suggesting benefit to routine infection control practices. Diagram of clonal isolate clusters Potential relationships and modes of transmission between clonal isolates in each cluster. *PJI: prosthetic joint infection; MSIS: Musculoskeletal Infection Society; SNP: single-nucleotide polymorphism; Disclosures Mohamad Sater, PhD, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Stocks/Bonds Ian C. Herriott, Bachelor's of Science, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Stocks/Bonds Miriam H. Huntley, PhD, Day Zero Diagnostics: Employee|Day Zero Diagnostics: Ownership Interest.

Salivary carcinosarcoma: insight into multistep pathogenesis indicates uniform origin as sarcomatoid variant of carcinoma ex pleomorphic adenoma with frequent heterologous elements.

The formal pathogenesis of salivary carcinosarcoma (SCS) remained unclear, both with respect to the hypothetical development from either preexisting pleomorphic adenoma (PA) or de novo and the clonal relationship between highly heterogeneous carcinomatous and sarcomatous components. We performed clinicopathological and molecular (targeted RNA sequencing) analyses on a large series of 16 cases and combined this with a comprehensive literature search (111 cases). Extensive sampling (average 11.6 blocks), combined with immunohistochemistry and molecular studies (PA-specific translocations including PLAG1 or HMGA2 proven in 6/16 cases), enabled the morphogenetic identification of PA in 15/16 cases (93.8%), by far surpassing a reported rate of 49.6%. Furthermore, we demonstrated a multistep (intraductal/intracapsular/extracapsular) adenoma-carcinoma-sarcoma-progression, based on two alternative histogenetic pathways (intraductal, 56.3%, versus myoepithelial pathway, 37.5%). Thereby, early intracapsular stages are identical to conventional carcinoma ex PA, while later extracapsular stages are dominated by secondary, frequently heterologous sarcomatous transformation with often large tumour size (>60 mm). Our findings strongly indicate that SCS (almost) always develops from PA, with a complex multistep adenoma-carcinoma-sarcoma-sequence, based on two alternative histogenetic pathways. The findings from this novel approach strongly suggest that SCS pathogenetically is a rare (3-6%), unique, and aggressive variant of carcinoma ex PA with secondary sarcomatous overgrowth. In analogy to changes of terminology in other organs, the term "sarcomatoid carcinoma ex PA with/without heterologous elements" might be more appropriate.

Distribution of emm, superantigen and other virulence genotypes and detection of phylogenetic relationships in group A streptococcal isolates.

Group A streptococci are important pathogens with various virulence factors, such as M protein, superantigens, hemolysins, deoxyribonuclease, and proteases. The aims of this study are to investigate the detection of emm genotypes and other virulence genes, such as SAgs, DNase, protease, antibiotic resistance, and phylogenetic relationships in GAS strains isolated from clinical samples.Test strains were obtained from Çukurova University Balcalı Hospital and regional hospitals in Adana province. The M proteins were detected by sequence analysis of emm genes. SAgs and other virulence gene profiles were determined using the Multiplex-PCR method. The antibiotic susceptibility of the isolates was performed by the disc diffusion method and evaluated according to CLSI criteria. The PFGE method was used to determine the clonal relationship between the strains.The emm gene was positive in 86 isolates. The most common emm genotypes were emm28 (22%), emm1 (18.6%), emm12 (13.9%), and emm3 (11.6%). Also, the most common virulence genes were speG (58.1%), speC (56.9%), sdaB (53.4%), and mac (53.4%). The rates of resistance to erythromycin, clindamycin, levofloxacin, ciprofloxacin and telithromycin were 19.8%, 16.3%, 4.7%, 3.5%, and 3.5%, respectively.As a result, additional regional studies on the detection and prevalence of GAS virulence factors in Turkey are required. We believe that this study will provide valuable information for epidemiological studies on emm sequences, Sags, and other virulence factors of Streptococcus pyogenes in Turkey.

Early fate bias in neuroepithelial progenitors of hippocampal neurogenesis.

Hippocampal adult neural stem cells emerge from progeny of the neuroepithelial lineage during murine brain development. Hippocampus development is increasingly well understood. However, the clonal relationships between early neuroepithelial stem cells and postnatal neurogenic cells remain unclear, especially at the single-cell level. Here we report fate bias and gene expression programs in thousands of clonally related cells in the juvenile hippocampus based on single-cell RNA-seq of barcoded clones. We find evidence for early fate restriction of neuroepithelial stem cells to either neurogenic progenitor cells of the dentate gyrus region or oligodendrogenic, non-neurogenic fate supplying cells for other hippocampal regions including gray matter areas and the Cornu ammonis region 1/3. Our study provides new insights into the phenomenon of early fate restriction guiding the development of postnatal hippocampal neurogenesis.

Genetic Relationship between Salmonella Isolates Recovered from Calves and Broilers Chickens in Kafr El-Sheikh City Using ERIC PCR.

A prevalent bacterial intestinal infection with severe economic damage is salmonellosis. Our study was carried out to diagnose Salmonella from chickens and calves, to determine its resistance to antimicrobials' phenotypic and genotypic characterization of integrons and β lactamase genes in the multidrug resistance of different Salmonella serotypes, and to detect the genetic relationship between Salmonella isolates collected from different origins using an ERIC PCR. In total, 200 samples from diseased chicken and diarrheic calves were obtained from 50 various farms from Kafr El-sheikh, Egypt. Salmonella poultry isolates were characterized as S. Typhimurium (3/8), S. Enteritidis (3/8), and S. Kentucky (2/8), but Salmonella isolates from cattle were S. Enteritidis (1/2) and S. Kentucky (1/2). When antibiotic susceptibility testing was completed on all of the isolates, it showed that there was multidrug resistance present (MDR). A PCR was applied for identifying the accompanying class 1 integrons and ESBLs from MDR Salmonella isolates (two isolates of S. Kentucky were divided as one from calf and one from poultry). Our results detected blaTEM and class 1 integron, but were negative for bla IMP, bla VIM, and bla SHV. An ERIC PCR was conducted for understanding the clonal relation between various β-lactamase-producing MDR Salmonella isolates. The same four previously mentioned isolates were also tested. The two isolates of S. Enteritidis isolated from poultry and calves had 100% similarity despite indicating that there were interactions between broilers and calves living on the same farm that caused infection from the same Salmonella strains, while the other two isolates of S. Kentucky showed only 33% serovarities.

The association between Acinetobacter baumannii infections and the COVID-19 pandemic in an intensive care unit

We aimed to describe the increased rate of Acinetobacter baumannii infections during the COVID-19 pandemic and define its significance within the last five years. This study was performed in a tertiary hospital with 280 beds and included all patients infected with A. baumannii in the intensive care unit between January 1, 2018, and June 30, 2022. A. baumannii-infected patients in the intensive care unit 27 months before the pandemic and 27 months during the pandemic were included. Pulsed-field gel electrophoresis was performed to assess clonal relatedness. The infection control measures were specified based on the findings and targeted elimination. In total, 5718 patients were admitted to the intensive care unit from January 1st, 2018, to June 30th, 2022. A. baumannii infection was detected in 81 patients. Compared to the pre-pandemic era, the rate of A. baumannii infection during the pandemic was 1.90 times higher (OR: 1.90, 95% CI: [1.197, 3.033]). Clonality assessment of multidrug-resistant A. baumannii samples revealed eight clusters with one main cluster comprising 14/27 isolates between 2021 and 2022. The case fatality rate of the pre-pandemic and pandemic era was not different statistically (83.33% vs. 81.48%, p = 0.835). Univariate analysis revealed the association of mechanical ventilation (p = 0.002) and bacterial growth in tracheal aspirate (p = 0.001) with fatality. During the COVID-19 pandemic, potential deficits in infection control measures may lead to persistent nosocomial outbreaks. In this study, the introduction of enhanced and customized infection control measures has resulted in the containment of an A. baumannii outbreak.

DETECCIÓN CLONAL DE CEPAS PARENTALES DE Streptococcus agalactiae Lehmann y Neumann POR AMPLIFICACIÓN ALEATORIA DE ADN POLIMÓRFICO

Streptococcus agalactiae (GBS) causes invasive infections in newborns, being the most frequent the maternal transmission. Epidemiological studies use molecular techniques that assess genetic diversity, including random amplification of polymorphic DNA (RAPD) that is found to be accessible, sensitive and uses arbitrary primers to amplify polymorphic segments of DNA by PCR. The objective was to determine the clonal relationship between GBS strains recovered from mothers and their respective newborns. Four pairs of GBS isolates obtained from vaginal-rectal swabs of mothers and blood cultures of their newborns were studied with RAPD. Primers OPS11, OPB17 and OPB18 were used to select one with the ability to discriminate between non-genetically related strains. The Hunter-Gaston formula that establishes the discrimination index (D) was used; when D>0.90, it is considered that the isolates belong to different clones. The amplification profiles for the eight isolates, using each primer independently, allowed to calculate a D=1 for OPS11, and D=0.84 for OPB17 and OPB18. Therefore, OPS11 was selected for the study of the clonal relationship of the isolates, and similar amplification profiles were found by RAPD for each mother-newborn pair of GBS isolates. Different amplification profiles were observed between pairs of mother-newborn strains, which reveals the discrimination between unrelated strains, confirmed by pulsating field electrophoresis (PFGE). These results indicated vertical transmission for each studied case and robustness of the OPS11 primer. Appropriate conditions of the RAPD trial were found, which is useful for epidemiological studies. Key words: Streptococcus agalactiae, neonatal disease, molecular epidemiology, RAPD technique, vertical transmission

The Occurrence and Genomic Characteristics of mcr-1-Harboring Salmonella from Retail Meats and Eggs in Qingdao, China.

Salmonella are widely distributed foodborne pathogens and are often associated with food animal products. Colistin resistance mediated by mcr-1 is an increasing threat; however, data on the characteristics of mcr-1-harboring Salmonella among retail foods are still lacking. In this study, retail meats from 24 supermarkets and eggs from nine markets in Qingdao city were investigated to determine the presence and genomic characteristics of mcr-1-harboring Salmonella. We found the retail meats and eggs were highly contaminated by Salmonella, with detection rates of 17.5% (31/177) and 12.3% (16/130), respectively. A total of 76 Salmonella isolates were obtained in this study, and 77.6% showed multidrug resistance (MDR). The MDR proportion of egg isolates (97.5%) was significantly higher than that in meat isolates (55.6%) (p < 0.05). The most prevalent Salmonella serotypes were Typhimurium (56.6%) and Enteritidis (17.1%). Of the 76 Salmonella isolates, 40 possessed mcr-1. All 40 mcr-1-positive isolates were ST34 S. Typhimurium and were from eggs of eight brands. Different mcr-1-harboring isolates existed in the same egg, and some isolates from different egg samples or brands showed clonal relationships. The mcr-1 was located on similar IncHI2/HI2A MDR non-conjugative plasmids lacking transfer region, resulting in the failure of conjugation. The phylogenetic tree using genome sequences showed that the mcr-1-positive isolates from eggs clustered together with mcr-1-positive isolates from chicken and humans in China, revealing that mcr-1-positive egg-borne Salmonella might be derived from chicken and could potentially trigger outbreaks in humans. The high occurrence of mcr-1-harboring Salmonella in fresh eggs is alarming, and there is an urgent need to monitor mcr-1-harboring Salmonella in retail meats and eggs. We report for the first time the role of retail eggs in disseminating mcr-1-positive Salmonella and the risk of transmission of these MDR pathogens from retail food to humans should be evaluated comprehensively.

015 Clonal sharing of skin T cells within different skin compartments and blood

The human skin harbors a heterogenous pool of cytotoxic tissue-resident memory T (TRM) cells seeded during infections and inflammation. TRM cells are retained at the site of viral infections and provide local immunity. These cells persist for years in the skin after bone marrow transplantation but circulating ex-TRM cells indicate several potential fates. Here, we explored the clonal relationships between human T cells in different skin compartments and circulation. 10x Genomics 5 prime immune profiling coupled with feature barcoding technology was employed to investigate the clonal relationship, transcriptomic and proteomic profiles of single T cells sorted from blood, subcutis, dermis, and epidermis.

Evaluation of ERIC-PCR and MALDI-TOF as typing tools for multidrug resistant Klebsiella pneumoniae clinical isolates from a tertiary care center in India.

Multidrug resistant Klebsiella pneumoniae is associated with nosocomial infections in both outbreak and non-outbreak situations. The study intends to evaluate the potential of enterobacterial repetitive intergenic consensus- polymerase chain reaction (ERIC-PCR), a genomic based typing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) proteomic-based typing techniques for clonal relatedness among multidrug resistant Klebsiella pneumoniae isolates. Multidrug resistant clinical isolates of Klebsiella pneumoniae (n = 137) were collected from March 2019 to February 2020. Identification and protein-based phylogenetic analysis were performed by MALDI-TOF MS. Genomic typing was done by ERIC-PCR and analyzed by an online data analysis service (PyElph). Dice method with unweighted pair group method with arithmetic mean (UPGMA) program was used to compare the ERIC profiles. The samples were also evaluated by PCR for the presence of genes encoding carbapenemases, extended spectrum beta lactamases (ESBLs) and mobile colistin resistance-1 (mcr1). The study presents ERIC-PCR as more robust and better discriminatory typing tool in comparison to MALDI-TOF for clonal relatedness in multidrug resistant K. pneumoniae clinical isolates. Isolates were typed into 40 ERIC types, and six groups by MALDI-TOF-MS. PCR-based analysis revealed that all the strains harbored two or more ESBL and carbapenemase genes. None of the isolates revealed the presence of the plasmid mediated mcr-1 gene for colistin resistance.

Abstract PR007: Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5 year survival rate of 11%. PDAC arises from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). Molecular analysis of human PanINs is critical to understand early pancreatic tumorigenesis, which could inform risk stratification, early detection, and cancer prevention approaches. Because PanINs are microscopic, it is challenging to determine their full extent and anatomic relationships using two dimensional histological sections. To determine the density and connectivity of PanINs, we utilized a novel machine learning algorithm (CODA) for three-dimensional (3D) reconstruction and cellular quantification. Large blocks of grossly normal pancreas were harvested from 39 surgical pancreatectomy specimens, followed by formalin fixation, paraffin-embedding, and complete serial sectioning. 3D reconstruction using CODA revealed striking multifocality of PanINs within the pancreata of most analyzed patients, with more than 600 spatially PanINs being modeled. Next, to determine whether these multifocal PanINs arose as independent neoplasms or via intraductal spread of a single PanIN, we assessed their clonal relationships using somatic multi-region DNA next generation sequencing (NGS). To do so, 8 additional blocks of grossly normal pancreatic tissue were harvested and 3D modeled, yielding 37 anatomically distinct PanINs for genomic analysis. Each spatially unconnected PanIN was separately microdissected in five different regions to assess both intra-PanIN and inter-PanIN genetic heterogeneity. 99 samples from the 37 PanIN lesions underwent targeted NGS using a panel covering major drivers of pancreatic ductal neoplasia. For PanINs with sufficient DNA, whole exome sequencing and deep sequencing of KRAS was also performed. Across the 8 blocks, 10 PanINs shared no mutations with other PanINs in the same block, indicating independent clonal origin. In addition, 8 PanINs shared only KRAS hotspot mutations with numerous other unshared mutations, likely indicating independent PanINs that shared hotspot mutations by chance. Six spatially separate low grade PanINs shared both driver and passenger mutations with at least one other spatially unconnected PanIN, demonstrating the ability of PanIN cells to dissociate from one lesion and establish unconnected, genetically related PanINs nearby. Furthermore, 5 PanINs harbored multiple KRAS mutations within a single PanIN, suggesting a polyclonal origin. One PanIN lacked any driver gene mutations. The genetic origins of the remaining 7 spatially separate PanINs could not be resolved due to a lack of discrete mutations found by targeted sequencing. Our 3D genomic analysis of anatomically distinct PanINs demonstrates that the unexpectedly large number of PanINs in normal pancreas most often arise independently, providing new foundations for our understanding of early pancreatic tumorigenesis. Determining the mechanisms for this multifocal neoplasia is an important direction for future research. Citation Format: Alicia M. Braxton, Ashley Kiemen, Rachel Karchin, Yuchen Jiao, Pei-Hsun Wu, Ralph H Hruban, Denis Wirtz, Laura D. Wood. Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR007.

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

Tissue resident (TR) immune cells play important roles in facilitating tissue homeostasis, coordinating immune responses against infections and tumors, and maintaining immunological memory. While studies have shown these cells are distinct phenotypically and functionally from cells found in the peripheral blood (PB), the clonal relationship between these populations across tissues has not been comprehensively studied in primates or humans. We utilized autologous transplantation of rhesus macaque hematopoietic stem and progenitor cells containing high diversity barcodes to track the clonal distribution of T, B, myeloid and natural killer (NK) cell populations across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract, in comparison with PB longitudinally post-transplantation, in particular we focused on NK cells which do not contain endogenous clonal markers and have not been previously studied in this context. T cells demonstrated tissue-specific clonal expansions as expected, both overlapping and distinct from blood T cells. In contrast, B and myeloid cells showed a much more homogeneous clonal pattern across various tissues and the blood. The clonal distribution of TR NK was more heterogenous between individual animals. In some animals, as we have previously reported, we observed large PB clonal expansions in mature CD56-CD16+ NK cells. Notably, we found a separate set of highly expanded PB clones in CD16-CD56- (DN) NK subset that were also contributing to TR NK cells in all tissues examined, both in TR CD56-CD16+ and DN populations but absent in CD56+16- TR NK across all tissues analyzed. Additionally, we observed sets of TR NK clones specific to individual tissues such as lung or GI tract and sets of TR NK clones shared across liver and spleen, distinct from other tissues. Combined with prior functional data that suggests NK memory is restricted to liver or other TR NK cells, these clonally expanded TR NK cells may be of interest for future investigation into NK cell tissue immunological memory, with implications for development of NK based immunotherapies and an understanding of NK memory.