mutL Gene Research Articles

UV-C Exposure Enhanced the Cd2+ Adsorption Capability of the Radiation-Resistant Strain Sphingomonas sp. M1-B02

Microbial adsorption is a cost-effective and environmentally friendly remediation method for heavy metal pollution. The adsorption mechanism of cadmium (Cd) by bacteria inhabiting extreme environments is largely unexplored. This study describes the biosorption of Cd2+ by Sphingomonas sp. M1-B02, which was isolated from the moraine on the north slope of Mount Everest and has a good potential for biosorption. The difference in Cd2+ adsorption of the strain after UV irradiation stimulation indicated that the adsorption reached 68.90% in 24 h, but the adsorption after UV irradiation increased to 80.56%. The genome of strain M1-B02 contained antioxidant genes such as mutL, recA, recO, and heavy metal repair genes such as RS14805, apaG, chrA. Hydroxyl, nitro, and etceteras bonds on the bacterial surface were involved in Cd2+ adsorption through complexation reactions. The metabolites of the strains were significantly different after 24 h of Cd2+ stress, with pyocyanin, L-proline, hypoxanthine, etc., being downregulated and presumably involved in Cd2+ biosorption and upregulated after UV-C irradiation, which may explain the increase in Cd2+ adsorption capacity of the strain after UV-C irradiation, while the strain improved the metabolism of the antioxidant metabolite carnosine, indirectly increasing the adsorption capacity of the strains for Cd2+.

MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray–based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein–coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.

Faecalibacterium taiwanense sp. nov., isolated from human faeces.

Two Gram-stain-negative, straight rods, non-motile, asporogenous, catalase-negative and obligately anaerobic butyrate-producing strains, HLW78T and CYL33, were isolated from faecal samples of two healthy Taiwanese adults. Phylogenetic analyses of 16S rRNA and DNA mismatch repair protein MutL (mutL) gene sequences revealed that these two novel strains belonged to the genus Faecalibacterium. On the basis of 16S rRNA and mutL gene sequence similarities, the type strains Faecalibacterium butyricigenerans AF52-21T(98.3-98.1 % and 79.0-79.5 % similarity), Faecalibacterium duncaniae A2-165T(97.8-97.9 % and 70.9-80.1 %), Faecalibacterium hattorii APC922/41-1T(97.1-97.3 % and 80.3-80.5 %), Faecalibacterium longum CM04-06T(97.8-98.0% and 78.3 %) and Faecalibacterium prausnitzii ATCC 27768T(97.3-97.4 % and 82.7-82.9 %) were the closest neighbours to the novel strains HLW78T and CYL33. Strains HLW78T and CYL33 had 99.4 % both the 16S rRNA and mutL gene sequence similarities, 97.9 % average nucleotide identity (ANI), 96.3 % average amino acid identity (AAI), and 80.5 % digital DNA-DNA hybridization (dDDH) values, indicating that these two strains are members of the same species. Phylogenomic tree analysis indicated that strains HLW78T and CYL33 formed an independent robust cluster together with F. prausnitzii ATCC 27768T. The ANI, AAI and dDDH values between strain HLW78T and its closest neighbours were below the species delineation thresholds of 77.6-85.1 %, 71.4-85.2 % and 28.3-30.9 %, respectively. The two novel strains could be differentiated from the type strains of their closest Faecalibacterium species based on their cellular fatty acid compositions, which contained C18 : 1 ω7c and lacked C15 : 0 and C17 : 1 ω6c, respectively. Phenotypic, chemotaxonomic and genotypic test results demonstrated that the two novel strains HLW78T and CYL33 represented a single, novel species within the genus Faecalibacterium, for which the name Faecalibacterium taiwanense sp. nov. is proposed. The type strain is HLW78T (=BCRC 81397T=NBRC 116372T).

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors.

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

2180. Pseudomonas aeruginosa epidemic high-risk clones and their association with multidrug-resistant

Abstract Background Pseudomonas aeruginosa (PAE) is an opportunistic pathogen associated with a variety of hospital infections. Treatment of infections caused by PAE is challenging due to the emergence of multidrug-resistant (MDR), which is associated with increased morbidity, mortality, and healthcare costs. Clonal spread and the presence of resistance genes probably explain the successful propagation of MDR. In Ecuador, data on the molecular epidemiology, as well as circulating clones, are limited. Therefore, the objective of this study is to know the population structure of PAE by identifying clones in clinical samples in Quito-Ecuador. Methods A significant set (45) of randomly selected clinical PAE isolates, including multidrug and non-multidrug resistant isolates, were assigned to sequence types (STs) and compared them with their antibiotic susceptibility profile. The genetic diversity was assessed by applying the multilocus sequence typing (MLST) scheme (Figure 1) and the genetic relationships between different STs were corroborated by the phylogenetic neighbor-net network (Figure 2).Figure 1.Analysis of the allelic profiles using eBURST algorithm.Figure 2.Splits Tree showing the distribution of all the sequence types obtained for the clinical Pseudomonas aeruginosa isolates studied. The Splits Tree was based on the analysis of the allelic profiles of the acsA, aroE, guaA, mutL, nuoD, ppsA and trpE genes. Asterisk mark (*) indicates the MDR isolates Results The analysis of seven loci, acsA, aroE, guaA, mutL, nuoD, ppsA and trpE, identified 24 different ST and the most prevalent STs were ST-3750 and ST-253. A SplitsTree was constructed with all the isolates analyzed. The majority of the MDR isolates were included in ST-3750 and ST-253, also 3 singleton ST was identified in to MDR isolates. The 20 different ST was found to non-MDR isolates, and only 3 ST were found in more the one isolates. Conclusion The population structure of clinical PAE presents in these isolates indicate a significant association between MDR isolates and the clonal types: all ST-3750 and ST-253 isolates were MDR. ST-3750 is closely related strains to the ST111 (CC111), and ST-253 and ST111 are a group of successful high-risk clones and widely distributed worldwide. The multiresistant-isolates studied are grouped in the most prevalent sequence types found, and the susceptible isolates correspond mainly to singleton sequence types. Therefore, these high-risk clones and their association with multidrug resistance phenotypes are contributing to the spread of MDR in Quito-Ecuador. Disclosures Jeannete Zurita, n/a, Pfizer: Grant/Research Support

Lacticaseibacillus huelsenbergensis sp. nov., isolated from grass silage and corn silage.

Two rod-shaped, facultative anaerobic, Gram-stain-positive lactic acid bacteria were isolated from corn silage and grass silage. They were characterized using a polyphasic approach and designated as HO 1656T and HO 0673. Analysis of 16S rRNA gene sequence of both strains indicated that they belong to the Lacticaseibacillus group. The most closely related species, Lacticaseibacillus casei DSM 20011T and Lacticaseibacillus zeae DSM 20178T, have digital DNA–DNA hybridization (dDDH) values of 63.9 and 53.4%, respectively, with the novel strains. In contrast, the dDDH value between strains HO 1656T and HO 0673 is 99.3 %, clearly showing that these two isolated strains belong to the same species. According to analysis of the housekeeping genes (dnaK, mutL and pheS), both strains form a distinct cluster within the Lacticaseibacillus group. Strains HO 0673 and HO 1656T could produce acid from d-arabinose, adonitol, ribose, rhamnose, dulcitol, sorbitol, turanose, l-fucose and l-arabitol, unlike their nearest phylogenetic neighbour L. casei DSM 20011T. The major cellular fatty acids of both strains are C16 : 0 and C18 : 1 ω9c. The G+C content of the genomic DNA of both strains is 48.0 mol%. Thus, strains HO 1656T and HO 0673 represent a novel species based on their chemotaxonomic, phenotypic and phylogenetic characteristics. The name Lacticaseibacillus huelsenbergensis sp. nov. is proposed with the type strain HO 1656T (=DSM 115425T=NCIMB 15466T).

Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

485. A hypermutator strain of Pseudomonas aeruginosa in a COVID-19 patient with ventilator-associated pneumonia

Abstract Background P. aeruginosa is a cause of hospital-acquired and ventilator-associated pneumonia. Hypermutator P. aeruginosa strains have been described in patients with cystic fibrosis and chronic respiratory infections but are rare in patients with acute P. aeruginosa infection. This case describes a hypermutator strain of P. aeruginosa found in a patient with COVID-19-associated acute respiratory distress syndrome (ARDS). Methods Serial respiratory and blood cultures were collected. Short-read sequencing libraries were prepared using the Illumina Nextera XT kit, and whole-genome sequencing was performed using the Illumina NextSeq platform. Long-read sequencing libraries were prepared from unsheared genomic DNA using ligation sequencing kit SQK-LSK109 and sequenced on the Oxford MinION platform. Single nucleotide variants were identified by aligning reads from each isolate to the complete genome of the first available clinical isolate. Hypermutator assays were performed by measuring the mutation frequency rate for rifampin resistance. Antibiotic minimal inhibitory concentrations (MICs) were performed. Growth curves were performed with a starting OD600 of 0.1 with measurements taken every 30 minutes for 24 hours. Results Seventeen respiratory and five blood isolates were obtained throughout 62 days of hospitalization. Fourteen of the 22 isolates exhibited hypermutator phenotypes by rifampin resistance assays, which demonstrated rapid accumulation of mutations. All five bloodstream isolates were hypermutators. MIC testing noted increased resistance to aminoglycosides, fluoroquinolones, and aztreonam in the hypermutator isolates. All bloodstream isolates descended from a single progenitor noted on whole-genome sequencing. Each hypermutator strain contained a mutation in the mismatch repair gene mutL, previously associated with the hypermutator phenotype. Genetic Tree of Patient Isolates The genetic tree highlights hypermutator versus non-hypermutator single nucleotide variants Conclusion This case was notable for multiple isolates of hypermutator P. aeruginosa that persisted over weeks. The patient’s COVID-19 infection and acute respiratory distress syndrome may have facilitated persistence of the P. aeruginosa lineage, allowing a hypermutator lineage to emerge. Disclosures All Authors: No reported disclosures.

Genomic heterogeneity underlies multidrug resistance in Pseudomonas aeruginosa: A population-level analysis beyond susceptibility testing.

BackgroundPseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time.MethodsTwenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time.ResultsThe majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits.ConclusionsThe overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Maritimibacter alexandrii sp. nov., a New Member of Rhodobacteraceae Isolated from Marine Phycosphere

Marine phycosphere hosts cross-kingdom algae-bacteria interactions playing a variety of crucial roles in aquatic ecosystems especially for the prevention and control of harmful algal blooms (HABs). During the investigation of structural composition of phycosphere microbiota (PM) of diverse marine HAB dinoflagellates, a Gram-negative, strictly aerobic, non-motile and rod-shaped bacterium designated LZ-17T was isolated from the phycosphere of highly toxic Alexandrium catenella LZT09. The 16S rRNA gene sequence analysis and the multilocus sequence analysis (MLSA) based on five protein-coding housekeeping genes(atpD, gyrB, mutL, topA and rpoD)indicated that strain LZ-17T was affiliated to the genus Maritimibacter within the family Rhodobacteraceae, and closely related to Maritimibacter alkaliphilus HTCC2654T (99.1%), 'Maritimibacter harenae' DP07T (97.9%) and M. lacisalsi X12M-4T (95.7%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain LZ-17T and the type strain of M. alkaliphilus were 96.9% and 74.7%. However, strain LZ-17T could be clearly distinguished from its closest by the phenotypical and phenotypical characteristics. Strain LZ-17T contained Q-10 as its major isoprenoid quinone, and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0 and C16:0 2-OH as the predominant fatty acids (>10%). The major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. The DNA G + C content was 64.3mol%. Based on the polyphasic taxonomiccharacterization, strain LZ-17T represents a novel species of the genus Maritimibacter, for which the name Maritimibacter alexandrii sp. nov. is proposed, with the type strain LZ-17T (=CCTCC 2019005T = KCTC 72193T).

The mutL Gene as a Genome-Wide Taxonomic Marker for High Resolution Discrimination of Lactiplantibacillus plantarum and Its Closely Related Taxa.

The current taxonomy of the Lactiplantibacillus plantarum group comprises of 17 closely related species that are indistinguishable from each other by using commonly used 16S rRNA gene sequencing. In this study, a whole-genome-based analysis was carried out for exploring the highly distinguished target genes whose interspecific sequence identity is significantly less than those of 16S rRNA or conventional housekeeping genes. In silico analyses of 774 core genes by the cano-wgMLST_BacCompare analytics platform indicated that csbB, morA, murI, mutL, ntpJ, rutB, trmK, ydaF, and yhhX genes were the most promising candidates. Subsequently, the mutL gene was selected, and the discrimination power was further evaluated using Sanger sequencing. Among the type strains, mutL exhibited a clearly superior sequence identity (61.6–85.6%; average: 66.6%) to the 16S rRNA gene (96.7–100%; average: 98.4%) and the conventional phylogenetic marker genes (e.g., dnaJ, dnaK, pheS, recA, and rpoA), respectively, which could be used to separat tested strains into various species clusters. Consequently, species-specific primers were developed for fast and accurate identification of L. pentosus, L. argentoratensis, L. plantarum, and L. paraplantarum. During this study, one strain (BCRC 06B0048, L. pentosus) exhibited not only relatively low mutL sequence identities (97.0%) but also a low digital DNA–DNA hybridization value (78.1%) with the type strain DSM 20314T, signifying that it exhibits potential for reclassification as a novel subspecies. Our data demonstrate that mutL can be a genome-wide target for identifying and classifying the L. plantarum group species and for differentiating novel taxa from known species.

Antibiotic Resistance Characteristics of Pseudomonas aeruginosa Isolated from Keratitis in Australia and India.

This study investigated genomic differences in Australian and Indian Pseudomonas aeruginosa isolates from keratitis (infection of the cornea). Overall, the Indian isolates were resistant to more antibiotics, with some of those isolates being multi-drug resistant. Acquired genes were related to resistance to fluoroquinolones, aminoglycosides, beta-lactams, macrolides, sulphonamides, and tetracycline and were more frequent in Indian (96%) than in Australian (35%) isolates (p = 0.02). Indian isolates had large numbers of gene variations (median 50,006, IQR = 26,967–50,600) compared to Australian isolates (median 26,317, IQR = 25,681–33,780). There were a larger number of mutations in the mutL and uvrD genes associated with the mismatch repair (MMR) system in Indian isolates, which may result in strains losing their efficacy for DNA repair. The number of gene variations were greater in isolates carrying MMR system genes or exoU. In the phylogenetic division, the number of core genes were similar in both groups, but Indian isolates had larger numbers of pan genes (median 6518, IQR = 6040–6935). Clones related to three different sequence types—ST308, ST316, and ST491—were found among Indian isolates. Only one clone, ST233, containing two strains was present in Australian isolates. The most striking differences between Australian and Indian isolates were carriage of exoU (that encodes a cytolytic phospholipase) in Indian isolates and exoS (that encodes for GTPase activator activity) in Australian isolates, large number of acquired resistance genes, greater changes to MMR genes, and a larger pan genome as well as increased overall genetic variation in the Indian isolates.

MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her-2/PI3K/AKT Signaling.

The rapid onset of resistance to cetuximab (CTX) limits its clinical utility in colorectal cancer (CRC) patients. This study aims to understand a potential role of mismatch repair gene mutL homolog 1 (MLH1) in CTX response. Functional analysis of MLH1 in Her‐2/phosphoinositide 3‐kinases (PI3K)/PKB protein kinase (AKT)‐regulated CTX sensitivity is performed using human CRC specimens, CRC cell lines with different MLH1 expression levels, and a subcutaneous xenograft model. Overexpression, knockdown, small interfering RNA, and inhibitors are used to examine the role of MLH1 and HER‐2 downstream signaling and apoptotic targets in CTX sensitivity. Reduced MLH1 expression is correlated with unfavorable prognosis in cetuximab‐treated patients. MLH1 loss decreases CTX sensitivity through Her‐2/PI3K/AKT signaling and apoptosis resistance in culture and in xenografts, while MLH1 overexpression increases CTX sensitivity. Blocking Her‐2 signaling increases CTX sensitivity of microsatellite instability CRC in vitro and in vivo. MLH1 loss induces activation of Her‐2/PI3K/AKT signaling and leads to cetuximab resistance in colon cancer.

Genome-based reclassification of Lactobacillus casei: emended classification and description of the species Lactobacillus zeae.

Taxonomic relationships between Lactobacillus casei, Lactobacillus paracasei and Lactobacillus zeae have long been debated. Results of previous analyses have shown that overall genome relatedness indices (such as average nucleotide identity and core nucleotide identity) between the type strains L. casei ATCC 393T and L. zeae ATCC 15820T were 94.6 and 95.3 %, respectively, which are borderline for species definition. However, the digital DNA‒DNA hybridization value was 57.3 %, which was clearly lower than the species delineation threshold of 70 %, and hence raised the possibility that L. casei could be reclassified into two species. To re-evaluate the taxonomic relationship of these taxa, multilocus sequence analysis (MLSA) based on the concatenated five housekeeping gene (dnaJ, dnaK, mutL, pheS and yycH) sequences, phylogenomic and core genome multilocus sequence typing analyses, gene presence and absence profiles using pan-genome analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling analysis, cellular fatty acid compositions, and phenotype analysis were carried out. The results of phenotypic characterization, MLSA, whole-genome sequence-based analyses and MALDI-TOF MS profiling justified an independent species designation for the L. zeae strains, and supported an emended the description of the name of Lactobacillus zeae (ex Kuznetsov 1956) Dicks et al. 1996, with ATCC 15820T (=DSM 20178T=BCRC 17942T) as the type strain.

Genes and Proteomes Associated With Increased Mutation Frequency and Multidrug Resistance of Naturally Occurring Mismatch Repair-Deficient Salmonella Hypermutators.

The emergence of antibiotic-resistant Salmonella through mutations led to mismatch repair (MMR) deficiency that represents a potential hazard to public health. Here, four representative MMR-deficient Salmonella hypermutator strains and Salmonella Typhimurium LT2 were used to comprehensively reveal the influence of MMR deficiency on antibiotic resistance among Salmonella. Our results indicated that the mutation frequency ranged from 3.39 × 10–4 to 5.46 × 10–2 in the hypermutator. Mutation sites in MutS, MutL, MutT, and UvrD of the four hypermutators were all located in the essential and core functional regions. Mutation frequency of the hypermutator was most highly correlated with the extent of mutation in MutS. Mutations in MMR genes (mutS, mutT, mutL, and uvrD) were correlated with increased mutation in antibiotic resistance genes, and the extent of antibiotic resistance was significantly correlated with the number of mutation sites in MutL and in ParC. The number of mutation sites in MMR genes and antibiotic resistance genes exhibited a significant positive correlation with the number of antibiotics resisted and with expression levels of mutS, mutT, and mutL. Compared to Salmonella Typhimurium LT2, a total of 137 differentially expressed and 110 specifically expressed proteins were identified in the four hypermutators. Functional enrichment analysis indicated that the proteins significantly overexpressed in the hypermutators primarily associated with translation and stress response. Interaction network analysis revealed that the ribosome pathway might be a critical factor for high mutation frequency and multidrug resistance in MMR-deficient Salmonella hypermutators. These results help elucidate the mutational dynamics that lead to hypermutation, antibiotic resistance, and activation of stress response pathways in Salmonella.

Effect of dihydrotestosterone fluctuation on mismatch repair gene mutL homolog 1 in prostate epithelial cells and its mechanism

Objective To investigate the relationship between the fluctuation of dihydrotestosterone concentration and the expression of mutL homolog 1 (MLH1) in mismatch repair system in prostate epithelial cells (RWPE-2 cell line) and its mechanism. Methods RWPE-2 cells were randomly divided into blank group [anhydrous ethanol solvent and dihydrotestosterone (DHT) concentration of 0 nmol/L], constant concentration group (concentration of DHT: 1, 10 nmol/L) and fluctuating concentration group (concentration of DHT: 1, 10 nmol/L, fluctuating every 24 h). After different time (0, 24, 48 and 72 h), cell counting kit-8 was use to examine cell proliferation and the target genes in androgen receptor (AR) signaling pathway [prostate specific antigen (PSA), FK506 binding protein 51 (FKBP5)], protein kinase B (Akt) signaling pathway [Cyclin D1, B cell lymphoma/leukemia-2 (bcl-2)] and MLH1 was detected by Western blotting, real-time quantitative polymerase chain reaction (Real-time PCR) and immunofluorescence. Graph Pad 7.0 statistical software was used for analysis and differences between groups were analyzed by t test. Results The morphological changes of RWPE-2 cells had a certain concentration-dependent relationship with DHT: constant group and fluctuating group had more cells and better growth than blank group. After RWPE-2 cells were stimulated with DHT for 72 h, as compared with the blank group (6.904±0.143), the relative A values of the constant group [(7.073±0.103) and (8.508±0.187)] and the fluctuation group [(8.662±0.327) and (9.239±0.167)] increased statistically (t=6.805, 4.922, 10.6, P<0.01). Western blotting and RT-qPCR results confirmed that the mRNA and protein levels of every target gene were increased significantly in constant group and fluctuating group (P<0.05) as compared with blank group. Meanwhile, the difference in fluctuating group was more significant than constant group. Whereas, FKBP5 mRNA decreased in fluctuating group as compared with constant group (t=7.101, 10.760, 8.289, 8.088, 8.519, 9.157, 11.330, P<0.05). The distribution of above genes increased in the nucleus after DHT intervention with more significant difference in fluctuating group. Conclusion DHT can up-regulate the expression of the mismatch repair protein MLH1 in RWPE-2 cells, meanwhile, DHT fluctuations can increase this trend and AR and Akt signaling pathways-mediated proliferation is one of the mechanisms. Key words: Dihydrotestosterone fluctuation; Cell proliferation; Mismatch repair; Androgen receptor signal pathway; Protein kinase B signal pathway

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

635. Genomic Evolution and Progression of Antimicrobial Resistance in a Series of Extensively Drug-Resistant Pseudomonas aeruginosa (XDR-Pa) Isolates from a Cystic Fibrosis Lung Transplant Recipient

BackgroundChronic respiratory infection due to extensively drug-resistant Pseudomonas aeruginosa (XDR-Pa) is a significant cause of mortality in cystic fibrosis (CF) patients. The CF respiratory anatomy, chronic antibiotic use, and PA colonization creates a milieu for high evolutionary pressure and genetic diversity. We sought to explore the progression of antibiotic resistance and genome evolution of XDR-Pa in a longitudinal series of isolates collected from an18-year-old CF patient who underwent lung transplantation.MethodsConsecutive respiratory isolates were collected from December 2016 to March 2018. Standard disk diffusion methods were used to evaluate antimicrobial susceptibility. Whole-genome sequencing (WGS) data were obtained on an Illumina NextSeq and assembled. Variants were identified using the GATK HaplotypeCaller and their functional impact was determined using snpEff. Maximum likelihood phylogenetic trees were constructed using MEGA and BEAST. Panther was used to test for enrichment of Gene Ontology functional categories among mutated genes.ResultsPhylogenetic analysis of complete genome sequences showed that 18 isolates formed a monophyletic group. Analysis using BEAST showed that genomes shared a common ancestor that was present prior to transplant. Over 300 single nucleotide variants and small insertion-deletion mutations were found, in comparison with a reconstruction of the ancestral sequence (Figure 1). Shared patterns of antibiotic susceptibility profiles were largely concordant with phylogenetic clustering and trended toward a decrease in susceptibility over time. Two different frameshift mutations in the DNA mismatch repair gene mutL were found in 15 genomes and these exhibited an increased rate of transition to transversion mutations, consistent with a hypermutator phenotype.ConclusionWGS of XDR-Pa identified variations in antibiotic resistance and virulence genes. Changes in mutL likely accelerated the accumulation of mutations. Multiple related sub-groups of strains appear to have been circulating prior to transplant and continued to diverge during the treatment period. Correlating antibiotic pressure, susceptibility profiles, and WGS in XDR-Pa from a single patient reveals the clinical impact of genomic evolution in CF. Disclosures All authors: No reported disclosures.

The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression

Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.

Preventive potential of dietary inclusion of Brachystegia eurycoma (Harms) seeds on N-methyl-N-nitrosourea-induced colon carcinogenesis in Wistar rats

Ethnopharmacological relevanceBrachystegia eurycoma seed is used as dietary condiment and as part of recipes for treating colorectal disorders, while laboratory studies have established that it contains crude fiber and polyphenols which are important in cancer prevention. Aim of the studyTo establish the efficacy of a Nigerian diet in colon cancer prevention, a study was conducted to evaluate dietary inclusion of Brachystegia eurycoma seed in experimental colon carcinogenesis. MethodsRats undergoing intra-rectal instillations of N-Methyl-N-nitrosourea (MNU) were fed B. eurycoma included diets at 0%, 2.5%, 5% and 10% for a period of ten (10) weeks following which they were sacrificed; blood and tissues were monitored for biochemical, histological and immunohistochemical parameters. ResultsBrachystegia eurycoma significantly (P < 0.05) prevented MNU-induced elevation of malondialdehyde and carcinoembryonic antigen (CEA) as well as reduced activities of catalase and superoxide dismutase. The colon showed deep mucosal ulceration with moderate inter-glandular inflammation in the MNU control group, but only mild or no inflammation was observed in the colon of the MNU groups fed experimental diets. Similarly, colon immunohistochemistry assay showed that the dietary inclusion significantly prevented MNU-induced damage to mismatch repair gene (MutL homolog1). Positive relationship existed between fiber content of B. eurycoma seeds and MutL homolog1 protein expression while that between polyphenol/flavonoids contents of diets and CEA was negative. ConclusionThese data suggest that both dietary fiber and polyphenol/flavonoids contribute synergistically or additively to the potential preventive effect of B. eurycoma seeds in colon carcinogenesis, presumably through mechanisms that involve limiting the extent of oxidative stress and preventing or delaying the onset of pro-carcinogenic inflammatory processes.