Serum Resistance Research Articles

Effect of Drug Resistance on the Pathogenicity and Virulence of Salmonella Typhimurium

Various strains of E. coli. and Salmonella typhimurium were studied in vitro for the presence of plasmids controlling haemolysin, drug resistance, serum resistance and colonization factor etc. They were also tested in mice to determine the probable role of the plasmids on the host pathogenicity. To determine the effect of associated R-factor on the pathogenicity and virulence of S. typhimurium, antibiotic sensitive and resistant strains were tested for virulence in mice. The experiments showed that drug resistance plasmids enhance the lethality of their host. This causes a serious problem in effective treatment of patient suffering with Salmonella and E. coli infections.

Exogenous glycine promotes oxidation of glutathione and restores sensitivity of bacterial pathogens to serum-induced cell death

Pathogenic strains of bacteria are often highly adept at evading serum-induced cell death, which is an essential complement-mediated component of the innate immune response. This phenomenon, known as serum-resistance, is poorly understood, and as a result, no effective clinical tools are available to restore serum-sensitivity to pathogenic bacteria. Here, we provide evidence that exogenous glycine reverses defects in glycine, serine and threonine metabolism associated with serum resistance, restores susceptibility to serum-induced cell death, and alters redox balance and glutathione (GSH) metabolism. More specifically, in Vibrio alginolyticus and Escherichia coli, exogenous glycine promotes oxidation of GSH to GSH disulfide (GSSG), disrupts redox balance, increases oxidative stress and reduces membrane integrity, leading to increased binding of complement. Antioxidant or ROS scavenging agents abrogate this effect and agents that generate or potentiate oxidation stimulate serum-mediated cell death. Analysis of several clinical isolates of E. coli demonstrates that glutathione metabolism is repressed in serum-resistant bacteria. These data suggest a novel mechanism underlying serum-resistance in pathogenic bacteria, characterized by an induced shift in the GSH/GSSG ratio impacting redox balance. The results could potentially lead to novel approaches to manage infections caused by serum-resistant bacteria both in aquaculture and human health.

Subinhibitory Concentrations of Antibiotics Alter the Response of Klebsiella pneumoniae to Components of Innate Host Defense.

Carbapenem-resistant Klebsiella pneumoniae isolates classified as multilocus sequence type 258 (ST258) are a problem in health care settings in many countries globally. ST258 isolates are resistant to multiple classes of antibiotics and can cause life-threatening infections, such as pneumonia and sepsis, in susceptible individuals. Treatment strategies for such infections are limited. Understanding the response of K. pneumoniae to host factors in the presence of antibiotics could reveal mechanisms employed by the pathogen to evade killing in the susceptible host, as well as inform treatment of infections. Here, we investigated the ability of antibiotics at subinhibitory concentrations to alter K. pneumoniae capsular polysaccharide (CPS) production and survival in normal human serum (NHS). Unexpectedly, pretreatment with some of the antibiotics tested enhanced ST258 survival in NHS. For example, a subinhibitory concentration of mupirocin increased survival for 7 of 10 clinical isolates evaluated and there was increased cell-associated CPS for 3 of these isolates compared with untreated controls. Additionally, mupirocin pretreatment caused concomitant reduction in the deposition of the serum complement protein C5b-9 on the surface of these three isolates. Transcriptome analyses with a selected ST258 isolate (34446) indicated that genes implicated in the stringent response and/or serum resistance were upregulated following mupirocin treatment and/or culture in NHS. In conclusion, mupirocin and/or human serum causes changes in the K. pneumoniae transcriptome that likely contribute to the observed decrease in serum susceptibility via a multifactorial process. Whether these responses can be extended more broadly and thus impact clinical outcome in the human host merits further investigation. IMPORTANCE The extent to which commensal bacteria are altered by exposure to subinhibitory concentrations of antibiotics (outside resistance) remains incompletely determined. To gain a better understanding of this phenomenon, we tested the ability of selected antibiotics (at subinhibitory concentrations) to alter survival of ST258 clinical isolates in normal human serum. We found that exposure of ST258 to antibiotics at low concentrations differentially altered gene expression, capsule production, serum complement deposition, and bacterial survival. The findings were isolate and antibiotic dependent but provide insight into a potential confounding issue associated with ST258 infections.

Characterization of virulence factors and antibiotic resistance pattern of uropathogenic Escherichia coli strains in a tertiary care center.

Background: Urinary tract infections (UTI) are the most prevalent bacterial infection in humans. The uropathogenic E. coli (UPEC) expresses a range of virulence factors that contribute to their pathogenicity . The emergence of multidrug resistance (MDR)-associated UTI is increasing. This study monitors the distribution of virulence factors among UPEC strains to note the antibiogram, outcome and type of associated UTI. Methods: A prospective cross-sectional time-bound study of six months was done on clinically significant urinary isolates of Escherichia coli. Detection of haemolysin production and serum resistance was done by phenotypic methods. Genotypic characterization of the virulence genes ( papC, iutA, hlyA, cnf1) was done by multiplex PCR. Demographic data, clinical history, antibiogram and type of UTI was collected from clinical case records. Results:75 E.coli isolates from patients with suspected UTIs were included. Females had a higher preponderance of UTI (66.7%). 93% of patients were adults and the remaining 7% were from paediatrics. 24 (32%) isolates showed haemolysis by plate haemolysis and all isolates were serum-resistant. Out of 75 isolates, 65 were positive for at least one of four targeted genes, while remaining ten isolates were negative for all four genes. Multidrug resistance was found in 40 (53.3%) isolates. 97.4% of the UTI cases had a favourable clinical outcome at discharge. Mortality due to urosepsis was 2.6%. Conclusion: Association of hemolysin production with resistance to imipenem and norfloxacin in UPEC strains was significant. Presence of hlyA gene is positively associated with ceftazidime resistance. Nitrofurantoin, piperacillin, tazobactam, and cefaperazone sulbactam are possible candidates for empirical therapy of UTIs. Drugs like aminoglycosides, carbapenems and fosfomycin may be used as reserve drugs in the treatment of MDR-UTI. However, inappropriate usage can increase antibiotic resistance. Hence proper selection of antibiotics in hospitals taking into account the local antibiogram is needed to reduce the emergence of antibiotic resistance.

Transcriptomic and Metabonomic Profiling Reveals the Antihyperlipidemic Effects of Tartary Buckwheat Sprouts in High-Fat-Diet-Fed Mice.

Flavonoids are known for potent antioxidant activity and antihyperlipidemia. As a result of the few antinutritional factors and high bioactive substances, such as flavonoids, sprouts of tartary buckwheat (Fagopyrum tataricum, STB) have become healthy food. This study aims to unravel the antihyperlipidemic effects of STB in vivo and its potential mechanism through transcriptomic and metabonomic analysis. The physiological parameters of mice administered the high-fat diet with or without 2.5 and 5% of STB for 10 weeks were recorded. Liquid chromatography-tandem mass spectrometry and RNA sequencing were applied to obtain the serum lipid metabolomic and hepatic transcriptomic profiling, respectively. Results revealed that STB could significantly alleviate the increase of body weight, liver, and abdominal adipose while ameliorating the lipid content in serum and insulin resistance of mice fed with a high-fat diet. Notably, the metabonomic analysis identified the core differential metabolites mainly enriched in the pathways, such as fat digestion and absorption, insulin resistance, and other processes. Transcriptomic results revealed that STB significantly altered the expression levels of PIK3R1, LRP5, SLC10A2, and FBXO21. These genes are involved in the PI3K-AKT signaling pathway, digestion and absorption of carbohydrates, and type II diabetes mellitus pathways. In this study, STB exhibited remarkable influence on the metabolism of lipids and glucose, exerting antihyperlipidemic effects. STB have the potential for the development and application of a lipid-lowering health food.

FHUSPA2/10 is a bactericidal monoclonal antibody targeting multiple repeated sequences of Moraxella catarrhalis UspA2

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.

An outbreak of extensively drug-resistant and hypervirulent Klebsiella pneumoniae in an intensive care unit of a teaching hospital in Southwest China.

Extensively drug-resistant and hypervirulent Klebsiella pneumoniae (XDR-hvKp) is a new problem for patients in Intensive Care Unit (ICU) and can become an even more severe threat if resistant to tigecycline, considered one of the ‘last lines of defense’ drugs. This study collected seven non-replicated tigecycline-resistant XDR-hvKp from seven patients and performed genome analysis and epidemiological investigation using whole genome equencing (WGS) and other methods. All strains in this study were identified as ST11-KL64 and showed high resistance to antibiotics such as β-lactams, aminoglycosides, quinolones, and tigecycline, and one strain was also resistant to colistin. All strains were determined to be hvKp by the results of serum resistance assay and Galleria mellonella infection models. All strains had resistance genes bla CTX-M-65,bla KPC-2,bla LAP-2,bla TEM-1B, rmtB, and qnrS1 and virulence factors such as rmpA, rmpA2, and aerobactin (iucABCD, iutA). The expression of the AcrAB-TolC efflux pump was upregulated in all strains, and the expression levels of the gene pmrK was significantly upregulated in colistin-resistant strain DP compared to colistin-sensitive strain WT in this study. In conclusion, we described an outbreak caused by tigecycline-resistant XDR-hvKp in the ICU of a teaching hospital in southwest China. The spread of these superbugs poses a great threat to patients and therefore requires us to closely monitor these XDR-hvKp and develop relevant strategies to combat them.

Comparison of Virulence between Two Main Clones (ST11 and ST307) of Klebsiella pneumoniae Isolates from South Korea.

In this study, we investigate the characteristics of two main clones of carbapenemase-producing Klebsiella pneumoniae isolates from South Korea, ST11 and ST307, including carbapenem-susceptible isolates. Antibiotic susceptibility, serotype or wzi allelic type, the presence of virulence genes, and virulence with respect to serum resistance and macrophage internalization were determined for ST11 and ST307 isolates. ST11 isolates had a wide range of characteristics, including serotype and virulence, compared with those of homogeneous ST307 isolates. The wzi14 or K14 type had higher virulence than that of other serotypes among the ST11 isolates, and the homogeneous ST307 isolates showed similar virulence level as that of the wzi14-type ST11 isolates. Our data suggest that it is necessary to monitor not only the introduction and spread of a specific clone, but also its detailed serotype.

Impaired pancreatic beta-cell function after a single dose of oral iron: A before-and-after (pre-post) study.

Although in vitro and animal studies have shown that iron loading in pancreatic beta cells impairs insulin secretion, no human studies have documented the acute effects of oral iron on beta-cell insulin secretory capacity. In the present study, we determined beta-cell insulin secretory capacity at baseline and after a single oral dose of iron (ferrous sulphate, 120 mg elemental iron) in healthy male individuals. Fifteen healthy male volunteers underwent an oral glucose tolerance test (OGTT) to document baseline glucose tolerance and insulin secretion kinetics (baseline OGTT). One week later, the same subjects underwent a second OGTT, 2 h after an oral dose of ferrous sulphate (120 mg of elemental iron) (post-iron OGTT). Changes in disposition index, insulin secretion kinetics, glucose tolerance, insulin resistance, insulin clearance and iron-related parameters in serum were determined. Compared to baseline OGTT, the areas under the curve (AUC) for serum iron and transferrin saturation increased by 125% and 118%, respectively, in the post-iron OGTT. The disposition index decreased by 20% (p = 0.009) and the AUC for glucose concentrations increased by 5.7% (p < 0.001) during the post-iron OGTT. The insulin secretion rate was marginally lower during the first hour (-3.5%, p = 0.63), but became significantly higher during the second hour (22%, p = 0.005) of the post-iron OGTT. Insulin resistance and insulin clearance rate were not affected by iron intake. The decrease in disposition index and glucose tolerance observed after the oral dose of iron points to an acute iron-induced impairment in pancreatic beta-cell insulin secretory capacity.

Correlation of phenotypic and genotypic virulence markers, antimicrobial susceptibility pattern, and outcome of Pseudomonas aeruginosa sepsis infection.

Pseudomonas aeruginosa (PA) possesses several virulence genes that enable them to evade the immune system and to cause injury in the host tissue. However, the number of studies that characterized the virulence genes profile in PA sepsis is limited. The main objective of this study was to identify and characterize virulence genes in PA causing sepsis, as well as investigate the relationship between virulence genes, antimicrobial susceptibility patterns, and infection outcomes. A prospective study, conducted between October 2020-October 2021, isolates were recovered from blood samples and identified using standard microbiological procedures. Phenotypic techniques were used to screen for capsule, siderophore production, biofilm formation, serum resistance, hemolysin production, and protease. Molecular techniques were performed to screen for alginate D (alg D), exoenzyme S (Exo S), exotoxin A (tox A), phospholipase H (plc H), phospholipase N (plc N), and elastase B (las B). Kirby-Bauer disc diffusion method was used to determine the antimicrobial susceptibility pattern of isolates, which was then interpreted according to the CLSI 2021 guidelines. Out of the n=215 Gram-negative bacteria recovered from sepsis patients during our study, n=20 were Pseudomonas aeruginosa. PA isolates were susceptible to all antibiotics tested except for 3 of the isolates that were resistant to gentamycin, 2 to imipenem, and 1 to ceftazidime, cefepime, meropenem, tobramycin, and amikacin. The most prevalent virulence genes present were capsule (100%), siderophore production (100%), alg D (100%), Las B (100%), and Tox A (100%). Our study found that PA causing sepsis harbours a high level of virulence genes. However, the high presence of virulence factors was not statistically associated with antimicrobial susceptibility, as most isolates in our study were susceptible to the antibiotics tested.

Beta-lactam induced morphological changes in serum of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae blood isolates

Klebsiella pneumoniae is an opportunistic pathogen, which frequently causes bacteremia. Ceftazidime and meropenem, two important beta-lactam antibiotics for treatment of K. pneumoniae infections, induce morphological changes in bacteria when examined in vitro. Thirty clinical Klebsiella spp. Bacteremia isolates were analyzed for antimicrobial resistance and serum resistance. To determine whether complement influenced the resistance to ceftazidime of extended-spectrum beta-lactamase producing-isolates and sensitivity to meropenem, one serum resistant and one partly serum sensitive isolate were analyzed in normal human serum, heat-inactivated human serum, and growth medium with addition of beta-lactam antibiotics.HA391 was resistant to ceftazidime and had identical minimum inhibitory concentrations for meropenem in normal human serum, heat-inactivated serum and RPMI. In normal human serum, HA233 was inhibited by ceftazidime and had lower inhibitory concentrations of meropenem. Morphological changes induced by serum and beta-lactam antibiotics were analyzed by light- and electron microscopy. Light microscopy showed elongation of bacteria treated with ceftazidime. By electron microscopy membrane attack complexes were observed for HA233 in normal human serum, thereby facilitating beta-lactam antibiotics access to the periplasmic space and the peptidoglycan layer, explaining the increased killing of HA233 by beta-lactam antibiotics. Complement did not enhance beta-lactam killing of HA391, underlining the importance of serum susceptibility.

Intestinal Tract of Broiler Chickens as a Reservoir of Potentially Pathogenic Curli Producing ESβL Escherichia coli

Commensal enteric E. coli in healthy birds have virulence genes which enable them to be pathogenic under suitable conditions. A total of 80 E. coli isolates recovered from the intestine of healthy broiler chickens were tested for some virulence characters based on Congo red (CR) binding assay, hemolysis, serum resistance and biofilm formation. The virulent isolates were identified serologically by slide agglutination test and O1 (25%) was the most prevalent enteric E. coli serogroups. The antibiogram profile revealed that all isolates were resistant to at least three different antibiotics. Amoxicillin and ceftriaxone showed the highest resistance (100%), followed by lincomycin, kanamycin, streptomycin and sulfamethoxazole-trimethoprim (96%). In contrast, 80 and 52% of the isolates were sensitive to amikacin and fosfomycin, respectively. The PCR revealed that all isolates harbored virulence genes (int1, ampC, fimH, iss and eaeA), in addition to 91, 16 and 16% of isolates had mcr1, pic and tsh genes, respectively, proving that avian intestinal reservoir of potentially pathogenic curli producing ESβL Escherichia coli.

Increased sensitivity of Aggregatibacter actinomycetemcomitans to human serum is mediated by induction of a bacteriophage.

Aggregatibacter actinomycetemcomitans, a Gram-negative oral pathobiont causing aggressive periodontitis and systemic infections, demonstrates serum resistance. We have identified a dsDNA-tailed bacteriophage, S1249, which was found to convert from this microorganism inducible by human serum into a lytic state to kill the bacterium. This phage demonstrated active transcripts when exposed to human serum: 20% of genes were upregulated more than 10-fold, and 45% of them were upregulated 5-10-fold when the bacterium was grown in the presence of human serum compared to without the presence of human serum. Transcriptional activation when grown in equine serum was less pronounced. This phage demonstrated a tail with inner rigid tubes and an outer contractile sheath, features of Myoviridae spp. Further characterization revealed that the lysogenized integration of the phage in the chromosome of A. actinomycetemcomitans occurred between the genes encoding cold-shock DNA-binding domain-containing protein (csp) and glutamyl-tRNA synthetase (gltX). Both phage DNA integrated lysogeny and nonintegrated pseudolysogeny were identified in the infected bacterium. A newly generated, lysogenized strain using this phage displayed similar attributes, including 63% growth inhibition compared to its isogenic phage-free strain when in the presence of human serum. Our data suggest that bacteriophage S1249 can be induced in the presence of human serum and enters the lytic cycle, which reduces the viability of infected bacteria in vivo.

7,8-Dihydroxyflavone alleviates Endoplasmic Reticulum Stress in cafeteria diet-induced metabolic syndrome

AimsProlonged Endoplasmic Reticulum Stress (ERS) is involved in the pathogenesis of metabolic syndrome, including type-2 diabetes mellitus, cardiovascular diseases, atherosclerosis, obesity, and fatty liver disease. There have been significant efforts to discover molecules to treat ERS and/or to ameliorate associate symptoms. In this study, we investigated the effect of 7,8-Dihydroxyflavone (7,8-DHF) on ERS in liver and pancreas tissues in a cafeteria (CAF) diet induced metabolic syndrome model. Main methodsMale C57BL/6 mice were fed CAF diet for 16 weeks and 7,8-DHF was administered intraperitoneally (5 mg/kg/day) for last four weeks. 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) in liver and pancreas tissues, insulin and interleukin-1β (IL-1β) in serum were analyzed by ELISA method and serum biochemistry parameters were analyzed with autoanalyzer. GRP78 and CHOP gene expression levels were determined by qRT-PCR. In addition, histopathological analyzes were performed on liver and pancreas tissues. Key findingsFindings revealed that CAF diet caused metabolic abnormalities, insulin resistance and inflammation in serum and triggered ERS in pancreas and liver tissues. 7,8-DHF treatment significantly reduced metabolic abnormalities by reducing serum biochemical parameters, HOMO-IR and IL-1β levels. qRT-PCR and ELISA results indicated that 7,8-DHF treatment down-regulated GRP78 and CHOP expression and protein levels in the liver and GRP78 expression in pancreas. Efficiency of 7,8-DHF in these tissues was also demonstrated by histopathological tests. SignificanceIn conclusion, CAF diet-induced metabolic syndrome model, 7,8-DHF suppressed ERS and ERS-induced metabolic disorders in both liver and pancreas. Therefore, 7,8-DHF may potentially be a novel therapeutic compound to ameliorate ERS and related metabolic symptoms.

Antibiogram profiles and virulence factors associated with water-borne gram-negative bacteria from Ogbese river, Edo State, Nigeria

Surface waters such as flowing rivers, fresh water lakes and hand dug wells are the major sources of water for most rural dwellers in Nigeria. These are often consumed without testing their potability. This study was conducted to assess the bacteriological quality of surface water sampled from five (5) sampling locations on Ogbese river in Ogbese and Olumoye towns, Ovia North East Local Government Area, Edo State. Water samples were collected in duplicates from the sampling locations during the months of June 2018 to February 2019. Bacteriological analysis of the water samples was conducted using the membrane filtration technique and isolates identified by standard cultural and genomic procedures. Disc diffusion technique was utilized to determine the antibiotic sensitivity profiles of the identified bacteria. Other microbiological analysis which comprised of screening for enterotoxin production, invasiveness, hemolysin production and serum resistance, which are all virulence factors were performed using relevant methods. The total heterotrophic bacterial count varied from 1.0 ± 32.0 ×104𝑐𝑓𝑢/𝑚𝑙 and 1.0 ±0.12 ×104𝑐𝑓𝑢/𝑚𝑙 to 9.7 ± 2.23 ×104𝑐𝑓𝑢/𝑚𝑙. The total coliform count ranged from 0.1 ± 0.02 ×102𝑐𝑓𝑢/𝑚𝑙 to 5.5 ± 0.05 ×102𝑐𝑓𝑢/𝑚𝑙. The observed differences between the mean heterotrophic bacterial and coliform counts recorded for Ogbese and Olumoye sampling points was significant (P&lt; 0.05). The identified bacterial isolates included; Escherichia coli, Enterobacter cloacae, Shigella flexneri, Salmonella enterica, Vibrio cholerae, Klebsiella pneumoniae and Pseudomonas aeruginosa Both E. coli (60.0%) and E. cloacae (60.0%) were the most frequently isolated water borne isolates cultured from Ogbese sampling point. All E. coli and S. enterica isolates exhibited sensitivity towards ciprofloxacin and streptomycin. All S. flexneri isolates were invasive and serum resistant. There is an urgent need by relevant Governmental and Non –Governmental agencies to conduct advocacy programs targeted at these communities on the necessity of treating and disinfecting abstracted water sourced directly from the water body prior to drinking.

Characterisation of clinical carbapenem-resistant K1 Klebsiella quasipneumoniae subsp. similipneumoniae strains harbouring a virulence plasmid

The continuous emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great challenge to human health owing to the associated extremely high morbidity and mortality. Klebsiella quasipneumoniae is a newly described bacterial species that is often misidentified as K. pneumoniae. Clinical K. quasipneumoniae strains have been reported worldwide, among which multidrug-resistant lineages have become a severe health problem, while less has been understood on this important pathogen. In this study, we characterised three clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates belonging to sequence type 367 (ST367) and capsular type K1 and containing several virulence genes, including salmochelin (iroBCDN), aerobactin (iucABCDiutA) and regulator of mucoid phenotype (rmpA/A2), as well as some resistance genes, including blaKPC-2, blaTEM-1, blaOKP-B-9 and oqxAB. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains containing virulence genes exhibited a higher level of virulence and serum resistance than a classical K. pneumoniae strain, while their virulence levels were slightly lower compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reports for the first time the genetic and virulence characterisation of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained blaKPC-2 and virulence genes, contributing to a better understanding of their resistance and pathogenicity as well as for epidemic surveillance worldwide.

Strain Specific Variations in Acinetobacter baumannii Complement Sensitivity.

The complement system is required for innate immunity against Acinetobacter baumannii, an important cause of antibiotic resistant systemic infections. A. baumannii strains differ in their susceptibility to the membrane attack complex (MAC) formed from terminal complement pathway proteins, but the reasons for this variation remain poorly understood. We have characterized in detail the complement sensitivity phenotypes of nine A. baumannii clinical strains and some of the factors that might influence differences between strains. Using A. baumannii laboratory strains and flow cytometry assays, we first reconfirmed that both opsonization with the complement proteins C3b/iC3b and MAC formation were inhibited by the capsule. There were marked differences in C3b/iC3b and MAC binding between the nine clinical A. baumannii strains, but this variation was partially independent of capsule composition or size. Opsonization with C3b/iC3b improved neutrophil phagocytosis of most strains. Importantly, although C3b/iC3b binding and MAC formation on the bacterial surface correlated closely, MAC formation did not correlate with variations between A. baumannii strains in their levels of serum resistance. Genomic analysis identified only limited differences between strains in the distribution of genes required for serum resistance, but RNAseq data identified three complement-resistance genes that were differentially regulated between a MAC resistant and two MAC intermediate resistant strains when cultured in serum. These data demonstrate that clinical A. baumannii strains vary in their sensitivity to different aspects of the complement system, and that the serum resistance phenotype was influenced by factors in addition to the amount of MAC forming on the bacterial surface.

Abstract 901: Sox2 and Oct4 activity identify CSC populations in ovarian cancer

Abstract High Grade Serous Ovarian Cancer is the most lethal gynecological cancer, mainly due to late-stage diagnosis and prevalence of chemotherapy-resistant disease. Tumors are comprised of heterogenous cancer cells, which makes targeted therapies challenging. Studies have identified a subset of cancer stem-like cells (CSCs) that, unlike bulk tumor cells, can evade chemotherapy and induce relapse. Cell surface markers such as CD133, CD44, CD117, or ALDH enzyme activity are typically used to identify and isolate CSCs; however, these markers are inconsistent across cell lines and patient samples making them unreliable for isolating CSCs. Consistent indicators of CSCs may be activity of stem cell transcription factors, such as SOX2, OCT4, and NANOG, which are known to promote long-term self-renewal and asymmetric division, processes required for tumor repopulation. We hypothesize that a reporter responding to SOX2 and OCT4 activity will represent a functionally relevant and reliable marker for identifying CSCs capable of enabling relapse. We have stably transduced a reporter (termed SORE6), which detects SOX2 and OCT4 activity, into OV90 and ACI23 ovarian cancer cells. SORE6+ cells, isolated using fluorescence assisted cell sorting (FACS), have 2 to 15-fold increases in SOX2/OCT4/NANOG transcripts, relative to SORE6- cells. Comparison of CSC markers in chemotherapy versus vehicle conditions suggests that SORE6 more consistently identifies the CSC population. Specifically, flow cytometry analysis of ACI23 cells showed a 2-fold increase in SORE6+ cells, 7-fold increase in CD117+ cells, and a 1.2-fold increase in CD133 cells in carboplatin treated cells relative to vehicle. For OV90, SORE6+ increases again by 2-fold, while CD117+ increases by 5-fold and CD133 increases by 1.5-fold. SORE6+ cells also showed significantly enhanced spheroid formation efficiency in low serum conditions and resistance to carboplatin and paclitaxel chemotherapies, relative to SORE6- cells. Finally, in vivo limiting dilution studies in mice showed a significant difference in tumorigenicity of SORE6+ cells at low dilutions in ACI23 cells, but not for OV90 cells. Flow cytometry analysis of disassociated tumors showed that tumors created using pure SORE6- cells remained SORE6-, while tumors created using pure SORE6+ cells maintained only 10% SORE6+, indicating the ability of SORE6+ cells to asymmetrically divide to create a heterogeneous tumor. In conclusion, these data suggest that the SORE6 reporter identifies a CSC population within ovarian cancer cell lines and could be a useful tool for consistent isolation of CSCs. Use of this reporter will enhance our understanding of mechanisms, such as SOX2 and OCT4 activity, that support chemoresistance and tumor repopulation. More reliable identification of CSCs will enable the design of therapies to overcome chemotherapy resistance and disease recurrence. Citation Format: Luisjesus S. Cruz, Mikella Robinson, Samuel F. Gilbert, Omar Lujano-Olazaba, Logan A. Alexander, Alex Horkowitz, Carrie D. House. Sox2 and Oct4 activity identify CSC populations in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 901.

Cellular RNA Targets of Cold Shock Proteins CspC and CspE and Their Importance for Serum Resistance in Septicemic Escherichia coli.

ABSTRACTThe RNA chaperones, cold shock proteins CspC and CspE, are important in stress response and adaptation. We studied their role in the pathogenesis of a virulent Escherichia coli, representative of extraintestinal pathogenic E. coli (ExPEC) which are serum resistant and septicemic. We performed a global analysis to identify transcripts that interact with these cold shock proteins (CSPs), focusing on virulence-related genes. We used CLIP-seq, which combines UV cross-linking, immunoprecipitation and RNA sequencing. A large number of transcripts bound to the CSPs were identified, and many bind both CspC and CspE. Many transcripts were of genes involved in protein synthesis, transcription and energy metabolism. In addition, there were virulence-related genes, (i.e., fur and ryhB), essential for iron homeostasis. The CLIP-seq results were validated on two transcripts, clpX and tdcA, reported as virulence-associated. Deletion of either CspC or CspE significantly decreased their transcript levels and in a double deletion mutant cspC/cspE, the transcript stability of tdcA and clpX was reduced by 32-fold and 10-fold, respectively. We showed that these two genes are important for virulence, as deleting either of them resulted in loss of serum resistance, a requirement for sepsis. As several virulence-related transcripts interact with CspC or CspE, we determined the importance of these proteins for growth in serum and showed that deletion of either gene significantly reduced serum survival. This phenotype could be partially complemented by cspE and fully complemented by cspC. These results indicate that the two RNA chaperones are essential for virulence, and that CspC particularly critical.IMPORTANCE Virulent Escherichia coli strains that cause infections outside the intestinal tract—extraintestinal pathogenic E. coli (ExPEC)—constitute a major clinical problem worldwide. They are involved in several distinct conditions, including urinary tract infections, newborn meningitis, and sepsis. Due to increasing antibiotic resistance, these strains are a main factor in hospital and community-acquired infections. Because many strains, which do not cross-react immunologically are involved, developing a simple vaccine is not possible. Therefore, it is essential to understand the pathogenesis of these bacteria to identify potential targets for developing drugs or vaccines. One of the least investigated systems involves RNA binding proteins, important for stability of transcripts and global gene regulation. Two such proteins are CspC and CspE (“cold shock proteins”), RNA chaperones involved in stress adaptation. Here we performed a global analysis to identify the transcripts which are affected by these two chaperones, with focus on virulence-associated transcripts.

Contributions of Yersinia pestis outer membrane protein Ail to plague pathogenesis.

Pathogenic Yersinia have been a productive model system for studying bacterial pathogenesis. Hallmark contributions of Yersinia research to medical microbiology are legion and include: (i) the first identification of the role of plasmids in virulence, (ii) the important mechanism of iron acquisition from the host, (iii) the first identification of bacterial surface proteins required for host cell invasion, (iv) the archetypical type III secretion system, and (v) elucidation of the role of genomic reduction in the evolutionary trajectory from a fairly innocuous pathogen to a highly virulent species. The outer membrane (OM) protein Ail (attachment invasion locus) was identified over 30 years ago as an invasin-like protein. Recent work on Ail continues to provide insights into Gram-negative pathogenesis. This review is a synopsis of the role of Ail in invasion, serum resistance, OM stability, thermosensing, and vaccine development. Ail is shown to be an essential virulence factor with multiple roles in pathogenesis. The recent adaptation of Yersinia pestis to high virulence, which included genomic reduction to eliminate redundant protein functions, is a model to understand the emergence of new bacterial pathogens.