ompC Genes Research Articles

Genomic Analysis of Enterobacter Species Isolated from Patients in United States Hospitals.

We analyzed the whole genome sequences (WGS) and antibiograms of 35 Enterobacter isolates, including E. hormaechei and E. asburiae, and the recently described E. bugandensis, E. kobei, E. ludwigii, and E. roggenkampii species. Isolates were obtained from human blood and urinary tract infections in patients in the United States. Our goal was to understand the genetic diversity of antimicrobial resistance genes and virulence factors among the various species. Thirty-four of 35 isolates contained an AmpC class blaACT allele; however, the E. roggenkampii isolate contained blaMIR-5. Of the six Enterobacter isolates resistant to ertapenem, imipenem, and meropenem, four harbored a carbapenemase gene, including blaKPC or blaNDM. All four isolates were mCIM-positive. The remaining two isolates had alterations in ompC genes that may have contributed to the resistance phenotype. Interpretations of cefepime test results were variable when disk diffusion and automated broth microdilution results were compared due to the Clinical Laboratory and Standards Institute use of the "susceptible dose-dependent" classification. The diversity of the blaACT alleles paralleled species identifications, as did the presence of various virulence genes. The classification of recently described Enterobacter species is consistent with their resistance gene and virulence gene profiles.

Characterization of genes related to the efflux pump and porin in multidrug-resistant Escherichia coli strains isolated from patients with COVID-19 after secondary infection

BackgroundEscherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin.MethodsA total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated.ResultsThe E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin.ConclusionsThe antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

Phagomagnetic separation in combination with real‐time PCR assay for detection of Salmonella Enteritidis and Typhimurium in dairy products

AbstractSalmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are the most commonly reported serotypes detected for the occurrence of Salmonellosis through foodborne transmission in recent years. In this study, a phagomagnetic separation in combination with qPCR (PhMS‐qPCR) based on phage T102 as a recognition element was developed for rapid enrichment and detection of Salmonella in dairy products. Phage T102 was coated onto magnetic beads for capturing Salmonella from samples through magnetic separation, with a capture efficiency of ~90% demonstrated. During the subsequent qPCR process, captured Salmonella was identified by detecting the OmpC gene, and specific sdf and STM4495 genes to further distinguish the serotypes of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST), respectively. The whole procedure can be performed in 2.5 h with a low detection limit of 10 CFU/mL in PBS. Subsequently, this detection strategy was successfully applied for the detection of Salmonella and serotype identification in spiked milk samples. This cell separation and detection system offers a promising alternative for rapid and accurate detection of Salmonella or Salmonella spp.

Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

Phenotypic and genomic characterization provide new insights into adaptation to environmental stressors and biotechnological relevance of mangrove Alcaligenes faecalis D334

Alcaligenes faecalis D334 was determined in this study as a salt-tolerant bacterium isolated from mangrove sediment. In response to 6% (w/v) NaCl, strain D334 produced the highest ectoines of 14.14 wt%. To understand adaptive features to mangrove environment, strain D334 was sequenced using Pacific BioScience platform, resulting in a circular chromosome of 4.23 Mb. Of note, D334 genome harbored 81 salt-responsive genes, among which two membrane-associated genes ompc and eric were absent in 3 selected A. faecalis genomes. Apart from that, a complete pathway for ectoine and 5-hydroxyectoine synthesis was predicted. To resist 40 mM H2O2, 46 genetic determinants contributing to oxidative stress response were employed. Moreover, two operons involved in polyhydroxyalkanoate (PHA) production were identified in the D334 genome, resulting in maximum PHA content of 5.03 ± 0.04 wt% and PHA concentration of 0.13 ± 0.001 g/L. A large flagellar biosynthesis operon contributing to swimming motility was found to be conserved in D334 and 8 other A. faecalis genomes. These findings shed light for the first time on the high versatility of A. faecalis D334 genome to adapt to mangrove lifestyle and the possibility to develop D334 as an industrial platform for PHA and 5-hydroxyectoine production.

Imipenem Induced Transcriptional Modulation of Multidrug Efflux Pumps and Porin Genes in Carbapenem Resistant Escherichia coli Isolates of Animal Origin

Background: The emergence of a multidrug resistance phenotype is most likely outcome of co-existing multiple mechanisms. The underlying molecular mechanism of carbapenem resistance is poorly understood in bacterial isolates of animal origin. The present study investigates the resistance mechanism for reduced susceptibility to carbapenem in E. coli. Methods: The study includes 12 multidrug resistant E.coli isolates showing decreased susceptibility to imipenem. Carbapenemase production was evaluated by the mCarba-NP test. The presence of the carbapenem resistance gene was confirmed by multiplex PCR. Efflux pump activity was accessed by broth microdilution method with or without selective efflux pump inhibitor. We analyzed differential expression of the outer membrane porin genes (ompF, ompC) and efflux pump genes (acrA, acrB) by quantitative real-time PCR analysis. Result: We observed increased expression of acrA and acrB genes in all the E. coli isolates under imipenem stress. A positive correlation (p less than 0.001) was evident between imipenem MIC and acrA and acrB over-expression in these isolates. A significant decrease in ompF and ompC expression was observed in these isolates under imipenem stress. The change in MIC was not correlated with the corresponding OmpC and OmpF gene expression. The results suggest the importance of efflux pump and porins proteins in the emergence of multidrug resistance besides carbapenemase production.

Shigella viruses Sf22 and KRT47 require outer membrane protein C for infection

Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops.

1285. Identification of KPC Omega Loop Variant R163S Conferring Ceftazidime-Avibactam Resistance

BackgroundWe report on a 56 year-old male with prolonged COVID-19 pneumonia who initially improved with dexamethasone and intubation but quickly decompensated. Clinical and radiologic features were consistent with VAP. Tracheal aspirate cultures grew carbapenem-resistant Enterobacter cloacae; meropenem (MEM) MIC was >8 ug/ml (resistant) while ceftazidime-avibactam (CZA) MIC was 2/4 ug/ml (susceptible). Lateral flow antigen assay detected a KPC enzyme. The patient was treated with CZA with steady improvement in respiratory function over the next two weeks. He then experienced an episode of tachycardia, prompting repeat culture. At this point the patient had been extubated: sputum culture grew KPC+ E. cloacae that now showed CZA-resistance (MIC >8/4 ug/ml) and paradoxical decrease in MEM MIC (4 ug/ml); meropenem-vaborbactam (< 2/8 ug/ml) was susceptible.MethodsThe pre- & post-CZA therapy E. cloacae isolates underwent whole genome sequencing using the Illumina 150bp paired end protocol; sequences were quality trimmed and compared.ResultsA point mutation in the plasmid blaKPC3 gene was identified in the post-CZA therapy isolate, an R163S mutation in the omega loop of the enzyme. ompC and ompF porin genes were analyzed to rule-out decreased influx as a mechanism for CZA-resistance: the pre- and post-CZA isolates had identical porin sequences.ConclusionThis case highlights emerging mutations within KPC carbapenemases that lead to resistance to ‘last-line’ antimicrobials like CZA. The presumptive mechanism is increased KPC active site promiscuity due to increased omega loop flexibility, allowing increased ceftazidime binding and hydrolysis, and decreased avibactam binding and beta lactamase inhibition. Paradoxically, MEM susceptibility improves after such omega loop mutations, likely due to decreased active site binding affinity, a ‘seesaw’ effect between MEM and CZA. While authors have reported MEM MICs falling into the ‘susceptible’ category after an omega loop variant, these bacteria invariably develop secondary mutations leading to MEM treatment failure. Fortunately, given our patient’s improved respiratory status, the post-CZA E. cloacae isolate was felt to reflect colonization and the patient was discharged home without antimicrobial therapy.Disclosures Romney Humphries, PhD D(ABMM), Accelerate Diagnostics (Individual(s) Involved: Self): Consultant, Shareholder; IHMA (Individual(s) Involved: Self): Consultant; Melinta (Individual(s) Involved: Self): Consultant; Momentum (Individual(s) Involved: Self): Grant/Research Support; Pattern (Individual(s) Involved: Self): Consultant; QPex (Individual(s) Involved: Self): Consultant; ThermoFisher (Individual(s) Involved: Self): Consultant; Torus (Individual(s) Involved: Self): Consultant

Display of Mycobacterium Tuberculosis Binding Peptide on Escherichia Coli Cell Surface

A recombinant expression vector was constructed to fuse the gene of small molecule peptide PK34 isolated from mycobacteria phages with the E. coli outer membrane protein OmpC gene, express recombinant PK34 and histidine labels on the outer membrane using the OmpC own promoter, and establish a surface display system with magnetic beads,and the surface display system was establisted with magnetic beads. The results showed that the peptide PK34 from mycobacteriophage could be displayed on the surface of E. coli by adopting the characteristic of outer membrane protein OmpC. Mycobacterium could be isolated through effective binding of PK34 within the surface display system. This study provides a new insight of the isolation and screening of Mycobacterium from clinical trial in the future.

Molecular Characterization and Survive Abilities of Salmonella Heidelberg Strains of Poultry Origin in Brazil.

The aim of the study was to evaluate the genotypic and phenotypic characteristics of 20 strains of S. Heidelberg (SH) isolated from broilers produced in southern Brazil. The similarity and presence of genetic determinants linked to virulence, antimicrobial resistance, biofilm formation, and in silico-predicted metabolic interactions revealed this serovar as a threat to public health. The presence of the ompC, invA, sodC, avrA, lpfA, and agfA genes was detected in 100% of the strains and the luxS gene in 70% of them. None of the strains carries the blaSHV, mcr-1, qnrA, qnrB, and qnrS genes. All strains showed a multidrug-resistant profile to at least three non-β-lactam drugs, which include colistin, sulfamethoxazole, and tetracycline. Resistance to penicillin, ceftriaxone (90%), meropenem (25%), and cefoxitin (25%) were associated with the presence of blaCTX–M and blaCMY–2 genes. Biofilm formation reached a mature stage at 25 and 37°C, especially with chicken juice (CJ) addition. The sodium hypochlorite 1% was the least efficient in controlling the sessile cells. Genomic analysis of two strains identified more than 100 virulence genes and the presence of resistance to 24 classes of antibiotics correlated to phenotypic tests. Protein-protein interaction (PPI) prediction shows two metabolic pathways correlation with biofilm formation. Virulence, resistance, and biofilm determinants must be constant monitoring in SH, due to the possibility of occurring infections extremely difficult to cure and due risk of the maintenance of the bacterium in production environments.

Roles of the EnvZ/OmpR Two-Component System and Porins in Iron Acquisition in Escherichia coli.

Escherichia coli secretes high-affinity Fe3+ chelators to solubilize and transport chelated Fe3+ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe2+ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe2+ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon.IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ, which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Regulation of biofilm formation by marT in Salmonella Typhimurium.

In this study, we aimed at identifying the regulatory role of marT gene, known as the regulator of misL, on 15 different biofilm-related genes in S. Typhimurium 14028 strain. We also tested the strains for their ability to form biofilm and determined the adherence characteristics of the wild type and the mutant strains of the organism on Caco-2 and HEp-2 cells. For comparative analyses of the candidate genes, individual gene mutations were created via antibiotic gene cassette insertion into each gene of interest. marT gene was cloned behind an arabinose inducible BAD promoter in order to control marT expression. This recombinant plasmid was transfer into each of the 15 mutant strains to investigate the level of expression of each single gene in the presence and absence of marT induction. Besides determination of variations in biofilm formation by each mutant strain, the attachment characteristics of them onto Caco-2 and HEp-2 cell lines were also reported. As a result of attachments experiments on polystyrene surfaces, it was determined that the biofilm production capacity of each mutant strain decreased in a statistically significant manner (p < 0.05). QRT-PCR trials indicated that the marT gene regulates the expression of 14 genes, namely fimA, fimD, fimF, fimH, stjB, stjC, csgA, csgD, ompC, sthB, sthE, rmbA, fliZ and yaiC, in a positive manner. QRT-PCR studies were also revealed that the MarT protein positively regulates its own promoter. When the adherence characteristics of the mutant strains and the wild-type were investigated by using Caco-2 and HEp-2 cells, it was determined that the single gene mutations did have no effect on bacterial adhesion. In view of our mutational analyses and biofilm formation studies, it was concluded that fliZ, ompC, rmbA, stjB and stjC genes are related with biofilm formation in Salmonella, besides other cellular functions of them. Taken together, our data suggested that the regulatory role of MarT protein is not only restricted to the regulation of misL gene expression, but it rather acts as a general regulator on the biofilm-related genes in Salmonella.

Tylosin exposure reduces the susceptibility of Salmonella Typhimurium to florfenicol and tetracycline

BackgroundAntibiotics exposure has been implicated in the emergence of bacterial strains that are resistant to structurally related or unrelated antibiotics. Tylosin is a macrolide antibiotic that has been administered to treat respiratory pathogenic bacteria in swine. Thus, this study was undertaken to evaluate the impact of exposure to a constant (3 μg/mL) and decreasing concentrations of tylosin on the susceptibility of Salmonella enterica serovar Typhimurium to various antibiotics.ResultsS. Typhimurium strains exposed to tylosin for 12 and 24 h in the in vitro dynamic model demonstrated at least an eight-fold increase in the minimum inhibitory concentrations (MICs) of florfenicol and tetracycline. Exposure to tylosin extended the lag-time of the growth curve and enhanced the generation of reactive oxygen species. Gene expression analysis demonstrated up-regulation of the acrAB and tolC Salmonella efflux pump genes and its global regulators (marA and soxS). Besides, the expression of ompC gene was down-regulated in tylosin exposed S. Typhimurium.ConclusionExposure to decreasing concentrations of tylosin could reduce the susceptibility of S. Typhimurium to florfenicol and tetracycline.

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli

BackgroundCarbapenem-resistant Enterobacteriaceae are considered by WHO as “critical” priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes.MethodsWe first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing.ResultsIn 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination.ConclusionsLineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.

Faropenem resistance causes in vitro cross-resistance to carbapenems in ESBL-producing Escherichia coli

ObjectiveFaropenem is an oral penem drug with activity against Gram-positive and Gram-negative bacteria, including CTX-M-15-type extended spectrum beta-lactamase (ESBL)-producing Enterobacteriales and anaerobic bacteria. As there are structural similarities, there is concern for the development of carbapenem cross-resistance; however, there are no studies confirming this. This study examined whether in vitro development of faropenem resistance in Escherichia coli isolates would result in cross-resistance to carbapenems. MethodsFour well-characterized E. coli isolates from the US Centers for Disease Control and Prevention antibiotic resistance isolate bank were utilized. Three isolates (NSF1, NSF2 and NSF3) are ESBL producers (CTX-M-15) and one (NSF4) is pan-susceptible. Faropenem minimum inhibitory concentrations (MICs) were determined and resistance was induced by serial passaging in increasing concentrations of faropenem. Susceptibility to carbapenems was determined and whole-genome sequencing (WGS) was performed to identify the underlying genetic mechanism leading to carbapenem resistance. ResultsFaropenem MIC increased from 1 mg/L to 64 mg/L within 10 days for NSF2 and NSF4 isolates, and from 2 mg/L to 64 mg/L within 7 days for NSF1 and NSF3 isolates. Reduced carbapenem susceptibility (ertapenem MIC ≥8 mg/L, doripenem/meropenem ≥2 mg/L and imipenem ≥1 mg/L) developed among three CTX-M-15-producing isolates that were faropenem-resistant, but not in NSF4 isolate that lacked ESBL enzyme. WGS analysis revealed non-synonymous changes in the ompC gene among three CTX-M-15-producing isolates, and a single nucleotide polymorphism (SNP) in the envZ gene in NSF4 isolate. ConclusionInduced resistance to faropenem causes cross-resistance to carbapenems among E. coli isolates containing CTX-M-15-type ESBL enzymes.

Expression and purification of an immunogenic SUMO-OmpC fusion protein of Salmonella Typhimurium in Escherichia coli

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.

Exposure to Al2O3 nanoparticles facilitates conjugative transfer of antibiotic resistance genes from Escherichia coli to Streptomyces

The spread of antibiotic resistance genes (ARGs) has become a global environmental issue; it has been found that nanoparticles (NPs) can promote the transfer of ARGs between bacteria. However, it remains unclear whether NPs can affect this kind of conjugation in Streptomyces, which mainly conjugate with other bacteria via spores. In the present study, we demonstrated that Al2O3 NPs significantly promote the conjugative transfer of ARGs from Escherichia coli (E. coli) ET12567 to Streptomyces coelicolor (S. coelicolor) M145 without the use of heat shock method. The number of transconjugants induced by Al2O3 particles was associated with the size and concentration of Al2O3 particles, exposure time, and the ratio of E. coli and spores. When nanoparticle size was 30 nm at a concentration of 10 mg/L, the conjugation efficiency reached a peak value of 182 cfu/108 spores, which was more than 60-fold higher than that of the control. Compared with nanomaterials, bulk particles exhibited no significant effect on conjugation efficiency. We also explored the mechanisms by which NPs promote conjugative transfer. After the addition of NPs, the intracellular ROS content increased and the expression of the classical porin gene ompC was stimulated. In addition, ROS enhanced the mRNA expression levels of conjugative genes by inhibiting global regulation genes. Meanwhile, expression of the conjugation-related gene intA was also stimulated, ultimately increasing the number of transconjugants. Our results indicated that Al2O3 NPs significantly promoted the conjugative transfer of ARGs from bacteria to spores and aggravated the diffusion of resistance genes in the environment.

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.