In recent years, an increase of uropathogenic Escherichia coli (UPEC) strains with Multidrug-resistant (MDR) and Extensively Drug-resistant (XDR) profiles that complicate therapy for urinary tract infections (UTIs) has been observed and has directly impacted costs and extended hospital stays. The aims of this study were to determine MDR- and XDR-UPEC clinical strains, their virulence genes and their phylogenetic groups and to ascertain their relationship with integrons and genetic diversity. From a collection of 500 UPEC clinical strains, 103 were selected with MDR [79.61% (82/103)] and XDR [20.38% (21/103)] characteristics. MDR-UPEC clinical strains were mainly associated with phylogenetic groups D (54.87%) and B2 (39.02%) with a high percentage (≥70%) of several fimbrial genes (ecpA, fimH, csgA, and papGII), an iron uptake gene (chuA), and a toxin gene (hlyA). In addition, a moderate frequency (40-70%) of other genes (iutD, tosA, and bcsA) was observed. XDR-UPEC clinical strains were predominantly associated with phylogenetic groups B2 (47.61%) and D (42.85%), which grouped with ≥80 virulence genes, including ecpA, fimH, csgA, papGII, iutD, and chuA. A moderate frequency (40-70%) of the tosA and hlyA genes was observed. The class 1 and 2 integrons that were identified in the MDR- and XDR-UPEC strains were associated with phylogenetic groups D, B2 and A, while the XDR-UPEC strains that were associated with phylogenetic groups B2, D and A showed an extended-spectrum beta-lactamase (ESBL) phenotype. The modifying enzymes (aadA1, aadB, aacC, ant1, dfrA1, dfrA17 and aadA4) that were identified in the variable region of class 1 and 2 integrons from the MDR strains showed resistance to gentamycin (56.25% and 66.66%, respectively) and trimethoprim-sulfamethoxazole (84.61% and 66.66%, respectively). The MDR- and XDR-UPEC strains were distributed into seven clusters and were closely related to phylogenic groups B2 and D. The diversity analysis by PFGE showed 42.68% of clones of MDR-UPEC and no clonal association in the XDR-UPEC clinical strains. In conclusion, phylogenetic groups including virulence genes are widely associated with two integron classes (B2 and D) in MDR- and XDR-UPEC clinical strains.

World Health Organization (WHO) has prioritized the infectious emerging diseases such as Coronavirus Disease (COVID-19) in terms of research and development of effective tests, vaccines, antivirals, and other treatments. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), the etiological causative agent of COVID-19, is a virus belonging to risk group 3 that requires Biosafety Level (BSL)-3 laboratories and the corresponding facilities for handling. An alternative to these BSL-3/-4 laboratories is to use a pseudotyped virus that can be handled in a BSL-2 laboratory for study purposes. Recombinant Vesicular Stomatitis Virus (VSV) can be generated with complementary DNA from complete negative-stranded genomic RNA, with deleted G glycoprotein and, instead, incorporation of other fusion protein, like SARS-CoV-2 Spike (S protein). Accordingly, it is called pseudotyped VSV-SARS-CoV-2 S. In this review, we have described the generation of pseudotyped VSV with a focus on the optimization and application of pseudotyped VSV-SARS-CoV-2 S. The application of this pseudovirus has been addressed by its use in neutralizing antibody assays in order to evaluate a new vaccine, emergent SARS-CoV-2 variants (delta and omicron), and approved vaccine efficacy against variants of concern as well as in viral fusion-focused treatment analysis that can be performed under BSL-2 conditions.

Acinetobacter baumannii is an opportunistic pathogen and is one of the primary etiological agents of healthcare-associated infections (HAIs). A. baumannii infections are difficult to treat due to the intrinsic and acquired antibiotic resistance of strains of this bacterium, which frequently limits therapeutic options. In this study, five A. baumannii strains (810CP, 433H, 434H, 483H, and A-2), all of which were isolated from a child with leukemia M2, were characterized through antibiotic susceptibility profiling, the detection of genes encoding carbapenem hydrolyzing oxacillinases, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), adherence and invasion assays toward the A549 cell line, and the whole-genome sequence (WGS). The five strains showed Multidrug resistant (MDR) profiles and amplification of the bla_OXA-23 gene, belonging to ST758 and grouped into two PFGE clusters. WGS of 810CP revealed the presence of a circular chromosome and two small plasmids, pAba810CPa and pAba810CPb. Both plasmids carried genes encoding the Sp1TA system, although resistance genes were not identified. A gene-by-gene comparison analysis was performed among the A. baumannii strains isolated in this study and others A. baumannii ST758 strains (HIMFG and INCan), showing that 86% of genes were present in all analyzed strains. Interestingly, the 433H, 434H, and 483H strains varied by 8–10 single-nucleotide variants (SNVs), while the A2 and 810CP strains varied by 46 SNVs. Subsequently, an analysis using BacWGSTdb showed that all of our strains had the same resistance genes and were ST758. However, some variations were observed in relation to virulence genes, mainly in the 810CP strain. The genes involved in the synthesis of hepta-acylated lipooligosaccharides, the pgaABCD locus encoding poly-β-1-6-N-acetylglucosamine, the ompA gene, Csu pili, bap, the two-component system bfms/bfmR, a member of the phospholipase D family, and two iron-uptake systems were identified in our A. baumannii strains genome. The five A. baumannii strains isolated from the child were genetically different and showed important characteristics that promote survival in a hospital environment. The elucidation of their genomic sequences provides important information for understanding their epidemiology, antibiotic resistance, and putative virulence factors.

The Hospital Infantil de México Federico Gómez (HIMFG) is a tertiary care hospital in Mexico City where Escherichia coli is frequently isolated from the urine samples of pediatric patients with urinary tract infections. A collection of 178 urinary Escherichia coli (UEc) isolates associated with complicated and uncomplicated urinary tract infections were evaluated in this study. The patterns of resistance to 9 antibiotic classes showed that 60.7% of the UEc isolates had a highly multidrug-resistant (MDR) profile. Genetic diversity analyses of the UEc isolates showed a high variability and revealed 16 clusters associated with four phylogenetic groups, namely, groups A, B1, B2, and D. Phylogenetic group B2 was widely associated with the 16 clusters as well as with virulence and fitness genes. The virulence and fitness genes in the UEc isolates, which included fimbriae-, siderophore-, toxin-, and mobility-associated genes, were grouped as occurring at a low, variable, or high frequency. Interestingly, only the papF gene could be amplified from some UEc isolates, and the sequence analysis of the pap operon identified an insertion sequence (IS) element and gene loss. These data suggested pathoadaptability and the development of immune system evasion, which was confirmed by the loss of P fimbriae-associated agglutination in the UEc isolates. E. coli clone O25-ST131 had a prevalence of 20.2% among the UEc isolates; these isolates displayed both a highly MDR profile and the presence of the papGII, fimH, papGIII, iutD, sat, hlyA, and motA genes. In conclusion, the UEc isolates from complicated urinary tract infection (cUTI) were characterized as being MDR, highly genetically diverse, and associated with phylogenetic group B2 and many virulence and fitness genes. Additionally, gene loss and IS elements were identified in some UEc isolates identified as clone O25-ST131.

Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion in the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged fusion proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. The FimH, CsgA, and PapG stimulated the release of 372 to 398 pg/mL IL-6; interestingly, the FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, the FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies generated against FimH, CsgA, PapG, FC, and FCP blocked the adhesion of the CFT073 E. coli strain to HTB5 bladder cells. In conclusion, the FC and FCP proteins were highly stable, demonstrated antigenic properties, and induced cytokine release (IL-6 and IL-8); furthermore, antibodies generated against these proteins showed protection against bacterial adhesion.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to β-lactam and non-β-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.