Project description:Incomplete antibiotic removal in pharmaceutical wastewater treatment plants (PWWTPs) could lead to the development and spread of antibiotic-resistant bacteria (ARBs) and genes (ARGs) in the environment, posing a growing public health threat. In this study, two multiantibiotic-resistant bacteria, Ochrobactrum intermedium (N1) and Stenotrophomonas acidaminiphila (N2), were isolated from the sludge of a PWWTP in Guangzhou, China. The N1 strain was highly resistant to ampicillin, cefazolin, chloramphenicol, tetracycline, and norfloxacin, while the N2 strain exhibited high resistance to ampicillin, chloramphenicol, and cefazolin. Whole-genome sequencing revealed that N1 and N2 had genome sizes of 0.52 Mb and 0.37 Mb, respectively, and harbored 33 and 24 ARGs, respectively. The main resistance mechanism in the identified ARGs included efflux pumps, enzymatic degradation, and target bypass, with the N1 strain possessing more multidrug-resistant efflux pumps than the N2 strain (22 vs 12). This also accounts for the broader resistance spectrum of N1 than of N2 in antimicrobial susceptibility tests. Additionally, both genomes contain numerous mobile genetic elements (89 and 21 genes, respectively) and virulence factors (276 and 250 factors, respectively), suggesting their potential for horizontal transfer and pathogenicity. Overall, this research provides insights into the potential risks posed by ARBs in pharmaceutical wastewater and emphasizes the need for further studies on their impact and mitigation strategies.
Project description:With the global increase in the use of carbapenems, several gram-negative bacteria have acquired carbapenem resistance, thereby limiting treatment options. Klebsiella pneumoniae is one of such notorious pathogen that is being widely studied to find novel resistance mechanisms and drug targets. These antibiotic-resistant clinical isolates generally harbor many genetic alterations, and identification of causal mutations will provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance, in which mutated genes that also show significant expression changes among their functionally coupled genes become more likely candidates. For network-based analyses, we developed a genome-scale co-functional network of K. pneumoniae genes, KlebNet (www.inetbio.org/klebnet). Using KlebNet, we could reconstruct functional modules for antibiotic-resistance, and virulence, and retrieved functional association between them. With complementation assays with top candidate genes, we could validate a gene for negative regulation of meropenem resistance and four genes for positive regulation of virulence in Galleria mellonella larvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antimicrobial resistance and virulence of human pathogenic bacteria with genomic and transcriptomic profiles from antibiotic-resistant clinical isolates.
Project description:Background: It remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in its progression to becoming carbapenem resistant. Methods: Carbapenem mutation frequency was measured in multiple subclades of extended-spectrum β-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. Results: All thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P<1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific adaptations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. Conclusions: ESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2 subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.
Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB) is a recognized nosocomial pathogen with limited therapeutics options. Lactic acid bacteria (LAB) constitute a promising therapeutic alternative. Here we aimed to study the antibacterial properties of a collection of LAB strains using phenotypic and transcriptomic analysis against A. baumannii clinical strains. One strain, Lacticaseibacillus rhamnosus CRL 2244, exerts a strong inhibitory capacity on A. baumannii with a strong killing activity. Scanning electron microscopy images showed changes in the morphology of A. baumannii with an increase formation of outer membrane vesicles. Significant changes in the expression levels a wide variety of genes were observed. Interestingly, most of the modified genes were involved in metabolic pathway known to be associated with bacterial survival. The paa operon, Hut system, and fatty acid degradation were some of the pathways that have been induced. The data reveals the impact of Lcb. rhamnosus CRL 2244 on A. baumannii response, resulting in bacterial stress and subsequent cell death. These findings highlight the antibacterial properties of Lcb. rhamnosus CRL 2244 and its potential as an alternative or complementary strategy for treating infections. Further exploration and development of this LAB as a treatment option could provide valuable alternatives for combating CRAB infections.