Association of blaVIM-2, blaPDC-35, blaOXA-10, blaOXA-488 and blaVEB-9 ß-Lactamase Genes with Resistance to Ceftazidime-Avibactam and Ceftolozane-Tazobactam in Multidrug-Resistant Pseudomonas aeruginosa.

Ceftazidime-avibactam and ceftolozane-tazobactam are approved for the treatment of complicated Gram-negative bacterial infections including multidrug-resistant (MDR) Pseudomonas aeruginosa. Resistance to both agents has been reported, but the underlying mechanisms have not been fully explored. This study aimed to correlate ß-lactamases with phenotypic resistance to ceftazidime-avibactam and/or ceftolozane-tazobactam in MDR-P. aeruginosa from Qatar. A total of 525 MDR-P. aeruginosa isolates were collected from clinical specimens between 2014 and 2017. Identification and antimicrobial susceptibility were performed by the BD PhoenixTM system and gradient MIC test strips. Of the 75 sequenced MDR isolates, 35 (47%) were considered as having difficult-to-treat resistance, and 42 were resistant to ceftazidime-avibactam (37, 49.3%), and/or ceftolozane-tazobactam (40, 53.3%). They belonged to 12 sequence types, with ST235 being predominant (38%). Most isolates (97.6%) carried one or more ß-lactamase genes, with blaOXA-488 (19%) and blaVEB-9 (45.2%) being predominant. A strong association was detected between class B ß-lactamase genes and both ceftazidime-avibactam and ceftolozane-tazobactam resistance, while class A genes were associated with ceftolozane-tazobactam resistance. Co-resistance to ceftazidime-avibactam and ceftolozane-tazobactam correlated with the presence of blaVEB-9, blaPDC-35, blaVIM-2, blaOXA-10 and blaOXA-488. MDR-P. aeruginosa isolates resistant to both combination drugs were associated with class B ß-lactamases (blaVIM-2) and class D ß-lactamases (blaOXA-10), while ceftolozane-tazobactam resistance was associated with class A (blaVEB-9), class C (blaVPDC-35), and class D ß-lactamases (blaOXA-488).

ß-lactamase-mediated resistance in MDR-Pseudomonas aeruginosa from Qatar.

BACKGROUND: The distribution of ß-lactam resistance genes in P. aeruginosa is often closely related to the distribution of certain high-risk international clones. We used whole-genome sequencing (WGS) to identify the predominant sequence types (ST) and ß-lactamase genes in clinical isolates of multidrug-resistant (MDR)-P. aeruginosa from Qatar METHODS: Microbiological identification and susceptibility tests were performed by automated BD Phoenix™ system and manual Liofilchem MIC Test Strips. RESULTS: Among 75 MDR-P. aeruginosa isolates; the largest proportions of susceptibility were to ceftazidime-avibactam (n = 36, 48%), followed by ceftolozane-tazobactam (30, 40%), ceftazidime (n = 21, 28%) and aztreonam (n = 16, 21.3%). All isolates possessed Class C and/or Class D ß-lactamases (n = 72, 96% each), while metallo-ß-lactamases were detected in 20 (26.7%) isolates. Eight (40%) metallo-ß-lactamase producers were susceptible to aztreonam and did not produce any concomitant extended-spectrum ß-lactamases. High risk ST235 (n = 16, 21.3%), ST357 (n = 8, 10.7%), ST389 and ST1284 (6, 8% each) were most frequent. Nearly all ST235 isolates (15/16; 93.8%) were resistant to all tested ß-lactams. CONCLUSION: MDR-P. aeruginosa isolates from Qatar are highly resistant to antipseudomonal ß-lactams. High-risk STs are predominant in Qatar and their associated MDR phenotypes are a cause for considerable concern.

Molecular profiling of multidrug-resistant river water isolates: insights into resistance mechanism and potential inhibitors.

Polluted waters are an important reservoir for antibiotic resistance genes and multidrug-resistant bacteria. This report describes the microbial community, antibiotic resistance genes, and the genetic profile of extended spectrum ß-lactamase strains isolated from rivers at, Pune, India. ESBL-producing bacteria isolated from diverse river water catchments running through Pune City were characterized for their antibiotic resistance. The microbial community and types of genes which confer antibiotic resistance were identified followed by the isolation of antibiotic-resistant bacteria on selective media and their genome analysis. Four representative isolates were sequenced using next generation sequencing for genomic analysis. They were identified as Pseudomonas aeruginosa, Escherichia coli, and two isolates were Enterobacter cloacae. The genes associated with the multidrug efflux pumps, such as tolC, macA, macB, adeL, and rosB, were detected in the isolates. As MacAB-TolC is an ABC type efflux pump responsible for conferring resistance in bacteria to several antibiotics, potential efflux pump inhibitors were identified by molecular docking. The homology model of their MacB protein with that from Escherichia coli K12 demonstrated structural changes in different motifs of MacB. Molecular docking of reported efflux pump inhibitors revealed the highest binding affinity of compound MC207-110 against MacB. It also details the potential efflux pump inhibitors that can serve as possible drug targets in drug development and discovery.

Comparative distribution of extended-spectrum beta-lactamase-producing Escherichia coli from urine infections and environmental waters.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli have been reported in natural environments, and may be released through wastewater. In this study, the genetic relationship between ESBL-producing E. coli collected from patient urine samples (n = 45, both hospitalized patients and out-patients) and from environmental water (n = 82, from five locations), during the same time period, was investigated. Three independent water samples were collected from the municipal wastewater treatment plant, both incoming water and treated effluent water; the receiving river and lake; and a bird sanctuary near the lake, on two different occasions. The water was filtered and cultured on selective chromID ESBL agar plates in order to detect and isolate ESBL-producing E. coli. Illumina whole genome sequencing was performed on all bacterial isolates (n = 127). Phylogenetic group B2 was more common among the clinical isolates than the environmental isolates (44.4% vs. 17.1%, p < 0.01) due to a significantly higher prevalence of sequence type (ST) 131 (33.3% vs. 13.4%, p < 0.01). ST131 was, however, one of the most prevalent STs among the environmental isolates. There was no significant difference in diversity between the clinical isolates (DI 0.872 (0.790-0.953)) and the environmental isolates (DI 0.947 (0.920-0.969)). The distribution of ESBL genes was similar: blaCTX-M-15 dominated, followed by blaCTX-M-14 and blaCTX-M-27 in both the clinical (60.0%, 8.9%, and 6.7%) and the environmental isolates (62.2%, 12.2%, and 8.5%). Core genome multi-locus sequence typing showed that five environmental isolates, from incoming wastewater, treated wastewater, Svartån river and Hjälmaren lake, were indistinguishable or closely related (≤10 allele differences) to clinical isolates. Isolates of ST131, serotype O25:H4 and fimtype H30, from the environment were as closely related to the clinical isolates as the isolates from different patients were. This study confirms that ESBL-producing E. coli are common in the aquatic environment even in low-endemic regions and suggests that wastewater discharge is an important route for the release of ESBL-producing E. coli into the aquatic environment.

Prevalence and Diversity of Antibiotic Resistance Genes in Swedish Aquatic Environments Impacted by Household and Hospital Wastewater.

Antibiotic-resistant Enterobacteriaceae and non-lactose fermenting Gram-negative bacteria are a major cause of nosocomial infections. Antibiotic misuse has fueled the worldwide spread of resistant bacteria and the genes responsible for antibiotic resistance (ARGs). There is evidence that ARGs are ubiquitous in non-clinical environments, especially those affected by anthropogenic activity. However, the emergence and primary sources of ARGs in the environment of countries with strict regulations for antibiotics usage are not fully explored. The aim of the present study was to evaluate the repertoire of ARGs of culturable Gram-negative bacteria from directionally connected sites from the hospital to the wastewater treatment plant (WWTP), and downstream aquatic environments in central Sweden. The ARGs were detected from genomic DNA isolated from a population of selectively cultured coliform and Gram-negative bacteria using qPCR. The results show that hospital wastewater was a reservoir of several class B ß-lactamase genes such as bla IMP-1 , bla IMP-2, and bla OXA-23, however, most of these genes were not observed in downstream locations. Moreover, ß-lactamase genes such as bla OXA-48, bla CTX-M-8, and bla SFC-1, bla V IM-1, and bla V IM-13 were detected in downstream river water but not in the WWTP. The results indicate that the WWTP and hospital wastewaters were reservoirs of most ARGs and contribute to the diversity of ARGs in associated natural environments. However, this study suggests that other factors may also have minor contributions to the prevalence and diversity of ARGs in natural environments.

Related carbapenemase-producing Klebsiella isolates detected in both a hospital and associated aquatic environment in Sweden.

Carbapenem antibiotics are one of the last-resort agents against multidrug-resistant (MDR) bacteria. The occurrence of carbapenemase-producing Enterobacteriaceae (CPE) in wastewater and aquatic environments is an indication of MDR bacteria in the community. This study evaluated CPE in aquatic environments and compared them to the local hospital isolates in Sweden. Phenotypic and genotypic analyses of antibiotic resistance of environmental and clinical CPE were performed. The relatedness of the isolates and possible clonal dissemination was evaluated using phylogenetic and phyloproteomic analysis. Klebsiella oxytoca carrying carbapenemase genes (blaVIM-1, blaIMP-29) were isolated from wastewater and the recipient river, while K. oxytoca (blaVIM-1) and Klebsiella pneumoniae (blaVIM-1, blaOXA-48, blaNDM-1, blaKPC-3) were isolated from patients at the local clinics or hospital. The K. oxytoca classified as sequence type 172 (ST172) isolated from the river was genotypically related to two clinical isolates recovered from patients. The similarity between environmental and clinical isolates suggests the dispersion of blaVIM-1 producing K. oxytoca ST172 from hospital to aquatic environment and the likelihood of its presence in the community. This is the first report of CPE in aquatic environments in Sweden; therefore, surveillance of aquatic and hospital environments for CPE in other urban areas is important to determine the major transfer routes in order to formulate strategies to prevent the spread of MDR bacteria.

Comparative Analysis of Stress Induced Gene Expression in Caenorhabditis elegans following Exposure to Environmental and Lab Reconstituted Complex Metal Mixture.

Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used Caenorhabditis elegans to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in C. elegans whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.