Dissemination Routes of Carbapenem and Pan-Aminoglycoside Resistance Mechanisms in Hospital and Urban Wastewater Canalizations of Ghana

Abstract

Wastewater has a major role in antimicrobial resistance (AMR) dynamics and public health. The impact on AMR of wastewater flux at the community-hospital interface in low- and middle-income countries (LMICs) is poorly understood. Therefore, the present study analyzed the epidemiological scenario of resistance genes, mobile genetic elements (MGEs), and bacterial populations in wastewater around the Tamale metropolitan area (Ghana). Wastewater samples were collected from the drainage and canalizations before and after three hospitals and one urban waste treatment plant (UWTP). From all carbapenem/pan-aminoglycoside-resistant bacteria, 36 isolates were selected to determine bacterial species and phenotypical resistance profiles. Nanopore sequencing was used to screen resistance genes and plasmids, whereas, sequence types, resistome and plasmidome contents, pan-genome structures, and resistance gene variants were analyzed with Illumina sequencing. The combination of these sequencing data allowed for the resolution of the resistance gene-carrying platforms. Hospitals and the UWTP collected genetic and bacterial elements from community wastewater and amplified successful resistance gene-bacterium associations, which reached the community canalizations. Uncommon carbapenemase/β-lactamase gene variants, like bla_DIM-1, and novel variants, including bla_VIM-71, bla_CARB-53, and bla_CMY-172, were identified and seem to spread via clonal expansion of environmental Pseudomonas spp. However, bla_NDM-1, bla_CTX-M-15, and armA genes, among others, were associated with MGEs that allowed for their dissemination between environmental and clinical bacterial hosts. In conclusion, untreated hospital wastewater in Ghana is a hot spot for the emergence and spread of genes and gene-plasmid-bacterium associations that accelerate AMR, including to last-resort antibiotics. Urgent actions must be taken in wastewater management in LMICs in order to delay AMR expansion. IMPORTANCE Antimicrobial resistance (AMR) is one the major threats to public health today, especially resistance to last-resort compounds for the treatment of critical infections, such as carbapenems and aminoglycosides. Innumerable works have focused on the clinical ambit of AMR, but studies addressing the impact of wastewater cycles on the emergence and dissemination of resistant bacteria are still limited. The lack of knowledge is even greater when referring to low- and middle-income countries, where there is an absence of accurate sanitary systems. Furthermore, the combination of short- and long-read sequencing has surpassed former technical limitations, allowing the complete characterization of resistance genes, mobile genetic platforms, plasmids, and bacteria. The present study deciphered the multiple elements and routes involved in AMR dynamics in wastewater canalizations and, therefore, in the local population of Tamale, providing the basis to adopt accurate control measures to preserve and promote public health.

Keywords: antibiotic resistance; environmental microbiology; genomic analyses; plasmid-mediated resistance; public health; wastewater treatment.

FIG 1

Geographical locations and bacterial counts of wastewater sampling points. (A) Map of Ghana, indicating the location of Tamale (black star), and map of the metropolitan area of Tamale. Sampling locations: TWH, Tamale West Hospital; TCH, Tamale Central Hospital; TTH, Tamale Teaching Hospital; and UWTP, Urban Waste Treatment Plant. Sampling points are displayed by different colored spots in each sampling location (see the legend in the bottom right corner): 1, canalization before the hospital/first treatment pond in UWTP; 2, drainage of the hospital/last treatment pond in UWTP; and 3, canalization after the hospital/drainage of the UWTP. Geographical coordinates of sampling points are indicated in Table S1. An interactive map is available at the Microreact website (https://microreact.org/project/2sBABj7YhfhoLZi8trXvSv). (B) Bacterial counts (CFU/mL) of total bacteria, carbapenem-resistant bacteria, and pan-aminoglycoside-resistant bacteria in wastewater samples from canalizations of Tamale. Total bacteria encompass the entire bacterial population, including carbapenem- and pan-aminoglycoside-resistant bacteria. Sampling locations and points are indicated according to panel A.

FIG 2

Tree of carbapenem/pan-aminoglycoside-resistant isolates based on its total resistance gene content. Bacterial species are indicated by leaf labels and colored sections in the outer ring (legend in the bottom right corner of the figure). Sampling points are indicated by colored leaf nodes in the tree (see the legend in the top right corner). Sampling locations: TWH, Tamale West Hospital; TCH, Tamale Central Hospital; TTH, Tamale Teaching Hospital; and UWTP, urban waste treatment plant. Sampling points: 1, canalization before the hospital/first treatment pond in UWTP; 2, drainage of the hospital/last treatment pond in UWTP; and 3, canalization after the hospital/drainage of the UWTP. An interactive visualization, together with the map of Tamale, is available at the Microreact website (https://microreact.org/project/2sBABj7YhfhoLZi8trXvSv).

FIG 3

Carbapenem/pan-aminoglycoside-resistant bacterium data. Trees are constructed based on the similarity between total resistance gene content. The sampling point from where each isolate was recovered is specified by different colored shades in the isolate code. Sampling points: 1, canalization before the hospital; 2, drainage of the hospital; and 3, canalization after the hospital. Bacterial species and sequence types are indicated following the isolates’ codes. NT, nontypeable ST. Phenotypic resistance to tested antibiotics is indicated by filled colored squares and phenotypic susceptibility by empty squares. Antibiotics: GEN, gentamicin; AMP, ampicillin; FOT, cefotaxime; TAZ, ceftazidime; MERO, meropenem; CHL, chloramphenicol; TMP, trimethoprim; AZI, azithromycin; COL, colistin; CIP, ciprofloxacin; NAL, nalidixic acid; TET, tetracycline; TGC, tigecycline; and SMX, sulfamethoxazole. The presence of antibiotic resistance genes and plasmid incompatibility groups is indicated by filled colored squares, and the absence of those genetic elements is indicated by empty squares. (A) Tamale West Hospital (TWH). (B) Tamale Central Hospital (TCH). (C) Tamale Teaching Hospital (TTH).

FIG 4

Genetic organization of carbapenemase genes strongly associated with specific mobile elements and bacterial species. Colored arrows indicate the functional group for each gene (bottom legend) and sequence direction. Manually curated genes are shown by labels (except for the Regulation/Metabolism and Hypothetical Protein groups). (A) P. putida chromosome region surrounding the novel genetic variants bla_VIM-71 and bla_CARB-53. (B) P. stutzeri novel plasmid pPS-VIM-5 harboring bla_VIM-5. (C) P. putida ST125 novel plasmid harboring bla_DIM-1.

FIG 5

Genetic organization of bla_NDM-1 and its multiple carrying structures. Colored arrows indicate the functional group for each gene (bottom legend) and sequence direction. Manually curated genes are shown by labels (except for the Regulation/Metabolism and Hypothetical Protein groups). (A) P. rettgeri pPrY2001-like plasmid pPR-NDM-1A harboring bla_NDM-1 and rmtC. (B) C. werkmanii IncC plasmid pCW-NDM-1 harboring bla_NDM-1 and armA. Gray shading highlights the plasmoid element when it is integrated in the IncC plasmid. (C) Enterobacteriaceae plasmoid element harboring bla_NDM-1.

FIG 6

Genetic organization of diverse plasmid structures harboring bla_CTX-M-15 and bla_OXA-1. Colored arrows indicate the functional group for each gene (bottom legend) and sequence direction. Manually curated genes are shown by labels (except for the Regulation/Metabolism and Hypothetical Protein groups). Closer visualizations of the region surrounding resistance genes in the plasmid are displayed at the bottom of each gene. (A) C. werkmanii pKPC-CAV1321-like plasmid pCW-CTX-M-15A. (B) C. youngae IncHI1B/IncFIB (pNDM-MAR-like) plasmid pCY-CTX-M-15.