Municipal wastewater monitoring revealed the predominance of
bla
GES
genes with diverse variants among carbapenemase-producing organisms: high occurrence and persistence of
Aeromonas caviae
harboring the new
bla
GES
variant
bla
GES-48
Authors: Tanabe Mizuki , Sugawara Yo , Denda Tomohiro , Sakaguchi Kanae , Takizawa Shino , Koide Shota , Hayashi Wataru , Yu Liansheng , Kayama Shizuo , Sugai Motoyuki , Nagano Yukiko , Nagano Noriyuki 
Published: October 2023

We characterized carbapenemase-producing organism (CPO) detected in municipal wastewater to better understand the epidemiology of CPOs in the community. In total, 36 samples were collected at six sampling sites every other month from December 2020 to October 2021. CPOs were not recovered from influent taken from inlet A receiving separated sewer line, treated effluents, and river water samples upstream and downstream of the effluent outlet. By contrast, 75 CPOs were detected in all influent samples taken from inlets B and C receiving combined sewer lines collecting both domestic/industrial wastewater and rainwater runoff.
Aeromonas caviae
was the dominant species (25/75, 33.3%), and the other 11
Aeromonas
spp. together accounted for 48% of CPOs. The remaining 39
Enterobacterales
strains mainly comprised 17
Klebsiella
spp. and 10
Raoultella
spp. CPOs carrying
bla
GES
carbapenemase genes were overwhelmingly dominant, accounting for 72 of 75 isolates, including two isolates harboring both
bla
GES-24
and
bla
IMP-1
(96%), followed by three
bla
IMPs
-positive isolates, where those carbapenemase genes were mainly carried in diverse class 1 integrons. Among
bla
GES
variants, including six new variants (
bla
GES-47
,
bla
GES-48
,
bla
GES-49
,
bla
GES-50
,
bla
GES-51
, and
bla
GES-54
),
bla
GES-5
was detected in 28 CPOs, with
Aeromonas
spp. accounting for 53.6% of these organisms. Quantitative analysis revealed that the repeated detection of
bla
GES-48
-positive
A. caviae
ST1056 from both inlets B and C ranked the total number of this bacterial clone highest in the wastewater influent. In summary, our study revealed the high prevalence and persistence of diverse
bla
GES
carbapenemase genes among CPOs isolated from influent inlets connected to combined sewer systems.

IMPORTANCE

The emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of
bla
GES
genes, including six new
bla
GES
variants, and less frequent
bla
IMP
genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were
Aeromonas
spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.