NDM-1- and OXA-23-producing Acinetobacter baumannii in wastewater of a Nigerian hospital

ABSTRACT Carbapenem-resistant Acinetobacter baumannii spp. are increasingly important pathogens with limited treatment options, and there is limited knowledge on the environmental factors contributing to their spread. We determined the occurrence of carbapenem-resistant A. baumannii in hospital wastewater and their phylogenetic relationships with clinical A. baumannii isolates. Grab samples of raw and treated hospital wastewater were collected monthly at the University College Hospital, Ibadan, Nigeria, between March 2021 and February 2022. Acinetobacter baumannii strains were selectively isolated and identified using VITEK2, and their whole genomes were sequenced on an Illumina platform. We performed antimicrobial susceptibility testing and in silico genomic characterization of the strains and determined their phylogenetic relationships to previously characterized clinical A. baumannii strains from Nigeria. A. baumannii complex isolates were recovered from wastewater throughout the study. Of the 82 isolates identified based on whole-genome sequences, 77 were A. baumannii. A. baumannii isolates had high resistance rates (≥48.1%) to 10 of 12 antimicrobials tested, and majority (42/77, 54.5%) were resistant to carbapenems, with bla NDM-1 being the most common (24/77, 31.2%) carbapenem resistance gene detected, followed by bla OXA-23 (n = 22, 28.6%). There was no statistically significant difference in carbapenem resistance rates or carbapenem gene carriage between the raw and treated wastewater isolates. Most of the isolates belonged to novel or sparsely described lineages, some of which were closely related to clinical isolates. The release of inadequately treated hospital wastewater into the environment may contribute to the increased spread of carbapenem-resistant and clinically important A. baumannii lineages in Ibadan, Nigeria. IMPORTANCE Acinetobacter baumannii is a leading cause of hospital-associated infections globally. A. baumannii reservoirs outside hospital settings are still unknown, and their occurrence in the environment is linked to clinical and anthropogenic activities. Although the risk of transmission of A. baumannii from environmental sources to humans is not fully understood, these sources pose significant risks for the continued dissemination of A. baumannii and their resistance traits. This study provides evidence that diverse and clinically relevant A. baumannii strains, many of which are resistant to carbapenems, are constantly being discharged into the environment through inadequately treated hospital wastewater. We further elucidate potential transmission routes between the environment and clinical infections and demonstrate the high prevalence of carbapenem resistance genes on highly mobile transposons among these strains. Our findings highlight the pressing need to address hospital wastewater as a crucial factor in curtailing the spread of carbapenem-resistant A. baumannii.


MATERIALS AND METHODS
This study was conducted at the University College Hospital (UCH), Ibadan, Oyo State, Nigeria.The University College Hospital, Ibadan, is a tertiary hospital with an 1229-bed capacity (38).The wastewater treatment plant at the Environmental Health Department, UCH, processes 28,000 L of wastewater per day and employs a multi-step wastewater treatment process.Gross solids are first removed at the preliminary treatment step using a skimming tank, after which the remaining solids are removed in the primary treat ment step using a combination of sedimentation, mechanical flocculation, and chemical coagulation methods.This is followed by aerobic oxidation treatment with activated sludge and anaerobic digestion.The digested sludge is separated and stabilized, and the wastewater is held in oxidation ponds and chlorinated before disposal.The final treated wastewater is discharged into the downstream Dandaru Reservoir, which is interconnec ted with a vast river network.

Collection of hospital wastewater and isolation of Acinetobacter baumannii
Monthly grab samples (i.e., single, discrete samples collected at a particular time) of untreated and treated hospital wastewater were collected between (March 2021) and February 2022 from the wastewater treatment plant at UCH. Untreated (raw) wastewater samples were collected from the inlet flow point into the wastewater treatment plant, while treated samples were collected from the final treated wastewater effluent at the point of discharge.Raw and treated wastewater samples were collected on the same day each month in sterile 1-L wide-neck plastic containers and transported on ice to the laboratory for processing typically within 1 hour of collection.All samples were collected between 9 a.m. and 12 noon.
Ten-fold serial dilutions were prepared from the water samples and plated onto freshly prepared CHROMagar Acinetobacter media with CHROMagar MDR Supplement CR102 (CHROMagar, Paris, France) additionally supplemented with 2 µg/mL of cefotax ime to increase the chances of recovering cephalosporin-resistant A. baumannii.Plates were incubated at 37°C for 24 hours, after which all presumptive A. baumannii colonies (up to a maximum of 20 distinct colonies for each sample) were selected based on their colony morphology (red colonies) and sub-cultured onto antibiotic-free CHROMAgar plates and incubated at 37°C for 24 hours to obtain pure cultures.Pure cultures were cryopreserved at −80°C prior to further analyses.

Identification of bacteria from wastewater
Presumptive A. baumannii isolates were identified using the GN ID (reference number: 21341) cards on the VITEK two automated system (bioMérieux, Inc., Marcy-l'Étoile, France) following the manufacturer's instructions.Isolates identified as Acinetobacter baumannii complex were stored for downstream characterization.

Antimicrobial susceptibility testing
The susceptibility of the A. baumannii complex isolates to selected antimicrobials was determined using the VITEK two automated system with the antimicrobial susceptibility testing (AST) N281 (reference number: 414531) cards according to the manufacturer's instructions.The antimicrobials tested included cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, minocycline, piperacillin/tazobactam, ticarcillin/clavulanic acid, and tigecycline.Minimum inhibitory concentration (MIC) values were interpreted using the AMR R package version 1.8.1 (https://msberends.github.io/AMR/)according to the clinical breakpoints of the Clinical Laboratory Standards Institute (CLSI) (39), except for the tigecycline MIC values as the current guidelines by CLSI and the European Committee on Antimicrobial Susceptibility Testing (40) do not contain breakpoints for interpreting tigecycline MIC values for A. baumannii.Tigecycline MICs of 2 µg/mL were reported as resistant (41).All non-suscepti ble isolates are reported as resistant in the analyses.

Whole-genome sequencing
Overnight cultures of A. baumannii complex isolates in tryptone soy vroth (Oxoid, Basingstoke, United Kingdom) were centrifuged at 6,000 revolutions per minute for 5 min, and the pellets were harvested for use as starting material for the DNA extraction using the FastDNA Spin Kit for Soil (MP Biomedicals, Irvine, CA, United States) according to the manufacturer's instructions.Whole-genome sequencing libraries were prepared from the extracted DNA using the NEBNext Ultra II FS DNA library kit for Illumina (New England Biolabs, Ipswich, MA, United States) according to the manufacturer's instruc tions, and the genomic libraries were sequenced on an Illumina MiSeq platform with 150-bp paired-end chemistry (Illumina, San Diego, CA, United States).

Clinical A. baumannii isolates
We sought to assess the clinical significance of the wastewater isolates by determining their phylogenetic relationships to previously characterized clinical isolates in Nigeria.
To do this, we retrieved the whole-genome sequences of 89 clinical A. baumannii isolates obtained from hospitals or laboratories across southwestern Nigeria between 2016 and 2022 .These included the genomes of 86 A. baumannii isolates characterized in our previous study (19) and 3 other additional isolates obtained from UCH, Ibadan.In total, 21 of the clinical isolates (including 18 previously characterized isolates) were from the UCH health facility, where the wastewater samples were collected, while the remaining 68 were from other healthcare facilities in different states within the same southwestern region of Nigeria.These facilities were Lagos University Teaching Hospital, Idi-Araba, Lagos State; Clina-Lancet Laboratories, Victoria Island, Lagos State; EL-LAB Medical Diagnostics, Festac, Lagos State; Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Osun State; University of Ilorin Teaching Hospital, Ilorin, Kwara State, and Babcock University Teaching Hospital, Ilishan-Remo, Ogun State.All raw reads of the clinical isolates are available in the European Nucleotide Archive (https:// www.ebi.ac.uk/ena) with study accession number PRJEB29739.Antimicrobial susceptibil ity data obtained using the same methodology as described for the wastewater isolates were available for 79 of the clinical isolates and were included in the analyses.The remaining 10 clinical isolates could not be resuscitated for repeat AST.

Bioinformatics analyses
De novo genome assembly, species identification, and quality assessment of the generated assemblies were performed using the de novo assembly pipeline described in detail in the Genomic Surveillance of Antimicrobial Resistance (GHRU) Retrospective 1 Bioinformatics Methods version 4 (https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bp2l6b11kgqe/v4).Only assemblies with N50 values greater than 15,000 and no more than 400 contigs were included in downstream analyses.Contamination was assessed using Confindr, and genomes containing >5% contaminating single-nucleotide variants of selected core genes were also excluded from downstream analyses.
To estimate phylogenetic relationships between the wastewater and clinical isolates, we included the genomes of the 89 previously characterized clinical A. baumannii isolates.All genomes were first annotated using Bakta (42) version 1.6.1, after which core genes were identified and aligned using Panaroo (43) version 1.3.2.The filtered core gene alignment, which excluded outlying genes identified based on the Tukey outlier test, was then used as input into RAxML-NG (44) version 1.1.0for phylogenetic tree inference with the GTR + G model, 50 distinct starting trees, and "bootstopping." Bootstrapping converged after 300 replicates.To determine the genetic similarity between genomes in specific clades of interest, we selected close reference genomes for each clade from the RefSeq database [accessions: GCF_001908295.1 (clade A), GCF_000828935.1 (clade B), GCF_000828935.1: (clade C), and GCF_000021245.2 (clade D)] and recon structed clade-specific reference-based phylogenies using the GHRU mapping-based phylogeny pipeline (https://www.protocols.io/view/ghru-genomic-surveillance-of-antimicrobial-resista-bp2l6b11kgqe/v4).Single-nucleotide polymorphism (SNP) distances were determined from the resulting alignments using snp-dists version 0.8.2.
To characterize the population structure and identify possible transmission pairs between clinical and wastewater A. baumannii isolates, a t-distributed two-dimensional stochastic cluster embedding analysis was conducted to identify hierarchical [Hierarch ical Density-based Spatial Clustering of Applications with Noise (HDBSCAN)] clusters using Mandrake (45) version 1.2.2 with the gene presence/absence matrix output from the Panaroo program as input and the following parameters: kNN = 100, perplexity = 40, and maxIter = 300,000,000.
Multi-locus sequence types (MLST) were predicted from the raw reads using the ARIBA (46) software version 2.14.4 with the A. baumannii Oxford (47) and Pasteur (48) typing schemes in the PubMLST database.Novel allele sequences were extracted from the assemblies using MLSTar (49) version 0.1.5,and the novel alleles and profiles were submitted to the PubMLST database (50) for allele and sequence type (ST) assignment.Using the BURST software within the PubMLST database, sequence types were assigned to one of the nine classified international clones (ICs) if they had no more than two locus variations from representatives of each clone (14,51,52).Antimicrobial resistance genes were detected using AMRfinderplus (53) version 3.10.24with database version 2022-04-04.Only genes in the "core" database and with an "element type" of "AMR" were included in the analyses."Partial" hits (<90% of the length of the reference database sequence) determined to not be truncated by a contig boundary were also excluded.The intrinsic bla OXA-51 -like and bla ADC -like genes were also not reported.To determine the genetic contexts of the detected carbapenem resistance genes, we mapped both the assemblies and the reads of the wastewater isolates to previously characterized mobile genetic elements carrying acquired carbapenem resistance genes in A. baumannii (16,19,(54)(55)(56) using Gview server (https://server.gview.ca/)with default parameters (for the assemblies) and BWA MEM (57) version 0.7.17 (for the reads).Duplicate reads were marked and removed using Picard version 3.0.0(http://broadinstitute.github.io/picard).

Data analyses
Statistical analyses and data visualizations were carried out using R version 4.2.1.The resistance rates to each antimicrobial were compared between the clinical and wastewater A. baumannii isolates using Pearson's chi-squared test with false discovery rate correction for multiple testing.The Wilcoxon rank-sum test with false discovery rate correction was used to compare the number of resistance genes conferring resistance to unique antimicrobial classes between strains with and without at least one carbapenem resistance gene.P values less than 0.05 were considered statistically significant.

Isolate collection
A total of 24 wastewater samples (12 untreated and 12 treated) were collected for the study.A total of 90 isolates from treated and untreated wastewater samples (range: 0-16 isolates per sample) were identified as A. baumannii complex using the VITEK 2 system and subjected to Illumina sequencing.Overall, at least one isolate belonging to the A. baumannii complex was recovered in all the months of sampling except June 2021.A. baumannii complex isolates were recovered from untreated wastewater samples in 10 of the 12 months of sampling and in eight of the 12 months from the treated waste water samples.Eighty-two of the 90 sequenced isolates passed the sequence quality checks and were included in the analyses.Based on the whole-genome sequences, 77 of the 82 isolates were identified as A. baumannii, while the remaining 5 isolates were Acinetobacter pittii.Of the confirmed 77 A. baumannii isolates, 33 were isolated from the raw/untreated wastewater, while 44 were isolated from the treated effluent.
Most of the STs were detected only transiently throughout the study period.Of the 31 distinct STs detected, most (25/31, 78.1%) were detected in only one of the 12 sampling months (Fig. 2).Five of the remaining six STs were recovered in two successive months (ST2803, March and April 2021; ST2089, September and October 2021; ST231, October and November 2021; ST2798, January and February 2022; and ST351, January and February 2022).ST472 was the only ST detected in two non-successive months (August 2021 and February 2022).Six of the 31 distinct STs were detected in raw wastewater but not in treated wastewater; 4 of these were detected only once.

Phylogenetic relatedness between the wastewater and clinical isolates
To determine whether the wastewater isolates were genetically related to clinical isolates, we constructed a maximum likelihood phylogeny of the wastewater isolates and 89 previously characterized clinical isolates from across southwestern Nigeria.The A. baumannii isolates recovered from wastewater were highly phylogenetically diverse and occupied distinct, deeply branching clades (Fig. 6).There were 28 distinct HDBSCAN clusters (clusters 0-27) generated from the pangenome gene presence/absence matrix using Mandrake, and these mostly correlated with the distinct clades on the phyloge netic tree.In general, there were a few phylogenetic and/or HDBSCAN clusters of clinical and wastewater isolates.Five bla OXA-23 -carrying ST2151 (IC8) isolates obtained from raw (n = 3) and treated (n = 2) wastewater in February 2022 clustered together (cluster 17) with three clinical isolates from the same healthcare facility, one of which was isolated from blood in May 2019 (clade A).Despite this clustering, these three clinical isolates, which were all identical (0 SNPs), were not phylogenetically identical to any of the five wastewater isolates (between 603 and 953 SNPs).Conversely, three isolates (two ST862 and one non-typeable ST) also recovered from raw and treated wastewater in February 2022 were phylogenetically similar (30-66 SNPs) to three blood isolates that were isolated back in November 2018 in a neighboring city, Ile-Ife, Osun State (clade B).
All six wastewater and clinical strains co-carried bla NDM-1 and bla OXA-23 .
Multiple isolates from final treated wastewater were phylogenetically identical to other isolates from raw (untreated) wastewater and to clinical isolates.Two ST919 isolates from treated wastewater in February 2022 carrying both bla NDM-1 and bla OXA-23 were phylogenetically identical (differed by two to four SNPs) to two clinical isolates subse quently isolated from a tracheal aspirate sample of a patient in the same healthcare facility in May 2022 (clade C).Within clade D, which comprised the different IC1 isolates, there were two sub-clades containing clusters of isolates from clinical and wastewater bla NDM-1 bla OXA-23 bla OXA-58 samples.The first sub-clade (cluster 4, ST231) included five clinical isolates that were closely related to two isolates from raw and treated wastewater.All seven isolates carried both bla NDM-1 and bla OXA-23 .The other sub-clade comprised two clinical isolates from blood, three isolates from raw hospital wastewater, and four isolates from treated wastewater, all of which carried bla OXA-23 and belonged to HDBSCAN cluster 3.
In terms of antimicrobial resistance, the clinical A. baumannii isolates had higher resistance rates than almost all the tested antimicrobials (except imipenem and meropenem) compared to the 33 isolates from raw wastewater, but these differences were not statistically significant (Fig. 3).

DISCUSSION
Wastewater treatment plants, particularly those that receive clinical waste, are known to be important sources of drug-resistant pathogens, including A. baumannii (25).In this study, we investigated treated hospital wastewater as a source of clinically relevant A. baumannii strains.A. baumannii isolates were recovered throughout the 1-year study period, consistent with previous reports of the high prevalence of A. baumannii in hospital wastewater elsewhere (22,25,58).It is uncertain to what extent the repor ted chlorination of the treated wastewater before discharge impacted occurrence and resistance levels in A. baumannii.
Notably, there was a high diversity of A. baumannii lineages in hospital wastewater, most of which belonged to novel or sparsely described STs.A few STs belonged to the major international clones IC1, IC6, and IC9, but the majority were either singletons or belonged to none of the globally disseminated clones.Despite the recovery of A. baumannii complex isolates in all but 1 of the 12 sampling months, specific A. baumannii sequence types were only detected transiently throughout the study, suggesting the absence of a reservoir for these STs in the hospital environment and reflecting the previously identified diversity of A. baumannii lineages in clinical settings in southwest ern Nigeria (19).Interestingly, no strain belonging to IC2, which is the most predominant Has ¡ 1 gene conferring carbapenem resistance Number of AMR genes in unique classes FIG 5 Comparison of the number of genes conferring resistance to unique antimicrobial classes in A. baumannii isolates with at least one carbapenem resistance gene versus isolates without a carbapenem resistance gene.AMR, antimicrobial resistance.clinical A. baumannii lineage in Nigeria (19) and has been previously reported in hospital wastewater in Germany (25), was detected throughout the 12 months.
The detection of so many novel STs in hospital wastewater may indicate missed/ unreported infections or hospital environment or patient colonization.A. baumannii spp.mostly exist as hospital environment or human colonizers and cause infections primarily in immunocompromised patients (5,59,60), but colonization in itself is associated with increased risk of subsequent infection among patients (14,61).One interesting finding was the detection of two isolates in treated wastewater in February 2022 that were nearly identical to two clinical isolates from a hospitalized patient in the same hospital 3 months later in May 2022.These four ST919 wastewater and clinical strains were clonal, differing by four to nine SNPs, and all carried both bla NDM-1 and bla OXA-23 .The two wastewater isolates were both resistant to cefepime, ceftazidime, ciprofloxacin, doripenem, imipenem, meropenem, levofloxacin, piperacillin/tazobactam, and ticarcillin/clavulanic acid, but there were no antimicrobial susceptibility data for the clinical isolates.Based on the available data, we cannot determine the association or direction of transmission between these strains, but they illustrate the likely existence of A. baumannii transmission links between the hospital environment and the human population.We identified a few phylogenetic and/or HDBSCAN clusters of clinical and wastewater isolates, indicating the potential clinical significance and likely clinical source of the wastewater isolates.The low numbers of these clusters may be due to the preponder ance of novel STs among the wastewater isolates and the fact that Acinetobacter spp.causing clinical infections are likely under-detected in our setting (19).Importantly, the clustering and close phylogenetic relationships between the clinical and wastewater isolates, including those from the final treated effluent, illustrate the presence of possible transmission chains from the hospital environment or even patients to the environment receiving the final treated wastewater.This release of A. baumannii into the environ ment via treated wastewater represents a significant public health concern requiring intervention as there is evidence that clinically relevant A. baumannii can persist in low-nutrient and even oxygen-deprived environments of the receiving water bodies for up to 50 days, which is potentially long enough to reach the human population (23,29).The subsequent transmission of carbapenemase-producing strains via lotic water has also been demonstrated previously (62).More so, the likelihood of these transmissions and their public health implications are even worse in low-and middle-income regions where poor water sanitation and hygiene conditions pervade and transmission routes are abundant (63,64).
Antimicrobial resistance rates were high among the wastewater A. baumannii isolates, with at least 50% resistance reported for 9 of the 12 tested antimicrobials.The continued spread of carbapenem-resistant A. baumannii is a huge health challenge globally as the remaining treatment options for these strains are severely limited due to resistance, in vivo efficacy, toxicity, cost, and pharmacokinetic issues associated with the other recommended therapeutic options such as the polymyxins, tetracyclines, and sulbactam (11,20,21).Carbapenemase gene carriage, which we determined to be significantly linked to carriage of multiple antimicrobial resistance determinants, was also notably high among the wastewater isolates.This is consistent with previous observations that carbapenem-resistant A. baumannii are often resistant to multiple antimicrobial classes (21).The exact reason for this phenomenon is unclear, but a possible reason is the fact that plasmids, large genomic islands, and other mobile genetic elements carrying carbapenem resistance genes in A. baumannii are often found associated with genes conferring resistance to multiple other antimicrobial classes (65).It is noteworthy that the rates of resistance to carbapenems and other antimicrobials and the number of resistance genes were lower among the final effluent isolates compared to isolates from raw wastewater, which is consistent with previous reports that the proportion of resistant isolates is likely to be reduced in final treated wastewater (23,25).Nevertheless, the carbapenem resistance rate observed among final effluent isolates in this study was still notably high (45.5%).The release of carbapenem-resistant A. baumannii into the environment via hospital wastewater may play a role in the continued spread of drug-resistant A. baumannii, and the relative importance of wastewater as a source for transmission and exposure to humans needs to be assessed.A recent study investigat ing the presence of multi-drug-resistant A. baumannii in various livestock and human wastewater sources found that only those from hospitals contained multi-drug-resistant isolates of A. baumannii, suggesting a lesser role of the livestock industry in the spread of these clinically relevant strains (25).
This study confirms the increasing prevalence of bla NDM-1 among A. baumannii isolates reported in recent studies, primarily in Africa and the Middle East (17,19,(66)(67)(68)(69).About a third of the wastewater isolates carried the bla NDM-1 gene, and almost half of these were isolates from the final treated wastewater released into the environment.The distribution of bla NDM-1 and bla OXA-23 predominantly among isolates in distinct phylogenetic clusters suggests that clonal expansion of carbapenem-resistant clones is the primary driver of increasing carbapenem resistance prevalence among A. baumannii.This is consistent with previous reports, and the clonal spread of carbapenem-resistant A. baumannii lineages both geographically and within hospital settings is well described in the literature (8,(70)(71)(72)(73)(74)(75)(76).Nevertheless, the seemingly lower frequency of horizontal acquisition of carbapenem resistance genes is still interesting as A. baumannii spp.have highly plastic genomes and a high tendency to acquire resistance genes on plasmids and other mobile genetic elements like transposons (3,8,77).One explanation for this may be the frequent carriage of carbapenem resistance genes on chromosomes by A. baumannii.In our previous study characterizing clinical A. baumannii isolates in the southwestern region of Nigeria, in all the isolates where the location of bla NDM-1 and bla OXA-23 genes were determined, both genes were chromosomally located (19).In these clinical strains, bla OXA-23 was carried entirely on Tn2006 or Tn2006-like transposons, as observed for the wastewater isolates in this study.Similarly, 20 of the 24 bla NDM-1 -posi tive wastewater isolates (belonging to nine distinct STs) carried the gene on the Tn125 transposon, which is a highly mobilizable transposon believed to be the primary means of horizontal dissemination of bla NDM-1 among A. baumannii and other Gram-negative pathogens (19,56,78).Wastewater contains sub-inhibitory concentrations of antimicro bials and is regarded as a potential hotspot for the increased exchange and uptake of antimicrobial resistance genes, and could thus accelerate the horizontal spread of these genes and mobile elements both between A. baumannii and to other species (79).Further studies are needed to understand the dynamics of carbapenem resistance gene acquisition among A. baumannii lineages and other bacterial populations in wastewater and related aquatic environments.

Limitations
The monthly samples and lack of a composite sampling technique preclude any definitive conclusions on the longitudinal trends and incidence of carbapenem-resistant A. baumannii in raw and treated wastewater in the facility.Similarly, due to the lack of an efficient method for the selective recovery of A. baumannii isolates from water samples, as has been previously described (25), we could not meaningfully interpret, and thus present, isolate count data, which would have been a useful denominator.We included 2 µg/mL of cefotaxime in the primary isolation plates to facilitate the selective isolation of A. baumannii, but both beta-lactam-resistant and beta-lactam-sensi tive A. baumannii isolates, as well as non-target species, were recovered throughout, a phenomenon that is common during A. baumannii isolation even with higher concentra tions of antibiotic supplements (22,25).Despite this, the cefotaxime supplement may have overestimated the reported beta-lactam resistance rates.Furthermore, the primary plates recovered isolates with morphologies similar to Acinetobacter baumannii complex isolates that were subsequently determined to belong to other species, including Aeromonas hydrophila, Pseudomonas putida, Pseudomonas stutzeri, and Stenotrophomo nas maltophila.Thus, plate counts could not be reliably reported as A. baumannii counts.Another limitation was our inability to concurrently obtain a larger number of clinical isolates from the same hospital that would have allowed more robust interpretations of the transmission links between the hospital wards and the environment.Finally, as only wastewater from one health facility was sampled, these results cannot be generalized to other health facilities in Nigeria.

Conclusions
Carbapenem-resistant A. baumannii strains belonging to diverse lineages can survive the hospital wastewater treatment process and can be released into the environment.This necessitates the improvement of hospital wastewater treatment processes to more effectively eliminate these pathogens.The importance of such treated wastewater as a contributor to the increasing prevalence of A. baumannii lineages carrying carbape nem resistance genes is uncertain and needs further studies.Addressing this challenge requires interventions guided by a robust genomic surveillance of these high-priority pathogens using a One Health approach.

28 FIG 2
FIG 2 Sequence type distribution of wastewater A. baumannii isolates according to date of isolation.*Novel sequence type.

FIG 4
FIG4 Distribution of carbapenem resistance genes among A. baumannii isolated from raw and treated hospital wastewater.The black circles denote gene presence.IC, international clone; ND, not determined.

FIG 6
FIG 6 Maximum likelihood phylogeny of 77 A. baumannii isolates obtained from hospital wastewater between March 2021 and February 2022, and 89 A.baumannii isolates obtained from various clinical samples between 2016 and 2020.IC, international clone; NA, not available.