key: cord-0284253-dcacoaxm
authors: Markkanen, M. A.; Haukka, K.; Pärnänen, K.; Dougnon, V. T.; Bonkoungou, I. J. O.; Garba, Z.; Tinto, H.; Sarekoski, A.; Karkman, A.; Kantele, A.; Virta, M.
title: Metagenomic analysis of antimicrobial resistance genes in wastewaters in Benin and Burkina Faso indicates a serious health risk from untreated hospital wastewaters in low-income countries
date: 2021-10-22
journal: nan
DOI: 10.1101/2021.10.19.21265183
sha: fe548c6ee56837325dcbfeea8dbd91b7b03a9348
doc_id: 284253
cord_uid: dcacoaxm

Antimicrobial resistance (AMR) is one of the greatest global threats to human health, but substantial gaps in AMR data exist in West African countries. To obtain in-depth data, we explored the presence of antimicrobial resistance genes (ARGs) in the hospital wastewaters (HWW) of nine hospitals in Benin and Burkina Faso and, for comparison, of four hospitals in Finland. The highest total relative abundance of ARGs in HWWs was observed in Benin and the lowest in Finland. HWW resistomes were more similar to each other in Benin and Burkina Faso than in Finland. Different clinically relevant carbapenemases were detected in varying abundances, especially in HWWs from Burkina Faso and Finland. The most widespread carbapenemase gene in the Beninese hospitals, blaGES, was also found in water used for handwashing. blaNDM was present in the HWW of one Beninese hospital and was also detected in the stools of a hospitalized patient. Mobile colistin ARGs were detected in the HWWs of all the three studied countries, with mcr-5 variants being the most common. These and other mcr genes were observed in very high abundance in treated wastewater released into rivers in Burkina Faso. In Benin, only little is done to treat wastewaters, including HWWs. The results provide evidence for public health decision-makers in Benin and Burkina Faso for the dire need to increase wastewater treatment capacity, with particular attention to HWWs.

The high-risk ESKAPEEc bacteria 6 (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, 83 Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli) 15 

As a consequence of the increased prevalence of extended-spectrum beta-lactamase-producing 97 Enterobacteriaceae (ESBL-PE), the use of broad-spectrum antibiotics has increased in clinical practise 2,21,22 .

Tracking down the spread of ESBL genes is, in many situations, no longer feasible or relevant, and attention has 99 been directed to plasmid-mediated genes that encode carbapenemases. The carbapenemases blaGES, blaIMP, 

Metronidazole is an example of an empirically widely used first-line treatment in many African countries 27 . This 106 antimicrobial drug is effective against various protozoan parasites and anaerobic bacteria 28 . Although acquired 107 resistance (mediated mainly by nim genes) towards metronidazole is considered to be relatively rare, an increasing 108 trend in its emergence has been reported 28 . Colistin is a last-resort antibiotic used for treating infections caused 

Although AMR is of global concern, the crisis affects most dramatically LICs, such as those in West Africa 3,4,32-113 34 . Lack of research data is a major factor hindering the development of solutions to tackle the AMR problem in 114 these countries 4,32,35,36 . Despite gaps in resistance surveillance data, it is well known that the level of AMR is 115 highest in many LICs, such as in many African countries 21,33,37,38 . By contrast, in Northern European countries, 116 such as Finland, AMR occurrence is among the lowest globally, both at the community level 38,39 and in the data 117 provided by healthcare-associated AMR surveillance 40 .

As hospital wastewater (HWW) from healthcare facilities is considered to be at the frontline of AMR emergence 120 and spread 41 , we set out to investigate the AMR situation and the characteristics of the resistomes in nine hospitals 121 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint collected from four different hospitals (hospitals K-N) in January of 2020. In Burkina Faso, the samples were 133 mainly from septic tanks or sewers in the hospital area. In Benin, none of the hospitals was connected to a sewer 134 system, and the samples were from septic tanks or sumps (unstructured wastewater wells), which were never 135 emptied to our knowledge. In most cases the toilet water was not directed into these sumps. For comparison, some 136 other types of samples were included: samples from river waters used for drinking in a remote countryside village 137 in the community of Savalou in central Benin (n=3); river water receiving treated wastewater in Burkina Faso 138 (n=2); and fecal samples of in-patients in Benin (n=4). In addition, the following samples were analyzed; well 139 water, treated tap water, water for handwashing, water in a municipality channel receiving biologically treated 140 wastewater, water and sediment from a street gutter next to the hospital, an empty HWW septic tank, and the soil 141 between HWW septic tanks. The four fecal samples were obtained from patients staying at hospital B with 142 diagnoses mainly associated with musculoskeletal injuries. No clinical data concerning bacterial infections were 143 reported for these patients, and we have no information on their possible use of antibiotics. Patient age varied 144 from 24 to 50 years old, and at the time of sampling, patients had stayed in the hospital for one to two months.

All sample details are provided in the Supplementary Material, 

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In addition, some technical and biological replicates (e.g., multiple samples collected from the same septic tank, 171 sump, or drain) were collected and analyzed. These were excluded from the statistical analyses when comparing 172 HWWs from different countries.

All samples were analyzed using an in-house Snakemake workflow. Snakemake enables reproducible analysis for 175 large datasets through stable version control of the bioinformatic programs used 42 . The program versions and 176 parameters applied for each program can be viewed on the project's GitHub page.

Briefly, the quality control steps included in the workflow were performed using FastQC 43 and MultiQC 44 , with 179 adapter and low-quality read removal using Cutadapt 45 . Nucleotide sequence reads were mapped using Bowtie2 46 180 against the ResFinder database (version 3.2, downloaded on 2020-06-28) 47 . The reads were sorted and filtered 181 using SAMtools 48 such that the reads mapping as pair or alone were calculated as a single count. Mobile genetic 

The accuracy of the normalization method was validated by comparing two distinct methods using the 16S rRNA 192 and rpoB housekeeping genes. The rpoB counts were obtained using the translated sequence reads described above 193 as input for hmmsearch 52 against a rpoB specific HMM profile (pf04563) from the Pfam-A database 53 . Hits for 194 forward reads (R1) were counted. The correlation between the ARGs normalized to both 16S rRNA and rpoB was 195 significant (R 2 = 0.61, p = 4.8 × 10 -8 ), and we proceeded to use 16S rRNA genes for normalization.

To study plasmid-mediated colistin resistance genes (mcr genes) and their genetic environments for their is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Visualizing the genetic environments of contigs harboring mcr-5. 1 

To visualize the co-localization of multiple ARGs with Bandage 58 , the assembled contigs were converted into 

Pearson correlation coefficients and adjusted p-values for each ARG for class 1 integron and qacED genes were is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

We analyzed hospital wastewater-associated resistomes from Benin, Burkina Faso, and Finland. On average, 31 258 million sequence reads per sample were analyzed. Our definition of ARGs followed the scheme used by the 259 ResFinder database, which relies on confirmed association between ARGs and their resistance phenotype 47 .

HWWs from Benin showed the highest and from Burkina Faso the second-highest abundance of all detected 262 ARGs normalized to bacterial 16S rRNA genes, here referred to as the relative sum abundance of ARGs. The Table   266 S2A).

There were no significant differences in the relative sum abundance of all MGEs between the HWWs of the three Table S2A ). Instead, country-wise differences for the gene intI1 of 

In total, 2,331 different ARGs were detected in HWWs from Benin, Burkina Faso, and Finland (Supplementary 275 Results, Figure S4 ). Of these, 1,280 were observed in at least one sample from every country. The resistomes of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Figure S4 ).

As the resistomes of West African countries and Finland were divergent, the drivers for the differences were 

The vertical line shows the median in boxplots, while the lower and higher hinges denote the 25 % and 75 % quantiles, respectively. In is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Table S3 ). In contrast, at the species level, Escherichia coli, Enterococcus faecium, and species of 316 Enterobacter cloacae and Pseudomonas aeruginosa complexes were present but rare in all the countries. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

There was no significant difference (adjusted p-value < 0.05) in the abundance of the genus Acinetobacter between 330 the HWWs from the three countries, but the abundances of different species of Acinetobacter varied significantly. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Figure S4 ).

Among the studied acquired oxacillinases, blaOXA-48 was detected solely in two samples in Burkina Faso. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint shown in Figure 1a) . First, correlations between the relative abundances of the detected ARGs and the two class 371 1 integron-associated genes, intI1 and qacED, were studied. These correlations were visualized for those ARGs 372 differentially abundant in HWWs from each country in pair-wise comparisons ( Figure 5) . Second, contigs carrying 373 the intI1 gene were studied for the putative co-localization of ARG sequences (Supplementary Material, Tables 374 S11A, and S11B). Third, fragments carrying multiple acquired ARGs were screened visually using assembly 375 graphs ( Figure 6 ).

The top 20 ARGs that were more abundant in the Beninese HWWs showed stronger positive correlations with 378 class 1 integron genes compared to the ARGs that were more abundant in Burkina Faso and Finland ( Figure 5) .

This suggests a stronger class I integron association of ARGs in Beninese HWWs than in the other two countries.

The detection of some of these ARGs in contigs carrying several other acquired ARGs (Figure 6 Tables S6, S7 , S11A, S11B, and S14). Table S12 ) and other ARGs (Figure 6c ).

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Pearson correlations between each ARG and the genes intI1 and qacED associated with class 1 integrons (Supplementary Material, Table   407 S11A, and Table S11B) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Mobile colistin resistance genes (mcr genes) were observed in several HWW samples in Benin, Burkina Faso, 419 and Finland ( Table 1) . Variants of mcr-5 (genes mcr-5.1 and mcr-5.2) were the most common as they were found 420 in HWWs from all except one hospital (hospital F) in the three countries. We were able to confirm the location of Table S13 ). 

In addition to the HWW samples, high relative abundances of mcr-5 genes were also detected in the immediate 429 and more distant surroundings of the hospitals, such as in the street gutter water 100 m away from hospital B and 430 in the soil between two HWW septic tanks in Benin (Supplementary Results, Table 2 ). For hospital C, the relative 431 abundance of mcr-5 in a puddle surrounding the surgery room HWW septic tank was greater than the average 432 relative sum abundance of mcr-5 in the actual HWW septic tanks of that hospital (Supplementary Results, Figure   433 S2c and Table S2 ).

In Burkina Faso, very high relative sum abundances of mcr-5 were observed in a municipality channel leading 436 out from hospital I after passing through a biological treatment process. Furthermore, the presence of mcr-5 was 437 also detected in nearby wetland where the processed wastewater is released (Supplementary Results, Table S2 ).

Similarly, in another Burkinabe city, mcr-5 was found in a sample before wastewater treatment and in the river 439 receiving the water after treatment (Supplementary Results, Table S2 ).

The next most prevalent mcr-genes, namely mcr-3, mcr-10, and mcr-7, were detected in certain cases at very high 442 relative abundances in the HWW samples ( Table 1 ). The gene mcr-3 was also present in the water used for is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Figure S2e , Supplementary Results, Table S2 ). The gene mcr-444 2 was not detected, and the lowest possible average relative sum abundance was detected for the gene mcr-1.

Other sample types within and outside the studied hospitals were investigated to obtain a more comprehensive (Figure 4b) and was also present in the HWWs of hospital B (Figure 4b and 4a ). Other Table S14 ).

As in the HWWs from Benin (Figure 4a) , blaGES carbapenemases were also prevalent in other sample types from 458 a hospital environment, such as in the water intended for handwashing (Supplementary Results, Figure S2e ) for 459 hospital C, soil between HWW septic tanks in the yard of hospital B, and street gutter water 100 meters away 460 from hospital B (Figure 4d) . blaGES carbapenemase genes were also found in various waters used for drinking, 461 including river waters in a remote countryside village (Figure 4c) . However, the relative sum abundance of ARGs 462 was low in the three rivers in central Benin (Figure 4c) . We also detected blaNDM in well water used for drinking 463 in Benin (Figure 4c ). Albeit at low relative abundances, blaGES, blaVIM, and blaOXA-58-like carbapenemases 464 were detected in natural waters receiving treated HWWs from Burkina Faso (Figure 4d ). Significantly higher 465 abundances of these ARGs were found in biologically treated wastewater before release into the river (Figure 4c ).

We characterized the bacterial community composition and resistome in 67 HWWs and 16 related samples from is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

The ARGs that were more abundant in HWWs from Benin compared to Finland appeared to be more commonly 492 carried by class 1 integrons. They were strongly correlated with class 1 integron-associated genes (intI1 and 493 qacED 10 ) and, in certain cases, detected in shared contigs with these genes. Moreover, we could detect gene 494 cassettes consisting of several different acquired ARGs linked to each other, likely referring to gene cassettes 495 carried by class 1 integrons 11 . Furthermore, a higher abundance of ARGs intrinsically carried by specific species, 496 such as blaOXA-211 genes by A. johnsonii 76,77 , was a distinctive feature of the HWW resistomes from Finland.

Intrinsic resistance is often chromosomally encoded and rarely mobile or related to horizontal gene transfer 78 ,

whereas acquired resistance is typically carried by MGEs, such as class 1 integrons. As expected, no significant 499 correlations between these intrinsic blaOXA genes and class 1 integron associated genes (intI1 and qacED 10 ) were 500 found, supporting the observation that ARGs putatively carried by class 1 integrons had a smaller impact on 501 resistomes in HWWs from Finland compared to Benin. Based on these results, we speculate that the high sum 502 abundance of ARGs seen in HWWs from Benin may, in part, be explained by the mobilome, more specifically, 

We suspect that as blaGES carbapenemases are typically carried by class 1 integrons, as confirmed by previous 518 studies 79 , a co-selection phenomenon 3,80 might play a role in increasing their spread also in here. In this scenario, 519 residues of antibiotics other than carbapenems would cause co-selection of blaGES. We also found a high relative 520 abundance of plasmid-mediated quinolone ARGs in the HWWs from Benin. These qnrVC genes showed strong 521 correlations to class 1 integron associated genes (intI1 and qacED 10 ) and were in some cases found to be located 522 in the same contigs with blaGES-5 carbapenemase genes (Figure 6b) . Hence, selection targeting quinolone ARGs 523 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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Despite the lowest total relative abundance of ARGs detected in HWWs from Finland, some of the seven 529 carbapenemase genes of special interest seemed to have even higher abundances in Finland than in the surveyed 530 West African countries. However, the occurrence of these ARGs was not homogenous among the Finnish samples. 

The mec operon beta-lactamase genes carried by some Staphylococcus strains were detected only in HWWs from Table S1A ). In HMICs, the use of vancomycin is more common 91 , and VRE is increasing in Europe 40 .

Our observation of the high prevalence of metronidazole ARGs in Benin and Burkina Faso but not in Finland 558 most likely reflects differences in the consumption of this drug. In LICs, diarrheal infections caused by pathogens 559 such as Clostridioides difficile and other anaerobic bacteria, as well as protozoa, are common. The consumption 560 of metronidazole commonly used to treat these infections may be high in these countries as these drugs are being 561 prescribed, for example, both with and without a positive diagnostic test for Clostridioides difficile 92 . However,

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Compared to many HMICs such as those in Northern Europe, the use of antimicrobials in agricultural 93 and 566 clinical 4 settings is less controlled -or even entirely unregulated -in many African countries. For example, while 567 banned in many other countries, the use of colistin as a feed additive is allowed in many LICs 94 , such as those in 568 Africa 93 . In contrast to previous reports on the prevalence of mobile colistin resistance globally 30 and Africa 31,95 , 569 mcr-5 was the most commonly detected gene in the HWWs from our study. Instead, our observations are in line 570 with the results obtained in Germany by Kneis and colleagues using both metagenomics and qPCR 96 . This 571 discrepancy may be due to differences between the methodologies used to screen for colistin resistance. Colistin 572 ARGs other than the best known genes mcr-1, -2, and -3 are rarely targeted when screening for mobile colistin 573 resistance with conventional PCR 96 . Our study shows that to obtain a more realistic view of the colistin resistance 574 situation in Africa, screening should also be conducted for different mcr genes, as was recently done by Ngbede 575 and colleagues 97 .

We confirmed that the mcr-5 gene detected here was embedded in a Tn3-like transposase similar to previous 

For example, at a Beninese hospital, a water puddle in the hospital yard had a higher relative abundance of several 583 mobile colistin resistance genes, such as mcr-5, compared to the actual HWW septic tanks for that hospital. 

In Burkina Faso, a high amount of colistin resistance genes was released into natural waters (2.36 × 10 -4 ) after 591 wastewater treatment. For comparison, a similar relative abundance of mcr-5 genes (3.0 × 10 -4 ) was reported for 592 untreated municipal wastewater in Germany 96 . Although we cannot confirm the association between the mcr-5 593 genes detected in the natural waters and the hospital-associated wastewater in the Burkinabe hospital, our study 594 suggests the inability of the currently used wastewater treatment processes in Burkina Faso to remove colistin 595 resistance genes. Wastewater treatment systems have also earlier been described to be inadequate in Benin and 596 Burkina Faso 101 . This increases the risk of the further spread of colistin and other ARGs as inadequately treated 597 water is released into natural waters used for various purposes by local people. Furthermore, in many African 598 countries, untreated wastewater is used for irrigation in urban agriculture, possibly enabling the dissemination of 599 ARGs to fresh produce 102,103 . The situation appears to be especially critical in Cotonou, where the hospitals 600 involved in the study did not use any kind of wastewater treatment. Groundwater in the city is extracted from the 601 shallow aquifer, which is polluted due to unauthorized waste deposits, inadequate toilets, pit latrines, and septic 602 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted October 22, 2021. ; https://doi.org/10.1101/2021.10.19.21265183 doi: medRxiv preprint tanks prone to leakage and hydraulic failure 104 . Our study shows that untreated hospital wastewaters, which can 603 leak into groundwater and the environment, carry clinically dangerous antimicrobial-resistant bacteria and 604 resistance genes.

Taken together, based on our results from nine hospitals, we can state that clinically important ARGs are 607 circulating in West African hospitals and can spread to their surroundings. There was no significant difference in 608 ARG abundances based on the hospital section or different hospitals within these countries. Various carbapenem 609 ARGs were found in HWWs from Burkina Faso and Finland while among these blaGES seem to dominate in the 610 studied hospitals in Benin. Our results suggest that class 1 integron mediated co-selection could play an important 611 role in shaping the resistome in the Beninese samples. In addition to HWWs, carbapenemase genes and mobile 612 colistin resistance genes were detected in the vicinity as well as locations more distant from the hospitals. Finally, 613 there were significant differences between the HWW resistomes in Benin and Burkina Faso concerning the total 614 relative sum of ARGs and the prevalence of clinically relevant ARGs. Therefore, it is important to consider the 615 differences between African countries in AMR surveillance. 

Data availability

The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the 638 accession number PRJEB47975 and will be public upon article publication in a peer reviewed journal.

Code availability 641 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons? ISME 820 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted October 22, 2021. ; https://doi.org/10.1101/2021.10.19.21265183 doi: medRxiv preprint