Urban rats as carriers of invasive Salmonella Typhimurium sequence type 313, Kisangani, Democratic Republic of Congo.

BACKGROUND: Invasive non-typhoidal Salmonella (iNTS-mainly serotypes Enteritidis and Typhimurium) are major causes of bloodstream infections in children in sub-Saharan Africa, but their reservoir remains unknown. We assessed iNTS carriage in rats in an urban setting endemic for iNTS carriage and compared genetic profiles of iNTS from rats with those isolated from humans. METHODOLOGY/PRINCIPAL
FINDINGS: From April 2016 to December 2018, rats were trapped in five marketplaces and a slaughterhouse in Kisangani, Democratic Republic of the Congo. After euthanasia, blood, liver, spleen, and rectal content were cultured for Salmonella. Genetic relatedness between iNTS from rats and humans-obtained from blood cultures at Kisangani University Hospital-was assessed with multilocus variable-number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing (MLST) and core-genome MLST (cgMLST). 1650 live-capture traps yielded 566 (34.3%) rats (95.6% Rattus norvegicus, 4.4% Rattus rattus); 46 (8.1%) of them carried Salmonella, of which 13 had more than one serotype. The most common serotypes were II.42:r:- (n = 18 rats), Kapemba (n = 12), Weltevreden and Typhimurium (n = 10, each), and Dublin (n = 8). Salmonella Typhimurium belonged to MLST ST19 (n = 7 rats) and the invasive ST313 (n = 3, isolated from deep organs but not from rectal content). Sixteen human S. Typhimurium isolates (all ST313) were available for comparison: MLVA and cgMLST revealed two distinct rat-human clusters involving both six human isolates, respectively, i.e. in total 12/16 human ST313 isolates. All ST313 Typhimurium isolates from rats and humans clustered with the ST313 Lineage 2 isolates and most were multidrug resistant; the remaining isolates from rats including S. Typhimurium ST19 were pan-susceptible.
CONCLUSION: The present study provides evidence of urban rats as potential reservoirs of S. Typhimurium ST313 in an iNTS endemic area in sub-Saharan Africa.

1 Introduction

Worldwide, invasive non-typhoidal Salmonella (iNTS) infections account for 535,000 (95% uncertainty interval 409 000–705 000) invasive disease cases. Sub-Saharan Africa accounts for the largest proportion (79.9%), the highest incidence (34.5% (26.6–45.0)) and a fatality ratio of 15.8% (10.0–22.9%) [1]. Risk factors for iNTS infection in sub-Saharan Africa are age < 5 years old, Plasmodium falciparum malaria and HIV-infection [2,3]. Main invasive NTS serotypes are Salmonella enterica subspecies enterica Typhimurium and Enteritidis (hereafter named S. Typhimurium and S. Enteritidis), in particular S. Typhimurium ST313 [4-7]. This contrasts with high-income countries, where most frequent S. Typhimurium sequence type is ST19 [8,9], that causes self-limiting diarrhea with only occasional progression to bloodstream infection [10]. In addition to their virulence, iNTS display high prevalence of antimicrobial resistance [2,11,12].

In high-income countries, the reservoir of non-typhoidal Salmonella is zoonotic and transmission is mainly foodborne [10,13,14]. In contrast, iNTS infections in sub-Saharan Africa are believed to be anthroponotic [7,15,16]. In line with Salmonella Typhi and Paratyphi A causing enteric fever in humans [17], invasive S. Typhimurium and Enteritidis have genetically adapted to humans [2,11]. These invasive NTS are clinically specific: they are mainly causing bloodstream infections (and not diarrhea) and are associated with high mortality [18,19]. Further, household studies of index patients with NTS bloodstream infections in Kenya and Burkina-Faso have demonstrated NTS stool excretion in household members but not in household livestock [15,20]. However, reviewing the evidence of source attribution, several authors stated that an animal reservoir for NTS in sub-Saharan Africa is not excluded and needs further study [21-23].

The Democratic Republic of Congo (DR Congo) is highly endemic for iNTS disease. The National Institute of Biomedical Research (INRB) in Kinshasa coordinates, in collaboration with the Institute of Tropical Medicine (ITM) in Antwerp, Belgium, a nationwide microbiological surveillance network processing blood cultures in patients suspected of bloodstream infections. Kisangani University Hospital participates—through hospitals and health centers within and around Kisangani—in this network [24]. Over the past decade, NTS consistently represented the most frequent blood culture isolates in children attending the hospitals and health centers in this network, with S. Typhimurium and Enteritidis isolates representing respectively 63.1% and 36.4% of iNTS isolates of which 87.4% displaying MDR [19].

As a fast-growing densely populated metropolis, Kisangani provides a wide array of ecological niches attractive for rodents. Previously, Kisangani University had assessed rats trapped in marketplaces in Kisangani as reservoirs of Rickettsia typhi and Bartonella spp. [25]. Rodents have been described as a reservoir and vector of many pathogens including Salmonella [26] but to our knowledge, rats have not yet been assessed as a potential reservoir of NTS. Therefore, we aimed to assess rats present in marketplaces in Kisangani for carriage of NTS. Our objectives were to estimate the proportion of S. Typhimurium and Enteritidis carriers (rats with S. Typhimurium or Enteritidis isolates from liver, spleen, blood, and/or intestinal content) among rats, and to assess genetic relatedness between rat and human NTS isolates. A secondary objective was to assess the antimicrobial resistance profile of the Salmonella isolates.

2 Materials and methods

2.1 Ethics statement

Ethical approval for the blood culture surveillance study was granted by the Institute of Tropical Medicine Antwerp Institutional Review Board (approval number: 613/08), the Ethics committee of Antwerp University (approval number: 8/20/96) and the Ministry of Health of the Democratic Republic of the Congo (approval number: 8/20/96). For the rat carrier study, authorization by the health authorities of Tshopo Province (Division Provincial de la Santé) was obtained on 12 May 2016 and procedures were carried out according to the 2016 Guidelines of the American Society of Mammologists for the use of wild mammals in research and education [27]. Before starting rat trapping, the responsible in charge of security at the trapping site was contacted for permission and surveillance of the traps.

2.2 Study design

In an exploratory study, rats trapped alive in marketplaces in the Kisangani area were sampled for Salmonella in the microbiological laboratory at the University Hospital of Kisangani. Salmonella isolates were tested for serotype and antimicrobial susceptibility. S. Typhimurium and Enteritidis isolates were compared by molecular typing for genetic relatedness with clinical isolates obtained from blood cultures.

2.3 Rat sampling

2.3.1 Sample size calculation

Based on CLSI guidelines, a minimum sample size of 30 Salmonella spp. isolates was set for reliable data about serotype distribution and antimicrobial susceptibility profiles [28]. At an assumed NTS carrier ratio of 5–10% [29-32] we targeted to capture a total of 600 rats to have sample size of 30 rats carrying Salmonella.

2.3.2 Rat collection settings

The rat sampling was conducted between April 2016 and December 2018, with interruptions due to administrative and/or logistical issues from January to February 2017 and from April 2017 to April 2018 (S1 Fig). Kisangani counts approximately one million inhabitants; 70 to 80% are living below the poverty line [33]. Sampling sites were selected and described previously [25] (Fig 1). The Central Market is the largest market in Kisangani city, located in the city center, as is the case for Makolo-Ngulu market, Tshopo market and Christ-Roi market. Yaté Market is in the secondary port of Kisangani, at the Congo River. Merchants from the surrounding villages temporarily reside at the market. The Mangobo slaughterhouse, at the bank of the Tshopo river, is situated in a savannah habitat, close to a small port trading cattle imported from the eastern provinces (Ituri and Nord-Kivu). In all these sites, the goods are displayed on tables made of unpolished wood allowing easy access by rats. In some places, the products for sale are placed on the ground (e.g. rice or corn put to dry). There is no rodent control policy in the city of Kisangani.

Fig 1

Overview of sampling sites in Kisangani city.

Kisangani map was made using OpenStreetMap data, Stamen open source maps (https://stamen.com/open-source/) and the R-package ggmap [34].

2.3.2 Rat sampling procedure

Tomahawk live-capture traps (Tomahawk Trap Co., Tomahawk, WI, USA) (dimensions 490/178/173 mm) were used with smoked fish or meat as bait. The traps were installed in warehouses, rice mills, or sheltered places in the sampling sites, to prevent them from being stolen or misused. Traps were installed between 5–7 p.m. and collected the next day between 6–8 a.m. Using a global positioning system (GPS) (Garmin 60 Cx Southampton, Hampshire, UK), the location of each positive trap was recorded.

Trapping success was defined as the proportion of rats caught per number of traps installed in the trapping site. Trapped rats were transported to the Faculty of Sciences in Kisangani. At reception, rats were taken out of the trap using a single-use cloth bag for each animal. Still in the bag (to prevent scratches or bites), they were euthanized with an intraperitoneal injection of a lethal dose of ketamine (Ketalin, Shalina laboratories PVT LTD, Mumbai India, 0.5-1ml of 250mg/5ml solution). The delay between collection from the trapping site and euthanasia was less than 3 hours and the animal was dissected immediately after the euthanasia (less than 10 minutes). Rat species (Rattus norvegicus versus Rattus rattus) was determined on morphological characteristics. Sexual status, length and mass were recorded.

After euthanasia, rats were placed in a dissection tray and the abdomen was sprayed with 3-5ml of 80% ethanol to disinfect the skin. Using a sterile scalpel blade, an incision was made throughout the length of the abdomen. Different samples (2–3 g, each sampled with separate sterile instruments) were collected from the spleen and subsequently from the liver. From May 2018 onwards, additional blood (0.5-1ml) was sampled by intracardiac puncture, before sampling of spleen and liver. At the end of the procedure, the rectal content was collected. Internal organs collected were transported to the lab for preliminary analysis within 4 hours.

The samples of spleen, liver and rectal content were placed in a sterile Petri dish, sliced, and transferred into 10ml Selenite broth (Becton Dickinson and Company, Franklin Lakes, NJ, US) followed by vortex mixing. Blood was inoculated directly into the Selenite broth tube. Selenite broth tubes were transported to the microbiology laboratory within 4 hours after inoculation.

2.4 Salmonella identification, serotyping, and antibiotic susceptibility testing in rats

Laboratory processing was done as described previously for stool samples [35]. Selenite broth tubes were incubated at 35°C for 12–18 hours, whereafter 10 μl broth was inoculated on two Salmonella-Shigella (SS) agar plates (Lab M Limited, Lancashire, UK). These were incubated at 35°C and read after 18-24h. In case of no growth, the plates were evaluated after another 24 hours of incubation. In case of growth, up to five (to detect the presence of multiple seroptypes) colonies suspected of Salmonella were transferred to Kligler Iron Agar (KIA) tubes (Lab M Limited) and incubated for 18–24 hours at 35°C. Bacterial growth in the KIA tubes suggestive of Salmonella was biochemically confirmed by a panel of disk-based biochemical tests (DiaTabs, Rosco, Taastrup, Denmark). Isolates with a reaction pattern compatible with Salmonella were stored in 2 ml tubes of Trypticase Soy Agar (Oxoid, Basingstoke, UK) and shipped to the Institute of Tropical Medicine (ITM, Antwerp, Belgium) for serotyping and antibiotic susceptibility testing (AST).

Serotyping was done by commercial antisera (Vison, Pro-lab Diagnostics Inc., Richmond Hill, Ontario, Canada). AST was done by disk diffusion (Neo-Sensitabs, Rosco, Taastrup, Denmark) and, in the case of azithromycin and ciprofloxacin, by E-test macromethod (bioMérieux, Marcy L’Etoile, France) for assessment of minimal inhibitory concentration values (MIC-values) as previously described [19] (S3 Table). Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100-S31 criteria [36]. Multidrug resistance (MDR) was defined as combined resistance to amoxicillin, trimethoprim-sulfamethoxazole, and chloramphenicol [12].

2.5 Human sampling

Salmonella isolates were obtained and stored as part of this INRB-ITM microbiological blood culture surveillance network at Kisangani University Hospital as previously described [24]. Basic clinical and demographic data were recorded. To assess the genetic relatedness between Salmonella isolates from rats and humans, blood culture isolates obtained during the rat trapping period and six months before and 6 months after this period (01 October 2015–30 June 2019) were selected. Serotyping and AST of blood culture Salmonella isolates were done as described previously [19].

2.6 Whole genome sequencing

For all but one Salmonella spp. isolate, whole genome sequencing (WGS) and analysis was performed as previously described [37]. Total DNA was extracted from overnight cultures at 37°C in tryptic soy broth (TSB) (Oxoid) using the MagNAPure 96 system (Roche Diagnostics, Meylan, France). WGS was performed by the genomic platform of the Institut Pasteur, in Paris, France (“Plateforme de microbiologie mutualisée”, P2M). The libraries were prepared with the Nextera XT kit (Illumina) and sequencing was performed with the NextSeq 500 system (Illumina, San Diego, CA, USA), generating 150 bp paired-end reads. The short reads were assembled de novo with SPAdes version 3.6.0.23. Serotype prediction, multilocus sequence typing (MLST), and core genome MLST (cgMLST) were performed with various tools integrated into EnteroBase (https://enterobase.warwick.ac.uk/). The presence and type of antimicrobial resistance genes (ARGs) or ARG-containing structures were determined with ResFinder version 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/) and PlasmidFinder version 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) on SPAdes assemblies.

2.6.1 Genetic relatedness between Salmonella isolates from rats and humans

Genetic relatedness between the iNTS isolates (in case those belonging to S. Typhimurium serotype, see Results) was assessed by three different approaches: multilocus variable-number tandem repeat (VNTR) analysis (MLVA), MLST, and cgMLST.

MLVA was performed at Sciensano (Brussels, Belgium) as described previously [35]. MLVA profiles were established based on five loci ordered STTR9-, STTR5-, STTR6-, STTR10-, and STTR3-. Identical S. Typhimurium MLVA types (clusters) were defined as isolates with variation in none or one of the rapidly changing loci (STTR5, STTR6, and STTR10) but no variation in the stable loci (STTR3 and STTR9) [38].

For WGS, the 7-genes MLST scheme was applied, based on the sequences of the seven housekeeping genes: aroC, dnaN, hemD, hisD, purE, sucA, thrA [39,40]. Hierarchical clustering on cgMLST (HierCC) and minimum spanning trees (MS-trees) was performed on EnteroBase based on the sequences of 3002 loci for Salmonella [41]. On the HierCC scheme, HC2850 values allow to deduct the Salmonella subspecies (i.e. difference of ≤ 150 or less alleles from the 3002 loci in the cgMLST scheme); HC200 values provide a similar discrimination as the 7-genes MLST typing (≤ 200 alleles difference between genomes); and HC5 values can be considered a strong indicator of epidemiological relatedness (i.e. if no more than five alleles are different) [41,42].

2.6.2 Genetic relatedness between isolates from this study and the literature

We compared some of the isolates of S. Typhimurium and S. Enteritidis identified in this study with previous studies on iNTS in Africa [7,43] using EnteroBase. This database recovers regularly Salmonella genomes from the GenBank Sequence Read Archive (SRA). The “Custom View” utility allows to add metadata for genomes already present on the site. EnteroBase has recovered 564 genomes from the study on S. Typhimurium ST313 by Pulford et al. [7] and 503 genomes from the study on S. Enteritidis ST11 by Feasey et al. [43]. A “Custom View” was created to add the information on lineages and plasmid repertoire for the S. Typhimurium ST313 from the Pulford et al. study [7], that was next compared to the HierCC clustering on cgMLST by EnteroBase [41]. A “Custom View” previously created, including the hierBAPS clade/cluster data described by Feasey et al. [37] was used to compare the hierBAPS clustering with the EnteroBase HierCC clustering. A minimum spanning (MS) tree (MStree V2 or GrapeTree) based on the EnteroBase “cgMLST V2 + HierCC V1” scheme was produced for each serotype to estimate the allelic distances between isolates from this study and the genomes from Pulford et al. [7] and Feasey et al. [43].

For the S. Typhimurium ST313 in this study, four plasmids, previously associated with the main lineages circulating in Africa: pSLT-BT (accession: FN432031.1), pBT1 (accession: LS997975.1), pBT2 (accession: LS997976.1), and pBT3 (accession: LS997977.1) were searched, and this information was included in the S. Typhimurium ST313 “Custom View”.

For the S. Enteritidis ST11 in this study, plasmid pSEN-BT (accession: LN879484.1) described by Feasey et al. [43] as characteristic of the hierBAPS clade 9, also denominated Central/Eastern African clade was also searched and the information was included in the S. Enteritidis ST11 “Custom View”.

2.7 Data management and analysis, definitions

Geographic coordinates, morphometric measurements of rats and microbiological data were entered in an Excel database (Microsoft, Redmond, WA, USA). Rats with at least one isolate confirmed as Salmonella were categorized as Salmonella carriers. Several rats carried more than one Salmonella serotype. Only the first isolate per serotype per rat was used for the analysis of the distribution of serotypes and AST. For the display of MLVA typing results, all Salmonella Typhimurium isolates were assessed but only the first isolate per MLVA type per rat was presented. All genomes obtained by WGS and analyzed on EnteroBase are available at the ENA depository under project number PRJEB54047 (S1 Table).

3 Results

3.1 Rats sampling

During 55 outreach visits, 566 (34.3%) of 1,650 traps yielded 566 rats including 541 (95.6%) Rattus norvegicus (Norwegian or brown rat) and 25 (4.4%) Rattus rattus (roof rat or black rat). The highest trapping success was seen in Central and Yaté marketplaces (30.4% and 48.7% respectively). There were no incidents (e.g. tripping) with the traps reported and apart from one cat, no other non-rat species was captured. A total of 46 (8.1%) rats carried Salmonella spp. (Table 1). All captured rats were alert and active, and none showed signs of illness and weakness. Most rats (89.0%, 504/566) were sexually mature, with a male-to-female ratio of 0.91; median (Interquartile Range, IQR) length (without tail) and mass were 22.1 cm (8.3–28.4 cm) and 280 g (30–515 g) respectively; showing no difference between Salmonella carriers and non-carriers (S2 Table).

Table 1

Numbers of outreach visits, rats caught and Salmonella carriers over the six sampling sites in Kisangani 2016–2018.

	Central Market	Yaté Market	Slaughter house Mangobo	Christ-Roi Market	Makolo-Ngulu Market	Tshopo Market	Total
Outreach visits	14	26	5	2	3	5	55
Traps installed	420	780	150	60	90	150	1650
Rats caught (% of traps)	128 (30.4%)	380 (48.7%)	12 (8.0%)	4 (6.7%)	16 (17.8%)	26 (17.3%)	566 (34.3%)
Salmonella carriers (% of rats)	12 (9.4%)	30 (7.9%)	2 (16.7%)	1 (25.0%)	0 (0.0%)	1 (3.8%)	46 (8.1%)

3.2 Distribution of serotypes and specimens in rats

The 46 Salmonella rat carriers yielded a total of 254 Salmonella isolates, of which 253 were available for further analysis, one isolate was not viable after transport. Table 2 lists the Salmonella serotypes isolated per body specimen and rat. Thirteen rats yielded multiple serotypes and a total of 73 non-duplicate serotypes isolates were recovered. Most common serotypes were serotype II.42:r:- (found in 18 rats), Kapemba (12 rats), Weltevreden and Typhimurium (10 rats each), and Dublin (found in 8 rats). All Salmonella Dublin non-duplicate serotype isolates (n = 11) and all but one S. Typhimurium isolates (n = 12 in 10 rats) were obtained exclusively from deep organ samples (spleen or liver). S. Enteritidis was not isolated from rats.

Table 2

Serotypes of rats (n = 46) according to specimen.

First isolate per serotype per specimen. A total of 73 single non-duplicate Salmonella serotype isolates were recovered from 46 rats; multiple serotypes were observed in 13 rats. Specimens listed were sampled for each rat, except for blood which was sampled for 218/566 (38.5%) rats.

	Blood	Liver	Spleen	Rectal content	Total	Numbers (%) of rats carrying the serotype
Salmonella Dublin	0	5	6	0	11	8 (17.3%)
Salmonella II:42:r:-	2	6	2	10	20	18 (39.1%)
Salmonella Kapemba	2	6	6	3	17	12 (26.0%)
Salmonella Mikawasima	0	0	1	1	2	2 (4.3%)
Salmonella Orion	0	0	0	1	1	1 (2.17%)
Salmonella Typhimurium	1	6	4	1	12	10 (21.7%)
Salmonella Weltevreden	0	5	2	3	10	10 (21.7%)
Total	5	28	21	19	73	46

3.3 Salmonella serotypes obtained from human blood cultures

A total of 40 non-duplicate Salmonella isolates were recovered from a total of 2742 blood cultures performed. Two isolates were not viable upon retrieval from shipment and not available for serotyping. Main serotypes comprised Typhimurium (n = 18), and Enteritidis (n = 11). Other serotypes were Typhi (n = 6), Paratyphi C (n = 1), II.42:r:- (n = 1), and monophasic I.6,7:y:- (n = 1). Median age of patients infected with iNTS (n = 34) was 20.5 months (3 days– 48 years), 81.8% (n = 27/33) were less than 5 years old. Male-to-female ratio was 0.83.

3.4 S. Typhimurium isolates

Results of MLVA and MLST typing of S. Typhimurium isolates from rats (12 isolates from 10 rats) and humans (18 isolates from 18 patients available for MLVA typing, 16 isolates from 16 patients available for MLST typing) are listed in Table 3. Fig 2A and 2B depicts the clustering of those 32 isolates through a minimum spanning tree on cgMLST.

Table 3

MLVA types of Salmonella Typhimurium isolated from rats and human blood cultures.

NA corresponds to a locus on which no allele has been amplified. The two groups of grey shaded cells present MLVA clusters of rat (bold) and human isolates. Two rats (MC071 and MC025) had each two different MLVA types. Code numbers of rats refer to sampling site: AB = Slaughterhouse (Abattoir) Mangobo, MC = Marché Central, MY = Marché Yaté; and specimen type: S = spleen, L = liver, B = blood, ND = no data.

MLVA type	Rats			Humans
	Code number	ST	Date	Code number	ST	Date	Residence township (distance from rat trapping site)
2-5-8-8-210	-		-	5548/4	313	28/10/16	Mangobo (3.5 km)
2-5-9-8-210	MY302/S MY305/L	313313	08/10/1808/10/18	--	--	--
2-5-11-8-210	---	---	---	5232/46284/46436/4	313313313	22/05/1607/08/1806/09/18	Kabondo (3.7 km)Kabondo (3.7 km)Tshopo (3.5 km)
2-6-11-8-210	--	--	--	5248/45376/4	313313	26/08/1610/08/16	UnknownUnknown
2-8-10-8-210	--	--	--	5809/45810/4	313313	13/05/1713/05/17	Kisangani (2.9 km)Kisangani (2.9 km)
2-7-10-8-210	MC071/L+S	313	06/10/16	5403/45464/45598/45601/4	313313313313	27/08/1623/09/1622/11/1624/11/16	Kabondo (2.6 km)UnknownKisangani (1.2 km)Kabondo (2.6 km)
2-7-11-8-210	MC071/S	313	06/10/16	-		-
2-4-11-7-210	-	-	-	7281/4	Not done	13/06/19
2-7-12-10-210	-	-	-	4930/4	Not done	30/10/15
2-13-4-3-NA	-	-	-	5835/4	313	31/05/17
2-NA-12-7-210	---	---	---	5390/47279/47292/4	313313313	16/08/1610/06/1914/06/19
3-12-5-9-311	MC025/L+S	19	15/05/16	-	-	-
3-12-5-10-311	MC025/L+S	19	15/05/16	-	-	-
3-15-5-11-311	MY213/S	19	10/06/18	-	-	-
	MY218/L	19	10/06/18	-	-	-
	MY365/S	19	16/12/18	-	-	-
	MY373/L	19	23/12/18	-	-	-
	MY375/B	19	23/12/18	-	-	-
2-20-9-7-212	MY300/L	19	08/10/18	-	-	-

Fig 2

Clustering of S. Typhimurium ST313 human and rat isolates.

Minimum spanning tree created using the MSTree V2 component in EnteroBase, based on the allelic differences over the 3002 alleles that constitute the EnteroBase HierCC scheme on cgMLST [41]. The distances between leaves in the tree indicate number of alleles different between genomes. Genomes with common HC5 values are at five or less alleles, strongly indicating epidemic relatedness. Isolates from rats are indicated with an icon and prefix MC and MY (referring to Christ market and Yate market respectively. In panel A, colors are according to cgMLST HC5 values; in panel B, colors are according to MLVA type. Human and rat isolates are grouping in two clusters (MY305/302 and MC071 with each six human isolates. For details of date and place, see Table 3. Rat drawing by Francisca Arévalo from NounProject.com.

The 18 human S. Typhimurium isolates comprised nine different MLVA types and all 16 sequenced isolates belonged to MLST ST313. Among the 12 S. Typhimurium isolates recovered from 10 rats, four isolates (from three rats) belonged to ST313. The rat ST313 isolates represented 6.5% of Salmonella-carrying rats and 0.5% of all rats captured. Rat ST313 isolates belonged to three MLVA types. The remaining eight S. Typhimurium isolates from rats belonged to ST19 (four MLVA types, seven rats). The three rats carrying Salmonella ST313 did not show signs of illness and had length and mass in line with all the other rats in the study (S2 Table).

Two human-rat clusters were identified among the ST313 isolates, involving the three ST313 carrying rats and 12 out of 16 (75.0%) human isolates (Table 3, Fig 2A and 2B). In the first cluster, two rat isolates (Yate Market, October 2018) grouped with six human isolates obtained between May 2016 and September 2018 (28 months) from patients living between in a perimeter of 3.5 to 3.7 km from the marketplace. In the second cluster, two isolates from one rat (Central Market, October 2016) grouped with six human isolates obtained between August 2016 and May 2017 (8 months) from patients living within a perimeter of 1.2 to 2.9 km from the marketplace.

The ST313 S. Typhimurium human and rat isolates clustered with the ST313 Lineage 2 isolates described by Pulford et al. [7], at less than 50 alleles (by the EnteroBase cgMLST scheme), and at more than 200 alleles to the Lineage 1 and Lineage 3 isolates in their study (Fig 3). As to the plasmid repertoire described by Pulford et al.[7], there were two distinct profiles: pBT2 and pBT3 (n = 6 humans and n = 1 rat) and pBT3 alone (n = 7 humans and n = 2 rats) (Fig 4). The ten ST19 S. Typhimurium isolates (all eight rats captured at Yaté and Central markets) clustered both by MLVA and HC5 (Fig 5).

Fig 3

Clustering of S. Typhimurium ST313 human and rat isolates with ST313 Lineage 2 isolates described by Pulford et al. [7].

Minimum spanning tree (MSTree V2) comparing the S. Typhimurium ST313 genomes analyzed by Pulford et al. [7] in 2021 and the genomes in this study. The ST313 genomes in this study (circled in red, filled in white) cluster together with the Lineage 2 strains in the study by Pulford et al. [7], colored in green).

Fig 4

Plasmid profiles of S. Typhimurium ST313 human and rat isolates according to the plasmid profiles of ST313 Lineage 2 isolates described by Pulford et al. [7].

Minimum spanning tree (MSTree V2) comparing all S. Typhimurium ST313 in this study. 58 alleles separate the two mixed clusters (human and rats isolates) with different plasmid content: in orange, isolates harboring plasmids pBT2 and pBT3; in green, isolates harboring pBT3, only. Rat drawing by Francisca Arévalo from NounProject.com.

Fig 5

Clustering of S. Typhimurium ST19 rat isolates according to HC5-values (cgMLST V2 + HierCC V1, upper left) and MLVA type (lower right).

Minimum spanning tree (MSTree V2) comparing all ST19 in this study. In panel A, colors are according to cgMLST HC5 values; in panel B, colors are according to MLVA type.

3.5 Salmonella Enteritidis isolates

Among 11 human S. Enteritidis isolates available, 10 (90.9%) belonged to ST11. When compared on EnteroBase with the genomes available from the study Feasey et al. [43], they clustered at less than 50 alleles to the hierBAPS clade 9, also denominated Central/Eastern African clade (S2 Fig), and carried the expected plasmid pSENT-BT.

3.6 Antimicrobial resistance profile

S. Typhimurium from both rats (all 3) and humans (14 out of 18) displayed MDR, encoding for resistances to a variable number of families of antibiotics. The most frequent profile consisted of AMR determinants to beta-lactams (blaTEM-1), aminoglycosides (strA, strB, ant(3’)-Ia, aac(3’)-IId), sulfonamides (sul1, sul2), trimethoprim (dfrA1), tetracyclines (tetB), chloramphenicol (catA10). Among human isolates, occasional decreased ciprofloxacin susceptibility and resistance to azithromycin and ceftriaxone were also observed (Table 4, S3 Table). In contrast, S. Typhimurium ST19 isolates from rats and all other serotypes from rats were susceptible to all antibiotics tested.

Table 4

Antimicrobial resistance profiles of Salmonella Typhimurium isolates from rats and humans (blood cultures).

Data present numbers (%) of resistant isolates. Only the first isolate per serotype and rat is listed. MDR: multidrug resistant, i.e. resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. DCS = decreased ciprofloxacin susceptibility, i.e. ciprofloxacin MIC-value > 0.064 mg/L and < 1 mg/L.

	Rats			Humans
Resistance to:	Salmonella TyphimuriumST 313 (N = 3)	Salmonella TyphimuriumST 19 (N = 7)	Other Salmonella (N = 52)	Salmonella Typhimurium (N = 18)
Ampicillin	3	0	0	18
Trimethoprim-sulfamethoxazole	3	0	0	18
Chloramphenicol	3	0	0	14
Multidrug resistant (MDR)	3 (100.0%)	0 (0.0%)	0 (0.0%)	14 (77.8%)
Ceftriaxone	0	0	0	1
Azithromycin	0	0	0	4
Decreased ciprofloxacin susceptibility (DCS)	0	0	0	3
MDR + DCS	0	0	0	1

ST11 S. Enteritidis isolates, all recovered from human samples, encoded for AMR determinants to 5 to 6 families of antibiotics: beta-lactams (blaTEM-1), aminoglycosides (strA, strB), chloramphenicol (cat2), sulfonamides (sul2), trimethoprim (dfrA7) and tetracyclines (tetA).

4 Discussion

In a sub-Saharan Africa setting endemic for iNTS, the present study showed a Salmonella carrier ratio of 8.1% among rats captured in an urban area. Among the serotypes recovered, there was invasive S. Typhimurium ST313 in 3/46 Salmonella-carrying rats which clustered with 13/16 human isolates.

4.1 Rats as reservoirs of non-Typhoidal Salmonella

Rodents–in particular rats–are well-known reservoirs of zoonotic pathogens including Salmonella [26]. Prevalence of Salmonella in rats has mainly been assessed in high-income countries and varied widely: in the natural environment, prevalence was consistently low (0 to 1%) [26,44,45] but is was higher in the proximity of human settlements (5–20%) such as cities and layer or pig farms (up to 28%) [29-32,46-50]. Of note, studies also showed a high variation in Salmonella prevalence between rats in different cities; 15% in Yokohama (Japan) versus 1% in Chicago (US), 0.34% in Nairobi (Kenya) and 0% in Baghdad (Iraq) [32,51-53]. These differences can partly be ascribed to the urban landscape (presence of food and shelter, sanitary conditions); the highest reported prevalence (49.1%) has been observed in wet marketplaces in Udon Thani City, Thailand [54].

In the above-mentioned studies, Salmonella serotypes from commensal rats varied widely but there was a consistent overlap with human epidemiologically relevant serotypes. Examples are Salmonella Enteritidis and Infantis from layer farms [26,47,55] and Salmonella Weltevreden from rats in marketplaces in Thailand [54]. S. Typhimurium and–to a lesser extent–Enteritidis belong to the most observed serotypes [29,31,32,47,48,52,54,56]. Molecular typing allowing for sequence typing of the non-Typhoidal Salmonella was not performed so far. The aforementioned study in Japan showed that S. Typhimurium isolates obtained from urban rats belonged to the DT104 phage type, i.e., the world-wide distributed zoonotic S. Typhimurium causing diarrhea in humans [32].

4.2 Rats carried S. Typhimurium ST313, the predominant S. Typhimurium clone in Africa

The 8.1% Salmonella prevalence among rats in the present study fits into the prevalence ranges described above. Among the serotypes recovered, the main finding was the presence of S. Typhimurium ST313 in three rats. By MLVA as well as by comparison of genomes in EnteroBase, the ST313 isolates grouped in two clusters comprising each six human ST313 isolates recovered from blood cultures. ST313 is the main pathovariant of iNTS in Africa [7]. Both rat and human isolates clustered together with what Pulford et al. describe as Lineage 2, i.e. the most common and recent S. Typhimurium ST313 lineage in the African continent [7]. Close genetic relation was also supported by the identification of the pSLT-BT plasmid, encoding for the different AMR genes that most ST313 isolates from rats and humans in the present study harbored, particularly the cat1 gene encoding for chloramphenicol resistance, which is hallmark for Lineage 2 [7].

4.3 Finding of Salmonella Typhimurium ST313 in rats: laboratory animal models

So far, reservoir and transmission of iNTS including ST313 were hypothesized to be human, based on the genetic signature [4,5,22] and household, case-control, and environmental studies [15,16,20].

Finding ST313 in rats indicates that ST313 is not restricted to the human host. Previous laboratory animal models showed that ST313 was able to cause invasive infections in chickens, mice, and rhesus macaque infection models [6,57-59]. Findings differed according to the challenging strain and host animal but overall ST313 caused, in comparison with ST19, a more rapid onset of bacteremia and infestation of liver and spleen combined with less diarrhea and colonization of the intestinal tract. The fact that in the present study ST313 in rats was exclusively recovered from deep organs may point to an invasive disease too, but none of the rats captured was visibly sick and their length and mass were in line with the other rats. Moreover, although S. Typhimurium strains can cause deadly infections, asymptomatic infections of liver and spleen with intermittent fecal shedding can occur too [29,60,61] and may even constitute the majority of infections [62].

4.4 Attribution of rats to Salmonella transmission

Despite being acknowledged as a reservoir for Salmonella, there is only circumstantial and anecdotal evidence as to the attribution of rats to the transmission of Salmonella [29,61,63]. Rather than being an original reservoir for Salmonella, rats are considered to be a “sponge” reflecting the environment they are living in [26,45,51]. This has been demonstrated in layer farms, where proportions of Salmonella carrying rats were related to the percentage of Salmonella contaminated eggs and the intensity of the environmental contamination [47].

Rats shed Salmonella for 2–4 months after infection and have up to 40 droppings a day [29]; Salmonella survives long (up to 86 days) in rat droppings [30]. In this way, rats contaminate the layer hen’s food and thereby maintain and even amplify Salmonella infection in the food production chain [26,47,55]. Conducive to this is the social life of rats (allowing transmission among rats [64] and the long (> 1 year) environmental survival of Salmonella [65], although ST313 may have lower environmental persistence compared to the diarrhea-causing ST19 [11]. In addition, rat carcasses may also constitute a Salmonella reservoir on which other animals (stray cats, lizards, cockroaches, ants, etc.) can feed and further contribute to the spread of Salmonella [26].

A similar mechanism of indirect (“sponge”) reservoir may be postulated for wet markets. In the present study, this is supported by the observation that the marketplaces where ST313 carrying rats were captured (Central Market and Yaté market) were those with the highest rat density. Further, the market setting in a low resource setting such as Kisangani offers exposure of food and shelter in the absence of pest control. By consequence, these are high density populations of rats which was demonstrated by the high trapping success in the present study (one third of non-pre-baited traps) [45]. The present rat population consisted of nearly 90% sexually mature rats versus 63.9% in a rat population in urban Canada; also, length and mass were higher in Kisangani versus Canada (medians of 22.1 cm and 280 g versus 17.5 cm and 142.2 g respectively). This means that the Kisangani rats were probably older, which may increase the probability of rat associated zoonosis [45].

At the human side, socioeconomic factors fueling transmission in Kisangani are numerous: poor sanitary conditions, crowding, overnight stay of vendors, unsafe drinking water and cooking practices. The marketplaces sampled in the present study are easy to reach by the Kisangani population, either by walking or by motorcycle, and human mobility is compatible with the geographical perimeters of the rat-human clusters in the present study. Of note, rats can move too: urban rats are territorial [32,45] but may walk up to 3 km and further in one night [66,67]

4.5 The present findings point to the evidence of an animal reservoir for ST313

As only 6.5% of Salmonella carrying rats and 0.5% of all rats captured harbored ST313, it is unlikely that rats constitute a major or preferred reservoir of ST313. However, the rat ST313 isolates clustered with the majority (75.0%) of human isolates, pointing to a close interaction between both. Moreover, the occurrence of ST313 in rat populations postulates also other animals as potential reservoirs of ST313. Previous household studies assessing the iNTS reservoirs in sub-Saharan Africa sampled livestock and poultry but no indoor rodents [15,20,68]. Recently, two studies assessed the meat pathway as a potential source of Salmonella. A slaughterhouse study from Kenya and Malawi found a 12.7% Salmonella prevalence in pig carcasses but failed to detect iNTS clades [66]. A study from East Africa showed that S. Enteritidis ST11 was present in the meat pathway and clustered with human isolates. Other non-typhoidal Salmonella serotypes were observed in the meat pathway too, but not ST313 [23]. Furthermore, a study in DR Congo showed a direct association of rainfall with iNTS, pointing to a possible waterborne environmental reservoir [69].

The potential existence of an environmental reservoir and transmission of iNTS may have an impact on ongoing efforts on iNTS vaccine development, most of which target S. Typhimurium [70] and put more emphasis on water, sanitation, and hygiene as control measures. Moreover, ongoing deforestation and climate change are expected to favor both rodent populations and salmonellosis [65,71] with a potential increasing impact of the rat-Salmonella reservoir and transmission.

4.6 Other Salmonella serotypes and antimicrobial resistance

The other serotypes obtained from rats mainly comprised II:42:r:-, Kapemba, Weltevreden and Dublin. They were susceptible to all antibiotics tested (= pan-susceptible), as was also the case for the S. Typhimurium ST19 isolates. This pan-susceptibility might be explained by the fact that the non-ST313 Salmonella isolates most probably represent animal-confined isolates. Agriculture and livestock raising in the Kisangani area are mainly organized as small-scale subsistence farming with expected a lesser use of antibiotics compared to human medicine. A similar observation was noted in the aforementioned study about Salmonella in pigs in Kenya and Malawi: most isolates were susceptible to all antibiotics tested, in line with presumed low antibiotic exposure [72].

Salmonella Dublin is host-adapted to cattle but has been recognized as iNTS too in Mali [73]; it has not yet been detected as part of the microbiological surveillance in DR Congo [22,71]. Salmonella Weltevreden is a major cause of intestinal infections in Asia and was cultured from rats in marketplaces in Thailand and India in different environments as poultry farm, feed store and around residential quarters [48,54]. To our knowledge, Salmonella serotypes II:42:r:-, Kapemba, Mikawasima and Orion so far have not yet been described in rats.

4.7 Limitations and strengths

A limitation of the present study was the fact that only outdoor sampling was conducted. Indoor sampling would have been a valuable adjunct but in view of the public perception and acceptability, it was presently not done. Likewise, traps were not pre-baited whereas doing so could have increased trapping success as rats are neophobic [45,67]. Trapping success was however high and in line with a previous study [25]. Last, the molecular subtyping alone (as presently used) cannot provide information about the pathways of transmission (rat to human or vice-versa) [13].

Among the strengths were the multiple sampling sites which precluded rat territorial bias [74]. Further, trapping was not impacted by non-target species and, thanks to excellent communication with the city and market authorities, there was no tripping or stealing by the public [74]. The present sample size (n = 566) was large compared to other studies [48,51,72] and human clinical isolates from the same period and area were available for comparison. In addition, we collected physical data of the rats (which may relate to the presence of zoonosis [74] and sampled year-round, precluding seasonal variation. The co-located laboratory facilities allowed for expertized staff and short and regular transport. Deep organs were sampled, and they accounted for three quarters of the non-duplicate serotype isolates. If only stool/rectal swab or content would have been sampled (as done in many studies [32,45,54,75]), carriage would have been as low as 2.6% (15 rats) and ST313 would have been missed. Furthermore, as confirmed here, rats may carry more than one Salmonella serotypes [44,45], which is the reason why we processed multiple colonies per sample.

Further research should confirm and extend the present findings to other iNTS-endemic areas. Field sampling should include wet marketplaces but also indoor rodent sampling in households of iNTS index patients. In conjunction with rat studies, market retail products should be assessed for contamination with iNTS [76]. Laboratory experiments should establish the pathophysiology of iNTS in rats in particular intestinal colonization and fecal shedding. At a larger scope, further environmental research into the reservoir of iNTS should be conducted [6,21,23]. Apart from rats, small rodents such as mice should be assessed, as well as lizards and geckos which are notable carriers of Salmonella too [77]. Food and water should be further assessed as a vehicle for transmission, including fork-to-farm studies along the meat pathway [23,78], crops and green leaves [13,69]. Source attribution studies such as case-control and outbreak investigation studies should complement the picture of iNTS transmission [13,78].

In conclusion, the present study provided evidence of rats as carriers of S. Typhimurium ST313 clustering with human blood culture isolates in an iNTS endemic area in sub-Saharan Africa.

ENA depository genomes.

(DOCX)

Click here for additional data file.

Morphometric parameters of rats (Rattus rattus and Rattus norvegicus) related to the Salmonella carriage status in Kisangani 2016–2018).

(DOCX)

Click here for additional data file.

Antibiotic susceptibility data of human and rat Salmonella isolates.

(DOCX)

Click here for additional data file.

Timeline of human and rat sampling.

(DOCX)

Click here for additional data file.

Clustering of human Salmonella Enteritidis isolates with ST11 Salmonella Enteritidis isolates.

(DOCX)

Click here for additional data file.

21 Jun 2022

Dear Dr. Hardy,

Thank you very much for submitting your manuscript "Urban rats as carriers of invasive Salmonella Typhimurium sequence type 313, Kisangani, Democratic Republic of Congo." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please note editorial comments made by reviewer 1, along with suggestions for more detail on the rodent trapping methods used and defining “trapping success” in the methods section of the revised manuscript.

Both reviewers 1 & 3 asked for a map with rodent trapping sites indicated.

Reviewer 3 also requested an excel file to include data used from other groups/public databases. A separate excel file with detailed MIC data should also be provided with the revised manuscript.

All genomic data used in this study will need to be made available per PLoS NTDs guidelines.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Travis J Bourret

Associate Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Please note editorial comments made by reviewer 1, along with suggestions for more detail on the rodent trapping methods used and defining “trapping success” in the methods section of the revised manuscript.

Both reviewers 1 & 3 asked for a map with rodent trapping sites indicated.

Reviewer 3 also requested an excel file to include data used from other groups/public databases. A separate excel file with detailed MIC data should also be provided with the revised manuscript.

All genomic data used in this study will need to be made available per PLoS NTDs guidelines.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes.

-Is the study design appropriate to address the stated objectives?

Yes.

-Is the population clearly described and appropriate for the hypothesis being tested?

Yes.

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Yes.

-Were correct statistical analysis used to support conclusions?

Yes.

-Are there concerns about ethical or regulatory requirements being met?

No.

Other comments:

1. Sometimes nontyphoidal Salmonella (NTS) is qualified with ‘invasive’ unnecessarily or inappropriately. For example, as a redundancy when bloodstream isolates are being described, and perhaps incorrectly when carriage isolates are being described (i.e., isolates from rectal content, when a definition for invasion is not met).

2. The term ‘pan-susceptible’ is used without definition. Relatedly, a full listing of antimicrobial agents tested is not provided in the Methods.

3. There seems to be some conflation of carriage, infection, and disease terms in relation to rat isolates, with 'carriage' being used when infection or disease is not comprehensively excluded.

4. Please check all occurrences of the word ‘rate.’ On several occasions the word ‘rate’ is used to describe a proportion or prevalence, rather than a time-denominated measure of incidence. For example, ‘case fatality rate’ is used when ‘case fatality ratio’ would be more appropriate, and ‘high rate of antimicrobial resistance’ is used when ‘high prevalence of antimicrobial resistance’ is intended. A lot has been written about this (pedantic) point, but one recent example is by S Mantha 'Ratio, rate, or risk?' Lancet Infect Dis 2020; 21: 165-6.

5. Oxford (serial) commas would be helpful for clarity in places.

6. Nontyphoidal Salmonella cause in humans, anatomically and pathologically, predominantly entercolitis rather than gastroenteritis. If you wish to be anatomically specific, I would switch terms to enterocolitis. If you wish to avoid this issue, perhaps a clinical term like ‘diarrhea’ would be more accurate.

7. ‘Paratyphi’ is not a recognized serovar of Salmonella enterica, but Salmonella Paratyphi A, Paratyphi B, and Paratyphi C are.

8. It would be useful to say a little more about non-genomic evidence for NTS adaptation to humans in the Introduction.

9. A little more detail on rodent trapping methods would be helpful. For example, site selection process, numbers of traps set per night per site, dates of trapping (in relation to seasons), whether trapping was outdoor, or indoor, or both, etc.

10. What was the median (range) time in hours (or target) between trapping and euthanasia (i.e., lairage time)? What was the median (range) time in hours (or target) between euthanasia and necropsy? What storage conditions were rodents kept in after euthanasia and before necropsy? These points are relevant to onset of recent bacteremia in rats in prolonged lairage, and for postmortem translocation of organism from the gut to deep tissues.

11. Little description of how ‘signs of illness’ in rats was assessed, nor of gross findings at rat necropsy are provided. Were gross findings besides length and weight recorded? I assume no histopathology of liver and spleen to rule out signs of inflammation?

12. What was the rationale for selecting up to five colonies, rather than a higher or lower number?

13. A definition of a ‘carrier’ would be helpful. The case for calling isolation from spleen and liver ‘carriage’ is made, but I am concerned that they could represent disease isolates since histopathologic studies were not done, and details of gross necropsy findings were scanty in the report.

14. The term ‘antibiotic’ is used when ‘antimicrobial’ may be more appropriate.

Reviewer #2: The methods are adequate for the study question.

Reviewer #3: yes, technically sound with a clear aim.

Line 107-108, how the sample calculation was conducted?

Detailed excel sheet regarding the data used (from other groups or public datasets) are needed.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan?

Yes.

-Are the results clearly and completely presented?

Yes.

-Are the figures (Tables, Images) of sufficient quality for clarity?

Yes.

Other comments:

1. I suggest that numerators, proportions, and denominators be presented consistently in the conventional manner i.e., ‘x (y%) of z,’ or ‘Of z, x (y%)…’ Always give actual numbers for numerators and denominators. For example, I would avoid ‘one third’ as a proxy numerator.

2. ‘Trapping success’ is not defined in the Methods. Is this per trap, per site, or something else?

3. A map of greater Kisangani with trapping sites would be helpful. Adding relevant human participants would also be useful if geolocated and ethically acceptable.

Reviewer #2: The results are adequately describing the study outcomes.

Reviewer #3: An excel sheet with detailed samples as well as their meta data is needed.

Is there any rat samples having multiple Salmonella isolates or serovars?

Detailed excel sheet regarding the MIC data should also be included.

Further serovar based analysis regarding Salmonella Dublin, II:42:r:-, Kapemba, Weltevreden is needed, some are important and potential hazards in Africa countries and many others.

A map of sampling is needed.

All genomic data is not available.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented?

Yes.

-Are the limitations of analysis clearly described?

Yes.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Yes.

-Is public health relevance addressed?

Yes.

Other comments:

1. Since Salmonella Weltevreden was a common serovar isolated from rats, it would be useful to know whether the markets to which rodents had access in Kisangani sold fish, including farmed fish.

2. More discussion on the interpretation of isolation of Salmonella from liver and spleen would be helpful. In particular, could this represent recent onset bacteremia following prolonged lairage, or post-mortem translocation from the gut to the deep tissues?

3. The term ‘rat-to-rat’ transmission but be interpreted by some readers with an epidemiologic background as an indication of transmission by direct contact (c.f., fecally contaminated water or food, feces consumption, etc). I assume that is not the authors' intent, and wonder if ‘transmission among rats’ may be less likely to imply insights into the mode of transmission.

4. It is likely going beyond the scope of the findings, but would the authors care to speculate on possible modes of transmission from rodents to people, should rodents be a reservoir here? For example, we are not told whether human slaughter and consumption of rats and other rodents is practiced in Kisangani. I assume that contamination of water and food by rodent feces is likely, but not explicitly mentioned.

Reviewer #2: The conclusions are adequately describing the study outcomes.

Reviewer #3: Rats as reservoirs of non-Typhoidal Salmonella, and many more references should be included.

The references in the field should be updated.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Please see suggested modifications under Methods, Results, and Conclusions.

Reviewer #2: (No Response)

Reviewer #3: Minor revision

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is an extremely important study that sheds light on rats as possible non-human reservoirs for nontyphoidal Salmonella strains causing invasive disease in humans in Africa. I have made a number of relatively minor points for improvement outlined in my comments by section.

Reviewer #2: The study sheds some light on the transmission patterns of iNTS disease and it will be important to publish these results.

Reviewer #3: Dadu Falay and colleagues (PNTD-D-22-00687) presented a large scale investigation regarding the Salmonella carriage in Urban rats. The study sampled all positive Salmonella, which was subjected into WGS, and compared with the human isolates. The overall data is very interesting and could guide iNTS infection in Africa.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

22 Jul 2022

Submitted filename: 220718 Response to the reviewer.docx

Click here for additional data file.

13 Aug 2022

Dear Dr. Hardy,

We are pleased to inform you that your manuscript 'Urban rats as carriers of invasive Salmonella Typhimurium sequence type 313, Kisangani, Democratic Republic of Congo.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Travis J Bourret

Academic Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: My only remaining concern is with the use of the term 'carriage' to refer to infection of (or isolation of a pathogen from) a normally sterile site like liver, spleen, or blood. While the authors can define terms however they like, I think that this use of 'carriage' is beyond what most readers would consider conventional.

Reviewer #3: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: My only remaining concern is with the use of the term 'carriage' to refer to infection of (or isolation of a pathogen from) a normally sterile site like liver, spleen, or blood. While the authors can define terms however they like, I think that this use of 'carriage' is beyond what most readers would consider conventional.

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Yes.

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: None.

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The authors have done a very nice job of responding to my comments. My only remaining concern is with the use of the term 'carriage' to refer to infection of (or isolation of a pathogen from) a normally sterile site like liver, spleen, or blood. While the authors can define terms however they like, I think that this use of 'carriage' is beyond what most readers would consider conventional.

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #3: Yes: Min Yue

30 Aug 2022

Dear Dr Hardy,

We are delighted to inform you that your manuscript, "Urban rats as carriers of invasive Salmonella Typhimurium sequence type 313, Kisangani, Democratic Republic of Congo.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases