Serotype distribution and antimicrobial resistance of human Salmonella enterica in Bangui, Central African Republic, from 2004 to 2013.

BACKGROUND: Limited epidemiological and antimicrobial resistance data are available on Salmonella enterica from sub-Saharan Africa. We determine the prevalence of resistance to antibiotics in isolates in the Central African Republic (CAR) between 2004 and 2013 and the genetic basis for resistance to third-generation cephalosporin (C3G). METHODOLOGY/PRINCIPAL
FINDINGS: A total of 582 non-duplicate human clinical isolates were collected. The most common serotype was Typhimurium (n = 180, 31% of the isolates). A randomly selected subset of S. Typhimurium isolates were subtyped by clustered regularly interspaced short palindromic repeat polymorphism (CRISPOL) typing. All but one invasive isolate tested (66/68, 96%) were associated with sequence type 313. Overall, the rates of resistance were high to traditional first-line drugs (18-40%) but low to many other antimicrobials, including fluoroquinolones (one resistant isolate) and C3G (only one ESBL-producing isolate). The extended-spectrum beta-lactamase (ESBL)-producing isolate and three additional ESBL isolates from West Africa were studied by whole genome sequencing. The blaCTX-M-15 gene and the majority of antimicrobial resistance genes found in the ESBL isolate were present in a large conjugative IncHI2 plasmid highly similar (> 99% nucleotide identity) to ESBL-carrying plasmids found in Kenya (S. Typhimurium ST313) and also in West Africa (serotypes Grumpensis, Havana, Telelkebir and Typhimurium).
CONCLUSIONS/SIGNIFICANCE: Although the prevalence of ESBL-producing Salmonella isolates was low in CAR, we found that a single IncHI2 plasmid-carrying blaCTX-M-15 was widespread among Salmonella serotypes from sub-Saharan Africa, which is of concern.

Introduction

Salmonella enterica serotypes Typhi, Paratyphi A, Paratyphi B d-tartrate negative and Paratyphi C are grouped as typhoidal Salmonella, and other serotypes are described as non-typhoidal Salmonella (NTS). Human Salmonella infections are generally either typhoid and paratyphoid fever, systemic diseases caused by typhoidal Salmonella, or gastroenteritis caused by a large number of NTS serotypes. Although most cases of salmonellosis due to NTS are self-limiting, they have emerged as a prominent cause of life-threatening bloodstream infections in sub-Saharan Africa, with approximately 388 000 deaths in 2010 [1].

S. enterica serotype Typhimurium (referred to here as S. Typhimurium) sequence type (ST) 313 has been described as the primary cause of invasive salmonellosis in sub-Saharan Africa, with mortality rates > 25%. Whole-genome analysis revealed that these ST313 strains belong to two dominant genetic lineages, I and II, which emerged independently within the past 40–50 years and spread across sub-Saharan Africa, in close temporal association with the current HIV pandemic [2]. Clonal replacement of lineage I by lineage II was observed in the mid-2000s, perhaps due to acquisition of chloramphenicol resistance [2].

Currently, there is no commercially available anti-Salmonella vaccine for controlling invasive NTS. Prompt, effective management with antimicrobials remains the main option for management of the disease; however, antimicrobial resistance emerged several decades ago, initially to the traditional first-line drugs such as chloramphenicol, ampicillin and cotrimoxazole. Third-generation cephalosporins (C3G) and fluoroquinolones have since become standard first-line empirical treatment [2]. Recently, C3G-resistant (C3GR) Salmonella populations have emerged and spread in all continents, including Africa [3].

The Central African Republic (CAR) is a resource-limited country in equatorial Africa (ranked 188/189 on the Human Development Index in 2018). In a case–control study conducted in Bangui, the capital, between 2011 and 2013 among children under 5 years of age, S. enterica was isolated in 4% of children presenting with diarrhoeal illness; rotavirus and Cryptosporidium hominis/parvum were the two pathogens most frequently recovered [4]. The study’s primary objective was to report on the prevalence of resistance to antibiotics in S. enterica strains isolated from patients in CAR between 2004 and 2013 to determine whether recommendations for first-line empirical treatment based on C3G and fluoroquinolones are relevant. Secondary objectives were i) to investigate the genetic background of serotype Typhimurium to determine whether ST313 is the primary cause of invasive salmonellosis, iii) to determine the genetic basis for resistance to C3G using whole genome sequencing (WGS).

Material and methods

Ethical approval

This retrospective study is based on a retrospective data collection issued from the routine bacteriological diagnostic activity within the private clinical laboratory located at Institut Pasteur in Bangui. Data consisted of anonymised laboratory results devoid of individual patient information or identifiers. Ehical approval and individual informed consent were not necessary. We submitted the study protocol to the WHO representative in Bangui, the Institut Pasteur in Bangui and the Ministry of Public Health in CAR which gave their approval.

Bacterial strains, serotyping, susceptibility, transferability of extended-spectrum beta-lactamase

Our private clinical laboratory, the major one in the capital, performed biological analysis on specimens from its patients but also on samples from other hospitals and clinical laboratories. All non-duplicate S. enterica clinical isolates collected between January 2004 and December 2013 were included, encompassing those previously described [4]. If more than one isolate with the same serotype and antimicrobial resistance phenotype was recovered from the same patient, only the first was retained for the analysis. Date of isolation and the nature of the biological sample were recorded.

The strains were serotyped on the basis of somatic O and phase 1 and phase 2 flagellar antigens by agglutination tests with antisera (Bio-Rad, Marnes-La-Coquette, France), as specified in the White-Kauffmann-Le Minor scheme (preliminary serotyping at the Institut Pasteur in Bangui, complete serotyping at the Institut Pasteur in Paris). Susceptibility to amoxicillin (20 μg), amoxicillin-clavulanic acid (20–10 μg), ticarcillin (75 μg), cefalotin (30 μg), cefoxitin (30 μg), cefotaxime (5 μg), ceftazidime (10 μg), imipenem (10 μg), amikacin (30 μg), gentamicin (10 μg), spectinomycin (10 μg), streptomycin (10 UI), nalidixic acid (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), cotrimoxazole (1.25–23.75 μg) and tetracycline (30 μg) was tested by the disc diffusion method on Mueller-Hinton agar (Bio-Rad), and production of ESBL was detected by the double-disc synergy (DDS) test. Minimum inhibitory concentrations (MICs) were determined with E-test strips (BioMerieux, Marcy L’Etoile, France). Isolates were classified as resistant, intermediate or susceptible according to the 2018 guidelines of CA-SFM/EUCAST (http://www.sfm-microbiologie.org). Isolates of intermediate susceptibility were not classified with the resistant ones for the analysis of the data.

A conjugation experiment was performed as described previously [5].

Clustered regularly interspaced short palindromic repeats polymorphism (CRISPOL) typing and multilocus sequence typing (MLST) of S. Typhimurium isolates

Total DNA was extracted with the Instagene kit (Bio-Rad, Marnes-la-Coquette, France). CRISPOL typing and MLST was performed on the Luminex platform (Luminex Coporation, Austin, TX, USA) at the Institut Pasteur in Paris, as previously described [6].

Whole-genome sequencing

WGS was performed on the ESBL-producing S. Typhimurium isolate S1027072 identified in this study and on three Salmonella isolates (serotypes Grumpensis, Havana and Telelkebir) recovered from West Africa in 2007 and 2008 and previously described, at the Institut Pasteur in Paris [3] (Table 1). These three comparison isolates produced a CTX-M-15 ESBL carried on a large IncHI2 plasmid.

Table 1

Plasmids used for syntenic analysis versus pKST313.

Strain	Species/Serovar	Country	Origin	Year	Plasmid	Accession number	Size (bases)	Percentage identity vs pKST313
KST313	S.a Typhimurium	Kenya	Human	2009–2012	pKST313	LN794248	300 375	_
S1027072	S. Typhimurium	CAR	Human	2008	pCARST313	PRJNA540305	268606d	99.98
08–3663	S. Grumpensis	Senegal	Human	2008	p08-3663	PRJNA540305	280987d	99.98
07–0319	S. Havana	Mali	Human	2007	p07-0319	PRJNA540305	282330d	99.98
07–1331	S. Telelkebir	Mali	Human	2007	p07-1331	PRJNA540305	282370d	99.99
A54560	S. Typhimurium	Malawi	Human	2009	pSTm-A54650	NC_024983.1	309 406	99.76
EB-247	E.b bugandensis	United Republic of Tanzania	Human	2010	pEB247	LN830952.1	298 984	99.80
CRENT-193	Enterobacter sp.	Republic of Korea	Human	2013	pCRENT-193_1	NZ_CP024813.1	298 989	99.82
Ec21617	E.c coli	Taiwan	Human	2013	pEc21617-310	MG878867.1	309 608	99.67

aSalmonella

bEnterobacter

cEscherichia

dNot fully assembled

DNA extraction was performed with the MagNAPure 96 system (Roche). The libraries were prepared with a Nextera XT kit (Illumina) and sequencing with the NextSeq 500 system (Illumina), generating 150-bp paired-end reads, yielding a mean 105-fold coverage. Reads were trimmed and filtered with AlienTrimmer [7] and a quality Phred score threshold of 28 on a minimum length of 70 nucleotides.

Plasmid assemblies and syntenic analysis

In order to reconstruct plasmids in ESBL-producing S. Typhimurium ST313 isolate S1027072, reads were mapped with Bowtie 2 software [8] against two assembled, annotated plasmids found in an S. Typhimurium ST313 isolate from Kenya [9]: pKST313, an IncHI2 plasmid encoding ESBL CTX-M-15 and pSBLT, an IncFII plasmid similar to the previously described pSLT-BT found in strain D23580 isolated in Malawi in 2004 [2]. Selected mapped reads were assembled with SPAdes [10], and the quality of the assembly was checked with QUAST software [11]. Plasmidic contigs were ordered against the reference plasmids pKST313 and pSBLT with ABACAS software to produce the putative plasmids pCARST313 and pCAR_SBLT, respectively [12]. The contigs were further annotated with Prokka [13]. Comparison files between putative plasmids and their reference were generated with BRIG software [14].

To better describe worldwide circulation of blaCTX-M-15-carrying IncHI2 plasmids, we used the same approach to compare pCARST313 with IncHI2 plasmids found in three Salmonella isolates from West Africa (Table 1). In addition, the nucleotide sequence of pCARST313 was searched with Mash Screen [15] in the PLSDB database (a resource containing 16 168 plasmid sequences collected from the NCBI nucleotide database) [16]. Four additional IncHI2 plasmids recovered from PLSDB were used for the BRIG comparison analysis: pSTm-A54650 (S. Typhimurium A54560) from Malawi, pEB247 (Enterobacter bugandensis EB-147) from the United Republic of Tanzania, pCRENT-193_1 (Enterobacter sp. CRENT-193) from the Republic of Korea and pEc21617-310 (Escherichia coli Ec21617) from Taiwan (Table 1).

The GenBank accession numbers of all the genomes studied are listed in Table 1.

Statistical analysis

The chi-square test for trend was used to compare rates of resistance to antibiotics during the study period. P values < 0.05 were considered statistically significant. All analyses were performed with STATA 12.0 (Stata Corporation, College Station, TX, USA).

Results

Serotype distribution and antimicrobial susceptibility

A total of 582 non-duplicate isolates of Salmonella belonging to 113 serotypes were collected during the 10-year study period. The most common serotypes were Typhimurium (n = 180, 31% of the isolates), Enteritidis (n = 64, 11%) and Typhi (n = 35, 6%) (Table 2). No strains assigned to serotypes Paratyphi A and Paratyphi B were detected. Typhimurium was the most frequently recovered serotype each year, except in 2010 (rank 2) and in 2013 (not found). Isolates were from stool (n = 452, 77%), blood (n = 97, 17%), urine (n = 14, 2%), cerebrospinal fluid (n = 13, 2%), wound (n = 5, 1%), and pleural fluid (n = 1) samples. Of the blood isolates, 77 (79%) were NTS, and Typhimurium (n = 49, 64%% of the isolates) and Enteritidis (n = 15, 19%) were the predominant serotypes. The other NTS serotypes were Dublin (n = 6), Stanleyville (n = 3), Brancaster (n = 1), Hartford (n = 1), Mikawashima (n = 1) and Saintpaul (n = 1). Three blood isolates were not typables and 17 belonged to serotype Typhi. A total of 13 cases of meningitis due to Salmonella enterica were reported during the study period, and Typhimurium was involved in 9 cases (69%). The other serotypes were Enteritidis (n = 1), Poona (n = 1), Sainpaul (n = 1) and Typhi (n = 1).

Table 2

Distribution of the 10 most frequent Salmonella enterica serotypes in Central African Republic, 2004–2013.

2004n = 46	%	2005n = 81	%	2006n = 100	%	2007n = 97		2008n = 37	%	2009n = 61	%	2010n = 39	%	2011n = 63	%	2012n = 43	%	2013n = 15	%	2004–2013n = 582	%
Typhimurium	28	Typhimurium	40	Typhimurium	29	Typhimurium	50	Typhimurium	46	Typhimurium	31	Enteritidis	18	Typhimurium	10	Typhimurium	47	Liverpool	27	Typhimurium	31
Typhi	13	Enteritidis	12	Enteritidis	16	Enteritidis	15	Typhi	8	Enteritidis	11	Typhimurium	13	Onireke	8	Nchanga	9	Kibusi	13	Enteritidis	11
Enteritidis	11	Typhi	6	Typhi	7	Typhi	13	42:r:- a	5	Stanleyville	10	Stanleyville	8	Enteritidis	6	Mikawasima	5	Hartford	7	Typhi	6
Mgulani	4	Saintpaul	6	Stanleyville	5	Dublin	5	Mikawasima	5	Kibusi	5	Kibusi	8	Stanleyville	6	Stanleyville	5	Texas	7	Stanleyville	3
42:r:-a	2	Stanleyville	5	42:r:- a	5	Stanleyville	1	Amoundernes	5	Llandoff	5	42:r:- a	5	Give	6	Onireke	2	Hvittingfoss	7	42:r:- a	2
Ayton	2	Dublin	4	Infantis	4	42:r:- a	1	Stanleyville	5	Infantis	3	Hull	5	Chicago	5	Liverpool	2			Kibusi	2
Bovismorbificans	2	Hartford	2	Poona	4	Schwarzengrund	1	Enteritidis	3	Typhi	2	Saintpaul	5	Stanleyville	3	Sinstorf	2			Onireke	2
Budapest	2	42:r:- a	2	Hull	4	Leeuwarden	1	Schwarzengrund	3	42:r:- a	2	Typhi	3	Mikawasima	3	Kentucky	2			Saintpaul	2
Butantan	2	Leeuwarden	1	Saintpaul	3	Mikawasima	1	Mgulani	3	Amoundernes	2	Hadar	3	Kibusi	3	Llandoff	2			Stanleyville	2
Chile	2	Infantis	1	Hadar	2	Stanley	1	Aberdeen	3	Poona	2	Onireke	3	Ilugun	3	Kingston	2			Mikawasima	1

asubsp. salamae

Overall, the 582 S. enterica isolates displayed a low level of resistance to all antibiotics, except those used as traditional first-line drugs: tetracycline (18%), chloramphenicol (30%), cotrimoxazole (39%) and amoxicillin (40%) (Table 3). The most frequent phenotype of resistance in serotypes Enteritidis (22/64, 34%) and Typhi (9/38, 24%) was susceptibility to all the beta-lactams tested, except for amoxicillin and ticarcillin, to aminoglycosides, to fluoroquinolones, and resistance to chloramphenicol, cotrimoxazole and tetracyclin. The susceptibility of S. Typhi to antibiotics was not significantly different from that of NTS (P > 0.5).

Table 3

Percentage resistance to specific antibiotics in Salmonella enterica in the Central African Republic, 2004–2013.

Antibiotic	2004	2005	2006	2007	2008	2009	2010	2011	2012	2013	Total
	n = 46	n = 81	n = 100	n = 97	n = 37	n = 61	n = 39	n = 63	n = 43	n = 15	n = 582
Amoxicillin	46	51	41	62	51	26	5	13	44	29	40
Amoxicillin-clavulanic acid	4	1	3	0	3	2	0	0	2	0	2
Ticarcillin	46	51	41	62	51	26	5	13	44	29	40
Cefalotin	0	1	0	0	3	3	0	0	2	0	1
Cefoxitin	0	0	0	0	0	0	0	0	2	0	0
Cefotaxime	0	0	0	0	3	0	0	0	0	0	0
Imipenem	0	0	0	0	0	0	0	0	0	0	0
Amikacin	0	0	0	0	0	0	0	0	0	0	0
Gentamicin	2	0	2	0	3	2	5	2	0	0	1
Nalidixic acid	0	0	3	0	8	2	8	2	0	0	2
Ciprofloxacin	0	0	0	0	3	0	3	0	0	0	0
Chloramphenicol	28	33	32	54	43	25	26	19	40	20	30
Cotrimoxazole	41	52	44	61	51	25	3	11	44	27	39
Tetracycline	20	22	25	28	22	13	5	6	9	7	18

One stool/blood isolate with serotype Kentucky collected in 2010 was resistant to ciprofloxacin (MICs: > 12 mg/L, respectively). The blood Typhimurium isolate S1027072 was resistant to the beta-lactams tested (ceftazidime: MIC 32 mg/L, ceftriaxone: MIC >256 mg/L), except for cefoxitin and imipenem, to gentamicin, to chloramphenicol, cotrimoxazole, and tetracyclin, and susceptible to amikacin, nalidixic acid, and ciprofloxacin. The DDS test was positive, indicating production of an ESBL.

Genetic background of Salmonella Typhimurium and antibiotic susceptibility

CRISPOL typing was performed on a set of 68 S. Typhimurium isolates, including S1027072. They were chosen using a random number table and corresponded to around 38% of the isolates collected every year. They were from stools (n = 39, 57%), blood (n = 17, 25%), cerebrospinal fluid (n = 5, 7%), urine (n = 3, 4%), wounds (n = 3, 4%) and pleural fluid (n = 1). S. Typhimurium was isolated from both stools and blood in 13 cases.

A total of 66 isolates (96%) belonged to CRISPOL type (CT) 28 group, of which 63 (97%) were assigned to CT28, two to CT698 and one to CT526. It was shown previously that CT28 is associated with lineage II, whereas CT698 is associated with lineage I [17] (Table 4). To confirm the association of ST313 to the CT28 group, MLST was performed on 10 isolates: 7 assigned to CT28, one to CT698 and one to CT526. All the isolates belonged to ST313. In addition, the ESBL-S. Typhimurium belonging to CT28 by CRISPOL typing was assigned to ST313 by WGS. All these data confirmed the association between CT28 group and ST313 in Salmonella isolates from CAR.

Table 4

Characteristics of 68 randomly chosen Salmonella Typhimurium isolates in Bangui, 2004–2013.

Resistance type	Associated with sequence type (ST) 313
	Yes			No
	CRISPOL type (CT) 28a	CT526a	CT698b	CT96	CT301
ATC	1
ATSNalSXTC	1
ATSpSXTCTe	1
ATSSpKTSXT
ATSSpSXT			1
ATSSpSXTC	1		1
ATSSpSXTCTe	2
ATSSXT	3
ATSSXTC	35	1
ATSSXTCTe	11
ATSSXTTe	3
ATSXTCTe	1
ATSXTTe				1
SSpSXT	1
SSXT	2
SSXTCTe	1
Susceptible					1
Total	63	1	2	1	1

A, amoxicillin; C, chloramphenicol; Nal, Nalidixic acid; S, streptomycin; Sp, spectinomycin; SXT, cotrimoxazole; Te, tetracycline, T, ticarcillin.

aAssociated with ST313 lineage II

bAssociated with ST313 lineage I

Additional antibiotics susceptibility tests were performed to streptomycin and spectinomycin. The most common resistance phenotype was to amoxicillin, ticarcillin, cotrimoxazole, chloramphenicol and streptomycin (35/66, 53%). All but one of the CT28 group isolates were resistant to at least three antibiotics (Table 4).

All the invasive S. Typhimurium isolates except one (CT301) were assigned to ST313 (CT28).

Plasmid carriage in the ESBL-producing S. Typhimurium isolate S1027072

Plasmid Finder-based typing showed the presence of plasmids of three incompatibility groups, IncHI2, IncFIB and IncQ1. The IncHI2 plasmid was assigned to ST1. Use of a mapping approach between plasmids of the ESBL-producing S. Typhimurium published genome KST313 (pKST313 and pSBLT) and isolate S1027072 made it possible to form two putative plasmidic assemblies, pCARST313 (IncHI2) and pCAR_SBLT (IncF).

The first plasmidic assembly pCARST313 presents 99.9% nucleotidic identity with pKST313 (300 375 bp). Both plasmids encode: (i) resistance to heavy-metal ions, including mercury (mer and tni genes), tellurite (ter genes), arsenic (ars genes), copper (cusS and pcoE genes), nickel and cobalt (rcnR and rcnA genes), and to tellurite utilization genes (tel genes); and (ii) resistance to antimicrobials, including aminoglycosides (aac(6')-Ib), streptomycin (aadA1, strA and strB), beta-lactams (blaOXA-30, blaTEM-1 and blaCTX-M-15), chloramphenicol (catA1 and catB3), trimethoprim (dfrA14), sulfonamides (sulI and sulII), gentamicin (aacC3) and tunicamycin (tmrB), most of which are in a class 1 integron. Genes for conjugative transfer were also identified. The transferability of the plasmid was confirmed in the conjugation experiment. We did not find any spontaneous chromosomal mutations associated with resistance to antibiotics. To explore putative circulation of close blaCTX-M-15-carrying IncHI2 plasmids identical to pCARST313 in sub-Saharan Africa, we assembled this plasmid from the strains S. Havana 07–0319 and S. Telelkebir 07–1331 isolated in Mali in 2007, S. Grumpensis 08–3663 collected in Senegal in 2008 and S. Typhimurium A54560 collected in Malawi in 2009. The plasmid was recovered from all isolates with a minimal identity of 99% (Table 1) and was also found among Enterobacteriaceae species: Enterobacter bugandensis collected in the United Republic of Tanzania in 2010, Enterobacter sp. isolated in the Republic of Korea in 2013 and Escherichia coli isolated in Taiwan in 2013 (Fig 1).

Fig 1

Syntenic analysis of pKST313 vs 8 other IncHI2 plasmids.

The innermost black ring 1 represents the reference sequence of pKST313. Following rings correspond to pairwise comparison with IncHI2 plasmids: ring 2 represents pCARST313; ring 3, p08-3663; ring 4, p07-0319; ring 5, p07-1331; ring 6, pSTm-A54650; ring 7, pEB247; ring 8, pCRENT-193_1; ring 9, pEc21617-310. Last 4 rings represent genetic map of pKST313: antibiotic resistance genes are indicated by red arrows, heavy-metals resistance genes by brown arrows, others annotated genes by grey arrows and hypothetical proteins by black arrows.

The second plasmidic assembly pCAR_SBLT, found in the S1027072 strain, also presented > 99% nucleotide identity with pSBLT (128 925bp) found in ST313. Both plasmids encode genes involved in antibiotic resistance: catA1, sulI, sulII, blaTEM-1, strA, strB, dhfrI, aadA1 and qacED1. This plasmid was not found in other Salmonella isolates harbouring the pKST313 plasmid type.

Discussion

In this study of 582 non-duplicate S. enterica isolates investigated between 2004 and 2013, Typhimurium and Enteritidis were the two most frequently recovered NTS serotypes, in accordance with numerous other studies in sub-Saharan Africa [1–3, 18–23]. Surprisingly, they were not found in 2013, probably due to the small number of Salmonella isolates collected this year. Indeed, significant differences in the numbers of organisms collected were observed by year, due to the economic and political crisis during the study period, which further restricted patient access to health care facilities. Since 2013, the CAR has faced an ongoing civil war, plunging the country into a chaotic state of violence and an ensuing humanitarian crisis. The surveillance of Salmonella enterica isolates at the Institut Pasteur in Bangui was discontinued but a WGS approach-study could be coordinated when the laboratory-surveillance capacities will be strengthened locally.

Overall, the resistance rates to all antibiotic classes were low, except to the antibiotics used previously (amoxicillin, chloramphenicol, cotrimoxazole and tetracycline), confirming recommendations for first-line treatment based on C3G and fluoroquinolones. We report the first case of ESBL-producing Salmonella (serotype Typhimurium) in CAR, which is of concern. More new descriptions of ESBL-producing Salmonella isolates have been made across sub-Saharan Africa since the first, in 2006 in Ethiopia, Typhimurium being the most frequently isolated ESBL-producing serotype [3, 24–26]. The low prevalence (0–1.3%) described in sub-Saharan Africa may not, however, represent the situation at country level, as most countries do not have national surveillance systems based on networks of clinical laboratories.

S. Typhimurium ST313 was the primary cause of invasive salmonellosis during the study period, in agreement with data from Congo (Central Africa), Kenya, Malawi and Mozambique (East Africa) [17, 23, 27]. The high proportion of S. Typhimurium ST313 among stools in our study suggests that this clone causes also an appreciable burden of gastroenteritis. A human reservoir for this clone is plausible, especially as identical strains were recovered in asymptomatic siblings and parents of index case-patients in Kenya (carriage prevalence, 6.9%) [28]. Our data also confirm the substantial prevalence of multi-drug-resistant S. Typhimurium ST313 [22, 27]. This is of major concern, and further investigations are required to identify the reservoirs and risk factors for exposure and transmission in order to design hypothesis-driven preventive measures.

The invasive ESBL-producing S. Typhimurium strain S1027072 was assigned to the highly invasive ST313 genetic background. The blaCTX-M-15 gene and most antimicrobial resistance genes were present in a large conjugative IncHI2 plasmid, which was closely similar to the pKST313 plasmid found in an ESBL-producing S. Typhimurium ST313 isolate in Kenya [9] and also to ESBL-carrying plasmids found in three other Salmonella serotypes collected in Senegal, Mali and Malawi, highlighting its significant role in the spread of blaCTX-M-15 genes in Salmonella isolates in sub-Saharan Africa. Its significance may be even greater, as it has been recovered in various Enterobacteriaceae species in the United Republic of Tanzania and east Asia. In addition, this plasmid has numerous genes encoding resistance to heavy-metal ions. It is well known that the location of both metals and antibiotic resistance genes on the same mobile element play a major role in the persistence, selection and spread of antibiotic-resistant bacteria in anthropogenic environments heavily contaminated with detergents, heavy metals and other antimicrobials [29, 30]. In developing countries, rivers, lakes and lagoons are often contaminated with untreated hospital and industrial effluents and also by urban storm-water containing anthropogenic pollutants due to intensive uncontrolled urbanization. This is optimal conditions for bacterial development and the spread of antibiotic-resistant bacteria. Further active surveillance is needed to minimize the spread of this successful IncHI2 plasmid in order to control dissemination of antibiotic resistance among Salmonella isolates. Unsurprisingly, a plasmid similar to the pSLT-BT virulence plasmid usually found in S. Typhimurium ST313 lineage II harbouring integron-borne resistance determinants was found in the ESBL-producing S. Typhimurium isolate S1027072 in CAR [9].

Our results confirm high rates of invasive multidrug-resistant S. Typhimurium ST313 among Salmonella infections in CAR between 2004 and 2013. Although the prevalence of ESBL-producing Salmonella enterica isolates was very low, the wide distribution of a single IncHI2 plasmid scaffold among Salmonella serotypes in sub-Saharan Africa, the origin of dissemination of the blaCTX-M-15 gene, is of concern.

20 Aug 2019

Dear Dr Breurec:

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We hope to receive your revised manuscript by Oct 19 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit your revised files, please log in to https://www.editorialmanager.com/pntd/

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Elsio Wunder Jr, D.V.M., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Deputy 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: This study, including a large number of strains describe the epidemiology of Salmonella infections in CAR between 2004 and 2013. His strength is to be interested in strain genetics. Furthermore, data concerning the antimicrobial resistance in this part of the world are substancial.

The manuscript is clear and easy to read and objectives are clearly defined.

Reviewer #2: Line 99: Please clarify what is meant by "no additional specimens were collected for this study". At this status of the manuscript it is not clear/known to the reader which samples actually were collected.

L101: To which healthcare facility are authors referring to? Which type of patients (including their eligibility criteria) and infectious/chronic diseases are under the routine surveillance mentioned?

L111: From which biological samples were the isolates detected?

L114-117: Which antibiotic concentration were tested?

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

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: The analysis are in accord with the plan and the results are clearly presented and easy to read.

- The Table 2 could be enlarged

- It would be interesting to detail the various serotypes of Salmonella responsible for bacteremia and meningitis

- Can the authors specify in which site (stool, blood or other ) the Salmonella producing ESBL was isolated?

Reviewer #2: L 183-184: So, 9/13 patients with meningitis had a co-infection with S. Typhimurium?

L 193: Please clarify what is meant by low resistance. Were the isolates tested resistant to the antibiotics tested or did they possibly show some borderline/intermediate patterns? Which standards were used as a reference to classify isolates based on their susceptibility/resistant patterns?

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

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: The conclusions are supported by the data. These data concerning the epidemiology of Salmonella nfections in the the sub-Saharan Africa are significant. Antimicrobial resistance data are very important for first-line treatments.

Reviewer #2: NA

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

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: This study is well designed and well conducted and the manuscript is clear.

It can be accepted

Reviewer #2: NA

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

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: (No Response)

Reviewer #2: Congratulations to this well designed and written article. I have just some very few suggestions related to the methods and results sections.

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

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: Yes: Dr Josette Raymond

Reviewer #2: No

9 Sep 2019

Submitted filename: Breurec_Salmonella_Reviewer 2.docx

Click here for additional data file.

26 Sep 2019

Dear Dr Breurec:

Thank you very much for submitting your manuscript "10-year surveillance of Salmonella enterica in Bangui, Central African Republic, 2004–2013" (PNTD-D-19-00975R1) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file ); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) Appropriate Figure Files

Please remove all name and figure # text from your figure files upon submitting your revision. Please also take this time to check that your figures are of high resolution, which will improve both the editorial review process and help expedite your manuscript's publication should it be accepted. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plosntds/s/figures

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We hope to receive your revised manuscript by Nov 25 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit your revised files, please log in to https://www.editorialmanager.com/pntd/

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Florian Marks, Ph.D., MPH

Guest Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Deputy 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: The manuscript was corrected according to the reviewer's comments and can now be accepted

Reviewer #2: No further comments.

Reviewer #3: This a descriptive microbiological analysis of routine Salmonella isolates from an admittedly very understudied setting in the CAR with then some more detailed information on a single ESBL isolate. While the study thus provides some new data on Salmonella in this geographic area, it also suffers from a number of major issues

1) There is no clear testable hypothesis stated. The provides a descriptive analysis of routinely collected isolates from a single clinical microbiology lab.

2) A description of how the samples came into the Pasteur lab is missing. Is the Institut Pasteur lab associated with a health clinic, hospital, private GPs? Did some isolates derive from the mentioned case-control study conducted in 2011-13? If yes, how many isolates from each "source"? What type of cases (invasive, stool) are these isolates most likely to be representative of?

3) Given that the title mentions "surveillance", one would at least expect a summary epidemiological analysis including age distribution & sex of cases, in addition to providing microbiological data like serotyping and antibiotic resistance testing on non-duplicate isolates. Please revise title to include "serotyping", "antimicrobial resistance" and "human" isolates

4) The data collection period and the typing technique CRISPOL used are starting to be dated. Would it not be possible to extend the analysis to isolates also from the last 5 years, 2014-2018 and also provide more WGS results if possible. The authors need to say at least whether Salmonella "surveillance" is still ongoing now or was discontinued after 2013.

5) The sample size of isolates from 2013 in table 2 is rather small compared to other years (and the serotype distribution is quite different to previous years - was S Liverpool really the top serovar?). Reasons for this needs to be at least discussed in the manuscript.

6) randomization procedure for selecting S. Typhimurium isolates needs to be explained more.

7) It is not clear where the various lab testing done, was all serotyping and AMR testing done in Paris at the end of the study period ?

8) No description was given of how the MLST type (ST313) was obtained. I guess that the authors assumed that the CT28 is indicative of ST313, rather than having conducted MLST, i.e the ST was "deduced" based on results from a previous study in the Democratic Republic in Congo. This needs to be made clearer in methods. I recommend that authors change wording in abstract, replacing "associated with" by "indicative of". One of the problems with this approach is that the correspondence between CT28 and ST313 might be local to the Congo area. Ideally at least some CT28 S. Typhimurium isolates should be sequenced by WGS to make sure that they also correspond to ST313 in the CAR. For the ESBL isolate submitted to WGS, the authors should also individually report for completeness the full antibiotic resistance profile, the ST, as well as any non-ESBL resistance genes or mutations, for example using ResFinder or CARD.

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

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: The manuscript was corrected according to the reviewer's comments and can now be accepted

Reviewer #2: No further comments.

Reviewer #3: In table 1, it's not clear why the size information of the plasmid is not given for strains in lines 2 to 5 - is it because it is not a full assembly? The authors could report the size of the de novo assembly and then indicate as a footnote, that it might not be complete.

In most developed countries, resistance patterns are strongly associated with serotype, typically higher for S. Typhimurium and Typhi than for other serovars. The authors do not provide any detailed AMR data for serovars other than S. typhimurium, like S. Enteritidis and S. Typhi which are also quite common. A new table should be added with showing most frequent AMR profiles for most frequent serotypes, or at least mention most most frequent AMR profiles for most frequent serovars in the text.

In table 4, for the footnotes a & b, need to add citation to paper showing the association with ST313

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

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: The manuscript was corrected according to the reviewer's comments and can now be accepted

Reviewer #2: No further comments.

Reviewer #3: The authors interpretation that one isolate of 582 (0.2%) being an ESBL is a "major" concern is debatable. One could also sonclude that ESBL in Salmonella in this setting are quite rare, and probably rarer than other Enterobacteriacea in other African countries. Consider replacing "major concern" by "concern".

Line 295 the authors write that clinical features are unknown and they say in the same sentence that they reported diarrhea. This doesn't make sense. Either they have (some of) this data or they don't.

Line 300 the authors mention the "emergence" of MDR ST313 which suggests a change in frequency over time. The data presented herein does not support such a changing trend over time, so authors should consider replacing "emergence" with "substantial prevalence of". Also the last sentence about more frequent" is quite speculative as the data presented in this study does not show any increase over time.

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

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: The manuscript was corrected according to the reviewer's comments and can now be accepted

Reviewer #2: No further comments.

Reviewer #3: Acknowledgments: it's not usual to thank technicians for doing what appears to be "routine" work.

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

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: It is a well conducted study, clearly written. It allows to precise the epidemiology of infections due to Salmonella in Central Africa Republic

Reviewer #2: All comments and suggestions raised during the 1st review were very well addressed. The manuscript reads very well.

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

8 Oct 2019

Submitted filename: Breurec_Salmonella_Reviewer 3.docx

Click here for additional data file.

11 Nov 2019

Dear Dr Breurec,

We are pleased to inform you that your manuscript, "10-year surveillance of Salmonella enterica in Bangui, Central African Republic, 2004–2013", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please note: your manuscript will not be scheduled for publication until you have made the required changes.

IMPORTANT NOTES

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*Now that your manuscript has been provisionally accepted, please log into EM and update your profile. Go to http://www.editorialmanager.com/pntd, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

*Note to LaTeX users only - Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines [http://www.plosntds.org/static/latexGuidelines.action] in the meantime.

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

Best regards,

Florian Marks, Ph.D., MPH

Guest Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Deputy 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 #2: No further comments.

Reviewer #3: All points have been adequately addressed.

**********

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 #2: No further comments.

Reviewer #3: All points have been adequately addressed.

**********

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 #2: No further comments.

Reviewer #3: All points have been adequately addressed.

**********

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 #2: No further comments.

Reviewer #3: All points have been adequately addressed.

**********

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 #2: No further comments.

Reviewer #3: All points have been adequately addressed.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

26 Nov 2019

Dear Pr Breurec,

We are delighted to inform you that your manuscript, "Serotype distribution and antimicrobial resistance of human Salmonella enterica in Bangui, Central African Republic, from 2004 to 2013 ," 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,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases