First report on antimicrobial resistance and molecular characterisation of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola

Accepted Manuscript Title: First report on antimicrobial resistance and molecular characterization of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola Authors: Mois´es Francisco, Sofia Santos Costa, Adriana Belas, Jorge Ramos, Isabel Couto, Constanc¸a Pomba, Miguel Viveiros PII: DOI: Reference:

S2213-7165(18)30028-6 https://doi.org/10.1016/j.jgar.2018.02.001 JGAR 595

To appear in: Received date: Revised date: Accepted date:

20-6-2017 23-1-2018 2-2-2018

Please cite this article as: Mois´es Francisco, Sofia Santos Costa, Adriana Belas, Jorge Ramos, Isabel Couto, Constanc¸a Pomba, Miguel Viveiros, First report on antimicrobial resistance and molecular characterization of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola (2010), https://doi.org/10.1016/j.jgar.2018.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

First report on antimicrobial resistance and molecular characterization of Salmonella enterica serotype Typhi isolated from human specimens in Luanda, Angola

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Moisés Franciscoa,b,c, Sofia Santos Costac, Adriana Belasd, Jorge Ramosc, Isabel Coutoc,

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Constança Pombad, Miguel Viveirosc,*

Faculty of Medicine, University Agostinho Neto, Luanda, Angola

b

National Institute of Public Health, Luanda, Angola

c

Global Health and Tropical Medicine, GHTM, Unit of Medical Microbiology, Instituto

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U

a

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de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon,

Laboratory of Antimicrobial and Biocide Resistance, Interdisciplinary Centre of

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d

M

Portugal

Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon,

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Lisbon, Portugal

E-mails:

MF:

[email protected];

SSC:

[email protected];

AB

[email protected]; JR: [email protected]; IC: [email protected]; CP:

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[email protected] MV: [email protected]

* Corresponding author: Miguel Viveiros. Unit of Medical Microbiology, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da 1

Junqueira 100, 1349-008 Lisbon, Portugal. [email protected], Tel.+351213652653, Fax.+351-21-3632105.

Highlights

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  

First report on antimicrobial susceptibility and population structure of S. enterica ser. Typhi in Angola Characterization of resistance to first-line antibiotics Characterization of reduced susceptibility to ciprofloxacin Majority of isolates belong to the globally disseminated lineage ST2

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

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Abstract

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Objectives: Typhoid fever is a common infection in Africa and in spite of scarce

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surveillance reports, its incidence is commonly considered high by the Angolan Health

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system. Drug-resistant Salmonella enterica serotype Typhi has emerged, turning antimicrobial susceptibility testing essential to provide clinical guidance. This is the first

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report analyzing antimicrobial resistance patterns and population structure of the few S.

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enterica ser. Typhi isolated from patients with Typhoid fever in Luanda, Angola. Methods: Isolates were collected by the National Institute of Public Health of Angola,

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between September 2013 and May 2014. A total of 10 isolates were identified by API20E system and serotyping, and the genus confirmed by PCR. All isolates were tested for

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antibiotic susceptibility and the presence of resistance genes (blaCTX-M, blaSHV, blaTEM, blaOXA-1, several plasmid-borne genes encoding AmpC β-lactamases, sul and qnr genes, dfrIa, dfrA12, aac(6')- Ib, cmlA and floR) screened by PCR. Isolates were typed by PFGE and MLST.

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Results:

Several

isolates

were

identified

with

resistance

to

trimethoprim-

sulfamethoxazole (n=6), beta-lactams (n=5), chloramphenicol (n=1) and reduced susceptibility to ciprofloxacin (n=2). PFGE revealed eight closely related restriction patterns and MLST grouped these in three sequence types: ST1, ST2 and ST8, with ST2 being predominant.

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Conclusion: This first epidemiological report provides a preliminary portray of the S. enterica ser. Typhi strains that circulate in Luanda, Angola and emphasizes the demand

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for a continuous monitoring of this pathogen to provide information to implement better

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epidemiological strategies for the control of Typhoid fever in Angola.

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molecular typing, Angola, human specimens

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Keywords: Salmonella enterica serotype Typhi, Enteric Fever, antimicrobial resistance,

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1. Introduction Epidemiological data about Salmonella enterica serotype Typhi infections and nontyphoidal Salmonellosis is very scarce in Angola, especially regarding prevalence and drug-resistance patterns, due to the absence of regular clinical notification and no reported microbiological research. Official data, gathered by the Epidemiological Surveillance

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Data Center at the National Direction of Public Health, reported 703.000 cases of

suspected Typhoid fever with 381 associated deaths in the years 2014 and 2015

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(Epidemiological Report of the National Direction of Public Health, Angola, 2016).

Previously to the 1980s, the recommended empirical therapeutic regimens for Typhoid

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fever were based on the use of ampicillin, trimethoprim-sulfamethoxazole, or

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chloramphenicol [1]. In the last decades, it has been observed an emergence of resistant

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strains, and currently resistance to these three first-line antibiotics defines a multidrug-

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resistant S. enterica ser. Typhi strain [2]. As a consequence, alternative antibiotic classes, including fluoroquinolones, macrolides and third-generation cephalosporins are now

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widely used [1,3,4]. In Angola, over the recent years, emerging undocumented drug resistance has also limited the usefulness of the first-line antibiotics and presently, when

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available, the alternative classes of antibiotics are preferred for empiric therapy, with a

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preferencial use for fluoroquinolones since they are relatively less expensive and easy to acquire. As a result, susceptibility of S. enterica ser. Typhi to these antibiotics, particularly fluoroquinolones, is declining in Angola as well as globally, posing new and

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urgent clinical and public health challenges [3]. Implementation of any control strategy to combat infections caused by S. enterica ser. Typhi must gather knowledge on the epidemiology and resistance patterns of the etiological agent [4,5]. In this context, the aim of this study was to characterize, for the first time, the drug resistance phenotype and genotype, as well as the molecular 4

epidemiology, of the few S. enterica ser. Typhi isolated among other Salmonella spp. isolated from patients with suspected Typhoid fever in Luanda, collected by the National Institute of Public Health of Angola, between September 2013 and May 2014.

2. Material and methods

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2.1 Isolate collection and identification

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From September 2013 to May 2014, 10 S. enterica ser. Typhi were isolated in 42 samples from patients with ages between 11 and 69 years old that had been admitted in health unit services with suspected Typhoid fever (Clínica Sagrada Esperança; National Institute

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Public Health) in Luanda, Angola. Eight isolates were collected from blood specimens

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and two from faeces. All isolates were identified by standard biochemical tests and the

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API 20E System (BioMérieux, France) for Enterobacteriaceae and the identification of

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the genus Salmonella was confirmed by PCR amplification of the invA gene [6]. Isolates identified as S. enterica ser. Typhi were subjected to serotyping by slide agglutination

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Berlin, Germany) [7].

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using the sera anti-Salmonella GroupD, anti-O:9, anti-Vi and anti-Hd (Sifin Diagnostics,

2.2 Antimicrobial susceptibility testing Susceptibility phenotypes were evaluated by determination of minimum inhibitory

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concentrations (MICs) using the microdilution system MicroScan® -Neg MIC panel Type 44 (Siemens Healthcare Diagnostics, West Sacramento, CA, USA). MIC values of ciprofloxacin were determined by E-test (Oxoid, Basingstoke, UK) in Mueller-Hinton agar plates. Antimicrobial susceptibility phenotypes were interpreted according to the

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Clinical and Laboratory Standards Institute document M100-S26 [8]. Intermediate isolates were reported as resistant.

2.3 Detection of antimicrobial resistance genes

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Total DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations. All isolates presenting phenotypic

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resistance to antimicrobials were tested for the presence of antimicrobial resistance genes, except for the qnr genes which were screened in all isolates. Resistance genes were

screened by simplex or multiplex PCR using specific primers (Supplementary Material

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S1), namely the beta-lactamase genes blaCTX-M, blaSHV, blaTEM and blaOXA-1 and the

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plasmid-borne genes encoding AmpC β-lactamases (blaCIT, blaACC, blaLAT, blaACT, blaMIR,

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blaFOX, blaMOX, blaDHA) for beta-lactam resistance; the genes sul1, sul2, sul3, dfrIa and

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dfrA12; genes cmlA and floR for resistance to chloramphenicol, and the qnrA, qnrB, qnrS, qnrC, qnrD and aac(6')- Ib genes for resistance to fluoroquinolones. The occurrence of

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mutations in the quinolone resistance-determining region (QRDR) of the gyrA/B and

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parC/E was screened by PCR and sequencing [9].

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2.4 Molecular typing

S. enterica ser. Typhi isolates were characterized by MLST and PFGE. Internal fragments

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of the seven housekeeping genes aroC, dnaN, hemD, hisD, purE, sucA and thrA were amplified by PCR using the primers and conditions described previously (http://mlst.warwick.ac.uk/mlst/dbs/Senterica). PCR products were sequenced and the sequences obtained were submitted to the MLST database to retrieve an allelic profile and sequence type for each isolate. Clonality was also assessed by XbaI-PFGE typing (XbaI,

New

England

Biolabs),

according 6

to

the

Pulsenet

protocol

(https://www.cdc.gov/pulsenet/pathogens/pfge.html). The patterns were analyzed using BioNumerics v 6.6 software and the unweighted pair group method with arithmetic mean and the Dice similarity coefficient. The definition of a pulsed-field type cluster was based on a similarity cut-off value of ≥80% and a subtype on the value of ≥97%. The ten S. enterica ser. Typhi isolates were submitted to a PCR approach for identification of the

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globally disseminated H58 haplotype [10].

3. Results

A total of 10 isolates of S. enterica ser. Typhi were isolated from clinical samples of

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patients with suspected Typhoid fever, from September 2013 to May 2014, and identified

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by serotyping and the API20E system (Table 1, Figure 1). These isolates were

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characterized regarding their antimicrobial resistance patterns and clonal relatedness.

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Regarding antimicrobial resistance (Supplementary Material S2), the most common

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pattern observed was resistance to ampicillin, ampicillin/sulbactam, piperacillin and trimethoprim-sulphamethoxazole, displayed by 3 out of 10 isolates. Two isolates showed resistance

to

cefuroxime.

One

isolate

presented

resistance

to

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additionally

chloramphenicol and trimethoprim-sulphamethoxazole. In addition, two isolates

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presented reduced susceptibility to ciprofloxacin. None of the isolates displayed a MDR phenotype. A single isolate was fully-susceptible. No resistance to tetracyclines,

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aminoglycosides, nitrofurantoin, colistin or fosfomycin was detected. Of all the resistance determinants screened by PCR only the sul1 gene was detected. Resistance to trimethoprim-sulphametoxazole was observed in 6 out of 10 isolates and associated with the presence of sul1 gene. Only one isolate presented resistance to chloramphenicol, but none of the resistance genes screened (cmlA and floR) was detected.

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In addition, none of the -lactamase resistance determinants screened were detected in the isolates resistant to ampicillin, ampicillin/sulbactam, piperacillin and cefuroxime. Of the isolates displaying reduced susceptibility to ciprofloxacin, one presented the mutation S83Y in GyrA whereas the other carried the mutation S464F in GyrB. None of these

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isolates presented any of the qnr or aac(6')- Ib genes. Molecular typing by XbaI-PFGE of the ten S. enterica ser. Typhi isolates discriminated three pulsotypes, with one of the pulsotypes comprising eight of the isolates in study,

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grouped in five sub-types (Figure 1). MLST data corroborated XbaI-PFGE typing; three

different sequence types were identified based on a seven-gene MLST scheme. The

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majority of the isolates belonged to ST2 (n=8), whereas ST1 and ST8 were represented

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by a single isolate each (Figure 1). These three STs are single locus variants (SLVs),

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belonging to the same clonal complex, CC13, demonstrating that all ten S. enterica ser.

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Typhi isolates are closely related. All eight ST2 isolates were non-H58 haplotype,

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4. Discussion

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whereas the ST1 and ST8 isolates were non-typable by the PCR approach used.

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Typhoid fever in Africa is not well understood, largely due to poor resources for laboratory diagnostics and insufficient infrastructure to support epidemiological and clinical studies; these problems are manifestations of the challenges faced by a large

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continent with high burden of infectious diseases. For public health interest, this kind of studies provides new knowledge about the disease and its etiological agent, since phenotypic and genotypic characterization, epidemiology and resistance to antimicrobials are unraveled, contributing to an awareness of the real situation of the country, region or community. 8

Resistance due to the production of extended-spectrum β-lactamases (ESBLs) is an ever increasing problem and is a cause of serious concern worldwide. So far, these enzymes have been detected in many Enterobacteriaceae, including S. enterica ser. Typhi [11- 13]. In Africa, some studies have characterized the presence of ESBLs in Salmonella spp. isolates from human specimens and the aforementioned enzymes have been documented

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in different proportions [14]. However, none of the genes encoding these enzymes was

detected in this study. Nevertheless, the diversity of ESBLs suggests that its incidence in

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Salmonella spp. may become a public health concern, requiring a continuous monitoring. The detection of sul1 gene in six isolates corroborates the high prevalence of this gene in

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Enterobacteriaceae, and many studies have reported the presence of this gene associated

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with trimethoprim-sulfamethaxazole resistance in S. enterica ser. Typhi [15]. In our

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findings, no gene associated with chloramphenicol resistance was detected (cmlA and

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floR). Other determinants associated to chloramphenicol resistance have been identified in Salmonella spp. [13]. Resistance to chloramphenicol can also be acquired through

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plasmids harboring a cat gene, which encodes an acetyltransferase that inactivates the

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chloramphenicol molecule or by alterations occurring in cellular permeability. In this study, we found two isolates of S. enterica ser. Typhi with reduced susceptibility

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to ciprofloxacin (MICs of 0.125 and 0.25 mg/L) although susceptible to nalidixic acid; one of the isolates presented the S83Y mutation in GyrA while the other displayed the S464F alteration in GyrB. Both mutations have been described in literature, occurring in

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isolates from Asia and Africa and associated with this phenotype [9,16,17]. The set of isolates in study were collected from blood and faeces samples of patients of both genders, mainly with ages between 11 and 38 years. The patient age distribution is similar to reports from other studies [13,18]. The empiric therapeutic regimen provided was mainly based on the use of the fluoroquinolone ciprofloxacin and the majority 9

overcame the infection (Table 1). Nevertheless, our findings urge a need for a continued monitoring of the resistance traits among S. enterica ser. Typhi, since this pathogenic agent has a high impact in public health. Both PFGE and MLST revealed a high clonality of the ten S. enterica ser. Typhi isolates collected in Angola. The lineages ST1 and ST2, detected in this study, reflect the global

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distribution of this pathogen [19]. Particularly, ST2 strains are the most commonly reported worldwide [19]. The global MLST analysis confirms the predominance of these

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two sequence types (ST1 and ST2), co-existing in the endemic regions, whereas S. enterica ser. Typhi strains belonging to ST8 are currently confined within the African

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continent [19]. It has been hypothesized that the ST8 lineage may have diverged from the

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earlier ancestral ST1 and ST2 lineages. The ST2 lineage then lost some of its fitness

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advantages, leading to poor worldwide dissemination [19]. These sequence types have

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been found in previous studies in other African countries, such as Senegal and Democratic Republic of Congo [19]. In recent years, new SNP-typing schemes were developed to

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increase the discrimination of S. enterica ser. Typhi, leading to the identification of 85 haplotypes. Of these, H58 is widely distributed, with particular focus in Asia and several

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East African countries, and frequently associated with MDR phenotypes and/or low-level

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resistance to fluoroquinolones [20]. Nevertheless, our study shows that none of the isolates from Angola belong to this haplotype. This study is the first epidemiological report of S. enterica ser. Typhi from Angola in the

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literature. Herein, evidence is provided for the occurrence of strains resistant to first-line antibiotics and with reduced susceptibility to fluoroquinolones. The latter result is particularly worrisome as fluoroquinolones are part of critical alternative therapeutic regimens in developing countries, as exemplified by the therapeutic schemes prescribed to patients from which the isolates in study were collected. These results provide a 10

preliminary portray of the S. enterica ser. Typhi strains that circulate in Luanda, Angola and emphasize the need of continuous monitoring of this pathogen to detect any change in its epidemiological and molecular patterns and to provide information to implement

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better epidemiological strategies for the control of Typhoid fever.

Acknowledgements

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We would like to aknowledge the National Institute of Public Health, Angola, the Faculty of Medicine, University Agostinho Neto, Angola, and the Clínica Sagrada Esperança,

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Luanda, Angola for their kind support to this work.

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Declarations

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Funding: This work was partially supported by Fundação para a Ciência e a Tecnologia

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(FCT, Portugal), through funds to GHTM-UID/Multi/04413/2013 and through Project UID/CVT/00276/2013. S. S. Costa and A. Belas were supported by grants

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SFRH/BPD/97508/2013 and SFRH/BD/113142/2015, respectively from FCT, Portugal.

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Competing Interests: No

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Ethical Approval: Not required

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References 1. Kariuki S, Gordon MA, Feasey N, Parry CM. Antimicrobial resistance and management of invasive Salmonella disease. Vaccine 2015;33(S3):C21-C29. 2. World Health Organization (WHO). Background document: the diagnosis, treatment

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and prevention of typhoid fever. WHO, Geneva, 2003. 3. Al-Emran HM, Eibach D, Krumkamp R, Ali M, Baker S, Biggs HM et al. A

susceptibility

to

ciprofloxacin

in

Sub-Saharan

Africa.

Clin

Infect

Dis

U

2016;62(S1):S42-S46.

SC R

multicountry molecular analysis of Salmonella enterica serovar Typhi with reduced

N

4. Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World

A

Health Organization 2004;82:346-53.

M

5. Steele AD, Burgess DCH, Diaz Z, Carey ME, Zaidi AKM. Challenges and opportunities for Typhoid fever control: a call for coordinated action. Clin Infect Dis

ED

2016;62(S1):S4-S8.

PT

6. Malorny B, Hoorfar J, Bunge C, Helmuth R. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl Environ

CC E

Microbiol 2003;69:290-296. 7. Grimont PAD, Weill FX. Antigenic formulae of the Salmonella serovars. Institut

A

Pasteur & WHO Collaborating Centre for Reference and Research on Salmonella, Paris, France, 2007.

8. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; Twenty – sixth informational supplement, M100 – S26. CLSI, Wayne, PA, USA, 2016.

12

9. Chau TT, Campbell JI, Galindo CM, Hoang NVM, Diep TS, Nga TTT et al. Antimicrobial drug resistance of Salmonella enterica serovar Typhi in Asia and molecular mechanism of reduced susceptibility to the fluoroquinolones. Antimicrob Agents Chemother 2007;51:4315-4323.

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10. Murgia M, Rubino S, Wain J, Gaind R, Paglietti B. A novel broadly applicable PCRRFLP method for the rapid identification and subtyping of H58 Salmonella Typhi. J

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Microbiol Methods 2016;127:219-223.

11. Naiemi NA, MC, Roosendaal R, Debets-Ossenkopp YJ, Mulder JA et al. Extendedspectrum-beta-lactamase production in a Salmonella enterica serotype Typhi strain

U

from the Philippines. J Clin Microbiol 2008; 46:2794–2795.

N

12. Pokharel BM, Koirala J, Dahal RK, Mishra SK, Khadga PK, Tuladhar NR. Multidrug-

A

resistant and extended-spectrum beta-lactamase (ESBL)-producing Salmonella

M

enterica (serotypes Typhi and Paratyphi A) from blood isolates in Nepal: surveillance

ED

of resistance and a search for newer alternatives. Int J Infect Dis 2006;10:434-438. 13. Crump JA, Sjolund-Karlsson M, Gordon MA, Parry CM. Epidemiology, clinical

PT

presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial

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management of Salmonella infections. Clin Microbiol Rev. 2015; 28:901-937. 14. Usha G, Chunderika M, Prashini M, Adriaan S, Essack W, Yusuf S. Characterization

A

of extended-spectrum β-lactamases in Salmonella spp. at a tertiary hospital in Durban, South Africa. Diagn Microbiol Infect Dis 2008;62:86-91.

15. Akhtar S, Sarker MR, Jabeen K, Sattar A, Qamar A, Fasih N. Antimicrobial resistance in Salmonella enterica serovar typhi and paratyphi in South Asia - current status, issues and prospects. Crit Rev Microbiol 2015;41:536-545.

13

16. Gupta R, Gaind R, Wain J, Deb M, Singh LC, Basir SF. Characterization of nonclassical quinolone resistance in Salmonella enterica serovar Typhi: report of a novel mutations in gyrB gene and diagnostic challenges. Biomolec Detect Quantif 2014;2:30-34. 17. Gaind R, Paglietti B, Murgia M, Dawar R, Uzzau S, Cappucinelli P et al. Molecular

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characterization of ciprofloxacin-resistant Salmonella enterica serovar Typhi and

Paratyphi A causing enteric fever in India. J. Antimicrob Chemother 2006;58:1139-

SC R

1144.

18. Feasey NA, Archer BN, Heyderman RS, Sooka A, Dennis B, Gordon MA et al.

U

Typhoid fever and invasive Nontyphoid salmonellosis, Malawi and South Africa.

N

Emerg Infect Dis 2010;16:1448-1451.

A

19. Yap K-P, Ho WS, Gan HM, Chai LC, Thong KL. Global MLST of Salmonella Typhi

M

revisited in post-genomic era: genetic conservation, population structure, and

ED

comparative genomics of rare sequence types. Front Microbiol 2016;7:270. 20. Wong VK, Baker S, Pickard DJ, Parkhill J, Page AJ, Feasey NA et al.

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Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet

A

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2015;47:632-639.

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Figure legends Figure 1. Clonal relatedness of the S. enterica ser. Typhi isolates and correlation with antimicrobial resistance pattern and carriage of antimicrobial resistance determinants. Isolates were considered to belong to the same pulsotype when presenting a similarity value equal or higher than 80% (dashed line). A similarity value equal or higher than 97 % was considered as the threshold for identical sub-types. AMP: ampicillin; A/S: ampicillin-sulbactam; CXM:

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cefuroxime; PI: piperacillin; SXT: sulphamethoxazole-trimethoprim; C: chloramphenicol; CIP: ciprofloxacin; MIC: minimum inhibitory concentration; QRDR: quinolone resistance

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CC E

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A

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determining region; ST: sequence type; CC: clonal complex.

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Table 1. Clinical data and treatment outcome of the patients diagnosed with S. enterica ser. Typhi infection in Luanda, Angola. Patient origin

Biological Sample

Age (Y)

Gender

Clinical diagnosis

Therapeutic Regimen

Disease Outcome

S2

Ingombota

Blood

17

M

Fever (8days)

Clindamicin, quinine

Survived

S3

Ingombota

Blood

34

M

No data

No data

Unknown

S18

Ingombota

Blood

16

M

Fever, abdominal pain

Ciprofloxacin

Survived

S19

Ingombota

Blood

69

F

Astenia, M. disturb

Ciprofloxacin,ceftriaxone

Died

S26

Belas

Blood

11

F

Fever, convultions

Ceftriaxone, ciprofloxacin

Survived

S27

Rangel

Blood

16

F

Fever, abdominal pain

Ciprofloxacin

Survived

S28

Cacuaco

Blood

38

M

Fever, abdominal pain

Ciprofloxacin

Survived

S29

Ingombota

Blood

11

M

Fever

Not treated

Unknown

S32

Maianga

Faeces

14

F

Fever, abdominal pain

Ciprofloxacin

Survived

S34

Maianga

Faeces

20

F

Abdominal pain

Nalidixic Acid

Survived

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M

A

Isolate ID

A

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Y: years; M: male; F: female.

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