Drug resistance of Salmonella enterica serovar Typhi (Salmonella Typhi) to first-line antibiotics is emerging in Central Africa. Although increased use of fluoroquinolones is associated with spread of resistance, Salmonella Typhi with decreased ciprofloxacin susceptibility (DCS) has rarely been reported in Central Africa. As part of a microbiological surveillance study in the Democratic Republic of the Congo (DR Congo), Salmonella Typhi isolates from bloodstream infections were collected prospectively between 2007 and 2011. The genetic relationship of the Salmonella Typhi isolates was assessed by pulsed-field gel electrophoresis (PFGE). The antimicrobial resistance profile of the isolates was determined and mutations associated with DCS were studied. In total, 201 Salmonella Typhi isolates were collected. More than half of the Salmonella Typhi isolates originated from children and young adults aged 5–19. Thirty different PFGE profiles were identified, with 72% of the isolates showing a single profile. Multidrug resistance, DCS and azithromycin resistance were 30. 3%, 15. 4% and 1. 0%, respectively. DCS was associated with point mutations in the gyrA gene at codons 83 and 87. Our study describes the first report of widespread multidrug resistance and DCS among Salmonella Typhi isolates from DR Congo. Our findings highlight the need for increased microbiological diagnosis and surveillance in DR Congo, being a prerequisite for rational use of antimicrobials and the development of standard treatment guidelines.
Typhoid fever is endemic in the Democratic Republic of the Congo (DR Congo). Although antimicrobial resistance data are sparse for Central Africa, drug resistance to first-line antibiotics is clearly emerging [1]. Also in DR Congo, multidrug resistance (MDR) [defined as co-resistance to first-line antibiotics ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole (TMP-SMX) ] in Salmonella enterica serotype Typhi (Salmonella Typhi) has been observed [2], [3]. Facing widespread MDR, fluoroquinolones have become the drugs of choice for treating typhoid fever, but their increased use has been associated with a spread in low-level fluoroquinolone resistance - further referred to as decreased ciprofloxacin susceptibility (DCS) [4]. Salmonella Typhi with DCS is common in Asia and has been reported in East Africa and South Africa [5]–[7], but apart from a single case reported from Cameroon [8], and two cases in DR Congo, Salmonella Typhi with DCS has not yet been observed in the central African region [2], [3], [9]. The present study describes the antimicrobial resistance profile of a prospective collection of Salmonella Typhi isolates recovered as part of a microbiological surveillance study from blood cultures obtained from patients in DR Congo over the years 2007–2011. Mutations associated with DCS were studied and the genetic relationship of the Salmonella Typhi isolates was assessed.
Ethical approval was granted by the Ethical Committee of the University of Antwerp, Belgium and from the Ministry of Health in DR Congo. The present study complies with the World Health Organization and international guidelines (European Society of Clinical Microbiology and Infectious Diseases Study Group for Antimicrobial Resistance Surveillance and Clinical Laboratory Standards Institute) on antibiotic surveillance for which no recommendation for an informed consent has been issued. The diagnostic procedure – blood cultures – is part of the standard diagnostic work-up of patients with a suspicion of bacteremia. Clinical information -as presented- and information about use of antibiotics was the standard information present on the laboratory request form. Data have been reviewed and analyzed anonymously. Between April 2007 and January 2011, blood cultures were performed on 9,634 patients suspected of typhoid fever or other systemic infections. Patients were seen at health care facilities in seven out of 11 provinces in DR Congo: Kinshasa, Bas-Congo, Bandundu, Equateur, Kasai Occidental, Kasai Oriental and Oriental province (Figure 1). In Kinshasa, health care facilities involved in the detection and study of the epidemic increase of typhoid fever-associated peritonitis of 2004 were selected [3]. Health care facilities in other provinces were recruited based on the past or actual existence of microbiological laboratories, professional contacts and the accessibility to reliable shipment facilities. Criteria for blood culture sampling were clinical suspicion of bacteremia associated with a local (pneumonia, urinary tract infection, meningitis or other) or systemic (typhoid fever, endocarditis) infection diagnosed at consultation or admission. Typhoid fever was defined according to the case definitions of the Ministry of Health surveillance of communicable diseases [10]. At the start of the surveillance project, teams of clinicians and laboratory technicians were trained in indications and sampling of blood cultures. For children <14 years, 1–4 ml of blood was inoculated into a pediatric blood culture vial (Bact/ALERT FP; bioMérieux; Marcy L' Etoile; France). For adults, 2×10 ml of blood was inoculated into aerobic blood culture vials (Bact/ALERT FA; bioMérieux; Marcy L' Etoile; France). Age, gender, geographic origin, use of antibiotics prior to blood culture sampling and presumptive diagnosis (focus of bacteremia including suspicion of typhoid fever) were recorded. Inoculated vials were shipped to the Institut National de Recherche Biomédicale (INRB) in Kinshasa, incubated at 35°C and daily checked for growth by visual inspection of the chromatographic growth indicator. Grown cultures were Gram stained, subcultured and identified to the species level. Skin or environmental bacteria (coagulase negative staphylococci, Corynebacterium spp. , Propionibacterium acnes and Bacillus spp.) were categorized as contaminants, the other bacteria were considered as clinically significant organisms [11]. Suspected colonies of Salmonella were identified as Salmonella Typhi using standard biochemical methods (characteristic aspect on Kligler Iron Agar (acid from glucose, no gas, trace of H2S), negative tests for urease, oxidase, β-galactosidase and indole production tests, positive tests for lysine decarboxylase) and the serotype of Salmonella Typhi (O: 9; H: d; Vi+) was confirmed with commercial antisera (Remel, Lenexa, Kansas). Identity of Salmonella species isolates was confirmed using the Vitek II system (Card GN21 341, bioMérieux). At the National Reference Laboratory for Salmonella and Shigella (Institute of Public Health, Brussels), the serotype of the Salmonella isolates was re-confirmed by slide agglutination with commercial monospecific antisera (Sifin, Berlin, Germany), following the Kauffmann-White scheme [12]. For analysis in the present study, only the first isolate per patient was considered. Susceptibility tests for ampicillin, cefotaxime and TMP-SMX were performed using the Vitek II system (Card AB AST-N156, bioMérieux). For nalidixic acid, ciprofloxacin, chloramphenicol and azithromycin, minimal inhibitory concentration (MIC) values were determined using the E-test macromethod (bioMérieux). Breakpoints for resistance were respectively ≥32 mg/l for ampicillin, ≥4 mg/l for cefotaxime, ≥32 mg/l for naladixic acid, ≥16 mg/l chloramphenicol (considering intermediate susceptible isolates resistant) and ≥4/76 mg/l for TMP-SMX using Clinical and Laboratory Standards Institute definitions [13]. DCS was defined according to European Committee on Antimicrobial Susceptibility testing (EUCAST) V 2. 0. guidelines, i. e. a MIC-value for ciprofloxacin >0. 064 mg/l [14]. For azithromycin, EUCAST V. 2. 0. suggests MICs of >16 mg/l as defining resistance [14]. Multidrug resistance was defined as co-resistance to ampicillin, chloramphenicol and TMP-SMX. Screening for mutations causing DCS was performed by amplification and sequencing of the quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, and parC genes. The presence of the plasmid-mediated quinolone resistance qnr genes (qnrA, qnrB, and qnrS) was determined using PCR [15]. Pulsed-field gel electrophoresis (PFGE) was performed according to the PulseNet protocol for molecular subtyping of Salmonella [16], using XbaI as restriction enzyme (New England Biolabs, Leusden, The Netherlands). For cluster analysis, Bionumerics 5. 1 was used (Applied Maths NV, Sint-Martens-Latem, Belgium), with as comparison settings the Dice similarity coefficient and UPMGA dendrogram type (optimization 0. 50%, position tolerance 1. 50%). PFGE profiles obtained were compared to PFGE of Salmonella Typhi profiles stored: In 1° the Institute of Public Health (Brussels) database, originating from Belgium (n = 27), Morocco (n = 8), Egypt (n = 1), Burkina Faso (n = 1), Niger (n = 1), Cambodia (n = 20), India (n = 11), Pakistan (n = 7), Bangladesh (n = 3), Indonesia (n = 1), Sri Lanka (n = 1), and Thailand (n = 1); 2° the PFGE database of the Centre for Enteric Diseases (CED) of the National Institute for Communicable Diseases in South Africa, containing patterns for 730 Salmonella Typhi isolates. All data were entered in an Excel database (Microsoft Corporation, Redmond, Washington, USA). Proportions were assessed for statistical significance using the Chi square test, considering p<0. 05 as significant (Stata 10, StatCorp, Texas, USA).
From the 9634 blood cultures performed, 989 (10. 3%) clinically significant organisms were grown, including 201 isolates of Salmonella Typhi, representing respectively 2. 1% of all blood cultures and 20. 3% of all clinically relevant organisms. There were no annual or seasonal differences in isolation rates. The geographic site of cases, positive on blood cultures for Salmonella Typhi in DR Congo, is shown in figure 1. Among the 201 Salmonella Typhi blood cultures isolates, 110 (54. 7%) were recovered from Bas-Congo and 67 (33. 3%) from Kinshasa. In Kinshasa, the isolation rate of Salmonella Typhi (67/5465,1. 2%) was lower compared to all other provinces in DR Congo (134/4064,3. 4%, p<0. 001). The median age of patients infected with Salmonella Typhi was 15 years (interquartile range 8–25), but infection of young children was also common (Table 1). Over half of blood cultures were in children <10 years, yet 32. 8% of the Salmonella Typhi isolates recovered were from children in this age group. The most affected age group were persons aged 10–19 years, in whom nearly 60% of the organisms isolated were Salmonella Typhi. In addition, this age group contributed to only approximately 10% of the blood culture samples, yet accounted for 30% of all Salmonella Typhi isolates recovered. In patients with clinically significant organisms, presumptive diagnosis of typhoid fever was made in 53. 0% (524/989) of the cases. In the 201 patients from whom Salmonella Typhi was cultured, presumptive diagnosis of typhoid fever at the moment of sampling was made in 80. 6% (162/201) of the cases. A total of 21. 0% (34/162) of these patients suffered from abdominal distension and/or gastro-intestinal bleeding and were classified as complicated typhoid fever. Other presumptive diagnoses (for several patients more than one presumptive diagnosis was mentioned) included complicated urinary tract infection (14. 4%), pneumonia (7. 0%), meningitis (2. 0%), malaria (5%) and other non-specified causes of bacteremia (16. 4%); for three patients (1. 5%), no data were available. Nearly half (93/201,46. 3%) of the patients had received antibiotics within 48 hours prior to sampling of blood cultures, mostly first-line antibiotics. Resistance against ampicillin, chloramphenicol or TMP-SMX were observed in 64. 7% (130/201), 41. 3% (83/201) and 57. 7% (116/201) of isolates, respectively. MDR and DCS were observed in 30. 3% (61/201) and 15. 4% (31/201) of isolates respectively; combined MDR and DCS occurred in 7. 5% (15/201) of isolates. Isolates with DCS corresponded to nalidixic acid resistant isolates and vice versa. Only two (1. 0%) isolates had azithromycin MIC values exceeding 16 mg/l (i. e. 24 mg/l); both isolates were also resistant against ampicillin and TMP-SMX (one also combined with DCS and nalidixic acid resistance). No cefotaxime resistance was observed. Fifty-one isolates (25. 4%) were fully susceptible to all three first-line antibiotics and 50 (24. 9%) were susceptible to all seven antibiotics tested. There was no apparent relationship between antimicrobial resistance and patient age, year of isolation, province of isolation or administration of antibiotics prior to isolation. All 31 isolates with DCS were analysed for mutations in the QRDRs of the gyrA, gyrB, and parC genes. No mutations were detected in gyrB or parC genes. No qnrA or qnrB genes were detected. For one isolate, the qnrS gene was detected. Apart from a mutation at codon 133 in the gyrA gene (conferring a Glu to Gly change in the GyrA protein), which was also present in nalidixic acid susceptible strains, all 31 isolates had DCS-associated mutations in the gyrA gene, conferring the following amino acid mutations in the GyrA protein: (i) Ser83 changed into Phe or Tyr (n = 22), or (ii) Asp87 changed into Gly, Tyr or Asn (n = 9). Among 185 isolates tested for PFGE, 30 different profiles were observed (Supplemental file). In 132/185 isolates (71. 4%), an indistinguishable PFGE profile occurred. This profile was the main or single PFGE profile over time and geography, although its proportion was lower (p = 0. 02) in Kinshasa province (39/64 isolates, 60. 9%) compared to the other provinces in DR Congo (93/121,76. 9%). In Kinshasa, the highest variation of profiles - in total 22 - was noted. Of the isolates showing the main/predominant profile, 31. 1% (41/132) were MDR and 17. 4% (23/132) were DCS, while 11 (8. 3%) showed a combined MDR and DCS. Comparison of the PFGE profiles of Salmonella Typhi from DR Congo with isolates from other geographic origins revealed one PFGE profile from a Salmonella Typhi isolate from DR Congo that was indistinguishable from a PFGE profile from an isolate recovered in Belgium, and 5 Congolese PFGE profiles that were indistinguishable from PFGE profiles of 9 Salmonella Typhi isolates recovered in South Africa.
This study was funded by Directorate General of Development Cooperation of the Belgian Government through Institutional Collaboration INRB-ITM (Network Program on Laboratory Quality Management; Project 3. 21). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that they have no conflicting interests in relation to this work.
Typhoid fever, caused by infection with Salmonella enterica serovar Typhi (Salmonella Typhi), is an important health problem in sub-Saharan Africa. Multidrug resistance of Salmonella Typhi to the first line antibiotics is spreading and treatment of typhoid fever increasingly relies on fluoroquinolone antibiotics such as ciprofloxacin. Increased use of fluoroquinolones is however associated with spread of resistance as well. In sub-Saharan Africa, microbiological cultures to detect invasive bacterial diseases are frequently absent and the extent of the problem is poorly known. In the present study, 201 Salmonella Typhi isolates were collected between 2007 and 2011 in DR Congo, mainly from children and young adults. For the first time, widespread Salmonella Typhi multidrug resistance (30. 3%) and decreased ciprofloxacin susceptibility (15. 4%) is described in Central Africa. Decreased ciprofloxacin susceptibility was associated with point mutations in the quinolone resistance determining region of the gyrA gene. Resistance to azithromycin, an alternative for treatment of uncomplicated typhoid fever in the case of decreased ciprofloxacin susceptibility, was still rare (1. 0%). Our findings demonstrate emergence of multidrug resistance and fluoroquinolone decreased susceptibility in DR Congo, and highlight the need for increased microbiological diagnosis and surveillance, being a prerequisite for rational use of antimicrobials and the development of standard treatment guidelines.
medicine
bacterial diseases
infectious diseases
salmonellosis
salmonella
Salmonella Typhi in the Democratic Republic of the Congo: Fluoroquinolone Decreased Susceptibility on the Rise