Detection of Salmonella enterica Serotype Typhimurium DT104 in

Am. J. Trop. Med. Hyg., 79(6), 2008, pp. 918–920 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

Short Report: Detection of Salmonella enterica Serotype Typhimurium DT104 in Mozambique Joaquim Ruiz,* Silvia Herrera-Leon, Inacio Mandomando, Eusebio Macete, Laura Puyol, Aurora Echeita, and Pedro L. Alonso Centre de Recerca en Salut Internacional de Barcelona, Hospital Clinic/Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; Centro de Investigacíon Biomedica en Red Epidemiología y Salud Pública, Spain; Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Spain; Centro de Investigação em Saúde da Manhiça, Manhiça, Mozambique; Instituto Nacional de Saúde, Maputo, Mozambique; Direcçao Nacional de Saude, Maputo, Mozambique

Abstract. The spread of Salmonella enterica serotype Typhimurium definitive phage type DT104 in sub-Saharan Africa is a public health concern. We obtained two isolates of S. typhimurium DT104 from blood cultures of infants with malaria in Mozambique. Both isolates contained Salmonella genomic island 1A and had the same pulsed-field gel electrophoresis PulseNet pattern (STYMXB.0005). Results showed the need for continuous surveillance of Salmonella spp. serotypes circulating in this area. also had anemia and gastroenteritis, respectively, both of which were possibly related to diagnosis of malaria (Table 1). Approximately 1–3 mL of whole blood were inoculated into one pediatric blood culture bottle (Pedibact威; Becton Dickinson, Franklin Lakes, NJ) and was incubated in an automatic BACTEC威 9050 system (Becton-Dickinson) for four days. The microorganisms were identified by using conventional biochemical tests, serotyping, and phage typing. Antimicrobial drug susceptibility levels to ampicillin (Amp), chloramphenicol (Cm), tetracycline (Tc), amoxicillin plus clavulanic acid (AMC), cotrimoxazole, ceftazidime, nalidixic acid, and gentamicin were established using the disk-diffusion method in accordance with the standards of the Clinical Laboratory Standards Institute.9 Genetic loci encoding resistance to Amp, Tc, and Cm and the presence of integrons were detected by polymerase chain reaction (PCR). Briefly, a small amount of each strain grown on MacConkey agar was resuspended in 100 ␮L of sterile distilled water and boiled for 10 minutes. After a short centrifugation step at 15,000 × g, the supernatant was transferred to another tube, and 5 ␮L were used in each 50-␮L volume in the PCR. The primers, reaction mixture, and amplification conditions used for each specific PCR were those described by Cabrera and others.3 Amplified products were separated by agarose gel electrophoresis, recovered from gels, and sequenced as described.3 The presence of left-hand and righthand boundaries of SGI1-A was determined by PCR, and PCR mapping was performed to confirm the linkage between the genes as described.10 Clonal relationships among both isolates were established by pulsed-field gel electrophoresis (PFGE) following the PulseNet protocol (http://www .pulsenet-europe.org/docs.htm). Salmonella typhimurium was isolated from both patients. These isolates were identified as belonging to the DT104 phage type. To determine the level of DT104 phage type dissemination in the study area, 407 Salmonella spp. isolated between 2001 and 2003 were serotyped. Three hundred sixtyfive of the Salmonella spp. were identified as causes of bacteremia and 42 Salmonella spp. were identified as causes of infantile diarrhea. Of these, 237 isolates were identified as S. typhimurium and phage typed. The two Salmonella DT104 isolates were resistant to Amp, Cm, and Tc. One of them showed intermediate resistance to AMC. Resistance to Cm was associated with the presence of

Common diseases, such as schistosomiasis, or a positive status for human immunodeficiency virus (HIV) in subSaharan Africa have been recognized as factors that may facilitate bacteremia by non-typhi Salmonella spp. (NTS).1 The result is a high prevalence of NTS, mainly of the Typhimurium and Enteritidis serotypes, that cause bacteremia in this area.1,2 This situation poses a serious public health problem because the concomitant presence of these pathologies, or others such as malaria, with NTS bacteremia increases morbidity and mortality.2 Antimicrobial drug–resistance levels of NTS isolates have increased during recent years.3,4 Among these resistant NTS isolates, S. enterica serovar Typhimurium definitive phage type (DT) 104 is particularly relevant because of its multidrug resistance and worldwide dissemination.5 In S. typhimurium DT104, antibiotic resistance genes are clustered within a 43kb integrated element known as the Salmonella genomic island 1 (SGI1).6 Classically, it contains five antibiotic resistance genes, all located within the boundaries of a complex class 1 integron, designated In104. These genes encode resistance for ampicillin (blaP1), chloramphenicol (floR), tetracycline (tetG), streptomycin (aadA2), and sulfonamides (sul1).4 A number of variants of SGI1 containing different sets of resistance genes have also been identified in different Salmonella serotypes, and, recently, in other microorganisms.7 These variants appear to have gained, lost, or exchanged resistance genes and have been named SGI1-A through SGI1O.7 Limited information is available regarding the presence of S. typhimurium DT104 in sub-Saharan Africa. To our knowledge, its existence has been reported only in South Africa and Ethiopia.5,8 Thus, the present study was conducted to identify multidrug-resistant S. typhimurium DT104 as a cause of infant bacteremia in southern Mozambique. Two children, a boy four months of age and a girl 19 months of age, were admitted in December 2000 and January 2001, respectively, to the Manhiça District Hospital in Manhiça, Mozambique. Both children had fever (ⱖ 37.5°C) and had been diagnosed by microscopy as having malaria. They

* Address correspondence to Joaquim Ruiz, Laboratori 311 (Laboratori de Malaria), Laboratoris Institut d’Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, C/Casanovas 143, 08036 Barcelona, Spain. E-mail: [email protected]

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S. TYPHIMURIUM DT104 IN MOZAMBIQUE

TABLE 1 Characteristics of the two patients in the study Patient

Sex

Age, months

Malaria

Other pathology

Died

1 2

M F

4 19

Yes Yes

Anemia Gastroenteritis

No Yes

the floR gene, and resistance to Tc was associated with the presence of the tetG gene. Resistance to Amp was associated with the presence of the blaP1 gene, which was detected in an integron of approximately 900 basepairs. Another integron of approximately 1,100 basepairs containing an aadA2 gene was identified. Other ␤-lactamases, Tc-resistance determinants, and Cm-resistance determinants were not detected. The presence of the genomic island SGI1-A was demostrated in both isolates. In addition, the two isolates showed indistinguishable PFGE profiles corresponding to the PulseNet pattern STYMXB.0005. However, no other S. typhimurium DT104 were detected among the 237 S. typhimurium tested. SGI1 has also been described in other Salmonella serotypes,10 and recently in Proteus mirabilis.7 However, in a study designed to determine the mechanisms of antimicrobial drug resistance in Salmonella spp. isolated in the area of Manhiça, none of the analyzed isolates had more than one typical antimicrobial drug resistance gene (floR, tetG, and blaP1) suggestive of SGI1-A (Mandomando et al, unpublished data). These results show that phage type DT104 has a low prevalence in the study area that and that other Salmonella spp. serotypes in this area do not contain SGI1-A. In developing countries, access to antimicrobial drugs is often restricted by economic reasons.11 In sub-Saharan Africa, NTS infections especially affect children and HIVpositive patients, two particularly vulnerable populations. Thus, detection of S. typhimurium DT104 among specific causes of bacteremia is significant because of its virulence and its resistance to many of the antibacterial agents commonly used in developing regions.11 Both isolates showed the PulseNet pattern STYMXB.0005, which is an uncommon S. typhimurium DT104 pattern in Europe. In a study that assigned PFGE profiles to 1,060 isolates of S. typhimurium DT104 from different European countries, only 4 samples belonging to this type were recovered, showing that despite the presence of 28 different pulsotypes, 90% of the isolates were attributable to only 4 types.12 In subSaharan Africa, the relevance and prevalence of this or others pulsotypes of S. typhimurium DT104 remain unknown. It is highly probable that in sub-Saharan Africa the prevalence of the different pulsotypes will be different from prevalence in Europe or other areas. However, this hypothesis has not been tested. Because laboratory facilities for adequate diagnosis are limited, the occurrence and spread of this phage type in Mozambique, and possibly in all of sub-Saharan Africa, should not be ruled out. A study that analyzed worldwide relevance of S. typhimurium DT104 requested information from only five countries in Africa, and only one (South Africa) provided data.13 Furthermore, Africa does not have a PulseNet initiative to perform general surveillance of the prevalence and distribution of microorganisms. Clonal microorganisms in different and separate areas have been described in sub-Saharan Africa.14 Manhiça is located in Maputo province, bordering South Africa, and has a continu-

ous exchange of persons and commercial products. Furthermore, the population in Manhiça has been historically characterized by high emigration, mainly of its male population, to South Africa for temporary employment in the mining sector. The low prevalence of S. typhimurium DT104 and the absence of isolates belonging to other serotypes that carry SGI1-A detected in the area, together with the fact that both S. typhimurium DT104 isolates were recovered in a short period of time, suggest that the temporal appearance of this serotype was the result of the aforementioned exchange of persons and commercial products. However, we cannot conclude that the presence of S. typhimurium DT104 in Mozambique is only a recent occurrence. In summary, multidrug-resistant S. typhimurium DT104 carrying the genomic island SGI1-A has been detected as a cause of infant bacteremia in Mozambique. This finding demonstrates that this specific phage type is widespread and that global pathogen surveillance, including developing countries, should be conducted to better understand and prevent its spread. Our results suggest a possible relationship among isolation of S. typhimurium DT104 and continuous movement of persons and products between South Africa and the area of Manhiça in Mozambique. Received March 11, 2008. Accepted for publication July 7, 2008. Acknowledgments: We thank Mariano Sitaube and Ana Aladueña for support in isolating strains and phage typing and Carolyn Daher and Tamara Berthoud for suggestions and help in editing the text. Financial support: Joaquim Ruiz was supported by grants CP05/0130 and PI06/0204 from Fondo de Investigaciones Sanitarias, Government of Spain. Authors’ addresses: Joaquim Ruiz and Laura Puyol, Laboratori 311 (Laboratori de Malaria), Laboratoris Institut d’Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, C/Casanovas 143, 08036 Barcelona, Spain, Tel: 34-93-227-5400 ext. 3388, Fax: 3493-227-9853, E-mails: [email protected] and [email protected]. Silvia Herrera-Leon and Aurora Echeita, Laboratorio de Referencia de Salmonella y Shigella, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera de Majadahonda a Pozuelo Km 2.2, 28220 Majadahonda, Madrid, E-mails: [email protected] and aecheita@ isciii.es. Inacio Mandomando and Eusebio Macete, Centro de Investigaçao em Saude do Manhiça, Manhiça Sede, Rua 12, Maputo, Mozambique, E-mails: [email protected] and [email protected]. Pedro L. Alonso, Centre de Recerca en Salut Internacional, C/Villarroel 132, 4°, 2ª. 08036-Barcelona, Spain, Tel: 34-93-227-5400 ext. 2982, Fax: 34-93-227-9853, E-mail: [email protected].

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