INTRODUCTION

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Despite improvements in individual and collective sanitation as well as in the careful monitoring of food processing, both sporadic episodes and outbreaks of salmonellosis continue to occur with high frequency in industrial countries. Factors such as intensive pig and poultry production are contributing to the emergence of new clones of Salmonella pathogenic to humans. Others, such as the increase in international travel, the rapidly growing international food trade between countries with different levels of hygiene in the production and manufacture of foods and with different endemic pathogenic clones, are the cause of their dispersion among different geographic areas. On the other hand, the increase in patients with AIDS, other immunodeficiencies, and chronic disorders, are factors that contribute to putting new groups of people at increased risk of developing severe clinical forms of salmonellosis.

Salmonella is an enteroinvasive bacterium and causes infections that may have one of five different clinical presentations (4, 8). Gastroenteritis is the most common presentation in industrial countries and is considered as an emergent food-borne pathogen-caused disease. It is a self-limited illness of brief duration, usually characterized by diarrhea and fever, in which antibiotic treatment is rarely indicated for immunocompetent patients. Much less frequent but much more severe presentations, usually requiring antibiotic treatment, are bacteremia-septicemia without localized infection and focal infections. Focal infections may affect different sites in the body, causing different disorders, which frequently occur during or after Salmonella bacteremia but may also occur concomitantly with other syndromes. These three clinical presentations may be caused by many Salmonella serotypes considered zoonotic. Although any serotype can cause any of these presentations, certain serotypes have been associated with specific presentations, e.g., Salmonella enterica serotype Enteritidis and serotype Typhimurium are associated with gastroenteritis, while serotype Choleraesuis and serotype Dublin are associated with bacteremia (8, 23). Enteric fever, or typhoid fever, is due to serotype Typhi (which is adapted to human hosts; animals do not serve as a reservoir) but may also be caused by other serotypes (mainly Paratyphi A, B, and C), and antibiotic therapy clearly shortens the duration of the disease. Drinking water disinfection and other sanitation measures have lowered its frequency in industrial countries, but the frequency of this disease remains very high in developing countries (8). A fifth presentation is the chronic carrier state (enteric or urinary), defined as the excretion of salmonellas for months or years after the initial onset of disease, which is most frequent after Typhi infection.

The aim of this study was to carry out an evaluation of extraintestinal salmonellosis cases treated in the Hospital Central de Asturias (HCA), Oviedo, Spain, over a 6-year period (1991 to 1996). It was conducted by analyzing the phenotypic and genotypic traits of Salmonella organisms, clinical and other features of the patients, and the relationships between nontyphoid Salmonella genomic groups and clinical presentations.

	MATERIALS AND METHODS

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Samples processed and Salmonella characterization. Blood and other clinical samples from patients with clinically suspected bacteremia and focal infection were processed according to standard techniques (2, 24). The microorganisms from positive cultures were isolated and characterized by standard laboratory methods. The biochemical profile (biotype) was ascertained by the PASCO system (Difco). Serotyping was performed with Bacto-Salmonella antisera for serotypes Enteritidis and Typhimurium (Difco). Assignment to other serotypes, as well as phage typing of Typhimurium and Enteritidis strains was carried out in the Centro Nacional de Microbiología (CNM), Majadahonda, Madrid, Spain, by using established phage-typing systems (1, 25).

Antimicrobial susceptibility test. Antimicrobial susceptibility testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards for the microdilution susceptibility test (17a) by using the commercial antibiotic panel for Spain of the PASCO system (Difco), which includes ampicillin, ampicillin-sulbactam, aztreonam, cefazolin, cefotaxime, cefoxitin, ceftizoxime, ceftriaxone, cefuroxime, imipenem, piperacillin, ticarcillin, chloramphenicol, gentamicin, tobramycin, amikacin, ciprofloxacin, ofloxacin, nalidixic acid, fosfomycin, and trimethoprim-sulfamethoxazole. In addition, streptomycin and sulfadiazine were analyzed by the disk diffusion technique with commercial disks (Difco).

Genetic typing procedures. Chromosomal DNA isolation and ribotyping were conducted as described in references 10, 11, and 17 by using a DNA fragment carrying the rrnB operon of Escherichia coli as a probe. Hybridization was performed by using the nonradioactive DNA Labelling and Detection Kit of Boehringer GmbH (Mannheim, Germany) according to the manufacturer's instructions. The different patterns of bands containing all or part of the rRNA genes and flanking sequences (ribotypes) were designated with the first letter (or letters) of the serotype, followed by the first letter(s) of the restriction endonuclease(s) used (H for HincII and PS for the mixture of PstI and SphI) and a number assigned according to the order of detection in our laboratories.

Data from medical reports. Data obtained from the medical reports of patients included age, sex, underlying diseases, portal of entry, predisposing factors, fever, diarrhea, and leukocytosis, or leukopenia, which were categorized according to standard criteria (9, 16). Only two patients were hospitalized simultaneously, but they were in different hospital buildings. During the period 1991 to 1996 there was no suspicion of Salmonella hospital outbreaks, and no investigation into common vehicles of infection or other possible linkages among the patients was recorded.

	RESULTS

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Microbiological data on extraintestinal salmonellosis. During 1991 to 1996 in the HCA Microbiology Laboratories, a total of 970 Salmonella isolates were collected, 890 from feces (92%), 59 from blood (6%), and 21 from other clinical samples (2%). These isolates represented 57, 1.3, and 0.025% of bacteria collected from feces, blood, and other clinical samples, respectively. Eighty samples other than feces, from 68 patients, were registered as positive for Salmonella. All but one of the isolates belonged to subspecies 1 and were differentiated into three serogroups (B, C, and D) which included seven serotypes (Typhimurium, Brandenburg, Bredeney, and Derby in serogroup B, Hadar in serogroup C, and Enteritidis and Typhi in serogroup D). No Typhi or Derby organisms were available for further testing, but some data from these organisms were compiled from microbiological reports. Enteritidis and Typhimurium strains were the most frequent; they were isolated from different types of clinical samples and were associated with different clinical presentations (Table 1).

View larger version (100K): [in this window] [in a new window]   FIG. 1.   Ribotypes found in Salmonella strains causing extraintestinal diseases. ST, serotype; E, Enteritidis; T, Typhimurium; B, Brandenburg; H, Hadar; S, Subspecies III; Br, Bredeney. RT, ribotype; the arabic numeral corresponds to the order assigned to the ribotype in our laboratories. a, ATCC 13076; b, ATCC 14028. (a) H ribotypes generated by HincII. , lambda DNA cleaved with PstI; sizes of fragments (in kilobases) from top to bottom are 5.08, 4.65, 4.5, 2.84, 2.58, 2.14, 1.98, 1.2, 1.1, 0.8, 0.5, and 0.2. (b) PS ribotypes generated by a mixture of PstI and SphI. , lambda DNA cleaved with HindIII; sizes of fragments (in kilobases) from top to bottom are 27.5, 23.1, 9.4, 6.55, 4.36, and 2.32.

View larger version (90K): [in this window] [in a new window]   FIG. 2.   RAPD types of Salmonella strains causing extraintestinal diseases, obtained by using the primers labelled OPB-17 and Universal. ST, serotype; E, Enteritidis; T, Typhimurium; B, Brandenburg; H, Hadar; S, Subspecies III; Br, Bredeney. AT, amplified type; the arabic numeral corresponds to the order assigned to the RAPD type in our laboratories. a, ATCC 13076; b, ATCC 14028. , lambda DNA cleaved with PstI; sizes of fragments (in kilobases) from top to bottom are 5.08, 2.8, 2.1, 1.7, 1.15, 1.1, 0.8, 0.5, and 0.4.

Patient data associated with extraintestinal salmonellosis. The medical and microbiological reports of only 52 patients with extraintestinal salmonellosis were available for review; it was found that 6 episodes had been diagnosed as typhoid fever, 32 as bacteremia, and 14 as focal infections (5 in the urinary tract, four pleuropulmonary, three abdominal, one in the central nervous system, and one osteoarticular). The most relevant epidemiological and clinical features of the three groups of clinical presentations are summarized in Table 2, and some of these features are discussed here. Typhoid fever and urinary-tract infections were more frequent in females than in males (2:1 and 4:1, respectively), whereas bacteremia and nonurinary focal infections were more frequent in males than in females (2.2:1 and 3:1, respectively). In relation to age groups, all but one of the patients with typhoid fever were under 31 years of age without predisposing conditions. Two of the episodes in the pediatric age group were diagnosed as bacteremia, affecting an 8-month-old boy and an 11-month-old girl, both of whom had no underlying disease but had presented with diarrhea previously. Thirteen bacteremia episodes and one urinary-tract infection affected young adults (20 to 45 years of age), of whom seven had AIDS, one had neoplasia, one had diabetes, one had undergone gastrectomy, and the remaining four had no predisposing conditions but had suffered diarrhea previously. Fifteen cases of bacteremia (46.8%) and 11 focal infections (78.6%) corresponded to the geriatric group (over 61), of whom eight had diabetes.

	DISCUSSION

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HCA is a general hospital incorporating the former Hospital Covadonga of Oviedo and Hospital General of Asturias, with 1,522 beds and 80% bed occupancy; it serves a population of about 200,000 inhabitants and is the reference center for other hospitals in the Principality of Asturias. In the HCA the overall incidence of bloodstream invasion by nontyphoid salmonellas over the period 1991 to 1996 was 7.63%. The percent Salmonella-positive hemocultures was 1.3%, lower than the percentage reported in the previous decade (24) despite the increase in patients with AIDS and other immunodeficiencies. This is similar to the percentage reported by the Spanish Bacteremia Group (1.58%), which compiled data from six Spanish hospitals (9).

From the present study, three microbiological findings should be discussed. Firstly, in the HCA, over the period 1991 to 1996, the serotypes most frequently recorded as causing human salmonellosis were Enteritidis, Typhimurium, Virchow, and Hadar, with 534, 254, 28, and 8 isolates, respectively; no isolate was identified as Choleraesuis or Dublin. Enteritidis and Typhimurium isolates were also the most frequent causes of extraintestinal salmonellosis treated in the HCA, whereas no episodes attributable to Virchow and only one attributable to Hadar were recorded. These data are in line with those recorded in other Spanish hospitals (3, 18) but differ from those of studies in other countries, in which Choleraesuis (21), Dublin (14), and Virchow (26) were reported as the most frequent causes of bacteremia. A review of Salmonella bacteremia in England and Wales (23) reported that less than 2% of nontyphoid salmonellas isolated from humans were from blood culture and emphasized that while the greatest number of bloodstream isolates were Enteritidis and Typhimurium, the highest incidence of septicemias was attributable to Choleraesuis, Dublin, and Virchow. Secondly, the most frequent genomic group (Enteritidis GG I) causing extraintestinal infections was also the most frequent group associated with intestinal infections. It could be considered endemic in Spain because it has been circulating over the last decade, mainly associated with poultry and hen eggs as the infection vehicles (references 11 and 12 and unpublished data). Thirdly, 35% of the Salmonella isolates were resistant to one (ampicillin, cotrimoxazole, or chloramphenicol) or more (ampicillin and chloramphenicol; ampicillin, chloramphenicol, and cotrimoxazole; or ampicillin and cefonicid) antimicrobial drugs used in salmonellosis control; the rate of resistance to these was similar to the rate reported in other Spanish series (6, 19) but higher than that reported in the United States (13). Not all serotypes had similar frequencies of resistance, and the high percentage of multidrug-resistant Typhimurium isolates is in line with data from other works (5). Another interesting finding is the association of Enteritidis PT6 and PT6a with resistance to ampicillin (78% of the strains tested in our laboratories). These strains carried a 25-MDa plasmid and fell into different genomic groups, while no PT4 strain showed this resistance or carried 25-MDa plasmids.

Regarding the features of the patients with Salmonella bacteremia, the following observations could be noteworthy. (i) Bacteremia was recorded for all age groups but did not show the bimodal distribution, with a higher incidence at the two extremes, which is usually reported (3, 20), although the geriatric group showed the highest frequency (14 patients, most of whom had predisposing factors). The strains collected from geriatric patients belonged to seven genomic groups; one, GG I (Enteritidis), was implicated in eight episodes, and each of the other six groups was implicated in one episode. (ii) Six of the eight patients between 21 and 31 years of age had AIDS. Only salmonellas from four AIDS patients could be characterized, and these were assigned to three genomic groups: two Enteritidis groups (GG I and GG X) and one Typhimurium group (GG XIII). It has been reported that bacteremia is about 100 times more frequent in AIDS patients than in the general population (22). (iii) The most frequent bacterial portal of entry was the gastrointestinal tract (56% of the patients), and presumably most of the remaining patients (44%) could also have been infected by this route. It is also noteworthy that a high percentage of patients (72%) had predisposing factors that could have favored the development of blood infection; other authors report lower percentages, ranging from 27 to 62% (14, 18, 26).

With respect to focal infections, we found that those affecting the urinary tract were the most frequent, including five episodes caused by strains assigned to four genomic groups, three Enteritidis groups (GG I, II, and IX) and one Typhimurium group (GG XIV). The single pyelonephritis episode was caused by one Enteritidis GG I strain in an AIDS patient (a 27-year-old male) with previous and recurrent Salmonella bacteremia. In other reports (7), the most frequent Salmonella focal infections were osteoarticular, while in our series only one patient (a 63-year-old male with diabetes) had a lumbar spondylodiskitis caused by an Enteritidis GG II strain. The four patients with pulmonary infection had illnesses predisposing them to infection; two of these had malignancies. Symptoms and signs were typical of patients presenting with acute bacterial pneumonia, and two patients had Salmonella-positive blood cultures, which supported hematogenous dissemination; these data are in line with the review described in reference 4. Three episodes were caused by Enteritidis (GG I and GG IV), and the fourth was caused by a Typhimurium GG XV strain resistant to ampicillin, cotrimoxazole, chloramphenicol, and streptomycin.

Salmonella meningitis is frequently reported in children, while it is seldom described in adults and AIDS patients. The only episode in our series affected a 65-year-old female without predisposing illness and without a previous episode of gastroenteritis; it was caused by an Enteritidis GG XII strain. Finally, the only three episodes classified as intra-abdominal infections (one case of peritonitis, one of perirectal abscesses, and one of cholecystitis) were caused by three very different strains (Enteritidis GG I, Typhimurium GG XV, and Subspecies IIIb GG XXI, respectively). Infections directly related to the digestive tract had a pathologic significance different from that of the other focal infections by Salmonella and did not indicate severe immunosuppression.

In conclusion, this study revealed that extraintestinal salmonellosis more frequently affected patients with some predisposing factor and/or underlying disease than patients without these features (2:1 ratio). Furthermore, it was caused by genotypically and phenotypically heterogeneous Salmonella organisms, although many of these (43.1%) belong to a prevalent genomic group of Enteritidis. Organisms assigned to this group have also been found frequently in human feces, food samples, and water samples collected over the last decade in different Spanish regions and are considered prevalent and endemic in Spain (11, 12).