Genotypic and demographic characterization of invasive isolates of Salmonella Typhimurium in HIV co-infected patients in South Africa

Background: Non-typhoidal Salmonella are frequently food-borne zoonotic pathogens that may cause invasive disease in HIV-positive individuals. Methodology: Invasive isolates (n = 652) of Salmonella Typhimurium from human patients in Gauteng Province of South Africa were investigated for the years 2006 and 2007. Bacteria were identified using standard microbiological techniques and serotyping was performed using commercially available antisera. Susceptibility testing to antimicrobial agents was determined by the E-test. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis analysis of digested genomic DNA. Results: Forty-five clusters of isolates were identified, of which four (clusters 3, 5, 6 and 11) were major clusters. Most isolates originated from hospital H2 and most were isolated from patients in the age range of 15 to 64 years. Ninety-three percent (213/230) of patients with a known HIV status were HIV-positive. Most isolates showed resistance to multiple antibiotics. The most commonly expressed antibiotic resistance profiles were ACSSuNa (14%; 75/555) and ACSSuTNa (13%; 72/555). Some evidence was found for nosocomial acquisition of isolates. Of the isolates from the major clusters 3, 5, 6, and 11, 33% (8/24), 6% (7/117), 4% (1/26) and 6% (3/52) were of nosocomial origin, respectively. Conclusions: In South Africa, Salmonella Typhimurium remains a major opportunistic infection of predominantly HIV-positive patients. Clonally diverse strains that are resistant to multiple antibiotics may circulate in patients aged between 15 and 64 years over prolonged periods within the hospital environment, adding to the health care burden.


Introduction
Salmonellosis is primarily caused by the consumption of contaminated food and water, but Salmonella may also be transmitted via other routes including faecal-oral transmission (human-to-human) and direct contact with animals infected with Salmonella [1].In immunocompromised patients [2], once Salmonella strains have entered the small bowel, the organisms break through the bowel mucosa and enter the Peyer's patches and associated lymph nodes, where they multiply and spread [1].Immunocompromised persons are thus susceptible to life-threatening bacteraemia [3,4].AIDS patients in sub-Saharan Africa infected with non-typhoidal Salmonella bacteraemia have a reported mortality rate of 35% to 60% [5].Of the HIV-positive patients who survive, 25% to 45% suffer from recurrent nontyphoidal Salmonella bacteraemia about one to six months after the first non-typhoidal infection [5].
The burden of HIV disease in South Africa is extremely high [G.Pembrey, http://www.avert.org/aidssouthafrica.htm].Very little epidemiological data exists for human Salmonella isolates recovered in South Africa and the association with HIV.In South Africa in 1998 through to 1999, multidrug resistant Salmonella Typhimurium phage type DT104 strains were isolated from HIV-positive patients at the Chris Hani Baragwanath Hospital in Gauteng Province [Crewe-Brown et al., ICAAC conference proceedings, September 2000, Toronto, Canada].In 2004, a study by Kruger et al. on nontyphoidal Salmonella isolates collected from December 2002 to March 2003 showed increased resistance to extended-spectrum cephalosporins [6].In 2006, multidrug resistant Salmonella Isangi were isolated from patients who were admitted to a tertiary hospital in Durban, South Africa [7].
A contributing factor of human-to-human transmission of salmonellosis is nosocomial infection: secondary infection acquired while under medical care, 48 hours and more after the patient has been admitted to a long-term care facility or hospital [8].There has been an alarming increase in nosocomial outbreaks reported in the last 10 years [8].Nosocomial infections have been reported in Russia and Belarus in the 1990s through to 2003 [8], in the United States from 1996 to 1998 [9], in Italy from 1998 to 2000 [10], in Spain from 1999 to 2000 [11] and in Romania in 2002 [12].
The aims of this study were to clarify the molecular epidemiology of invasive Salmonella Typhimurium isolates in Gauteng Province, South Africa, for the years 2006 and 2007 in association with HIV, to enhance our understanding of the nosocomial nature of this organism, and to identify epidemiological clusters that may assist in the interventions to stop further spread of disease.

Case definition and selection of isolates
Invasive nosocomial salmonellosis was defined as a positive culture from a patient for Salmonella two or more days after admission from a normally sterile body site.Salmonella Typhimurium isolates from normally sterile body sites in human patients were collected from clinical laboratories in Gauteng.
Isolates were stored at -75°C in tryptic soy broth with 10% (vol/vol) glycerol (Diagnostic Media Products, Sandringham, South Africa).Surveillance officers appointed to four sentinel surveillance sites in Gauteng, South Africa, completed basic patient information by interviewing patients or reviewing patient records.Information was recorded in the surveillance database using the EpiInfo (version 6.04d) software (CDC, Atlanta, USA).

Bacterial identification and serotyping
Phenotypic and genotypic characterization of Salmonella Typhimurium isolates referred by laboratories in Gauteng was performed by the Enteric Diseases Reference Unit (EDRU) of the National Institute for Communicable Diseases (NICD) in Johannesburg, South Africa.Bacterial isolates were identified using standard microbiological techniques.Specific antisera (Statens Serum Institut, Copenhagen, Denmark; Remel Europe Ltd, Dartford, Kent, UK; and BioMérieux, Marcy-I'Étoile, France) were used to serotype Salmonella Typhimurium isolates, according to the Kauffman-White scheme.

Pulsed-field gel electrophoresis (PFGE) analysis of isolates
PFGE analysis was performed on isolates as previously described [13].Genomic DNA were digested with restriction enzyme XbaI (Roche Diagnostics GmbH, Mannheim, Germany) and thereafter separated on a 1% agarose gel (SeaKem Gold agarose, Lonza, Rockland, ME, USA).Electrophoresis was performed using the CHEF-DR electrophoresis systems (Bio-Rad Laboratories Inc., USA).The following run parameters were used: A voltage of 6 volts, at a run temperature of 14°C, a run time of 21 hours, an initial switch time of 2.2 seconds and a final switch time of 63.8 seconds.These patterns were then visualized by UV illumination after staining the agarose gels with ethidium bromide.Fingerprint patterns were analysed using BioNumerics (version 5.1) software (Applied Maths, Sint-Martens-Latem, Belgium).Patterns were normalized against the reference pattern for S. enterica serovar Braenderup (strain H9812).Dendrograms were produced by using the unweighted pair group method with arithmetic means.Analysis of the band patterns was performed with the dice-coefficient at an optimization setting and position tolerance setting of 0.5% and 1.5%, respectively.For the purpose of this study, three or more isolates with a similarity value of ≥ 90% was defined as a PFGE cluster.The clusters were numbered 1, 2, 3, etc. for referral purposes.

Statistical analysis
In addition to descriptive analysis, univariate logistic regression was performed to determine which individual explanatory variables were significantly associated with the outcome variables -HIV status and nosocomial infectionsby calculation of unadjusted odds ratios and 95% confidence intervals.

Discussion
Salmonella infections are common in a hospital environment due to overcrowding of patients and insufficient medical staff [14].In addition, Salmonella infections in hospitals may be associated with the use of contaminated medical equipment, as well as with the consumption of contaminated meals served at hospitals resulting from poor hand hygiene practices and inadequate cooking of meals by kitchen staff [14].In the absence of timely investigation, these factors may have accounted for possible routes by which the majority of clusters of Salmonella Typhimurium could have circulated in Gauteng hospitals.
The predominance of invasive salmonellosis in young patients (31%) is unsurprising, as young patients have an immature immune system and are more prone to Salmonella infections [2,15].Similarly, the number of infections and nosocomial infections in the 15 to 64 years age range correlates well with published HIV-positive incidence rates in South Africa [16].That the same four PFGE clusters (clusters 3, 5, 6 and 11) were represented in both of these age groups and at three hospitals allows us to speculate that nosocomial exposure, either through transfer of staff or of patients between hospitals and possibly in association with increased virulence of these Salmonella strains, permitted these strains to dominate.We have no data to confirm whether such transfers occurred, and cannot account for the ubiquitous presence of these clusters.Older patients are usually more predisposed to acquiring salmonellosis as a secondary infection as a result of their weakened immunity [1].Conversely, the results in this study suggest salmonellosis in elderly patients in Gauteng for 2006 and 2007 occurred less frequently.These results could possibly be accounted for by the underreporting of Salmonella cases in older patients caused by numerous undefined reasons [1] or to the overwhelming salmonellosis identified in patients of other age groups (less than 65 years) due to the high burden of HIV infection in these age groups [2,5].
HIV is a life-threatening epidemic in South Africa and accounts for up to 1,000 deaths of AIDS patients daily.Statistical  positive patients are 20 times more likely to acquire non-typhoidal Salmonella infection compared with immunocompetent patients [1,17].Patient demographic information, such as HIV status and age, was not available for all patients; the lack of this information may have skewed results and may have been a limitation of this study.Despite the unknown HIV status for 59% (325/555) of the patients in the current study, the results still suggest that Salmonella Typhimurium may be responsible for extensive comorbidity suffered by HIV-positive patients living in Gauteng Province.
Multidrug resistance was common in the nosocomial clusters.We showed that ESBL production was in fact a risk factor for invasive nosocomial salmonellosis.In clusters 3, 5, and 6, the majority of the isolates were multidrug resistant isolates ( > 75%); antimicrobial management of such patients with invasive Salmonella Typhimurium may be compromised, resulting in longer morbidity periods and possibly higher death rates among patients.Previous studies have shown that the pentaresistant ACSSuT pattern that we observed is frequently reported in Salmonella Typhimurium strains isolated in the United Kingdom (UK), France and North America [18,19].An increase in resistance to the quinolone class of antimicrobials (nalidixic acid), such as we noted, in addition to the pentaresistant pattern, has also been documented in an international survey performed on representative isolates of Salmonella Typhimurium for the years 1992 to 2001 [19].Cluster 11 included 36 isolates showing susceptibility to all six antibiotics and included 23 isolates showing resistance to sulfamethoxazole only.For this cluster of isolates, the treatment given to patients may be uncomplicated and morbidity may be less severe.
Compared with isolates in the other clusters, isolates in cluster 3 were the most likely to be nosocomial.Past studies have shown that nosocomial infections occurred most commonly when there was an over-population of patients and fewer health care workers.There tends to be a reduction in infection control practiced by health care professionals to comply with the demand of seeing and treating an increased number of patients [14], which could possibly account for the high number of nosocomial isolates in hospital H2, a large academic hospital which accommodates not only patients living in the surrounding areas in Gauteng Province, but also serves as a referral hospital for a large part of South Africa and neighbouring African countries.Hospital infections could also have been acquired through direct contact (person-to-person) or through contact with common contaminated surfaces in the hospital environment [1].
The predominance of HIV-positive patients in whom nosocomial infection was identified supports the observation of previous studies that immunocompromised patients, such as HIV-positive patients, are more susceptible to nosocomial infections [1].The extensive time period over which these isolates were sourced suggests that these nosocomial clusters were circulating in these three hospitals (H1, H2 and H49) for many months.
In conclusion, invasive Salmonella Typhimurium isolates in Gauteng demonstrated an extensive genetic diversity as shown by PFGE analysis, which segregated 555 isolates into 45 clusters.Most isolates showed resistance to multiple antibiotics, making these patients challenging to treat.Most isolates were from patients aged between 15 and 64 years, and patients were mostly HIV-positive.The occurrence of certain clusters over a prolonged period of time is cause for serious concern: it suggests that appropriate infection control measures have been inadequate in those hospitals for extended periods or are only intermittently followed, with resultant stresses both on health care systems as well patient morbidity.This hypothesis is supported by the extensive number of PFGE clusters, which suggests that these organisms are being repeatedly introduced into the hospital environment.In South Africa, invasive Salmonella Typhimurium remains an important opportunistic infection particularly associated with HIV-positive patients and is associated with nosocomial transmission.

Figure 1a .
Figure 1a.Number of patients in Gauteng hospitals, represented as a percentage, falling within each PFGE cluster.

Figure 1b .
Figure 1b.Age ranges of patients in Gauteng hospitals, represented as a percentage, falling within each PFGE cluster.

Figure 1c .
Figure 1c.HIV status of patients in Gauteng hospitals, represented as a percentage, falling within each PFGE cluster.
This study was funded by grants from the Medical Research Council of South Africa and in part by cooperative agreements from the HHS Centers for Disease Control and Prevention (CDC), the National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP), and the Global AIDS Program (GAP) Cooperative Agreement U62/PSO022901.Its contents are solely the responsibility of the authors and do not necessarily represent the official views of CDC.

Table 1 .
reports have shown that in South Africa at the end of 2007, approximately 5.7 million people were living with HIV [G.Pembrey, website publication, http://www.avert.org/aidssouthafrica.htm].Attributes of nosocomial isolates of Salmonella Typhimurium.Important findings are emphasised in bold face.A, Ampicillin; C, Chloramphenicol; S, Streptomycin; Su, Sulfamethoxazole, T, Tetracycline; Na, Nalidixic acid; susceptible, susceptible to all six of the former mentioned antibiotics. b