For publication in: Journal of Clinical Microbiology (Short form paper)

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1 JCM Accepts, published online ahead of print on 17 October 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 For publication in: Journal of Clinical Microbiology (Short form paper) 2 3 Extreme drug resistant Salmonella enterica Senftenberg infections from patients in Zambia Rene S. Hendriksen 1 *, Katrine Grimstrup Joensen 1, Chileshe Lukwesa-Musyani 2, Annie Kalondaa 2, Pimlapas Leekitcharoenphon 1, Ruth Nakazwe 2, Frank M. Aarestrup 1, Henrik Hasman 1, James C.L Mwansa 2 1 WHO Collaborating Centre for Antimicrobial Resistance in Food borne Pathogens and European Union Reference Laboratory for Antimicrobial Resistance, National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark 2 University Teaching Hospital, Pathology and Microbiology Department, P/B RW 1X, Lusaka, Zambia *: Corresponding author: Rene S. Hendriksen, National Food Institute, Technical University of Denmark Kemitorvet, Building 204, DK-2800 Kgs. Lyngby, Denmark Phone: Fax: rshe@food.dtu.dk Running title: Resistant Salmonella Senftenberg in Zambia 1

2 Key words: Salmonella Senftenberg, whole genome sequencing, SNP analysis, antimicrobial resistance genes, plasmid replicon typing, MIC determination, severe clinical cases, salmonellosis, Zambia Abstract Two cases of extreme drug resistant Salmonella Senftenberg isolated from patients in Zambia were investigated utilizing MIC determination and whole genome sequencing. The isolates were resistant to, and harboured genes towards, nine drug classes including fluoroquinolones and extended spectrum cephalosporins, contained two plasmid replicons, and differed by 93 SNPs. Text Infections with Salmonella that are resistant to multiple antimicrobials are associated with increased morbidity and mortality (6,19) and the global emergence is leaving clinicians with few, or no, treatment options (8). Recently, several studies have indicated the emergence and spread of multidrug resistant Salmonella clones in Africa (4,10,13,20). Often those clones have a different epidemiology than what is observed in developed countries, complicating control and prevention strategies (15). It is paramount to identify new multidrug resistant clones as early as possible to hamper further dissemination (6). Here, we describe two clinical cases of human salmonellosis in Zambia caused by extreme drug resistant (EDR) Salmonella enterica serovar Senftenberg. The genomes of the isolates were sequenced to determine the multilocus sequence type (MLST), to investigate the occurrence and genetic mechanisms of antimicrobial resistance, plasmid replicons, and genetic relatedness by single-nucleotide polymorphism (SNP). 2

3 On January 18 th 2012, a 34-year old male from Mazabuka (72 km South of the capital; Lusaka, Southern province of Zambia) was admitted to the Mazabuka District Hospital. Based on medical examination, the patient was diagnosed with gastroenteritis, and treated with ciprofloxacin and co-trimoxazole. Two days later, the patient was discharged continuing treatment on cephalexin and co-trimoxazole, but was readmitted with epistaxis and occipital pulsatile headache, and treated with adrenaline and vitamin K. The patient was discharged six days later to be reviewed. On February 6 th the patient was referred to the renal unit of the University Teaching Hospital (UTH) in Lusaka (the capital), being pale, dehydrated, afebrile, tachycardic, with a scaphoid abdomen, and later also developed ureamic encephalopathy. This time, the patient was diagnosed with sepsis and chronic renal failure. Among other tests that all were negative, the renal ultrasound was normal but the stool culture yielded Salmonella (#588). Three days later, the patient was unable to eat and was fed through a nasogastric tube and intravenous fluids. The patient was transfused four days after admission but no dialysis were initiated. The patient died the morning of February 11 th A second patient; a 30-year old male from the Chibolya compound (2 km West of Lusaka and 74 km away from case #1) and had spent most of his time in the compound. The patient was admitted to the UTH diagnosed with gastroenteritis with TB after having been referred from a local clinic on March 9 th The patient was three months prior to admission treated with antimicrobials due to sexually transmitted infections. Prior to admission on March 9 th 2012, the patient complained of headache, chills, fever, diarrhoea, and general weakness. On March the 13 th, a stool sample were collected and yielded Salmonella (#1028). The patient reported to have consumed vegetables bought from the local market. Based on chest x-ray, the patient was diagnosed with extrapulmonary TB and treated with rifampicin, isoniazid, pyrazinamide, 3

4 and ethambutol. On the 16 th of March, the patient was also diagnosed with HIV and received emtricitabine, tenofovir, efavirenz, and co-trimoxazole. The Salmonella isolates were shipped to the Technical University of Denmark (DTU) for further characterization. The isolates were serotyped followed by MIC determination as previously described including tigecycline (7). Both isolates belonged to Salmonella enterica serovar Senftenberg and had an almost identical antimicrobial susceptibility pattern conferring resistance to amoxicillin + clavulanic acid (MIC =16), ampicillin (MIC 32), cefepime (MIC 16), cefotaxime (MIC 64), cefpodoxime (MIC 32), ceftazidime (MIC = 128), ceftiofur (MIC 8), ceftriaxzone (MIC 128), chloramphenicol (MIC 64), ciprofloxacin (MIC 4), gentamicin (MIC 16), nalidixan (MIC 64), neomycin (MIC 32), spectinomycin (MIC 256), streptomycin (MIC 128), sulfamethoxazole (MIC 1024), tetracycline (MIC 32), and, trimethoprim (MIC 32). In addition, isolate #588 was also resistant to florfenicol (MIC 64). The isolates were susceptible to apramycin, cefoxitin, colistin, imipenem, meropenem, and tigecycline. However, one could question if those antimicrobials would be used for treatment since florfenicol and apramycin are only approved for animal usage, colistin is difficult to administer and has renal toxicity (19), cefoxitin group with 2 nd generation cephalosporins why it most likely will not have any effect due to the isolates already being resistant to 3 rd generation cephalosporin (3) and, carbapenems are too expensive considering that patients cover hospital expenses themselves. Treatment with tigecycline may be effective towards nontyphoid Salmonella but clinical trials needs to be further investigated to explore the full potential of using it for human treatment caused by multidrug resistant nontyphoid Salmonella. The isolates were sequenced on the Ion Torrent PGM TM (Life Technologies) following the manufacture s protocols for 200bp gdna fragment library preparation (Ion Xpress Plus 4

5 gdna and Amplicon Library Preparation), template preparation (Ion OneTouch TM System), and sequencing (Ion PGM TM 200 Sequencing kit). The sequence data were assembled and analyzed using the pipeline available on the Center for Genomic Epidemiology ( (12,22). The isolates belonged to MLST type ST14. The Resfinder tool detected the following resistance genes present either in both or in one of the isolates, as well as two mutations in gyra, and one mutation in parc responsible for high-level fluoroquinolone resistance (Table 1). Both isolates contained an inch12 plasmid replicon and isolate #588 contained an inca/c plasmid replicon. The genetic relatedness of the two isolates was examined identifying 93 high-quality SNPs (The informative SNPs were determined based on a minimum coverage of 20, base calling quality of 30 and a minimum distance of 10 bp between each SNP) between the two isolates, using S. Senftenberg SS209 reference genome (Bio project no:prjea82547) (5) and 530 and 521 SNPs between isolates; #1028 and #588 and the reference genome. There is currently insufficient data on the nucleotide diversity between clonally related and unrelated Salmonella isolates to determine whether this is indicative of separate or clonally related strains. WGS studies on Salmonella have indicated an accumulation rate of 1-2 SNP s per year (15). Thus, the 93 SNP differences observed here in combination with the differences in resistance profile and - genes, may suggest that the isolates have an unrelated origin. S. Senftenberg has also previously been the cause of serious human infections (9,11,16). S. Senftenberg is well recognized as being common among poultry (2) but has also been associated with infant formula (18), mussels (14) and vegetables (1,16,17). It is noteworthy to mention that one of the patients claimed to have consumed vegetables prior to onset. S. 5

6 Senftenberg has the ability to adhere to plant leaves perhaps contributing to the infections in these cases (1). A similar case of one resistant S. Senftenberg was recently reported from a traveller returning from Egypt, indicating the importance of this resistant serovar in Africa (21) In conclusion, we report cases from Zambia of EDR S. Senftenberg isolates causing severe human infections and leaving very few treatment options. We speculate if these clones are emerging and suggest for public health authorities to become alert of any further dissemination. Acknowledgement This work was funded by the Center for Genomic Epidemiology ( and by the World Health Organization Global Foodborne Infections Network ( Thanks to Ministry of Health, Zambia, for permission to send the isolates. Reference List 1. Berger, C. N., R. K. Shaw, D. J. Brown, H. Mather, S. Clare, G. Dougan, M. J. Pallen, and G. Frankel Interaction of Salmonella enterica with basil and other salad leaves. ISME.J. 3(2): Boumart, Z., S. M. Roche, F. Lalande, I. Virlogeux-Payant, C. Hennequet-Antier, P. Menanteau, I. Gabriel, F. X. Weill, P. Velge, and M. Chemaly Heterogeneity of persistence of Salmonella enterica serotype Senftenberg strains could explain the emergence of this serotype in poultry flocks. PLoS.One. 7(4):e Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. M100-S22. 22nd Informational Supplement Wayne, PA, USA. Ref Type: Generic 6

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9 Table 1. Distribution of antimicrobial resistance genes in Salmonella Senftenberg isolates from Zambia. Resistance gene classes, resistance Isolate ID genes, and resistance mutations Aminoglycoside aac(6 )-IIc aac(6 )-IIc aac(6 )-Iy aac(6 )-Iy aph(3')-ic aac(6 )-Ib-cr stra strb aada2 aac(6 )-Ib-cr stra strb Beta-lactam bla TEM-1 bla TEM-1 bla CTX-M-15 bla OXA-10 9 bla CTX-M-15 bla OXA-30 Low-level fluoroquinolone aac(6 )-Ib-cr aac(6 )-Ib-cr High-level fluoroquinolone gyra (S-83-Y and D-87-G) gyra (S-83-Y and D-87-G) parc (S-80-I). parc (S-80-I). Macrolide/lincosamide/streptogramin erea erea Phenicol catb3 cata2 cmla1 catb3 cata2 flor Rifampicin ARR-2 Sulphonamide sul1 sul1 sul2 sul2 Tetracycline teta tetd tetd Trimethoprim dfra14 dfra18 dfra23 dfra18