Laboratory based surveillance of travel-related Shigella sonnei and Shigella flexneri in Alberta from 2002 to 2007

Between 2002 and 2007, travel related cases of Shigella sonnei and S. flexneri in Alberta, Canada were acquired from Central America, the Indian subcontinent and North America. Of this group, resistance to ciprofloxacin and nalidixic acid was identified in isolates from patients who had travelled to the Indian subcontinent. This study provides a Canadian perspective to a growing body of literature linking ciprofloxacin and nalidixic acid resistance to travel to the Indian subcontinent. Shigella is a common cause of diarrheal illness in North America with a rate of 2.0 per 100,000 in Canada [1] and a rate of 3.2 per 100,000 in the United States [2,3]. Imported cases of Shigella infections have been reported in developed countries following travel to a foreign or developing country [4,5] and may be impacted by factors including socio-economic factors [6], food distribution networks [5] and microbiologic factors [7]. Across multiple geographic regions, high rates of antimicrobial resistance to multiple agents (e.g. sulfonamides, tetracycline, chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) have limited the choices for empiric antimicrobial therapy required to manage Shigella infections and reduce fecal excretion of the bacteria [8-10] with descriptions of shifting species dominance and changes in antimicrobial susceptibility [10,11]. Generally, Shigella flexneri and Shigella sonnei are the dominant species and are heavily impacted by changes in antimicrobial susceptibility [12,13]. This study identifies the global regions associated with travel-related cases of S. flexneri and S. sonnei in Alberta, Canada and compares antibiotic resistance patterns of these isolates for 2002 to 2007 inclusive. Specimens collected 2002-2007 (inclusive) from S. flexneri and S. sonnei infections in Alberta, Canada were included for study. Data collected at time of specimen submission included: date of specimen collection, outbreak association if present, travel history and antibiogram (data source-ProvLab Information Systems; Communicable Disease Report at Alberta Health and Wellness). Outbreaks were defined by public health officials as ≥ 2 epidemiologically related cases. Each outbreak was assigned a unique incident number. Repeat isolates received within six months of original case infections were excluded. Only one representative case for each outbreak was included, unless the isolates had different antibiotic susceptibility patterns. Based on travel history the origin of an isolate was grouped into corresponding regions and continents. Regions included in the study represented major travel destinations for individuals living in Canada. Domestic exposures were defined as "travel within North America."


Susceptibility testing
Susceptibility testing was performed using Sensititre panels (Trek Diagnostic Systems, Cleveland, OH) against the following antimicrobial agents: The minimum inhibitory concentrations (MIC) and breakpoints were determined in accordance with guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [16,17].

Data analysis
GraphPad Prism 5 software (GraphPad Software, Inc. La Jolla, CA) was used for statistical analysis.
Between 2002-2007, 578 Shigella isolates were received and confirmed by ProvLab. The overall  Amoxicillin/ clavulanic acid   resistant to AMP, CHL, NAL, STR, TET and SXT (Table 1). When median MICs were analyzed for all agents the following changes were identified as in Table 2. For S. flexneri median MICs were within two dilutions for most agents over the study period. Exceptions were for the following agents; AMP (increase), CHL (increase), SXT (increase and following drop), and SSS (decrease). For S. sonnei, median MICs were within two dilutions for most agents over the study period with the following exceptions; exception of AMP (decrease).
When data was combined for all years, the NAL and CIP resistance was 20.1% (33/164) and 14.9% (33/222) for S. flexneri and S. sonnei respectively. CIP resistance was identified only in S. flexneri isolates (4.9%, 8/164) when averaged over the six-year study period (Fisher's exact test, p = 0.001) (Figure 1a and 1b) CIP resistance in S. flexneri was not steady but instead was most evident in the years 2005, 2006, and 2007 (Figure 1a). Combined CIP and NAL resistance was related to travel to the Indian subcontinent for S. flexneri (84.8%, 28/37) and S. sonnei (80.0%, 20/25) (Fisher's exact test, p < 0.0001). The proportion of antibiotic resistance was constant over six years except for S. sonnei, where AMP resistance decreased from 83% in 2002 to 11% in 2007 (p < 0.0001, χ 2 = 36.52, df = 5) and NAL resistance increased from 0% in 2002 to 30% in 2007 (p = 0.0168, χ 2 = 13.82, df = 5). At the study onset, treatment guidelines suggested a fluoroquinolone for acute traveler's diarrhea regardless of travel location. It is possible that some CIP resistance was underestimated in 2002-2003 due to the smaller number of isolates tested. By 2009, treatment guidelines for acute traveler's diarrhea (outside of Latin America and Africa) suggested azithromycin or a fluoroquinolone [18,19]. Data also suggests that azithromycin resistance may be emerging and resistance rates of 16% have been recently described in Bangladesh [20]. These studies indicate that travel to the Indian subcontinent, in patients returning to Western Canada with traveler's diarrhea should be determined to guide initial empiric treatment options; especially for severe infections because the association of S. flexneri and S. sonnei isolates from this region with fluoroquinolone and potential macrolide resistance [13,21]. Although CIP resistance was described only in S. flexneri, we should remain vigilant for developing gyrA and parC mutations as well as the presence of plasmid mediated quinolone resistance determinants (PMQR) genes that may lead to increasing rates of CIP resistance in travel-related Shigella isolates which are beginning to emerge globally [4,22].
There are multiple factors that may have lead to CIP and NAL resistance in Shigella species originating from the Indian subcontinent [21]. It is possible that part of this emerging resistance may be associated with the increasing dominance of specific STs or clones of Shigella. Both this study and other work have identified a dominance of S. flexneri STs 2 and 6 in isolates of Indian origin and cases of traveler's diarrhea associated with the Indian subcontinent [23]. One factor driving multi-drug resistance in the Indian subcontinent may be the emergence of specific clones within these dominant STs [24]. Therefore, the identification of clonal groups within Alberta strains may be a powerful tool for tracking the development of drug-resistance in Shigella isolates from future cases of traveler's diarrhea.