Isolate confirmation

Presumptive Shigella isolates were confirmed using conventional biochemical tests [14]. Serotyping was done for S. flexneri and phagetyping was done for S. sonnei. Serotyping was performed using commercially available antisera (Denka Seiken USA Inc., Campbell, CA) for S .flexneri and the following serotypes (STs) were determined: 1-4, 6, SH-101, SH-104, and variants × or y [14]. Phage typing was performed on S. sonnei isolates following standard procedures at the National Microbiology Laboratory in Winnipeg, Manitoba [15]. For 2002 and 2003, there were representative but fewer numbers of isolates were available for testing. For example, in 2002 and 2003, only 24% and 58% of representative isolates were available respectively. From 2004-2007, representative isolates for each case of infection were available for susceptibility testing: 2004 (100%), 2005 (100%), 2006 (89%), 2007 (91%).

Susceptibility testing

Susceptibility testing was performed using Sensititre panels (Trek Diagnostic Systems, Cleveland, OH) against the following antimicrobial agents:

  • amikacin (AMI)

  • amoxicillin/clavulanic acid (AMC)

  • ampicillin (AMP)

  • cefoxitin (FOX)

  • ceftiofur (TIO)

  • ceftriaxone (AXO)

  • chloramphenicol (CHL)

  • ciprofloxacin (CIP)

  • gentamicin (GEN)

  • kanamycin (KAN)

  • nalidixic acid (NAL)

  • streptomycin (STR)

  • tetracycline (TET)

  • sulfisoxazole (SSS)

  • trimethoprim/sulfamethoxazole (SXT)

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 distribution of species included: S. sonnei 54.7% (n = 316); S. flexneri 33.9% (n = 196); S. boydii 7.6% (n = 44); S. dysenteriae 3.8% (n = 22). Twenty nine S. flexneri and 79 S. sonnei were not archived (stored and cataloged); three S. flexneri could not be cultured; 15 S. sonnei belonged to four outbreaks and were removed as they had the same antibiogram as the index isolate for each outbreak (nine S. sonnei isolates in 2006 and six S. sonnei isolates in 2007). All but four S. flexneri and S. sonnei isolates were isolates from stool specimens; two S. sonnei isolates from blood, and two S. flexneri isolates were from blood and urine. Of the 386 S. flexneri and S. sonnei isolates, 74.9% (n = 289) were associated with international travel; 12.7% (n = 49) associated with domestic exposure within North America; 12.4% (n = 48) unknown travel history or origin of acquisition.

Rate calculations from Alberta population data were utilized to ensure no bias to study. The data set lacks a true denominator for all specimens received and tested. S. flexneri rates ranged from 0.70 to 1.21 per 100,000, and S. sonnei rates ranged from 1.10 to 1.98 per 100,000 per annum. The majority of travel cases for S. flexneri were from Central America (32.3% [53/164]), the Indian subcontinent (22.6% [37/164]) and North America (8.5% [14/164]). The majority of S. sonnei cases were from Central America (39.2% [87/222]), North America (15.8% [35/222]), and the Indian subcontinent (11.3% [25/222]).

Of the 196 S. flexneri isolates, as described above 164 were available for analysis, while 29 were not archived and 3 did not grow. The most common ST for S. flexneri was ST2 (37.8% [62/164]) with 40.3% (25/62) of the ST2 isolates originating from Central America. Of the S. flexneri isolates from the Indian subcontinent the two most common STs were ST2 (40.5% [15/37]) and ST6 (35.1% [13/37]). The most common phage type for S. sonnei was S1 (65.8% [146/222]) with (38.4% [56/146]of S1 isolates from Central America.

Only 1.2% (n = 2) S. flexneri and 8.1% (n = 18) S. sonnei isolates were pan-susceptible to all antibiotics tested. All S. flexneri isolates were susceptible to AMI, GEN, AMC, KAN, FOX, TIO, AXO. All the S. sonnei were resistant to AMP, CHL, NAL, STR, TET and SXT (Table 1).

Table 1 Travel history and frequency of antimicrobial resistance of Shigella isolates in Alberta, 2002-2007A
Full size table

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).

Table 2 Median MICs of antimicrobial agents in S. flexneri and S. sonnei per year.
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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, χ2 = 36.52, df = 5) and NAL resistance increased from 0% in 2002 to 30% in 2007 (p = 0.0168, χ2 = 13.82, df = 5).

Figure 1
figure 1

Frequency of antimicrobial resistance of study isolates from Alberta 2002-2007: 1 a) S. flexneri (n = 164); and 1b) S. sonnei (n = 222).

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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.