Genetic Mechanisms behind the Spread of Reduced Susceptibility to Azithromycin in Shigella Strains Isolated from Men Who Have Sex with Men in Québec, Canada

Azithromycin has been considered a promising alternative treatment for multidrug-resistant Shigella infections (1). However, Shigella spp. with reduced susceptibility to azithromycin (RSA) have emerged, and the mphA and ermB plasmid-mediated genes were reported as determinants of RSA in these bacteria (2). Shigellosis has been frequently reported as a sexually transmitted infection (STI) among men who have sex with men (MSM). Resistant outbreaks involving Shigella spp. with RSA are more recent phenomena and are continually reported in North America (3–5) and in different parts of the world (6, 7).

The acquisition of antibiotic resistance genes involves various gene transfer systems, such as conjugative plasmids, transposons, and integrons (8). Recently, a plasmid carrying azithromycin resistance genes in Shigella flexneri serotype 3a was demonstrated to be involved in the rapid intercontinental dissemination of RSA among MSM (2).

To better understand the mechanisms and dissemination pathways of RSA among Shigella strains, a study was conducted on 60 isolates with RSA and six sequenced plasmids from different Shigella species and serotypes.

A total of 254 Shigella isolates (one strain from each patient), representing 69.2% of Shigella isolates reported in the notifiable diseases system in Québec, were received at the Québec Public Health Laboratory. One hundred thirty-one and 123 isolates were isolated in 2013 and in 2014, respectively. The identification at the genus, species, and serotype levels was performed as previously described (3). Shigella sonnei (n = 150; 59.1%) was the most frequently identified species, followed by Shigella flexneri (n = 101; 39.8%), Shigella boydii (n = 2; 0.8%), and Shigella dysenteriae (n = 1; 0.4%). In the city of Montreal, S. flexneri (55.6%) was the predominant species observed, followed by S. sonnei (43.7%).

Susceptibility testing against azithromycin was performed using the Epsilometer test (Etest) according to the manufacturer’s recommendations (bioMérieux, Saint-Laurent, QC, Canada) and was interpreted using epidemiological cutoff values (ECV) (9). The azithromycin resistance genes mphA and ermB were detected by PCR as previously described (10).

Of the 254 Shigella isolates, 194 isolates (76.4%) were susceptible to azithromycin (MIC, 0.25 to 8 μg/ml), and 60 isolates (23.6%) showed RSA (MIC, 32 to ≥256 μg/ml). None of the isolates had an MIC of 16 μg/ml.

Among Shigella isolates susceptible to azithromycin, S. sonnei was the most predominant (73.2% [142/194]), followed by S. flexneri (25.2% [49/194]). The proportions of S. sonnei susceptible isolates were almost equal between female (51.4% [73/142]) and male (48.6% [69/142]) subjects aged 1.14 to 88 years and 1.28 to 67 years, respectively. However, S. flexneri was predominantly isolated from male subjects (71.4% [35/49]) aged 0.02 to 66 years. Shigellosis patients under 5 years old represented only 8.2% (16/194).

The rate of Shigella with RSA doubled from 2013 to 2014, at 20/131 (15.3%) and 40/123 (32.5%) isolates with RSA, respectively. Most isolates with RSA were S. flexneri (86.7% [52/60]) serotypes 3a, 2a, 3b, and provisional 104, followed by S. sonnei (13.3% [8/60]), and displayed MICs of 32, 64, 128, and ≥256 μg/ml. An MIC of ≥256 μg/ml was the most frequently recorded for both S. flexneri (90.4% [47/52]) and S. sonnei (62.5% [5/8]). In S. flexneri, this MIC was predominant for serotype 3a (85.1% [40/47]), followed by serotype 3b (8.5% [4/47]).

Shigella spp. with RSA were mostly isolated from men (98.3%) 21 to 69 years old. One case of a woman 27 years old (MIC of 32 μg/ml) was observed. Strains with RSA were mainly isolated from Montreal (75% [45/60]) and from nearby regions. In Québec, the sexual orientation of the shigellosis cases was determined by questioning the patients according to the routine public health surveillance. In Montreal, we confirmed at least 31/45 (68.8%) cases as MSM with recent sexual activity and one case of a man as non-MSM; the sexual orientation for the remaining 13 cases was unknown.

Shigella isolates with RSA harbored at least one of the two genes previously identified as RSA determinants, namely, mphA and ermB (Table 1). Both genes were detected in 52/60 isolates (86.7%) displaying an MIC ≥256 μg/ml, while mphA alone was detected in 7/60 isolates (11.7%) and ermB alone in 1/60 isolates (1.7%).

Six strains with variable RSA, species, and serotypes were selected for plasmid sequencing. They were also chosen based on the presence of one or both of the azithromycin resistance genes: mphA and ermB. They included one S. sonnei isolate (ID127706, MIC of 64 μg/ml) and five S. flexneri isolates from four different serotypes: provisional 104 (ID134382, MIC of ≥256 μg/ml), 3b (ID134353, MIC of ≥256 μg/ml), 2a (ID116124, MIC of 32 μg/ml), and two isolates of serotype 3a (ID135719, MIC of ≥256 μg/ml; and ID137694, MIC of 32 μg/ml). After extraction and transformation in Escherichia coli DH10B, the plasmids carrying the RSA genes were extracted from the transformants previously selected on LB medium supplemented with 32 μg/ml of azithromycin.

Plasmid sequencing libraries prepared with the Nextera XT DNA library preparation kit were sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) and assembled using FLASH (Fast Length Adjustment of SHort reads, v1.2.11) and SPAdes v3.9. The plasmids assembled in three or fewer contigs and found to have identical flanking ends were circularized, and the Staden program Gap v4.10 was used to organize the contigs. We used PROKKAversion 1.12.0, BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), ISfinder (https://www-is.biotoul.fr/), and INTEGRALL (http://integrall.bio.ua.pt/) for sequence annotation. The alignment plasmid sequences were performed using Gview server (https://server.gview.ca/).

Plasmids from S. sonnei (ID127706), S. flexneri serotype 3b (ID134353), and provisional serotype 104 (ID134382) were circularized, while plasmids from S. flexneri 3a (ID135719 and ID137694) and 2a (ID116124) were assembled as pseudoplasmids. Plasmids from strains ID134382, ID134353, ID135719, ID116124, ID127706, and ID137694 were designated pSf1, pSf2, pSf3, pSf4, pSf5, and pSs1, respectively. The sizes of these plasmids were between 67.7 and 94.3 kb, and a sequence analysis showed that the six plasmids were distinguishable. The fragments of 53,669 and 6,948 bp of pSf3 exhibited an identity of 99% with the conjugative plasmid pKSR100 conferring high-level resistance to azithromycin found in S. flexneri 3a strain SF7955, which was detected in different intercontinental disseminated sublineages of S. flexneri 3a (2).

Interestingly, RSA determinants were located on antimicrobial resistance mosaic regions that exhibited a unique genetic structure for each plasmid (Fig. 1). The ermB and mphA genes were all associated with IS26, an insertion sequence mostly reported in resistance plasmids of clinical isolates that contributes significantly in the dissemination of the antibiotic resistance (11). The IS26-mphA-mrx-mphR(A)-IS6100 unit, previously described in the p2246-CTXM plasmid of a clinical strain of S. boydii (12), was detected in four plasmids (pSf1, pSf2, pSf3, and pSf4) and in pSs1, which was missing IS6100. In the plasmid pSf5, missing the mphA gene, IS26 was located upstream of ermB and its leading peptide gene ermB(L). Insertion sequences IS26 and IS6100 were also detected in the vicinity of mphA in an S. sonnei strain isolated in France (13). In addition, bla_TEM-1 conferring resistance to ampicillin and class 1 integrons associated with genes conferring resistance to trimethoprim (dfrA12 and dfrA17), aminoglycosides (aadA2 and aadA5), and sulfonamides (sul1) were detected, except on pSf5 of ID137694, which harbored only ermB.

Our study revealed that S. sonnei was the most frequently isolated Shigella species in Québec from 2013 to 2014. However, S. flexneri was the most predominant in Montreal. Shigella with RSA was mostly associated with S. flexneri serotypes (mainly 3a), with an increasing rate from 2013 to 2014. Shigella flexneri, which is the predominant species in developing countries, is characterized by a long-term persistence in regions of endemicity. The emergence of this species and its association with RSA among the MSM community in industrialized countries is of concern (3, 4, 7). Most isolates with RSA in the present study exhibited MICs of ≥256 μg/ml, and the RSA was encoded by plasmid-mediated genes mphA and ermB.

We showed that in addition to plasmid mobilization (2), the acquisition and dissemination of RSA among Shigella species and serotypes also potentially occurred through IS26 mobilization. We emphasize that the genetic organization of the structures described in the selected isolates cannot be extrapolated to all the isolates in this study.

Shigella with RSA is of a great concern, especially since RSA in combination with ciprofloxacin resistance has already been reported in Québec (14). Of note, the lack of effective orally administered antibiotics reduces further the treatment options of shigellosis (15). A provincial surveillance program should be established to get the real picture of Shigella with RSA, and studies should be extended to further elucidate the mechanisms of spread of this resistance in order to limit and prevent its emergence, mainly within MSM communities.

We thank Aleisha Reimer for the revision of the article.

We declare no conflict of interest.