Diarrhea in an infant due to Shigella flexneri 1 carrying multiple cephalosporinase-encoding genes

Background Infections caused by multidrug-resistant shigellae resistant to broad-spectrum cephalosporins are becoming more prevalent in the Middle East. We report a case of severe diarrhea due to a multiresistant Shigella flexneri 1 strain carrying four different ß-lactamase genes. Case presentation A one-year-old Syrian infant presented with severe acute diarrhea, vomiting and dehydration. She did not respond to empirical treatment with amoxicillin-clavulanic acid followed by cefotaxime. Later, stool culture revealed S. flexneri 1 resistant to both these drugs. The patient was successfully treated with meropenem to which S. flexneri 1 was susceptible. The isolate was resistant to eight classes of antibiotics, and the whole genome sequence (WGS) identified four ß-lactamase genes (blaCTX-M-15, blaEC-8, blaOXA-1, and blaTEM-1) along with genes mediating resistance to seven other antibiotic classes. The WGS also identified several virulence genes including senA that encodes ShET-2 which induces watery diarrhea. Phylogenetically, the isolate was closely related to isolates from South Asia. Conclusions This report highlights the emergence of extremely resistant Shigella that has acquired multiple resistance genes to cephalosporins rendering these drugs ineffective. Supplementary Information The online version contains supplementary material available at 10.1186/s13099-021-00413-9.


Background
Antibiotics are the cornerstone therapy for treating shigellosis [1]. However, over the years, shigellae have developed resistance to numerous antimicrobials making treatment difficult [2][3][4]. Shigella isolates in many countries have acquired resistance traits leading to the inefficacy of cephalosporins for treating patients [5][6][7][8][9]. Resistance to cephalosporins is mostly mediated by genes located on plasmids and encoding the so-called AmpC or extended-spectrum ß-lactamases (ESBLs) [4,10,11]. There are reports of Shigella isolates carrying two or three ß-lactamases [8,[12][13][14]. We describe here a case of shigellosis in an infant in Kuwait due to a multidrug-resistant Shigella flexneri 1 that carried four different ß-lactamase genes along with many other antibiotic resistance genes, resulting in a highly drug-resistant strain. We also performed a phylogenetic analysis of the isolate to find out its relationship with the isolates from other parts of the world.

Case presentation
A one-year old female Syrian infant was admitted at the Al-Sabah hospital, Kuwait, in April 2019, with an acute onset of diarrhea and vomiting, both 8-10 times a day, and fever. There was no macroscopic evidence of blood in the stool. She did not have a history of travel. She was severely dehydrated with a reduced skin turgor and sunken eyes, lethargic and tachypneic, with a body temperature of 40 °C, and a blood pressure of 97/58 mm. She

Open Access
Gut Pathogens *Correspondence: manuel.albert@ku.edu.kw was rehydrated with intravenous fluid containing saline, dextrose and KCl, and treated with intravenous amoxicillin-clavulanic acid (500 mg every 12 h). Her stool, urine and blood samples were analyzed for bacterial culture and susceptibility. Even though her general condition significantly improved after 24 h, fever (39.1 °C) and diarrhea continued. Amoxicillin-clavulanic acid was stopped and intravenous cefotaxime (275 mg every 6 h) was started. Fever (39.7 °C) and diarrhea continued through to the fourth day, when the stool culture and susceptibility report was received (see below). Based on the report, cefotaxime was stopped and intravenous meropenem was started (200 mg every 8 h). After 48 h, the child became afebrile and the frequency of diarrhea was reduced. After the completion of a 7-day course of meropenem, the child was discharged with a complete recovery from diarrhea. Urine and blood cultures were sterile (see below).
Stool, urine and blood samples were cultured for bacterial pathogens as described previously [15]. S. flexneri 1 (further designated as SF1) was cultured from stool with no bacteria cultured from other samples. The organism was tested for susceptibility to several antibiotics (HiMedia) by disk diffusion method and interpreted according to CLSI criteria [16]. The isolate was resistant to streptomycin, ampicillin, piperacillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, aztreonam, tetracycline, trimethoprim, sulfamethoxazole, cotrimoxazole, ciprofloxacin, chloramphenicol, cephalothin, cephazolin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, and cefepime, but susceptible to gentamicin, amikacin, piperacillin/tazobactam, azithromycin, colistin, fosfomycin, ertapenem, meropenem, imipenem, cefoxitin, and tigecycline. These results were corroborated with Etests for all antibiotics except for cephalothin and glycylglycine for which Etests were not available. The isolate was, therefore, multi-resistant to eight classes of antibiotics.
Chromosomal DNA was sequenced. Sequencing libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina) and the sequence read data were produced on the Illumina NextSeq instrument (paired end, 150 base reads). The reads were assembled using Skesa 2.3.0 [18]. The draft assembly contained 418 contigs and 4,412,722 total bases. The sequence type (ST) of the isolate was 245 which confirmed it as S. flexneri [19,20]. Comparative core genome phylogenetic analysis was performed using Nullarbor (https:// github. com/ tseem ann/ nulla bor). Abricate (https:// github. com/ tseem ann/ abric ate) was used to detect virulence genes by scanning with VFDB [21] and antibiotic resistance genes by scanning with the Resfinder database [22] and the National Database of Antibiotic Resistant Organisms (NDARO) (https:// www. ncbi. nlm. nih. gov/ patho gens/ antim icrob ial-resis tance/). Read data produced for the isolate, SF1 has been submitted to NCBI under BioProject, PRJNA556301.
The details of phylogroup 1 Shigella isolates [23] used for core genome phylogenetic analysis are shown in Additional file 1: Table S1. The phylogenetic tree is shown in Fig. 2. The isolate SF1 clustered with the isolates that had been recovered from Pakistan and Bangladesh. The resistance genes found in the WGS were  aadA1, aph(3′')-1b, aph (6)-1d, catA1, dfrA1, qnrS1, sul2,  tet(A), tet(B), bla CTX-M-15, bla EC-8, bla OXA-1, and bla TEM-1 . No mutation in gyrA, gyrB, parC and parE known to confer resistance to quinolone [24] was identified. The resistance genes of the isolates are shown on the right of Fig. 2. The number of resistance genes among the isolates from developing countries appeared to be higher than that in developed countries. A number of virulence genes were identified in SF1, namely ipaA-ipaD, spa29, ipaH, sepA and senA.
Conjugation experiments for resistance transfer were done by both liquid mating [25] and filter mating [26] with sodium azide-resistant E. coli J53 as the recipient strain. Bacterial suspension from the filter and culture after liquid mating were streaked on three Mac-Conkey agar (Oxoid) plates, each one with one of the drug combinations, (sodium azide [200 mg/L] + cefotaxime [2 mg/L], sodium azide [200 mg/L] + ampicillin [50 mg/L], and sodium azide + cefotaxime + ampicillin) to get isolated colonies. No colonies grew on these three selective agars after liquid and filter mating showing a lack of transfer of resistance.

Discussion and conclusions
Although shigellosis is a typically dysenteric illness with blood, pus and mucus in the stool [27], children often present with diarrhea only [28]. Our patient had watery diarrhea. Shigella enterotoxin 1 (ShET-1) and ShET-2 alter water and electrolyte transport in the small intestine, and cause diarrhea and dehydration [29]. Our isolate possessed the senA gene that encodes ShET-2 which likely contributed to the severe watery diarrhea. We detected ipaH and spa29 genes that are involved in the invasion of the colonic epithelial cells [30]. The genome also contained the sepA gene, which encodes serine protease A autotransporter, which is responsible for barrier disruption [31].
The bla CTX-M-15 gene is present in the chromosome or plasmid in shigellae [45,46]. The bla EC-8 gene was reported in Escherichia coli with the apparent location of the gene in the chromosome [47]. bla OXA-1 has been found in plasmid and integron in shigellae [48,49]. The bla TEM-1 is present in plasmid or the chromosome [6,50]. We could not transfer any of the cephalosporinase genes to the recipient E. coli by mating, indicating that their locations might be in the chromosome, even though SF1 had plasmids.
Although the child was of Syrian origin, her family had been living in Kuwait long-term with no history of recent travel outside of Kuwait, indicating that the infection was acquired locally. Phylogenetic analysis showed that SF1 was closely related to the isolates from Pakistan and Bangladesh. An expatriate population Fig. 2 A tree showing the relationship between the core genomes of 77 phylogroup 1 S. flexneri isolates from Connor et al. [23] and isolate SF1 (in blue) (metadata can be found in Additional file 1: Table S1). The S. flexneri 2a str. 301 genome sequence (Chr: NC_004,337 and pCP301: NC_004851) was used as a reference for the core genome alignment and is represented in the tree as the unlabeled outgroup. The tree was inferred using IQtree version 1.6.12. Approximately, 80% of the S. flexneri 2a str. 301 genome sequence was used for the core genome and the greatest pairwise SNP distance was 2151 bases. Taxon labels include serotype, isolate name, country (abbreviated using ISO3166 three letter code; note EU = Europe, and UNK = Unknown) and year of isolation. The close relationship of the Kuwaiti isolate with those from Pakistan and Bangladesh is indicated in purple lines. A genotypic antimicrobial resistance profile for 13 resistance genes is shown at from these countries resides in Kuwait. It appears that the clone(s) circulating in South Asia is circulating in Kuwait. The study of Connor et al. [23] suggested local acquisition of antimicrobial resistance determinant(s) by isolates in each country, and no evidence of intercontinental spread of antimicrobial resistant strains. However, our study suggested the possible spread of a resistant clone from South Asia to the Middle East. The present day intercontinental migration of refugees portends a widespread transfer of resistant strains. The occurrence of multiple β-lactamase-encoding genes in Shigella is novel which will render cephalosporin antibiotics ineffective for treating the disease.
Additional file 1: Table S1. The 77 phylogroup 1 S. flexneri 1 isolates used for construction of phylogenetic tree in Fig. 2 are listed. These isolates were taken from Connor et al. [23].