The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam

Abstract

Background: Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs) has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.

Methodology: We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M) encoding plasmid.

Principal findings: We show that two different bla(CTX-M) genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M) gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356) carried the bla(CTX-M-24) gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.

Significance: The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

Figure 1. Graph depicting an increase in number and proportion of ceftriaxone resistant Shigella spp. isolated between April 2007 and March 2009 at the hospital for tropical diseases in Ho Chi Minh City.

The thick black line with circles represents the number of ceftriaxone resistant Shigella isolates per month (x axis); the thin black line with triangles represents the total number of Shigella isolates per month (both related to the left y axis). The broken line represents the proportion of strains isolated in six month periods resistant to ceftriaxone (right y axis). The increasing proportion of ceftriaxone resistant organisms over six month periods is statistically significant (p = 0.024) as calculated using the chi-squared test.

Figure 2. Demonstration of the absence and presence of genes from DNA isolated from ceftriaxone resistant and ceftriaxone sensitive S. sonnei isolates using the ASParray.

Red boxes indicate presence of genes; green boxes indicate absence of genes. BLAST indicates reporter DNA identity (%) to the S. sonnei Ss046 genome. DNA was hybridized from isolates (left to right) DE0115, DE0477, DE0685, DE0891, DE1150, DE1198, DE1256, DE0611, EG0204, EG0373, EG0395, EG0430, EG1007, EG1008 and EG1009.

Figure 3. A schematic representation of the bla _CTX-M-24 encoding plasmid, pEG356.

pEG356 is a 70,275bp IncFI plasmid containing 104 coding sequences. The various features are highlighted by the various concentric circles according to the annotation of the of the plasmid (ac. FN594520). The outer colored circle represents coding sequences on the forward strand, the second circle represents coding sequences on the reverse strand. The coding sequences are coded by colour, red; plasmid replication, orange; conserved hypothetical, brown; pseudogene, dark blue; adaptation, grey; segregation, light blue; conjugation/transfer, light pink; transposition, dark pink; degradation/resistance and yellow; metabolism. The third concentric circle represents the location of pseudogenes and the fourth circle represents the four main modules of predicted function, red; replication, pink; transposition, dark blue; iron transport and light blue; conjugational transfer. The fifth and final coloured circle represents the location of the repeat sequences. The primary central graph (a) represents GC content, ranging from high (black) to low (grey) (mean 52%) and the secondary central graph (b) represents G/C coding bias ranging from high (black) to low (grey). The ISEcp1 type element carrying the bla _CTX-M-24 is distinguished by grey shading.

Figure 4. A schematic representation of ISEcp1-pEG356 and DNA sequence alignment highlighting corresponding DNA homology between pEG356 and pAPEC-O1-ColBM.

The DNA sequences for pEG356 and pAPEC-O1-ColBM were aligned and compared in Artemis comparison tool (ACT). The numbers on the diagram correspond to the respective plasmid sizes and the black integers highlight 10 Kbp intervals. The genetic backbone of the pEG356 and pAPEC-O1-ColBM is shown in yellow along with the various modules, red; rep (replication), pink; tn (transposition), dark blue; iro (iron uptake) and light blue; tra (DNA transfer/conjugation). Areas with high DNA homology between pEG356 and pAPEC-O1-ColBM are shown with grey shading and the pink shading corresponds to a magnified view of ISEcp1-pEG356. The numbers on ISEcp1-pEG356 correspond to the location of the element on the host plasmid, with integers representing 1 Kb intervals. The genes are functionally coded, pink; tn (transposition), orange; unk (unknown function), brown; psu (pseudogene) and dark pink; bla _CTX-M-24, primer locations for the transposon PCR are highlight by Tnp24F and TnpR. The location of the IS908D downstream of the bla _CTX-M-24 is highlighted The region is flanked by an inverted repeat (blue triangles) and contains an additional inverted repeat sequence flanking the transposase gene. Corresponding inverted repeats are linked by arrows.