The First Saudi Study Investigating the Plasmid-borne Aminoglycoside and Sulfonamide Resistance among Acinetobacter baumannii Clinical Isolates Genotyped by RAPD-PCR: the Declaration of a Novel Allelic Variant Called aac(6')-SL and Three Novel Mutations in the sul1 Gene in the Acinetobacter Plasmid (s)

Abstract

Background: Acinetobacter baumannii (A. baumannii) is one of the most important nosocomial pathogens responsible for a wide range of infections.

Aim: This study aimed to investigate the existence of the plasmidic genes encoding for aminoglycoside modifying enzymes (AMEs), 16S rRNA methyltransferases (RMT), and the altered dihydropetroate synthase (DHPS) encoded by the sul1 gene among A. baumannii clinical isolates collected from Taif, Kingdom of Saudi Arabia (KSA). The mutations in aac(6')-Ib and sul1 genes were also investigated.

Methods: Forty A. baumannii clinical isolates were investigated for their susceptibility to ten antibiotics. The plasmid DNA was extracted and screened for nine genes encoding for aminoglycoside resistance in addition to the sul1 gene. The clonal relatedness was determined by random amplified polymorphic DNA (RAPD)-PCR. Mutation in aac(6')-Ib and the sul1 genes were detected by capillary electrophoresis sequencing (CES).

Results: All isolates were A. baumannii in which 42.5% of them exhibited a high level of aminoglycoside resistance (HLAR). The most prevalent AMEs and RMT encoding genes were aph(3')-VI, the two aac(6') gene variants [aac(6')-Ib and aac(6')-SL], ant(3'')-I, and armA in which 90%, 87.5%, 85%, and 45% of isolates tested positive, respectively. The other investigated aminoglycoside resistant encoding genes, namely aac(3)-II, aac(6')-II, and rmtB, were not detected. Only 15% of isolates harbored the sul1 gene. RAPD-PCR classified the 40 isolates into three clusters in which cluster II was the main cluster. DNA sequencing revealed that 34.29% (12/35) of isolates tested positive for aac(6')-Ib were found to harbor a common missense mutation in position 102 indicating a novel allelic variant named aac(6')-SL. Also, DNA sequencing revealed three missense mutations in the sul1 gene.

Conclusion: This is the first Saudi study to investigate the plasmid borne aminoglycoside and sulfonamide resistance genes among A. baumannii clinical isolates. A novel allelic variant for aac(6')-Ib was detected in addition to novel mutations in the sul1 gene.

Keywords: 16S rRNA; A. baumannii; AMEs; RAPD-PCR; armA.

Figure 1

PCR products of amplified bla_OXA-51used in molecular confirmation of A. baumannii. L1; 100 bp DNA ladder supplied from Solis BioDyne, Estonia; lanes 60–65 and 66–72 represent the isolate number .

Figure 2

PCR products of the genes coding for aminoglycoside and sulfonamide resistance. L1; 100 bp DNA ladder supplied from Solis BioDyne, Estonia; lanes 1–5 represent the amplified resistance genes as stated above each PCR product.

Figure 3

Multiple sequence alignment of the amino acids sequences of the 12 mutated aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib] encoded by the novel aac(6')-SL allelic variant. The code AAG33663.1 represents the accession Id of the reference AAC(6')-Ib protein. The codes QZB49340-QZB4934051 represent the newly  accession Ids assigned  by the GenBank for the mutated AAC (6')-Ib protein detected in the current study.

Figure 4

Multiple sequence alignment of the amino acid sequences of the mutated sulfonamide-resistant dihydropteroate synthase (DHPS). The code WP_063855115.1 represents the accession Id of the reference protein sequence. The codes QZH81639- QZH8163942 represent the newly accession Ids assigned by the GenBank for the mutated sulfonamide-resistant DHPS detected in the current study.

Figure 5

DNA fingerprint profile generated by RAPD-PCR for A. baumannii clinical isolates. L1 and L2, represent 100 bp and 1 kb DNA ladders, respectively that were supplied from Solis BioDyne, Estonia. Lanes 94 to 99 and 100 to 110 represent the isolate number.

Figure 6

Clonal relatedness between A. baumannii clinical isolates based on the constructed unweighted pair-group method with arithmetic average (UPGMA) dendrogram for the generated RAPD-PCR banding profile. The aac(6')-Ib gene detected in the isolates Acb_73, Acb_75, Acb_80, Acb_81, Acb_83, Acb_84, Acb_90, Acb_91, Acb_97, Acb_108, Acb_109  was  termed more specifically as aac(6')-SL allelic variant in this study.