Detection of New arr-4 and arr-5 Gene Cassettes in Clinical Pseudomonas aeruginosa and Klebsiella pneumoniae Strains from Brazil

Abstract

New arr alleles emerged in class 1 integrons from a clinical Pseudomonas aeruginosa strain (arr-4) and four Klebsiella pneumoniae strains (arr-5) in Brazil/American continent. arr-4 was preceded by aacA7-catB3, whereas arr-5 was the unique cassette. The putative proteins shared 75% (Arr-5) and 78% (Arr-4) identities with Arr-2.Rifampin resistance is a notable global health problem concern (11), since it is the front line drug for treating tuberculosis (4) and preventing meningococcal diseases (13). More than 90% of rifampin resistance is due to mutational alterations in an 81-bp rifampin resistance determining region of the rpoB gene (9). However, resistance may also arise by horizontal acquisition of arr genes, which code for ADP-ribosyltransferases responsible for the drug inactivation. The arr-1 gene was first described in the Mycobacterium smegmatis chromosome (10). Subsequently, arr-2 and arr-3 alleles were described as gene cassettes in class 1 integrons present in gram-negative isolates from Europe and Asia (1,6,8,14), showing high-level rifampin MICs ranging from 32 to Ͼ256 g/ml.We report here the identification and characterization of two new arr alleles, arr-4 and arr-5, and the emergence of this class of gene in the American continent as gene cassettes from class 1 integrons present in clinical Pseudomonas aeruginosa and Klebsiella pneumoniae strains.One P. aeruginosa (PS1111) and four K. pneumoniae (K56, K224, K688, and K830) isolates recovered from different inpatients of a Brazilian hospital (Rio de Janeiro city) in 2005 tested positive for class 1 integron signatures (3). DNA macrorestriction using SpeI, followed by pulsed-field gel electrophoresis, was performed as previously described (3) and demonstrated four distinct profiles among the K. pneumoniae strains. Sequence analysis of the class 1 integron variable regions showed the presence of a 453-bp open reading frame, encoding a predicted protein of 150 amino acid residues in all integrons. These sequences from the K. pneumoniae strains and from isolate PS1111 shared 78% amino acid identity, and 75 and 78% amino acid identity, respectively, with ADP-ribosyltransferase Arr-2 (Fig. 1). Therefore, these open reading frames were named arr-4 (PS1111) and arr-5 (the K. pneumoniae strains). The genes were assigned to be parts of cassettes due to the presence of attC sites. The arr-4 gene cassette was preceded by a aacA7-catB3 array, which confer resistance to aminoglycosides and chloranphenicol, respectively, and arr-5 was the unique cassette inserted in all K. pneumoniae integrons. The arr-4 gene cassette is 516 bp long and presented an attC site only 22 bp in length due to the loss of 2L and 2R simple sites (Fig. 2). The arr-5 gene cassette is 555 bp, presenting a classical attC of 61 bp, encompassing the core site sequences that make up the left-hand and right-hand consensus sites composed of 1L, 2L, 1R, and 2R simple sites (Fig. 2) (12).arr-2 and arr-3 gene cassettes share 99% nucleotide identity and, moreover, present identical attC site sequence...

Molecular Epidemiology of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Isolates from Norway and Sweden Shows Import of International Clones and Local Clonal Expansion

“…The aacA4 gene cassette was present in both integrons, but they carried two different ␤-lactamase genes, bla OXA-35 and bla PSE-1 . The integron in isolate K34-73 also carried a new putative arr gene (named arr-7), encoding rifampin resistance, that was most similar to the Arr-4 gene (90% amino acid identity; GenBank accession number EF660562) (5).…”

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain

“…Here, we observed that this H-Y-[QED] motif is highly conserved in almost all hundreds of Arr sequences analyzed, indicating its importance in Arr activity, and suggesting that these Arr sequences could be functional. Indeed, the Arr enzymes already functionally characterized (Arr-ms, Arr-2 to Arr-8, Arr-cb, Arr-sc, Arr_Mab, Arr-wd1-4) 2 , 4 , 6 – 8 and those of the present study were observed along the phylogenetic tree, showing that despite the sequence diversity, even in the RIF binding sites, their activities against rifamycins are maintained. These patterns were observed in sequences carried by bacteria belonging to high and low antibiotic-impacted environments.…”

“…Highly conserved residues (≥ 90%), considering all Arr sequences analyzed in the phylogeny, are represented below the alignment by the fuchsia blocks. References: Arr-2, Arr-sc, and Arr-ms 1 ; Arr-4 and Arr-5 6 ; Arr-8 8 ; Arr-w1, Arr-w2, Arr-w3, and Arr-w4 4 ; ArrMab 7 ; Arr-cb 2 . …”

“…The genomic context of the arr gene might vary by organism; for instance, while Arr-ms is chromosomally encoded in the saprophyte organism M. smegmatis , Arr-2 has been associated with mobilizable and mobile elements (transposons and integrons) of Gram-negative pathogenic bacteria (e.g., Pseudomonas aeruginosa and Klebsiella pneumoniae ) 1 . Besides these Arr enzymes, few others (Arr-3 to Arr-8, Arr-cb, Arr-sc, Arr_Mab) have already been characterized 2 , 4 , 6 – 8 . Although Arr has been associated primarily with resistance to clinically important rifamycin antibiotics, some authors suggested that it may be able to act on other substrates and metabolic pathways 1 , 5 , 9 .…”

Integrative metagenomic and biochemical studies on rifamycin ADP-ribosyltransferases discovered in the sediment microbiome

“…1b ). Our results include the rifampin and NAD + binding sites reported in the previous studies of arr-ms from Mycobacterium smegmatis , arr-sc from S. coelicolor , and arr-2 and homologous genes ( arr -3, arr- 4, arr- 5, arr -7, and arr -8) from several gram-negative pathogenic bacteria 13 , 43 – 46 . In particular, H19 and Y49, which were identified for NAD + binding 13 , were highly conserved in our alignment.…”