Preferential carriage of class 2 integrons in Acinetobacter baumannii CC113 and novel singletons

Summary

Our understanding of the distribution of integrons associated with multidrug resistance in Acinetobacter baumannii isolates around the world remains incomplete. The association between the class 1 and 2 integron A. baumannii-positive isolates (n = 60), recovered since 1982 from 11 Argentinean hospitals, and the circulating lineages, was investigated. While class 2 integrons were highly significantly associated with clonal lineage CC113^B/CC79^P (P = 0·009) and novel singletons (P = 0·001), class 1 integrons were found not to be associated with CC109^B/CC1^P or other lineages. The study reveals a differential distribution of class 2 integrons in lineages, and suggests that the prevalence of intI2 in Argentina is related to the emergence of novel singletons in recent years and to the abundance of CC113^B/CC79^P, which has been the local dominant lineage for several decades.

Acinetobacter baumannii has emerged as a major relevant global nosocomial pathogen in the last two decades, causing a variety of serious infections such as pneumonia and bacteraemia, often associated with high mortality [1]. In a CDC report published last year, A. baumannii was considered as a microorganism of serious concern, since about the 63% of the isolates are multidrug resistant with about 500 deaths each year attributed to its infections in the USA [2].

The accumulation of antibiotic resistance mechanisms occurs frequently in this species, underlining its genomic plasticity to evolve to extensive multidrug resistance in the nosocomial environment. Class 1 and 2 integrons have been frequently found in a variety of resistance determinants [3, 4]. However, a clear differential distribution of both these integron classes in clinical isolates across continents has been reported by different groups with class 1 integrons being frequently identified from Europe, USA and Australia, while class 2 integrons are more common in several countries in South America [3–5].

This prompted us to investigate if there was a particular dissemination of integrons in different A. baumannii lineages. We therefore analysed the association between the circulating clones in South America and the presence of class 1 and 2 integrases in A. baumannii isolates recovered from 1982 to 2014 from 11 hospitals. Some isolates included in the present work, were from previously published studies (Table 1) [3, 6–8] but hitherto had not been investigated for association of strain lineages with the presence of class 1 and 2 integrons.

Table 1. Origin, clonal lineage and integron content of 60 isolates of Acinetobacter baumannii from 11 hospitals

CC, Clonal complex; PCR, polymerase chain reaction.

* ^B Bartual scheme; ^P Institut Pasteur scheme.

A total of 168 A. baumannii isolates was investigated by polymerase chain reaction (PCR) amplification of intI1 and intI2 genes using specific primers [3, 9]. Sixty isolates were positive either for class 1 (n = 17) or class 2 integrases (n = 35) or both (n = 8). Confirmatory DNA sequencing was performed on 23 of the 43 intI2 amplicons, and one intI1 amplicon [3]. All intI2 amplicons possessed the typical internal stop codon [3, 10], and the intI1 amplicon harboured one of the most common alleles of intI1 [9]. Isolates with class 1, or class 2 integrons were identified respectively in 9 and 10 of the 11 hospitals studied. Coexistence of both classes of integrons in the same strain was found in five hospitals from two cities, Buenos Aires and Rosario. Overall these results evidence the widespread presence of these genetic elements in nosocomial settings in Argentina.

The lineages of these 60 intI-positive isolates were determined by a pan-PCR assay [11] and multilocus sequence typing [12] which revealed the presence of six defined clonal complexes (CC) and at least nine singletons (Table 1) Statistical analysis was undertaken by the sign test which is a non-parametric test to confirm a zero median difference between continuous distributions of two random pairs. This showed high significant associations between intI2 and the CC113 lineage (P = 0·003) as well as between intI2 and the novel singletons (P = 0·01). In fact, 15/16 isolates of CC113^B/CC79^P and 15/17 isolates belonging to novel singletons harboured intI2.

Regarding class 1 integrons, 16 of the 25 intI1-positive isolates were assigned to lineage CC109^B/CC1^P. Only two isolates of CC113 harboured a class 1 integron and the other six intI1-positive isolates corresponded to singletons, and one to CC110. No statistical association between CC109^B/CC1^Pand class 1 integrons (P = 0·052345) was found. However, CC109 was infrequently reported in Argentinean isolates recovered during the period 1982–2010 [12]. Interestingly, eight isolates possessed both integrons and belonged to different CCs or singletons; three were of lineage CC109^B/CC1^P, one of CC113^B/CC79^P, and five of different singletons (Table 1). These last results suggest that novel singletons may play a role in the double acquisition of integrons.

In conclusion, we describe a marked association between intI2 and CC113^B/CC79^P, which is the most frequently isolated lineage of A. baumannii in Argentinean isolates during the years 1992–2009 and this explains the hitherto high frequency of intI2 observed in isolates from Argentina [3, 6, 7, 12]. In addition, several novel singletons frequently containing intI2, some harbouring both classes of integrons, were evident.