Carbapenem Resistance in Acinetobacter baumannii and Other Acinetobacter spp. Causing Neonatal Sepsis: Focus on NDM-1 and Its Linkage to ISAba125

Abstract

Carbapenem-resistant determinants and their surrounding genetic structure were studied in Acinetobacter spp. from neonatal sepsis cases collected over 7 years at a tertiary care hospital. Acinetobacter spp. (n = 68) were identified by ARDRA followed by susceptibility tests. Oxacillinases, metallo-β-lactamases (MBLs), extended-spectrum β-lactamases and AmpCs, were detected phenotypically and/or by PCR followed by DNA sequencing. Transconjugants possessing the bla NDM-1(New Delhi metallo-β-lactamase) underwent further analysis for plasmids, integrons and associated genes. Genetic environment of the carbapenemases were studied by PCR mapping and DNA sequencing. Multivariate logistic regression was used to identify risk factors for sepsis caused by NDM-1-harboring organisms. A. baumannii (72%) was the predominant species followed by A. calcoaceticus (10%), A. lwoffii (6%), A. nosocomialis (3%), A. junni (3%), A. variabilis (3%), A. haemolyticus (2%), and 14TU (2%). Fifty six percent of the isolates were meropenem-resistant. Oxacillinases present were OXA-23-like, OXA-58-like and OXA-51-like, predominately in A. baumannii. NDM-1 was the dominant MBL (22%) across different Acinetobacter spp. Isolates harboring NDM-1 also possessed bla (VIM-2, PER-1, VEB-2, CTX-M-15), armA, aac(6')Ib, aac(6')Ib-cr genes. bla NDM-1was organized in a composite transposon between two copies of ISAba125 in the isolates irrespective of the species. Further, OXA-23-like gene and OXA-58-like genes were linked with ISAba1 and ISAba3 respectively. Isolates were clonally diverse. Integrons were variable in sequence but not associated with carbapenem resistance. Most commonly found genes in the 5' and 3'conserved segment were aminoglycoside resistance genes (aadB, aadA2, aac4'), non-enzymatic chloramphenicol resistance gene (cmlA1g) and ADP-ribosylation genes (arr2, arr3). Outborn neonates had a significantly higher incidence of sepsis due to NDM-1 harboring isolates than their inborn counterparts. This study demonstrates the significance of both A. baumannii and other species of Acinetobacter in cases of neonatal sepsis over an extended period. Oxacillinases and bla NDM-1 are the major contributors to carbapenem resistance. The dissemination of the bla NDM-1 is likely linked to Tn125 in diverse clones of the isolates.

Keywords: 5′–3′ CS; ARDRA; Acinetobacter spp.; India; NDM-1; OXA-23; Tn125; neonatal sepsis.

Figure 1

Analysis of genetic relationship according to Dice's similarity coefficient and the unweighted pair group method with arithmetic mean (UPGMA) (the position tolerance and optimization were set at 1.5 and 1% respectively) of the ApaI patterns of (A) A. baumannii (n = 49), (B) A. calcoaceticus (n = 6), (C) A. lwoffii (n = 4) isolates, (D) A. baumannii (n = 6), and (E) A. calcoaceticus (n = 4) harboring NDM-1. More than 90% similarity in PFGE band pattern interpreted as indistinguishable. A few pairs of strains were found to be indistinguishable but could not be considered as episodes of cross-transmission as they were isolated from neonates who were not treated in the NICU during an interval of 15 days: (i) A_152 (DOB 06/01/2013) and A_153 (DOB 24/01/2013), (ii) A_162 (DOB 5/09/2013) and A_163 (DOB 21/10/2013), (iii) A_151 (DOB 10/01/2013) and A_155 (DOB 08/03/2013), (iv) A_108 (DOB 24/08/2008) and A_109 (DOB 24/11/2008), (v) A_147 (DOB 25/01/2012) and A_149 (DOB 01/04/2012). ^*One A. calcoaceticus isolate was untypable. A represents absence of NDM-1, P represents presence of NDM-1.

Figure 2

(A) Schematic representation of Tn125 carrying bla_NDM−1 gene in a representative transconjugant. Genes and their transcription orientation are indicated by arrows. The lengths of the target genes and their exact location of the target genes are not to scale. Gene names are abbreviated according to the corresponding proteins (Bonnin et al., 2012): cutA1 for divalent cation tolerence protein; groES, groEL for heat-chaperonin protein; insE for ISCR21 of tnpA family. Δ pac for truncated phospholipid acetyltransferase. IRL and IRR are for inverted repeat left and right, respectively. This structure was found in all of the transcojugants (except one transformant). (B) Diagram showing the linkage between bla_{OXA−58−like} with ISAba3 (A_114-J53, A_136-J53, and A_156-J53). (C) Diagram showing the linkage between bla_{oxa−23−like} with ISAba1 (A_134-J53, A_140-J53, and A_148-J53).

Figure 3

Schematic diagram of genes in between 5′CS and 3′CS of class 1 integron of 8 transconjugants of Acinetobacter spp. Three of the transconjugants (A_134-J53, A_137-J53, and A_156-J53) share similar structure. The genes are not to scale and vertical lines represent the boundaries of each gene. Gene names are abbreviated according to their corresponding proteins: aadB, aadA2, aacA4′− resistance to trimethoprim, gcuF, gcuE2, gcu8- unknown ORF, arr3, and arr2- ADP-ribosylation gene, carb-2- resistance to β-lactamases. attC site- attachment sites of each gene.