Coexistence of metallo-beta-lactamase-encoding genes in Pseudomonas aeruginosa

Abstract

Background & objectives: The emergence and rapid spread of carbapenem resistance mediated by metallo-beta-lactamase (MBL) in Pseudomonas aeruginosa is of major concern due to limited therapeutic options. This study was aimed at detecting the presence of MBL and its association with integrons in imipenem-resistant P. aeruginosa isolates and to determine their genetic relatedness.

Methods: A total of 213 P. aeruginosa isolates were collected from two tertiary care centres and tested against anti-pseudomonal antibiotics by antimicrobial susceptibility testing, followed by the detection of MBL production by combined disk method. Minimum inhibitory concentration (MIC) of meropenem was determined by E-test. Multiplex polymerase chain reaction (PCR) was performed for the detection of blaSPM, blaIMP, blaVIM, blaNDM, blaGIM and blaSIM. PCR was carried out to characterize the variable region of class 1 integron. Transcongujation assay was carried out for the confirmation of plasmid-mediated resistance. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR was performed for determining the genetic relatedness among P. aeruginosa isolates.

Results: Of the 213 P. aeruginosa isolates, 22 (10%) were found to be carbapenem resistant and these were from pus 18 (82%), urine 2 (9%), sputum 1 (5%) and tracheal wash 1 (5%). Among 22 isolates, 18 (81.8%) were found to be MBL producers by phenotypic method and MIC range of meropenem was 8 to >32 μg/ml. PCR amplification showed that 20 (91%) isolates carried any one of the MBL genes tested: blaVIM and blaNDM in seven (32%) and six (27%) isolates, respectively; blaVIM and blaNDMin three (14%); blaIMP and blaNDM in two (9%); blaVIM and blaIMP in one (5%) isolate. The blaVIM, blaIMP and blaNDM were found to co-exist in one isolate. None of the isolates were positive for blaSPM, blaSIM and blaGIM. All 22 isolates carried class I integron. Of the 20 MBL-positive isolates, transconjugants were obtained for 15 isolates. ERIC-PCR analysis showed all isolates to be clonally independent.

Interpretation & conclusions: Our results showed 10.3 per cent of carbapenem resistance among P. aeruginosa isolates, and the coexistence of MBL-encoding genes among P. aeruginosa mediated by class I integron.

Fig. 1

Multiplex polymerase chain reaction for detection of metallo-beta-lactamase-encoding genes. Lane 1, negative control; lane 2, 100 bp ladder; lane 3, PA32 (VIM positive); lane 4, PA44 (VIM, IMP, NDM positive); lane 5, PAE181 (IMP positive); lane 6, PA27 (VIM positive); lane 7, PAE180 (IMP, VIM positive).

Fig. 2

Polymerase chain reaction for detection of gene cassettes (5’-CS with 3’-CS). Lane 1, negative control; lane 2, 250 bp Ladder; lane 3-8, PA27, PA30, PA42, PA44, PAE120, PAE131.

Fig. 3

Dendrogram tree of unweighted pair group method with arithmetic mean method of enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR). The values on top of the horizontal lines represent the node identity and the values given below the horizontal lines represent the branch length. The bar at the bottom of the figure represents an amount of genetic change (0.05).