Misidentification of pan drug-resistant Klebsiella pneumoniae clinical isolates as a metallo-β-lactamase producers by the EDTA/DDST test

Abstract

Carbapenemase-producing Enterobacteriaceae may exhibit susceptibility to carbapenems. For this reason with the recent spread of NDM-1 among Enterobacteriaceae, the phenotypic detection of metallo-beta-lactamase (M␤L)-producing has been recommended by Brazilian Agency of Sanitary Surveillance (ANVISA). 1 In 2013, two pan drug-resistant Klebsiella pneumoniae (KPN1 and KPN2) isolates were recovered from urine (cystostomy) of a 75-year-old male patient hospitalized in a tertiary teaching hospital in Florianópolis, Santa Catarina, Brazil. Both isolates were phenotypically identified as M␤L producers by ethylenediamine tetraacetic acid (EDTA)/double-disk synergy test (DDST) and forwarded to our laboratory for further characterization. Identification of both isolates as K. pneumoniae was confirmed by MALDI-TOF MS (Bruker Daltonics, Germany), according to the manufacturer's recommendations. Both isolates showed an identical pattern by ERIC-PCR. The minimal inhibitory concentrations (MICs) for selected antimicrobial agents were determined by broth microdilution according to Clinical and Laboratory Standards Institute -CLSI. The MICs were interpreted according to the CLSI guidelines, 2 except for tigecycline, which interpretation was performed according to the European Committee on Antimicrobial Susceptibility Testing -EUCAST. Both isolates were fully resistant to all broad-spectrum cephalosporins, aztreonam, gentamicin, fluoroquinolones, meropenem, ertapenem, and polymyxin B as shown in Table 1. The isolates showed intermediate resistance to imipenem and amikacin. While KPN1 was susceptible to tigecycline, KPN2 became resistant to this agent.The isolates were also screened for ES␤L production by disk approximation and the synergism was observed only when amoxicillin/clavulanic acid disk was tested 15 mm a part of ceftriaxone, ceftazidime and cefepime disks. The phenotypic detection of M␤L was carried out by the EDTA/DDST and confirmed by ertapenem hydrolysis assay using MALDI-TOF MS (Bruker Daltonics, Germany) as previously reported. 3 Although both isolates showed an increase in the inhibition zone of ceftazidime/EDTA (6 mm) and meropenem/EDTA (7 mm) disks compared to the ceftazidime and meropenem disks, respectively, hydrolysis of ertapenem was not observed, suggesting that both isolates were not M␤L producers.The search for ␤-lactamase encoding genes was carried out by PCR followed by DNA sequencing of amplicons. Both K. pneumoniae isolates carried bla CTX-M-15 , and the narrow spectrum-␤-lactamases encoding genes, bla SHV-11 , bla TEM-1 and bla OXA-1 . The presence of the plasmid mediated qnrS1 gene and a mutation Ser83Ile in gyrA were also detected, justifying the quinolone resistance exhibited by both KPN isolates. The analysis of the outer membrane proteins profile by SDS-PAGE showed that both isolates lost the major porins OmpK35 and OmpK36. Sequencing analysis of the ompk35 and ompk36 genes revealed the presence of the insertion sequence IS1 between the promoter region and the start codon of the ompK35 gene, and ...

Molecular detection of virulence genes in Klebsiella Pneumoniae clinical isolates from Kurdistan Province, Iran

“…Generally, one of the classes of antibiotics used for treating K. pneumoniae is beta-lactams such as cephalosporin [17]. However, the presence of ESBL enzymes impairs the performance of these antibiotics [18]. The difference in sensitivity and drug resistance in different geographic regions can be associated with different patterns of antibiotic use in different areas [19].…”

Pandrug-resistant Gram-negative bacteria: a systematic review of current epidemiology, prognosis and treatment options

“…24, 37, 52-57, 59, 60, 63-72, 75 Summing the data from all studies, all-cause mortality in PDR GNB infection was 53% (n=75 of 142) but highly variable; excluding studies with < 5 patients, all-cause mortality ranged from 20% to 71%. 4,5,20,35,36,51,65,[73][74][75] Mortality was high irrespective of infecting pathogen or site of infection, ranging from about 20% in urinary tract infections to >40% in other sites ( Table 2).…”

“…Among the 75 reported deaths, mortality was judged by the authors to be directly attributable to the infection in 11 cases, 20,52,56,61,62,67,71,73,75 but there were at least 6 deaths known to be unrelated to the PDR infection 5,57,60,68,72 and for the rest of the cases this was not discussed.…”

“…The only available data come from studies, exclusively case series or case reports, involving n=105 patients; K. pneumoniae n=31, 5,49,52,53,57,63,64,66,68,69,71 P. aeruginosa n=45, 37,51,58,60,70,72,73,75,76 A. baumannii n=11, 4,24 Providencia stuartii n=15, 65 Chryseobacterium indologenes n=2 54 , Burkholderia cepacia n=1. 55 .…”

“…Synergistic combinations may also be useful according to two studies. 73,75 Amikacin (1g/day) with meropenem (2g q8h infused over 3 hours) resulted in both microbiological and clinical success in 4 cases of ventilator-associated pneumonia. 73 The combination was confirmed to be synergistic in vitro.…”