Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Clinical_Isolates: Algeria

Introduction

The prevalence of extended spectrum β-lactamase (ESBL) and carbapenemase-producing Klebsiella pneumoniae isolates is constantly rising in clinical settings. ¹ Consequently, colistin, a previously abandoned antimicrobial agent due to its nephrotoxicity and neurotoxicity in humans, was reintroduced in clinical settings for the treatment of infections caused by multidrug-resistant (MDR) organisms. ² Recently, there is an increasing number of reported cases of combined resistance to antibiotics of the last resort, carbapenems and colistin, in clinical settings.^3,4

In Algeria, the first report of a colistin-resistant isolate was published in 2015 and described as an Acinetobacter baumannii ST2 isolated from patients in the university hospital center of Béni-Messous in Algiers. This isolate presented with a deleterious insertion of an amino acid named “alanine” in the pmrB gene at position 163. ⁵ Thereafter, the mcr-1 plasmid-mediated colistin resistance gene was described in Escherichia coli after its isolation from animals as well as in clinical settings.^6,7 In this study we report the first detection of a colistin-resistant K. pneumoniae coharboring OXA-48 carbapenemase, which was isolated from a hospital in Algeria.

In 2016, three colistin-resistant K. pneumoniae isolates were recovered in Annaba University hospital, in Algeria, from three different patients who have in common a urological surgery antecedent (Table 1). The patients were admitted to the infectious diseases unit for recurrent urinary tract infection, where urine cytobacteriology and antibiotic susceptibility testing were performed. Of note, two of the aforementioned patients had previously received colistin for treatment of their recurrent urinary tract infection.

Identification of the isolates was done using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (Microflex; Bruker Daltonics). ¹⁶ Antibiotic susceptibility testing was performed by disk diffusion method. Interpretation of results was done according to the European Committee following the antimicrobial susceptibility testing guidelines. The three isolates were resistant to colistin, aztreonam, ceftazidime, cefotaxime, ceftriaxone, cefoxitin, gentamicin, ciprofloxacin, nalidixic acid, nitrofurantoin, and fosfomycin; however, they remained sensitive to amikacin, trimethoprim/sulfamethoxazole, tetracycline, and imipenem (Table 1). In addition, two of three K. pneumoniae strains were resistant to chloramphenicol and ertapenem (Table 1). The minimum inhibitory concentration (MIC) of colistin, imipenem, and ertapenem for isolates was determined by broth microdilution, which revealed that all isolates were resistant to colistin (MIC ≥16 μg/mL) with only two of them being also resistant to ertapenem (MIC = 8 μg/mL); all isolates were imipenem susceptible (Table 1). It is to be mentioned that sensitivity to imipenem was further tested using E-test. The latter revealed the presence of imipenem MICs of 0.25, 1.5, and 1 μg/mL in M5, M6, and M7 strains, respectively. The carbapenemase activity of the two carbapenem-resistant isolates (M6 and M7) was thereafter confirmed by a positive modified Carba-NP test performed as previously described. ⁵

Multilocus sequence typing (MLST) analysis, according to the Pasteur schemes available at the Institute Pasteur's MLST Website (www.pasteur.fr/mlst/), revealed that all of them belonged to the same sequence type “ST101.” Real-time PCR amplification of carbapenemase-encoding genes (bla_OXA-48, bla_NDM, bla_VIM, and bla_KPC) and ESBL-encoding genes (bla_CTX-M, bla_TEM, and bla_SHV) (Table 2) was performed. Later, standard PCR amplification and DNA sequencing of the positive ones showed that all isolates were bla_CTX-M-15, bla_TEM-183, and bla_SHV-106 positive, with two of them, which are ertapenem-resistant, coharboring bla_OXA-48. None of the isolates expressed bla_NDM, bla_VIM, or bla_KPC. The molecular mechanism of colistin resistance was investigated by PCR amplification and sequencing of the pmrA, pmrB, phoP, phoQ, mgrB, crrAB, mcr-1, and mcr-2 genes (Table 2). The plasmid-mediated colistin resistance genes mcr-1 and mcr-2 were absent in the three K. pneumoniae strains. Since CrrAB was not amplified in any single isolate by the PCR method, we concluded that this two-component system is absent in three analyzed isolates. As previously reported, the crrAB system was absent in some K. pneumoniae, associated with the probable insertion sequence-mediated deletion or substitution in the locus. ¹⁷ Sequence analysis revealed no mutations in the phoP and phoQ genes, but showed an inactivating insertion in the mgrB gene in one isolate (M5) at nucleotide 94 with 95% identity at the nucleotide level with IS903B insertion sequence (IS5 family of insertion sequences). The A217V pmrA substitution was observed in two strains (M5 and M6) with mutations in the pmrB gene for the three isolates (V212G, T256A, and T246A) (Table 1).

Colistin is the last-line antibiotic for treatment of infections by Gram-negative bacteria such as K. pneumoniae and the ongoing emergence of colistin and carbapenem resistance represents a serious problem for the management of infections caused by these bacteria. ¹⁸ This study is in accordance with recent studies that highlighted the emergence of colistin resistance in MDR K. pneumoniae arising from loss of function by inactivation of the mgrB gene and activation of the PmrA/B system inducing modification of the lipopolysaccharide.^17–19 The A217V pmrA mutation shown in this study was reported previously in colistin-resistant K. pneumoniae from Serbia, also belonging to ST101 and harboring bla_OXA-48. ⁴ In this study, the authors concluded that this mutation in the pmrA gene could play a role in the development of colistin resistance. These data would strengthen the presumption that this mutation was responsible for colistin resistance. The pmrB mutation T246A detected in this study was also shown in polymyxin B-resistant K. pneumoniae isolated from rectal swabs in Brazil. ²⁰ In this study, the authors suggest that the specific pmrB mutation (T246A) found was not capable of producing polymyxin resistance alone, since this mutation was also found in polymyxin-susceptible isolates and was considered not deleterious by PROVEAN software. To our knowledge, the other pmrB mutations detected in this study (V212G and T256A) have not been described previously.^2,21

There are only three reports of genomic investigation on OXA-48-producing K. pneumoniae ST101 that are also colistin resistant (red in Fig. 1). The strains from two of those reports (Serbia and Turkey) have amino acid changes in the pmrB gene.^4,21 The third study, from Tunisia, described that the colistin resistance of K. pneumoniae was due to an insertion sequence in the mgrB coding gene between the nucleotides 123 and 124. The inserted sequence does not match with any identified IS sequences. ⁴⁴ Thus, this is the first description of colistin- and carbapenem-resistant K. pneumoniae ST101 in Algeria.

OPEN IN VIEWER

FIG. 1.

Geographical distribution of the carbapenemase-producing Klebsiella pneumoniae ST101 with different colistin susceptibility phenotype. Other carbapenemases include KPC, NDM, and OXA-181. ColiR, colistin resistant.^4,9,21–43

The analysis results of M5 colistin-resistant strain with the mgrB truncation collected from a patient not treated with colistin showed that the clinical use of colistin may not be the only reason for the emergence of colistin resistance. Another possibility is the horizontal transmission between patients, who have in common a stay in the urological unit of the same hospital. However, this is probably not the case here since only one isolate with the mgrB truncation was isolated. Thus, a possible spread of nosocomial infections to a larger number of patients and healthy individuals should be prevented. It is urgent to establish a powerful monitoring system in each hospital with perfect coordination between all Algerian hospitals to detect as soon as possible an epidemic infection and prevent the spread of such MDR bacteria inducing infections that are difficult to treat. ⁴⁵