Non-clonal occurrence of pmrB mutations associated with polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae in Brazil

BACKGROUND Polymyxins are currently used as a “last-line” treatment for multidrug-resistant Gram-negative infections. OBJECTIVES To identify the major mechanisms of resistance to polymyxin and compare the genetic similarity between multi-drug resistant Klebsiella pneumoniae strains recovered from inpatients of public hospitals in the Mid-West of Brazil. METHODS 97 carbapenems non-susceptible K. pneumoniae were studied. β-lactamases (bla OXA-48, bla KPC, bla NDM, bla CTX-M, bla SHV, bla TEM, bla IMP, bla VIM) and mcr-1 to mcr-5 genes were investigated by polymerase chain reaction (PCR). Mutations in chromosomal genes (pmrA, pmrB, phoP, phoQ, and mgrB) were screened by PCR and DNA sequencing. Clonal relatedness was established by using pulsed-field gel electrophoresis and multilocus sequence typing. FINDINGS K. pneumoniae isolates harbored bla KPC (93.3%), bla SHV (86.6%), bla TEM (80.0%), bla CTX-M (60%) genes. Of 15 K. pneumoniae resistant to polymyxin B the authors identified deleterious mutations in pmrB gene, mainly in T157P. None K. pneumoniae presented mcr gene variants. Genetic polymorphism analyses revealed 12 different pulsotypes. MAIN CONCLUSIONS Deleterious mutations in pmrB gene is the main chromosomal target for induction of polymyxin resistance in carbapenem-resistant K. pneumoniae in public hospitals in the Mid-West of Brazil.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKp) and the increased use of polymyxin B to treat infections caused by these microorganisms may have contributed to the spread of polymyxin-resistant K. pneumoniae isolates (PRKP). (1,2,3) The polymyxin resistance is most commonly associated with the modification of the lipopolysaccharide (LPS) following the addition of 4-amino-4-deoxi-L-arabinose to lipid A. Modifications of Ara4N are regulated by two component systems: PhoP/ PhoQ, PmrA/PmrB and MgrB regulator. Mutations in genes involved in the production of these systems may result in lower antibiotic fixation. (4) Previous studies reported that disruption of mgrB gene is one of the major mechanisms of polymyxin resistance in K. pneumoniae. (2,5,6) Recently, mcr-1 gene and its variants (mcr-1 to mcr-8 genes) was described conferring resistance to polymyx-in in Enterobacteriaceae isolated from humans, animals and environmental samples worldwide, including Brazil. (7,8,9) The global dissemination of these genes may be a signal of a new era of pandrug resistant bacteria. (10) To date, there is no information about the mechanisms of polymyxin resistance in Gram negative bacilli in Mato Grosso do Sul state. The aims of this study were to investigate the mechanisms of resistance to polymyxin and to evaluate the genetic diversity in carbapenem and polymyxin-resistant K. pneumoniae strains recovered from patients admitted in intensive care units of public hospitals in the Mid-West of Brazil.

MATERIALS AND METHODS
Bacterial isolates and susceptibility tests -A total of 97 carbapenem non-susceptible K. pneumoniae isolated from patients admitted in intensive care units of three tertiary hospitals in Mato Grosso do Sul, Brazil (Hospital A, 592 beds; Hospital B, 271 beds and Hospital C, 352 beds) between 2013 and 2014 were included in this study. The identification and antimicrobial susceptibility testing were performed by Vitek 2 compact System (bioMérieux, Marcy L'Etoile, France). The minimum inhibitory concentration (MIC) of tigecycline was determined by using E-test ® strips (bioMérieux, Marcy L'Etoile, France) applied in Mueller-Hinton agar (Oxoid, England) according to the manufacturer's instructions.
The polymyxin B MICs were performed by broth microdilution test according the CLSI. (11) Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control. BrCast (12) breakpoints were used for interpretation of tigecycline (Susceptible ≤ 1 mg/L, Resistant > 2 mg/L) and polymyxin MIC results (Susceptible ≤ 2 mg/L, Resistant > 2 mg/L). (12) Multidrugresistance (MDR) was defined as non-susceptibility to at least one agent in three or more antimicrobial categories. Extensively drug-resistant (XDR) was defined as susceptible to only one or two categories in all. (13) Screening for genes of resistance -Resistant genes bla OXA-48like , bla KPC , bla NDM were investigate by multiplex polymerase chain reaction (PCR) and bla CTX-M , bla SHV , bla-TEM , bla IMP , bla VIM by simple PCR using primers as previously described. (14,15,16,17) The plasmid-mediated polymyxin resistance gene, mcr-1 to mcr-5, was determined by multiplex PCR using primers as previously described. (7) Mutations in chromosomal genes (pmrA, pmrB, phoP, phoQ, and mgrB) were screened by PCR and DNA sequencing. (18,19) DNA was extracted from fresh bacterial colonies using an AxyPrep Bacterial Genomic DNA Miniprep kit (Axygen Scientific, Union city, CA, USA). The amplification products were purified using DNA Illustra GFX 96 kit (GE Healthcare Life Sciences, UK Ltd., Buckinghamshire, UK) and sequenced using the 3730 DNA analyser (Applied Biosystems, CA, USA). Data were analysed using Geneious (6.1.8) software (Auckland, New Zealand) and BLASTN (NCBI) tool (www.ncbi.nlm.nih.gov/blast). The PROVEAN platform was used to predict alterations in biological functions of proteins using K. pneumoniae MGH 78578 (CP00647.1) as reference.
Ethics -This study was approved by the Plataforma Brasil Research Ethics Committee.
Genetic polymorphism analyses of PRKp revealed 12 different pulsotypes (A to L) by PFGE method with similarity below 85%. MLST analysis showed 11 ST among these isolates. ST 11 (belonging to CC258) was present in four PRKp isolates of all hospitals studied and ST13 in three PRKp, from two hospitals (A and C). The antimicrobial susceptibility profiles, resistance genes determinants and clonal patterns are shown in Table. DISCUSSION Infections caused by multidrug-resistant K. pneumoniae are currently a concern in public health. Polymyxins are often the last line of therapeutic options for the treatment. Consequently, a high mortality rate has been observed, especially in intensive therapy units. (22) It is well documented in the literature that the levels of resistance to antibiotics may vary according to the hospital characteristics and distinct geographic areas. In our study, PRKp isolates showed low resistance to tigecycline, fosfomycin, and amikacin suggesting these antibiotics would be successful as treatments in infections caused by carbapenem and polymyxin-resistant K. pneumoniae.
The results of this study show different mechanisms of antimicrobial resistance, including non-neutral mutations in pmrB gene (A66E, P272H, G318A, H58N, V67D) not previously associated to polymyxin resistance in K. pneumoniae.
Similar to previous Brazilian studies, (1,2,23) we also observed a high rate KPC-producing K. pneumoniae and described high genetic diversity among the isolates.
Almost 50% of K. pneumoniae studied was considered K. pneumoniae XDR. The high resistance to β-lactams observed (Table) may be related to the presence of genes as bla SHV , bla TEM and bla CTX-M , mainly bla KPC .
Recent Brazilian studies carried out in the Southeast region of Brazil demonstrate a temporal increase of resistance to polymyxin since 2009, ranging from 0 to 30.6%. (2,3,24) In our study, we documented that 15.5% of K. pneumoniae isolated between 2013 and 2014 were resistant to polymyxin, in the Brazilian Mid-West hos- pitals. This is the first description of resistance to polymyxin in this region. The genetic analysis demonstrates that in these hospitals the resistance to polymyxin are from chromosomal origin because none K. pneumoniae presented mcr gene variants.
Although previous studies report that alterations in the mgrB gene is the main mechanisms of polymyxin resistance in Brazil and worldwide, (2,5,6,25,26) in our study, only two PRKp presented deleterious mutations in this gene. Aires et al. (6) reported disruption in mgrB gene by IS903B, IS5, IS102, ISKpn26 (IS5 family), and IS10L (IS4 family) in different regions of Brazil, including two K. pneumoniae from the Mid-West region (Distrito Federal).
Different from what was expected the majority of PRKp isolates studied carried alterations in pmrB gene, mainly in T157P. To be best of our knowledge, this kind of alteration has not been described in Brazil prior to this study. Jayol et al. (19) showed this point mutation in the pmrB gene (T157P) leads to an upregulation to pm-rCAB and pmrHFIJKLM operons conferring resistance to polymyxins in K. pneumoniae isolates from South-Africa, Colombia and Turkey. (19) In our study, we identified four PRKp with non-neutral mutation (R256G) on PmrB protein as also described by Aires et al. (6) in the Southeast region of Brazil (6) Pitt et al. (26) detected R256G in polymyxin-susceptible and in polymyxin-resistant K. pneumoniae, suggesting that this mutation is not determinant for polymyxin resistance. The same authors related simultaneous alterations in pmrB (P158R, T140P) and in mgrB genes with increases the MIC values.
In the hospitals studied, no relationship was observed between MIC values and mutations in pmrB genes. However, in K. pneumoniae with mutations in mgrB gene we observed high level polymyxin-resistant (MIC = 32 µg/ mL), as also described by Aires et al. (6) The most prevalent mechanism of resistance to polymyxin observed in the Mid-West region is due to mutation in the pmrB gene, different from that observed (mutation in the mgrB gene) in other Brazilian states. (1,2,6) Antimicrobial pressures may be responsible for the evolution of different mutation profiles observed between different geographic regions.
The emergence and evolution of complex 258 in KPC-K. pneumoniae is carried by mobile transposable elements. Pereira et al. (1) described the clonal diversity in Brazil with prevalence of ST11 and ST340 in Northeast region, ST437 in South and Southeast regions and ST11 in Mid-West. Our results suggest that PRKp isolates belonging to ST11 and ST13 clones are adapted in Mid-West region and highlights the emergence of new ST in PRKp (ST70, ST273, ST449, ST323, ST2084, ST1075, ST1298, ST2687). Complementation assays should be done later to elucidate the role of these STs and polymyxin resistance.
In conclusion -Our results show that many K. pneumoniae not susceptible to carbapenem isolated in Brazilian Mid-West Hospitals are considered MDR and XDR, but still show low resistance to tigecycline, fosfomycin and amikacin. The high resistance to β-lactams observed may be related to the presence of genes as bla SHV , bla TEM and bla CTX-M , mainly bla KPC .
The resistance to polymyxin are from chromosomal origin because none K. pneumoniae presented mcr gene variants. The main mechanism of resistance to polymyxin in K. pneumoniae in the hospitals studied is due to a mutation in the pmrB gene.