Evaluation of carbapenem-resistant Enterobacteriaceae in a tertiary-level reference hospital in Rio Grande do Sul , Brazil

Introduction: The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) is a threat to the health system. Methods: We evaluated the antimicrobial susceptibility profiles of 70 CRE isolated in a tertiary hospital in Brazil between August and December 2015, and determined their resistance mechanisms. Results: The most prevalent microorganism was Klebsiella pneumoniae (95.7%); it showed high-level resistance to carbapenems (>98%), with sensitivity to colistin (91.4%) and amikacin (98.6%). The blaKPC gene was detected in 80% of the CRE isolates. Conclusions: Evaluation of bacterial resistance contributes to an appropriate treatment, and the reduction of morbimortality and dissemination of resistance.

Bacterial resistance is a serious health problem, leading to relapsing nosocomial infections, reduced drug efficacy, and expensive and imprecise treatment, and resulting in high morbidity and mortality rates 1 .The spread of carbapenemresistant Enterobacteriaceae (CRE) is threatening the health system, since the bacteria show multiple resistance mechanisms, such as the production of carbapenemases [e.g., Klebsiella pneumoniae carbapenemase (KPC); metallo-β-lactamases (MBL); and oxacillinases (OXA)] and efflux pumps, and porin loss 2 .
Since the spread of CRE is a worldwide problem, studies focusing on the characterization of resistance mechanisms and on the understanding of epidemiology at infection sites are required to inform an adequate empirical therapy and control the dissemination of resistance 3 .Therefore, the aim of the present study was to evaluate the antimicrobial susceptibility profiles of 70 CRE isolates obtained between August and December 2015 at the Hospital Universitário de Santa Maria (HUSM) (Santa Maria, Rio Grande do Sul, Brazil) and to determine their resistance mechanisms.The isolates were obtained from several clinical specimens and identified using the Vitek ® 2 automated system (bioMérieux, Marcy-l'Étoile, France).
Antimicrobial resistance of the isolates was evaluated against antimicrobial agents described in Table 1.The minimum inhibitory concentration (MIC) values were determined using the Advanced Expert System (AES) program in Vitek ® 2; these values were interpreted according to the criteria established by the Clinical and Laboratory Standards Institute (CLSI) and the technical standard no.01/2013 from the Agência Nacional de Vigilância Sanitária (ANVISA) 4,5 .Phenotypic methods, such as disc diffusion tests with enzymatic inhibitors [phenylboronic acid (PBA); ethylenediaminetetraacetic acid (EDTA)], biochemical tests Carba NP [Carbapenemase Nordmann-Poirel (CN)] and Blue-Carba (BC), and molecular methods [polymerase chain reaction (PCR)] were used to evaluate the resistance mechanisms.The disk diffusion method identifies KPC-producing strains using carbapenem disks supplemented with PBA; it identifies MBL-producing strains using the chelating agent EDTA 4 .The CN biochemical test is based on the hydrolysis of the β-lactam ring of imipenem.The strains were considered to be carbapenemase producers when a color change of a solution containing the phenol red indicator was observed.The BC test is based on the same principle as CN, except that the bromothymol blue indicator is used 6 .To confirm the production of carbapenemases, PCR was performed to detect the presence of the bla KPC , bla OXA-48 , bla NDM , and bla GES genes 7 .The sensitivity and specificity of the different phenotypic methods were calculated considering PCR as the standard methodology 6 .
During the study period, 70 isolates were obtained from patients infected or colonized by CRE, being that each isolate corresponded to only one clinical specimen per patient.K. pneumoniae was the prevailing microorganism (67 isolates; 95.7% of all CRE isolates), followed by Escherichia coli (1; 1.4%), Citrobacter freundii (1; 1.4%), and Enterobacter cloacae (1; Carbapenem-resistant gram-negative bacilli may exhibit low susceptibility to antimicrobials, mainly to β-lactam antibiotics, because the production of carbapenemases is associated with porin loss, overexpression of efflux pumps, and the production of another β-lactamase [e.g., extended-spectrum β-lactamases (ESBL) and AmpC β-lactamase] 8 .This may explain why the bla KPC gene was not detected in 14 (out of 70) isolates in the present study.Similarly, Ribeiro et al., observed highlevel resistance against β-lactams and ciprofloxacin, and sensitivity to polymyxin B and amikacin in Enterobacteriaceae isolates in southern Brazil.In the same study, the bla KPC gene was identified in only 14 (14/345) isolates and, among the non-carbapenemase-producing isolates, ESBL and AmpC phenotypes were observed 9 .Differentiating a carbapenemaseproducing CRE from a non-carbapenemase-producing CRE is important.The selective pressure caused by indiscriminate use of broad-spectrum antibiotics in treating hospital infections may lead to resistance of endogenous bacteria (from the intestinal microbiota), but the reduction of susceptibility may also be associated with a rapid horizontal transmission (e.g., via healthcare workers, equipment contamination, and mobile genetic elements), contributing to the spread of heterogeneous resistance 10 .
In recent years, multiple resistance mechanisms have reduced the available therapeutic options, leading to an extensive use of aminoglycosides and polymyxins 11 .Although colistin showed good activity against CRE, 6 (6/70) K. pneumoniae isolates were resistant to this antimicrobial agent (MIC ≥ 16µg/mL).Santos et al. described a similar resistance profile of colistin-resistant Pseudomonas aeruginosa (9/231) isolated at the same hospital (HUSM) as in the present study 12 .This phenotype may be due to the lipopolysaccharides changes, such as modifications in lipid A by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N)

TABLE 2
Phenotypic and molecular evaluation of the isolated clinical specimens.
and phosphoethanolamine (pEtN), and mgrB gene mutations, responsible for the regulation of the PmrAB and PhoPQ twocomponent systems, as well as through plasmid-mediated mcr-1 gene, responsible for horizontal transfer of colistin resistance, necessitating monitoring of colistin use during therapy and reinforcing control practices during hospital infections to avoid the dissemination of resistance and loss of efficacy of this drug 13 .
In the present study, all phenotypic methods (disk diffusion, CN and BC) detected the KPC enzyme activity with 100% sensitivity and specificity.Similarly, Giske et al. reported 100% sensitivity and 98% specificity of an assay that relied on the 3-aminophenylboronic acid for the detection of KPC in K. pneumoniae strains 14 .Further, García-Fernández et al. reported 100% specificity of both methodologies, with 100% and 98% sensitivity of CN and BC, respectively 15 .
As demonstrated, the disk diffusion (PBA and EDTA) method, and CN and BC tests are characterized by high sensitivity and specificity, similarly to PCR.They might be incorporated into the workflow of a clinical microbiology laboratory, as they are quick, practical, and low-cost.
Simultaneous use of two or more of these methods increases the reliability of CRE detection and, together with the antimicrobial susceptibility profile data, they may inform the adequate treatment, assisting in the reduction of mortality and dissemination of resistance.The phenotype found in the Institution is worrying and the adoption of nosocomial infection control and prevent measures are necessary.
This research presented limitations of the study in terms of non-detection of other carbapenemases enzymes (OXA, MBL), which could interfere with sensitivity and specificity.Posteriorly, mechanisms such as efflux pump and porin loss will be evaluated, as well as molecular typability for outbreak analysis.

Ethical considerations
This research was approved by the Ethical Research Committee, Universidade Federal de Santa Maria, under approval number 38850614.4.0000.5346.

TABLE 1
Antimicrobial susceptibility profile of 70 CRE isolated between August and December 2015 at the HUSM, Santa Maria/RS, Brazil.
CRE: carbapenem-resistant Enterobacteriaceae; HUSM: Hospital Universitário de Santa Maria; MIC: minimum inhibitory concentration.*MIC50,concentration required to inhibit the growth of 50% of microorganism.1.4%).The specimen type from which the CRE were isolated most frequently was the rectal swab used in the

Table 1 .
Genotypic analysis based on PCR revealed the presence of the bla KPC gene in 56 isolates (56/70; 80%), with the KPC activity also phenotypically demonstrated by the disk diffusion, CN, and BC techniques (

Table 2 )
. The OXA (bla OXA-48 ) and MBL (bla NDM and bla GES ) genes were not detected.All phenotypic methods (disk diffusion, CN and BC) detected the KPC enzyme with 100% sensitivity and specificity.