b-lactamase-producing Gram-negative bacteria in an intensive care unit in southern Brazil

The present study evaluated the antimicrobial susceptibility profile, b-lactamase production, and genetic diversity of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. using phenotypic identification, antimicrobial susceptibility testing, and b-lactamase phenotypic detection. Isolates were obtained from patients in an intensive care unit in a hospital in southern Brazil. Bacterial genomic DNA was extracted, followed by the genotypic detection of carbapenemases and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Fifty-six isolates (26 Klebsiella pneumoniae, five Escherichia coli, three Enterobacter aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were evaluated. The phenotypic extended spectrum b-lactamase (ESBL) test was positive in 53.8% of the K. pneumoniae isolates, 100.0% of the E. coli isolates, and 100.0% of the E. aerogenes isolates. Phenotypic and genotypic testing of K. pneumoniae carbapenemase (KPC) was positive in 50.0% of the K. pneumoniae isolates. Phenotypic and genotypic testing showed that none of the P. aeruginosa or Acinetobacter spp. isolates were positive for metallo-b-lactamase (MBL). The blaOXA gene was detected only in Acinetobacter spp. The lowest genetic diversity, determined by ERIC-PCR, was observed among the KPC-producing K. pneumoniae isolates and OXA-producing Acinetobacter spp. isolates, indicating the inadequate dissemination control of multidrug-resistant bacteria in this hospital environment.


INTRODUCTION
Healthcare-associated infections are among the leading causes of morbidity and mortality in patients and are associated with higher treatment costs (Sydnor, Perl, 2011).Some studies have reported the importance of controlling nosocomial infection to reduce the number of these infections, which provides economic benefits (Gastmeier et al., 2006;Rosenthal, Maki, Graves, 2008;Landelle, Marimuthu, Harbarth, 2014).
The emergence of multidrug resistance has hampered and limited treatment options.Prompt and adequate treatment is crucial.The management of these life-threatening infections and treatment decisions should be guided by reliable antimicrobial susceptibility testing (Gagliotti et al., 2014;Tängdén, Giske, 2015).
The increase in multidrug-resistant pathogens has Page 2 / 7 occurred concomitantly with a drastic reduction of the discovery and development of new antimicrobial agents, making these infections more difficult to control (Cassir, Rolain, Brouqui, 2014).
The identification of microorganisms that are involved in nosocomial infections and knowledge of their antimicrobial resistance profiles can guide physicians in choosing the appropriate therapy, playing a key role in epidemiology (Stuart, Leverstein-Van Hall, 2010).
The present study evaluated the antimicrobial susceptibility profile, b-lactamase production, and genetic diversity of Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.isolates that were obtained from patients in an intensive care unit (ICU) in a hospital in southern Brazil from March 2012 to August 2013.

METHODS
Enterobacteriaceae and non-glucose-fermenting Gram-negative (P.aeruginosa and Acinetobacter spp.) isolates were obtained from patients in an ICU in a hospital in southern Brazil, from March 2012 to August 2013.Only one isolate from each patient was selected and stored at -80 ºC in the Laboratory of Medical Bacteriology, Department of Clinical Analysis and Biomedicine, State University of Maringa.No personal data were taken from patients, the privacy was guaranteed as well the law concerning research with humans (Resolution 466/2012 Brazil National Health Council, Health Ministry).Phenotypic identification and antimicrobial susceptibility testing (AST) were carried out by AUTO-SCAN-4 automated system (Siemens Microscan, Inc., Deerfield, IL, USA).The susceptibility testing was interpreted according to the criteria of the Clinical and Laboratory Standards Institute (CLSI, 2015).
KPC phenotypic detection was carried out in Enterobacteriaceae, which had Minimal Inhibitory Concentration (MIC)≥0.5 mg/mL to meropenem by the modified Hodge test (MHT) (CLSI, 2015).K. pneumoniae isolates with and without bla KPC gene, kindly provided by the Central Laboratory of Parana (LACEN) were used as control.
C e f t a z i d i m e -r e s i s t a n t P. a e r u g i n o s a a n d Acinetobacter spp.isolates were evaluated for the presence of MBL by disc-approximation test, using 2-mercaptopropionic acid (2-MPA, Acros, New Jersey, USA) and EDTA (Invitrogen, Carlsbad, USA) (Arakawa et al., 2000).IMP-1-producing A. baumannii (A-3227) (Gales et al., 2003b) and P. aeruginosa ATCC ® 27853 were used as positive and negative controls, respectively.
Detection of bla KPC , bla OXA , bla SPM , bla IMP , bla GIM, and bla SIM β-lactamase genes were performed by PCR using Veriti 96-well thermal cycler instrument (Applied Biosystems at Life Technologies, Foster City, CA) and AmpliTaq Gold ® PCR master mix (Applied Biosystems at Life Technologies, Hammonton, NJ).The primer sequences and amplicon sizes are shown in Table I.

RESULTS
The studied institution is a small-size public hospital with 10 adult's ICU beds and pediatric.Fifty-six isolates (26 K. pneumoniae, five E. coli, three E. aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were tested for susceptibility profile and genotyped.
Resistance profiles for all isolates are shown in Table II.Most of the Acinetobacter spp.and K. pneumonia isolates were resistance to carbapenems.
Among Enterobacteriaceae, the phenotypic ESBL test was positive in 53.8% (14/26) of the K. pneumonia isolates, 100.0%(5/5) of the E. coli isolates, and 100.0%(3/3) of the E. aerogenes isolates.The phenotypic KPC test was positive in 50.0%(13/26) of the K. pneumoniae isolates.The KPC enzyme was not detected in the E. coli or E. aerogenes isolates.Most of the KPC-producing K. pneumoniae isolates were resistant to all of the antibiotics tested (only 15.4% were sensitive to amikacin).Among the non-KPC-producing K. pneumoniae isolates, 46.1% were resistant to ertapenem.All of the KPC-producing K. pneumoniae isolates, detected by the MHT test, were positive for the bla KPC gene.None of the other Enterobacteriaceae isolates were positive for bla KPC .
None of the ceftazidime-resistant P. aeruginosa isolates or Acinetobacter spp.isolates were positive for MBL.The bla KPC , bla SPM , bla IMP , bla GIM , and bla SIM genes were not detected in the P. aeruginosa or Acinetobacter spp.isolates that were resistant to ceftazidime.The bla OXA genes were detected in Acinetobacter spp.isolates, in which 100.0% (13/13) were positive for bla OXA-51 and 84.6% (11/13) were positive for bla OXA-23 .
The ERIC-PCR analysis of 26 K. pneumoniae isolates revealed DNA band patterns that could differentiate four isolates, and 84.61% (22/26) of the K. pneumoniae isolates formed three clusters, with four, eight, and 10 isolates each.For the five E. coli isolates, three were unique, and 2/5 (40%) were clustered.The three E. aerogenes isolates presented unique profiles.The nine P. aeruginosa isolates could be differentiated into one cluster with four isolates (4/9 [44.44%]) and five isolates with unique patterns.For Acinetobacter spp.isolates, 10/13 (76.92%) were clustered, and three had unique patterns.

DISCUSSION
Although sparse official data have been published, bacterial resistance in Brazil is a major challenge to antimicrobial therapy, especially in the southern and southeastern regions (Rossi, 2011;Toledo et al., 2012).The present study detected high rates of resistance to thirdand fourth-generation cephalosporins and a concerning increase in resistance to carbapenems in Gram-negative bacilli that were isolated from patients in an ICU in a hospital in southern Brazil.In Enterobacteriaceae, ESBL production is a decisive mechanism that determines resistance to broad-spectrum cephalosporins.Recent studies by Jones et al. (2013), Bonelli, Moreira, andPicão, (2014), andNogueira et al. (2014) corroborate our findings, indicating that this mechanism of resistance is especially common in K. pneumoniae, E. coli, and Enterobacter spp. in Brazil, with important implications for decisions regarding adequate antimicrobial therapy.
In the present study, ESBL production was high in K. pneumoniae.Because the co-production of KPC prevents interpretations of the results of ESBL phenotypic testing (Poulou et al., 2014), we did not test for ESBL genes.
The production of carbapenemases was important for antimicrobial resistance in the Enterobacteriaceae and non-glucose-fermenting Gram-negative isolates.The predominant carbapenemases include New Delhi metallo-β-lactamases (NDM), Oxacillinases (OXA), Verona integron-encoded metallo-β-lactamase (VIM), Imipenemase (IMP), and KPC, which are encoded by the genes bla NDM , bla OXA , bla VIM , bla IMP , and bla KPC , respectively (Nordman, Naas, Poirel, 2011).In Brazil, some types of carbapenemases have been detected more frequently, such as São Paulo metallo-β-lactamase (SPM) in P. aeruginosa isolates (Rossi, 2011;Rizek et al, 2014.),OXA-23 in Acinetobacter spp.isolates, and KPC in Enterobacteriaceae isolates (Rossi, 2011, Biberg et al., 2015;Rieger et al., 2016).The identification of carbepenemase production is currently the most successful way by which carbapenem resistance is determined among members of the Enterobacteriaceae family (Gupta et al., 2011).In the present study, the bla KPC gene was detected in 50% of the K. pneumoniae isolates, and bla OXA was detected in all of the Acinetobacter spp.isolates.The MBL genes (bla SPM , bla IMP , bla GIM , and bla SIM ) were not detected in any of the isolates tested.
The phenotypic MHT test presented high sensitivity in detecting KPC-producing K. pneumonia in the present study, which was confirmed by genotypic testing.Raghunathan, Samuel, and Tibbetts (2011) reported similar results, with sensitivity as high as 90%.However, Girlich, Poirel, and Nordmann (2012) found that the phenotypic MHT test had low sensitivity in detecting other carbapenemases, particularly MBL, such as NDM.
The presence of MBL is quite common in P. aeruginosa.However, despite the significant resistance of the isolates to carbapenems, this enzyme was not detected by our phenotypic test.Genotypic testing, which targets the most common bacterial MBL genes that are associated with resistance, was negative.These results suggest other mechanisms of resistance, such as the direct actions of efflux pumps or the loss of porins.
The bla OXA-51 gene was detected in all of the Acinetobacter spp.isolates, which may indicate that all of them belonged to the A. baumannii species, which naturally harbors this gene in its chromosome (Héritier et al., 2005).The OXA-51 family, similar to b-lactamases, comprises several members and has weak activity against carbapenems.When these enzymes are expressed in vivo, however, they can increase the MIC of carbapenems, leading to a resistant isolate.Although few studies have implicated OXA-51 in the resistance to carbapenems, these enzymes are nonetheless concerning and have been shown to confer resistance to carbapenems in A. baumannii (Evan, Amyes, 2014).Furthermore, over 80% of the Acinetobacter spp.isolates that were resistant to carbapenems were also positive for bla OXA-23 .The bla OXA-23 gene is able to confer resistance to carbapenems, although high levels of resistance (MIC≥32 µg/mL) were observed only when bla OXA-23 was associated with other mechanisms (Evan, Amyes, 2014).
ERIC-PCR revealed high genetic similarity among KPC-producing K. pneumoniae.This finding raises concerns about inadequate control in the studied hospital environment and the dissemination of multidrug-resistant bacteria.KPC-producing Enterobacteriaceae was first reported in Brazil in 2005 and has spread, becoming endemic in many hospitals (Bonelli, Moreira, Picão, 2014).A previous study in southern Brazil was based on a surveillance program for multidrug-resistant bacteria and found that KPC-producing Enterobacteriaceae increased from 17% in 2010 to over 80% in 2011 (Toledo et al., 2012).Of the KPC-producing K. pneumoniae and OXAproducing Acinetobacter spp., 12/13 (92.3%) and 10/11 (91.0%), respectively, belonged to the same cluster.This sharp increase in such a short period of time emphasizes the importance of this kind of antimicrobial resistance in Brazil.

CONCLUSION
High antimicrobial resistance to carbapenems, mainly by b-lactamase-producing Enterobacteriaceae and non-glucose-fermenting Gram-negative, was detected in isolates from patients in an ICU in southern Brazil.The genetic similarities among the KPC-producing K. pneumoniae isolates and among the OXA-producing Acinetobacter spp.should alert health professionals to the necessity of implementing suitable measures for infection control in this studied environment.

TABLE I -
Oligonucleotides used in the PCR reactions a F, sense primer; R, antisense primer

TABLE II -
Antimicrobial resistance profile of Gram-negative bacteria isolated from patients in an ICU in southern Brazil, from March 2012 to August 2013