Emergence of aph(3’)-VI and accumulation of aminoglycoside modifying enzyme genes in KPC-2-possessing Enterobacter aerogenes isolates from infections and colonization in patients from Recife-PE, Brazil

Enterobacter aerogenes is a gram-negative bacillus that belongs to the family Enterobacteriaceae. The bacterium is found in the environment and in the human gastrointestinal tract, and is an important opportunistic pathogen capable of colonizing and infecting hospitalized patients. E. aerogenes frequently causes healthcare-associated infections, such as pneumonia, meningitis, urinary tract infections, surgical site infections and sepsis^1,2.

Carbapenems were the first-line antibiotic in the treatment of infections caused by multidrug-resistant E. aerogenes³. However, resistance to carbapenems has emerged and the prevalence of carbapenem resistant isolates of E. aerogenes is increasing⁴.

One of the major causes of carbapenem-resistance in Enterobacteriaceae is the production of Klebsiella pneumoniae carbapenemase (KPC). This enzyme has been frequently reported in E. aerogenes^5,6. KPC-producing bacteria are often resistant to other antimicrobials, such as aminoglycosides, which may be due to the presence of the bla _KPC gene, genes encoding aminoglycoside modifying enzymes (AMEs), and 16S rRNA methyltransferase genes in the same plasmid^7,8.

Although studies have increasingly reported the emergence of KPC-producing gram-negative bacteria that are multidrug-resistant, little is known about the presence of bla _KPC-2 and genes for AMEs in colonized isolates. In China, one study described the presence of aph(3’)-VI associated with another carbapenemase, New Delhi metallo-β-lactamase (NDM)⁹. Another study based in Curitiba-PR, Brazil, reported aac(3)-III, aac(6’)-Ib, and ant(3”)-Ia genes in four E. aerogenes isolates, with none detected simultaneously with the bla _KPC-2¹⁰ gene. A study from Recife-PE, Brazil, described many E. aerogenes isolates that harbored beta-lactam resistance genes, including bla _KPC-2⁶. However, no AMEs genes were investigated. Therefore, the objective of this study was to characterize AMEs genes present in 29 isolates of E. aerogenes harboring the bla _KPC gene that were obtained from isolates that had colonized and infected patients at a public hospital in Recife-PE, Brazil.

The E. aerogenes isolates harbored the bla _KPC-2 gene and were resistant to one or more aminoglycosides. The isolates were from a tertiary hospital in Recife-PE, Brazil, from November 2011 to August 2012. The isolates were identified biochemically using the Bactec 9120 or Phoenix™ automated systems (BD). The presence of bla _KPC-2 in these isolates was previously reported⁶. The minimum inhibitory concentration for antimicrobials was determined using the Phoenix™ system, according to CLSI criteria¹¹.

Bacterial genomic DNA was extracted using the Kit ZR Fungal/Bacterial DNA Miniprep (Zymo Research), according to the manufacturer instructions for gram-negative bacteria and quantified using a NanoDrop 2000c UV-Vis spectrophotometer. AMEs genes aac(3)-Ia, aac(3)-IIa, aac(6’)-Ib, ant(2”)-Ia, and aph(3’)-VIwere amplified by PCR using specific primers. The PCR amplification reactions were performed in a total volume of 25 μl per tube, containing 1 μl of genomic DNA (10 ng/µl), 2.0 U of Taq DNA polymerase (Promega), 200 μM of deoxyribonucleotide triphosphate (dNTP) (Promega), 1.5 mM MgCl₂, and 1 µmol of the primers. The PCR amplifications were performed in a thermocycler (Biocycler Biosystems) for 5 minutes at 94°C for initial denaturation, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 55^oC (Table 1) and 1 minute at 72°C, followed by a final extension at 72°C for 5 minutes. The PCR products were subjected to electrophoresis on a 1% agarose gel. Representative isolates of positive PCR products for the tested genes were selected, purified using a commercial kit (Wizard® SV Gel and PCR Clean-Up System, Promega), and sequenced by the Sanger method to confirm the presence and variant of the genes. The nucleotide sequences were analyzed by BLAST (http://www.ncbi.nlm.nih.gov/) and Clustal W (European Bioinformatics Institute) (http://www.cbi.ac.uk/) and have been deposited in GenBank (accession numbers: KU899109, KU899110 and KU695892).

The isolates were from different patients admitted to different sectors of a public hospital in Recife-PE, Brazil. Of the 29 E. aerogenes clinical isolates, 15 (51.7%) were obtained from rectal swabs (surveillance culture), followed by blood (n=7), urine (n=3), tracheal secretion (n=2), catheter tips (n=1), and ascitic fluid (n=1) (Table 1).

All E. aerogenes isolates were resistant to amoxicillin-clavulanic acid, ampicillin, cefazolin, cefotaxime, cefoxitin, ceftriaxone, ciprofloxacin, ertapenem, levofloxacin, meropenem, and piperacillin-tazobactam. Resistance to imipenem reached 93% (27/29) and 90% (26/29) for cefepime, and 82% (24/29) trimethoprim/sulfamethoxazole. In addition, 100% (29/29) of the isolates were resistant to tobramycin, 86.2% (25/29) were resistant to amikacin, and 65.5% (19/29) were resistant to gentamicin. Three aminoglycoside resistance phenotypes were identified in the isolates (Table 1). Among those, the most predominant was the phenotype III, which presented resistance to all aminoglycosides tested. Phenotypes I (resistant to amikacin and tobramycin) and II (resistant to gentamicin and tobramycin) were also detected.

The most frequent aminoglycoside resistance gene was aph(3’)-VI, which was detected in 89.6% (26/29) of the isolates and were detected in all aminoglycoside resistance phenotypes. The aac(3)-IIa gene was found in 51.7% (15/29) of the isolates with resistance phenotypes II and III. The ant(2”)-Ia gene was detected in 41% (12/29 ) of the samples (phenotype I and III) (Table 1). The aac(6’)-Ib and aac(3)-Iagenes were not found. In phenotypes I and III, resistance to amikacin could be explained by the presence of the aph(3’)-VI gene, and the resistance to gentamicin and tobramycin by the presence of the genes aac(3)-IIa and ant(2”)-Ia.

Of the 29 isolates of E. aerogenes, 15 were colonization isolates and 14 were obtained from infections (Table 1). Among the colonization isolates, phenotype III was the most common, presenting resistance to the three aminoglycosides tested. Phenotype I (amikacin and tobramycin) was the most observed among the infection isolates. Colonization and infection isolates presented mainly the genetic profiles aac(3)-IIa/aph(3’)-VI or ant(2”)-IIa/aph(3’)-VI.

Patients hospitalized for prolonged periods, mainly in the intensive care unit, are most at risk of acquiring infections by E. aerogenes¹²,¹³. In the present study, the samples analyzed were obtained from patients admitted to different sectors of a public hospital.

It is alarming that most of the genes (bla _KPC, aph(3’)-VI, aac(3)-IIa, and ant(2”)-IIa) were detected in colonization isolates obtained from cultures of surveillance samples (swab rectal). A review in 2016 showed that patients colonized by carbapenem resistant Enterobacteriaceae had a 16.5% increased risk of developing an infection¹³. Therefore, it is essential to identify colonized patients by isolates carrying carbapenemases and other resistance genes, for example the AMEs genes, to prevent the transmission of resistance genes and adequately treat the patients. The present results highlight the need for medical authorities to institute rigorous detection and dissemination control methods for multi-drug resistant isolates in the hospital environment, since the intestinal tract provides a favorable locus for the transfer of genes that confer resistance.

Based on the results of the susceptibility profile, several isolates of E. aerogenes showed resistance to the three aminoglycosides tested, and were correlated with the presence of more than one of the AMEs. Most isolates showed resistance to amikacin, which explains the high percentage of the aph(3’)-VI gene. In China, a study showed the presence of the aph(3’)-VI gene in isolates of E. aerogenes associated with NDM¹⁰. To our knowledge, the present study is the first report of the aph(3’)-VI gene in E. aerogenes in Brazil, especially in isolates harboring the bla _KPC gene. This causes concern since amikacin is only recommended when resistance to gentamicin and tobramycin is observed¹⁴. Therefore, the presence of the aph(3’)-VI gene and the bla _KPC gene in the same isolate may hamper the treatment of infections caused by these microorganisms, since aminoglycosides and carbapenems will be inactivated by the encoded enzymes by these two genes.

In this study, all isolates encoding aac(3)-IIa were resistant to gentamicin, suggesting that the enzyme AAC (3)-II is a determinant of resistance to gentamicin in E. aerogenes¹⁴.

In Brazil, the gene ant(2”)-Ia has been found in other gram-negative bacteria, such as Pseudomonas aeruginosa¹⁵. The ANT(2”)-Ia enzyme confers resistance to gentamicin and tobramycin¹⁴. However, in this study, this enzyme seemed to be directly related to tobramycin resistance. The aac(6’)-Ib and aac(3)-Iagenes were not detected in this study, although they have already been described in E. aerogenes in Paraná, Brazil¹¹.

The simultaneous presence of resistance genes aph(3’)-VI, aac(3)-IIa and ant(2”) and bla _KPC in the same isolate, as demonstrated in this study, adds to the mechanisms of the dissemination of resistance to aminoglycosides and carbapenems in the hospital environment, and decreases the chances for therapeutic success of infections caused by E. aerogenes. Several AMEs have been already identified in different bacteria species in Brazil. However, the emergence of aph (3 ‘)-VI gene in E. aerogenes described here indicates the continuous dissemination of AMEs encoding genes in Brazil. These results highlight the importance in establishing rigorous methods for the surveillance of resistance genes, especially in colonizing patients.