During the last decade, carbapenem-resistant isolates have become a serious threat to public health worldwide. Since the first identification of the bla NDM-1 gene in Klebsiella pneumoniae in Sweden in 20091other bacterial species, including NDM-1 producing Acinetobacter spp., have been isolated in several parts of the world2. The first reports of NDM-1 in Brazil were in 2013 and 2014, in Enterobacteriaceae and Acinetobacter baumannii, respectively3–5. Here, we report the occurrence in 2012 of a clinical isolate of Acinetobacter pittii carrying bla NDM-1 in a hospitalized patient with no national and international travel history.
An 86-year-old woman was admitted for the first time to the emergency room at the Hospital Dona Helena (HDH), Joinville, in southern Brazil in 2005 with a suspected stroke. Imaging tests revealed an ischemic stroke, with severe motor sequelae, speech disorder, and difficulty swallowing.
In September 2012, she was attended in the emergency room at HDH with a decline in her general state of health. Broncho aspiration and hypoxia were confirmed. She was referred to the intensive care unit (ICU) and intubated due to worsening respiratory function and decreased oxygen saturation. After evaluation of the clinical signs and presence of secretion in the orotracheal tube, she was diagnosed with pneumonia and received empirical treatment with 12.5 mL of ampicillin/sulbactam (1 g/500 mg), every 8 hours, while awaiting blood and urine culture results. The urine was positive, with growth of Proteus mirabilis above 105 colony forming units (CFU)/mL. The isolate was sensitive to ampicillin/sulbactam, cefazolin, ceftriaxone, cefepime, ciprofloxacin, ertapenem, imipenem, and norfloxacin. Treatment with ampicillin/sulbactam was maintained, but the patient’s condition worsened after 2 days with development of tachycardia and hypotension. She was infused with 10 mL/hour of dobutamine (12.5 mg/mL) and her condition stabilized. There was rapid weight loss (10 kg) and worsening of swallowing during this period.
On the fifth day of ICU admission there was no improvement of orotracheal secretion. Bronchoalveolar material was sent for culture. Multidrug-resistant Pseudomonas aeruginosa (4.0 × 104 CFU/mL) and K. pneumoniae (8.0 × 104 CFU/mL) resistant only to ampicillin were detected. Aspiration pneumonia was diagnosed, and mechanical ventilation was necessary.
Following treatment, on day 19 of hospitalization, she underwent gastrostomy to reduce the risk of aspiration pneumonia and to improve food support. On day 26, mechanical ventilation was withdrawn and she was transferred to the inpatient ward with tracheostomy. However, on day 42, the clinical condition worsened, evolving to systemic inflammatory response syndrome. She returned to the ICU and treated with piperacillin/tazobactam 4 g/500 mg by prolonged (30-minute) intravenous infusion every 8 hours. She remained febrile with production of orotracheal secretion, and blood and bronchoalveolar lavage cultures were requested. Both cultures were positive for carbapenem-resistant bacteria of the Acinetobacter calcoaceticus – A. baumannii complex. The clinical option was to maintain the treatment. On day 50 severe diarrhea developed and she was medicated empirically with metronidazole (400 mg every 8 hours) while awaiting fecal culture results. After a positive result for Clostridium difficile, the piperacillin/tazobactam and metronidazole therapy was maintained for 7 days. Clinical improvement occurred and she was discharged.
Investigation of the bla NDM-1 gene was performed by polymerase chain reaction (PCR) using the NDM-F (5′-GGTGCATGCCCGGTGAAATC-3′) and NDM-R (5′-ATGCTGGCCTTGGGGAACG-3′) primers and previously described thermocycling conditions1. A 661 bp segment of the gene was confirmed after agarose gel electrophoresis. The amplicons were bidirectional sequenced using the Big Dye® Terminator v3.1 kit (Thermo Fisher Scientific, Waltham, MA, USA) on the ABI-Prism 3500 Genetic Analyzer platform (Thermo Fisher Scientific) and compared to reference nucleotide sequences of the variants of bla NDM gene available on GenBank (www.ncbi.nlm.nih.gov/genbank/) using the ClustalW software contained in the BioEdit program package version 220.127.116.11.
A. baumannii (5379RM) and K. pneumoniae (12491RM) strains carrying the bla NDM-1 gene, provided by the Central Laboratory of the State of Paraná, were employed as positive controls.
The bla NDM-1 gene was confirmed in A. calcoaceticus – A. baumannii isolated from blood and bronchoalveolar lavage samples of the same patient and collected on the same day of hospitalization as described above. The isolate harbored the ISAba1 insertion sequence and was negative for all bla OXAgenes analyzed, according to multiplex PCR6.
To speciate the bla NDM-1 positive isolate, approximately 500 bp segments of the 16S rRNA encoding gene were obtained via PCR and amplicons were purified with ExoSAP-IT® (USB Affymetrix). Sequencing was performed using the Microseq 500 16S rDNA kit and BigDye® XTerminator purification kit (Thermo Fisher Scientific) on the ABI 3100 analyzer (Thermo Fisher Scientific). The obtained sequences were compared with the bacterial sequences deposited using the Le Bibi® and Sepsis Blast® systems, which confirmed A. pittii. The isolate was also identified by matrix-assisted laser desorption-ionization time-of-flight spectrometry (Vitek-MS®, BioMerieux). The spectrograms were interpreted as previously described7 using the Vitek-MS® RUO mode of the SARAMIS ™ spectral archive and microbial identification system version 4.12 and enriched by exactly the same SuperSpectrum created based on the selection of 40 specific masses and validated by the authors. This procedure correctly identified species belonging to the A. calcoaceticus – A. baumannii complex.
Disk diffusion antibiotic susceptibility tests and automated testing (Vitek 2, BioMerieux®) revealed that A. pittii was resistant to meropenem, imipenem, ampicillin/sulbactam, piperacillin/tazobactam, and ceftazidime, and sensitive to polymyxin B, ciprofloxacin, amikacin, gentamicin, doxycycline, sulfamethoxazole/trimethoprim, and tobramycin, according Clinical and Laboratory Standards Institute guidelines8.
The study received ethical and methodological approval from the Research Ethics Committee of the Universidade da Região de Joinville – Univille (Protocol Nr. 788.455).
The bla NDM-1-positive A. pittii isolate from a hospitalized patient with no national or international travel history confirmed the occurrence of NDM-1 in Brazil since 2012. In our country, the first isolate harboring the bla NDM-1 gene was reported in 2013, in a diabetic patient hospitalized in Porto Alegre, Rio Grande do Sul, in tissue from a severely injured (and subsequently amputated) foot. P. rettgeri resistant to carbapenems was confirmed, with PCR identification of the gene. There were no previous trips to other countries3. In the same year, bla NDM-1 in isolates of Enterobacteriaceae with reduced susceptibility to carbapenems was reported from 17 hospitals in the same state4. Isolates were obtained from cultures of clinical and surveillance materials (rectal swabs and hospital environments). The bla NDM-1 gene was found in six isolates belonging to Enterobacter cloacae complex and in two Morganella morganii isolates. Most strains were derived from surveillance cultures.
In addition to Enterobacteriaceae, NDM-1 has been detected in non-fermenting gram-negative bacilli globally, including China, Egypt, Germany, and Israel9. In Brazil, the first A. baumannii strain carrying the bla NDM-1 gene was reported in 2014 from the urine of a 71-year-old male with obstructive respiratory disease admitted to the ICU of a hospital in Londrina, Paraná5. The bla OXA-51-like gene was confirmed, but bla OXA23-like, bla OXA-24-like, bla OXA-58-like and blaOXA-143 genes were absent in this isolate. So far, the only previously identified A. pittii (ST 119) carrying blaNDM-1 gene in Brazilian territory was reported in a colonizing isolate from a 66-year-old man with bladder carcinoma admitted to a tertiary hospital in Porto Alegre, Rio Grande do Sul10.
It is noteworthy that the bla NDM-1 positive A. pittii isolate reported here was initially identified as belonging to the A. calcoaceticus – A. baumannii complex, without definition of the species. Most of the phenotypic characteristics among A. baumannii, A. calcoaceticus, A. pittii, A. dijkshoorniae, and A. nosocomialis are very similar. Thus, identification at the species level inside the complex by conventional methods is difficult. So, it is reasonable that other A. pittii isolates carrying bla NDM-1 gene may be spreading in Brazilian hospitals as the majority of clinical laboratories do not determine the species that make up the A. calcoaceticus – A. baumannii complex employing molecular techniques11.
In conclusion, confirmation of the bla NDM-1 gene in A. pittii isolated from a hospitalized patient in 2012 demonstrates that this carbapenemase was present in Brazilian territory before the previously published date. This finding warns of the possibility of spread to the community, environment, and animals. It also highlights the need for a better understanding of clonal relationships to control dissemination in the hospital environment. The detection of A. pittii carrying the bla NDM-1 gene occurred in a patient with no national or international travel history or a transfer from another hospital center. This prompts concern about the lack of definition of its possible reservoirs.