Coexistence of blaTEM, blaCTX, blaKPC, blaNDM, blaSIM e blaOXA-48 in polymicrobial bloodstream isolates from a patient with acute myeloid leukemia

Background: Bloodstream infections are among the most frequent and serious complications in patients with haematological malignancies. Case presentation: A patient diagnosed with acute myeloid leukemia was admitted to the hospital for chemotherapy induction, developed several episodes of febrile neutropenia. Had bloodstream infection with at least four strains of Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumanii. The majority showed resistance to ampicillin, cefepime, ceftriaxone, ciprofloxacin and sulfamethoxazole/trimethoprim. blaTEM and blaSIM were detected in P. aeruginosa, blaTEM, blaCTX and blaOXA-48 in E. coli, blaCTX, blaKPC, blaNDM, blaSIM and blaOXA-48 in K. pneumoniae and blaOXA-48 in A. baumannii. Conclusions: The patient was treated with meropenem for 10 days, without progressing from fever episodes, evolved to death.


Introduction
The epidemiology of bloodstream infection in patients with haematological cancer has been changing over time.
Gram-negative bacteria infections are increasingly prominent in patients with hematological malignancies associated with antimicrobial resistance (Pagano et al., 2014).
Multidrug-resistant gram-negative bacterial infections are an increasing cause of mortality in acute myeloid leukemia (AML), compromising the success of antineoplastic therapy (Castañón et al., 2019). Patients with bloodstream infection (BSI), particularly those with multidrug-resistant bacteria (MDR), have a higher risk of mortality (Islas-Muñoz et al., 2018). As a result, the mortality rate in patients with hematological neoplasms increases with bloodstream infections. Bloodstream infections in carbapenem-resistant Enterobacteriaceae are appearing in patients with hematological malignancies and are associated with ineffective initial empirical therapy, long delays in the administration of active antimicrobials and high mortality rates (Satlin et al., 2013).

Methodology
We conducted a case report at a referral center for people with cancer in Pernambuco, Brazil. We analyzed all consecutive episodes of bloodstream infection in a cancer patient. Information on baseline characteristics, clinical characteristics, were prospectively collected from medical records. And the etiology, antimicrobial susceptibility, empirical antibiotic therapy and resistance genes were determined through phenotypic and molecular tests. The study was approved by the Research Ethics Committee of the Hospital do Câncer de Pernambuco.

Case Presentation
A 24-year-old woman with acute myeloid leukemia M3 subtype -LMA-M3 (promyelocytic leukemia) was admitted to the hematology department of the Pernambuco Cancer Hospital on 01/20/20 to start the first cycle of chemotherapy with daunorubicin and cytarabine, without any comorbidity, and used prophylactic sulfamethoxazole + trimethoprim and acyclovir.
The patient presented dyspnea associated with fever and productive cough, requiring supplemental oxygenation. Ceftriaxone 2g / day (D7) and azithromycin 500mg / day (D5) were started. During hospitalization, the patient had several peaks of febrile neutropenia, the first episode of fever was 02/05/20 with 38.2°C. The second episode was 02/14/20 with 38.4°C and the third episode on 02/17/20 with 38.4ºC. More than three episodes of NF were recorded during hospitalization. Made use of 300 mcg (30 UM / mL) of granulokine during hospitalization.
The complete blood count exams showed intense leukopenia, thrombocytopenia according to hemoglobin values 7.9 g/dL, leukocyte count of 220/mm³, neutrophils 201/mm³ and platelets of 23,000/mm³. Presenting a decrease in the values of red blood cells 2.59, hemoglobin 7.5 g/dL, leukocytes 150/mm³, neutrophils 38/mm³, and platelets 12,000/mm³. Biochemical laboratory tests showed serum levels of 143 mEq/L sodium (Na + ), 3.7 mEq/L potassium (K + ), 100 mEq/L chloride (Cl -), 2.1 mEq/L magnesium (Mg ++ ) and 31.6 mg/dL of C-reactive protein. Other tests were ordered, such as ferritin, 1650ng/mL, iron 63µg/dL, transferitin 103mg/dL, fibrinogen 412mg/dL. Serology for HBV, HCV, CMV, EBV, HIV, Herpes I and II, VDRL, were all negative. Computed tomography of the chest showed a small bilateral pleural effusion to a greater degree on the right, compressive atelectasis of the pulmonary segments adjacent to the effusion. There was no evidence of ground-glass opacity or consolidations between the parenchyma that would allow the detection of a disease of pulmonary infectious nature detectable by the method. In transitional sections with the upper abdomen, homogeneous hepatomegaly was observed, with no detectable lesions between the parenchyma.
At each episode of febrile neutropenia, blood was collected for blood culture. After blood collection for culture tests and antimicrobial susceptibility, empirical therapy of meropenem and vancomycin was administered for 10 days, in addition anidulafungin was administered for the same period. The febrile neutropenic patient with major impairment of general condition underwent transfusion of blood components. The result of blood culture revealed polymicrobial infection with the presence of Pseudomonas aeruginosa and Escherichia coli on the first day of collection 02/05/20. Only the antimicrobial meropenem was maintained with the result of the culture. On 02/14/20, the patient presented the second peak of NF at 38.4°C, whose blood culture result revealed the presence of Klebsiella pneumoniae and Escherichia coli in the bloodstream. At the third peak of NF (38.4°C) on 02/17/20, Acinetobacter baumanii was observed.
Species identification was carried out by automated testing and biochemical tests. The resistance genes were detected by the molecular technique (Table 1). And the antimicrobial profile was performed using the minimum inhibitory concentration (MIC) ( Table 1).
After the diagnosis, the therapy was adjusted according to the antimicrobial profile, with this, the use of meropenem was maintained, however the patient evolved to death due to the severity of her health status due to septic shock, febrile neutropenia, pneumonia and acute myeloid leukemia.

Antimicrobial susceptibility profile
The antimicrobial susceptibility test was tested using a disc fusion method and microdilution using the lowest inhibitory concentration. Most isolates were resistant to ampicillin, azithromycin, ceftazidime, ciprofloxacin, ceftriaxone and tetracycline. All showed resistance to sulfamethoxazole / trimethoprim by the disk diffusion method (table 1). Resistance to ampicillin, cefepime, ceftriaxone, ciprofloxacin and piperacillin / tazobactam was detected by the minimum inhibitory concentration in most isolates (Table 1).

Detection of the extended-spectrum beta-lactamase resistance genes (ESBL) and metallo-β-lactamase (MβL)
The bacterial isolates were identified and by automated tests and biochemical tests, then they were subcultured in Brain Heart Infusion (BHI) broth for 37ºC at 24 hours, to perform DNA extraction and amplification by Polymerase Chain Reaction (PCR) to detect the resistance genes. DNA extraction was performed using the boiling lysis method. The cell suspension of a culture overnight was boiled at 100 ° C for 10 min and immediately maintained at -20 ° C for at least 6 h. The supernatant was used as a model for PCR amplification and was stored at -20°C. Research, Society andDevelopment, v. 10, n. 5, e39310514985, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i5.14985 4 The presence of ESBL encoding genes was detected by monoplex PCR using primers specific for blaSHV, blaTEM, blaCTX was performed using the following protocol for amplification; initial denaturation 95ºC at 5 min, followed by 30 cycles of denaturation 95ºC at 1 min, annealing 60ºC at 1 min and extension 72ºC at 1 min. Foi realizada uma etapa de extensão final a 72ºC a 10 min, para cada um dos genes estudado. The presence of genes encoding MβL was detected by PCR using specific primers for blaSPM, blaIMP, blaVIM, blaNDM, blaKPC, blaGIM and blaSIM, using the following protocol for amplification; initial denaturation 95ºC at 5 min, followed by 25 cycles of denaturation 95ºC at 1 min, annealing 60ºC at 1 min and extension 72ºC at 1 min.
The strains were also subjected to PCR to detect class D carbapenemases, such as oxacillinase (blaOXA-48, blaOXA-58), using the following protocol for amplification; initial denaturation 94ºC at 5 min, followed by 30 cycles of denaturation 94ºC at 45 sec, annealing 52ºC at 45 sec and extension 72ºC at 1 min. A final extension step was performed at 72ºC at 6 min.
In the first episode of febrile neutropenia, blood culture showed two bacterial isolates in the same sample carrying the blaTEM genes in both isolates of P. aeruginosa and E. coli. The blaSIM and blaOXA-48 genes were also detected in the species, respectively.
In the second episode of NF, two microorganisms were detected the presence of blaKPC e blaNDM, blaSIM, blaOXA-48 e blaCTX cohabiting K. penumoniae and the presence of the blaTEM, blaCTX in E. coli. Genotypic detection of the isolates revealed the presence of at least two resistance genes for the bacterial isolates.

Discussion
Although polymicrobial infection is less frequent in patients with hematological malignancies (Rosa;Goldani, 2014), the risk of mortality is high in these patients. Chemotherapy treatment can induce neutropenia and fever, thereby temporarily decreasing the levels of leukocytes, neutrophils and platelets in the blood, increasing the risk of infection (Lubwama et al., 2019).
Adequate and early empirical therapy is a crucial factor in increasing the survival of these patients. In a study in Hunan, China (Tang et al., 2020), the rate of inadequate initial antimicrobial therapy was classified as a risk factor for early mortality in patients with hematological malignancies associated with febrile neutropenia caused by BGN-BSI.
The prophylaxis with sulfamethoxazole / trimethoprim did not reduce the incidence of infection by gram-negative bacteria. In contrast, the study by Castañón et al., (2019) fluoroquinolone significantly reduced the incidence of carbapenemase and GNB-producing species of Klebisella pneumoniae, resulting in a reduction in mortality in patients with acute myeloid leukemia.
Resistance to cefepime was widely detected in the patient's blood culture isolates, a similar result was observed by Chong et al. (2010). Although the utility of cefepime and piperacillin-tazobactam as an empirical therapy for Enterobactericeae bacteremia in patients with hematological malignancy is widely known in our region, most isolates showed resistance to these, making therapeutic options difficult. Empirical therapy with cefepime or piperacillin-tazobactam adjusted for carbapenems at the beginning of treatment was not linked to increased mortality, as documented in another study (Benanti et al., 2019).
Meropenem was used for the empirical treatment of infections. The effectiveness of meropenem was assessed in a study in Japan (Wakisaka et al., 2015) with febrile neutropenics (81.8%), patients with hematological malignancy (79.2%) and with solid tumor (91.8%). The increase in the use of 4th generation cephalosporins has led to increased resistance, which is often expressed with resistance to other classes of antibiotics needed in the treatment of febrile patients with hematological malignancies such as extended-spectrum cephalosporins and carbapenems.

Conclusion
In conclusion, we present a case of persistent polymicrobial bloodstream infection in a patient with acute myeloid leukemia caused by gram-negative bacteria resistant to β-lactams and carbapenemases. Empirical treatment with cefepime or meropenem has been routinely used in these cases. In the last cultures collected for diagnosis, resistance to meropenem has been observed. The extensive use of antibiotics active against carbapenemases-resistant Enterobactericeae should be avoided, and combinations of empirical antibiotics active against carbapenemases may be suggested as treatment in patients with hematological malignancies in febrile neutropenia. New antibiotics must be produced, as well as the combination of pharmaceuticals can be used to minimize antimicrobial resistance.