Successful Treatment of an AML Patient Infected with Hypervirulent ST463 Pseudomonas Aeruginosa Harboring Rare Carbapenem-Resistant Genes blaAFM-1 and blaKPC-2 Following Allogeneic Hematopoietic Stem Cell Transplantation

Background

As one of the most common nosocomial bacteria, Pseudomonas aeruginosa often causes infections associated with blood, urinary tract, skin and soft tissue.¹ In the treatment of multidrug-resistant P. aeruginosa (MDR-PA), carbapenems have proven to be the most effective antibiotics. However, the extensive use of carbapenems has resulted in a significant increase in carbapenem-resistant P. aeruginosa (CRPA) strains.² The increasing prevalence of CRPA has further elevated the mortality rate, with a 30% 30-day mortality rate even after appropriate treatment,² which was higher in hematological disorder patients with granulocytopenia.

Hematopoietic stem cell transplantation (HSCT) is a crucial treatment for curing hematologic malignancies. Infections often occur during the neutropenic phase of HSCT, which may lead to graft failure or life-threatening complications. In particularly, early-onset severe CRPA infection during HSCT will negatively impact the long-term prognosis because it poses a significant constraint on therapeutic strategies. Therefore, it is important to further understand the drug-resistance mechanisms of P. aeruginosa and make rational choices for anti-infection strategies. Herein, we isolated a strain of bla_KPC-2 ST463 CRPA, resistant to ceftazidime/avibactam, from the bloodstream of a HSCT patient. The final result of whole-genome sequencing (WGS) analysis indicated that this strain simultaneously produces Klebsiella pneumoniae carbapenemase (KPC) and Alcaligenes faecalis metallo-β-lactamase-1(AFM1) enzymes, exhibiting a high level of drug resistance. A literature review retrieved only a few papers describing a novel type of plasmid with multidrug-resistant (MDR) regions, and no case had been reported in HSCT with severe immune deficiency. To further understand these strains, we described the process of CRPA strain isolated from blood and the patient’s treatment course against CRPA to provide resources for clinicians.We also reviewed the available studies on CRPA carrying the blaAFM gene to further study the resistance mechanism of this bacterium.

Case Presentation

A 32-year-old Asian man with hyperleukocytosis was diagnosed with acute myeloid leukemia (AML) in March 2022 (FAB M1), accompanied with CREBBP and ALK mutations, positive fusion gene ETO and t (8;21) (q22; q22.1) cytogenetic abnormality. He achieved partial remission after the first round of induction chemotherapy (cytarabine 100 mg/m² days 1–7 and idarubicin 10 mg/m² days 1, 2 and 3), and achieved complete remission after an additional round of chemotherapy with HAD (homoharringtonine 2 mg days 1–7, idarubicin 10 mg/m² days 1, 2 and 3, and cytarabine 100 mg/m² days 1–7). Subsequently, consolidation therapy was administered with HAD and intermediate-dose cytarabine, however, the patient did not reach genetic minimal residual disease (MRD) negativity throughout the treatment course (Figure 1). Owing to hyperleukocytic AML and insufficient depth of remission, the patient was classified as “high-risk AML”, and allogeneic stem cell transplantation was indicated. A fully-matched unrelated donor was found in the China Bone Marrow Donor Registry for allogeneic-HSCT (allo-HSCT), and the patient underwent conditioning regimen with BUCY (Busulfan-Cyclophosphamide). On Day −5, the patient developed febrile neutropenia during the anti-thymocyte globulin (ATG) application. Laboratory tests revealed an elevated C-reactive protein (CRP) level of 81.45 mg/L (0–8 mg/L) and an increased procalcitonin (PCT) level of 11 μg/L (0–0.046 μg/L), which might also be observed after ATG administration.³ Considering the patient’s neutropenic state, we could not rule out that the fever was caused by an infection, and he was treated with meropenem for 2 days but showed inadequate response. Subsequently, tigecycline was added to cover a wider range of bacterial sources. Finally, the patient’s temperature, CRP and PCT returned to normal.

Figure 1 Minimal residual disease of AML1/ETO expression by quantitative RT-PCR assays. The expression level of fusion gene decreased from 407.55% at diagnosis to 0.00% (MRD negative) 19 months after allo-HSCT (cured).

On the evening of stem cell infusion, the patient developed fever again, with a peak temperature of 40.4°C, accompanied by mild perianal pain. Blood culture and metagenomic next-generation sequencing (mNGS) of peripheral blood specimen were performed immediately. The blood culture results were negative, but mNGS indicated the presence of P. aeruginosa carrying bla_KPC-2 gene. The antibiotics were adjusted to polymyxin B and ceftazidime/avibactam, considering the presence of KPC-producing P. aeruginosa. Additionally, hematopoietic growth factors and transfusion support were provided. The patient’s experienced persistent agranulocytosis and the condition deteriorated with persistent fever ranging from 39–40°C, despite aforementioned antibiotic treatment (Figure 2A and B). The CRP levels progressively increased to over 200 mg/L, and PCT reached a peak of 26 μg/L on Day +3. The perianal pain became increasingly severe, accompanied by local redness and swelling. Moreover, the perianal skin started rupturing and showed signs of suppuration (pus formation) (Figure 3). Due to uncontrolled infection, the patient developed signs of septic shock.

Figure 2 The condition of fever and agranulocytosis during stem cell engraftment. The patient experienced persistent fever (A) during the whole stage of agranulocytic, and the infection got gradually controlled since day +10 when neutrophil engrafted (B).

Figure 3 Changes of the infected perianal skin. (A) showed perianal purulent spot formed on day +6. (B) showed perianal abscess with local rupture on day +8. (C) showed giant perianal ulcer after infection control on day +21. (D), (E) and (F) showed the recovery of perianal ulcer.

Since there were no antibiotic susceptibility results to guide antibiotic regimen adjustments, we invited a colorectal surgeon to perform surgical debridement and provided aggressive life support treatment to stabilize the patient’s condition. On Day +9 after transplantation, a CRPA strain was isolated from a blood culture specimen, with enzyme testing indicating the production of carbapenemase (KPC). CRPA was also identified in the subsequent cultures of perianal secretions. The susceptibility testing (Table 1) showed that the CRPA was resistant to multi-drug including ceftazidime/avibactam and piperacillin-tazobactam, but was sensitive to polymyxin B. Antimicrobial disk combined susceptibility tests (Figure 4A) indicated that when combined with polymyxin B, there was a slight zone of inhibition observed, suggesting some degree of effectiveness against the strain. Therefore, the antimicrobial treatment was adjusted to a combination of polymyxin B, based on the susceptibility results. Ultimately, with aggressive surgical debridement, effective antimicrobial therapy and successful neutrophil engraftment, the infection was brought under control.

Table 1 Susceptibility to Antimicrobial Agents

Figure 4 The evidence of pathogenic bacterium and whole-genome sequencing (WGS) analysis. (A) showed the antimicrobial disk combined susceptibility tests. (B) showed Plasmid ring map of pE45, and (C) presented chromosome and plasmid straight lines.

To further elucidate the resistance mechanism of this KPC-producing P. aeruginosa which exhibited resistance to ceftazidime/avibactam, we performed whole-genome sequencing (WGS) (designated as E45). Results showed that the particular strain belonged to ST463 P. aeruginosa, and carried both the bla_AFM-1 and bla_KPC-2 genes (Figure 4B and C). Antimicrobial resistance genes bla_AFM-1 was detected on the chromosome of E45 located within a conserved region of ISCR29 unit, and bla_KPC-2 was detected on the plasmid located within a conserved region of IS26 unit. This result explained the reason for the extensive drug-resistance exhibited by this strain.

Discussion

P. aeruginosa is one of the most common nosocomial bacteria and often causes infections associated with blood, urinary tract, skin and soft tissue. Carbapenems have been the most efficient antibiotics for treating multidrug-resistant P. aeruginosa. However, with the extensive use of carbapenems, the incidence of CRPA has increased significantly. In recent years, CRPA have been identified as refractory pathogens because of their increased transmissibility and limited treatment options.^2,4

The main mechanisms of resistance to carbapenems in P. aeruginosa involve various factors, including the loss of OprD porin, overexpression of the effector pump and chromosomal β-lactamase AmpC, and acquisition of carbapenemases (such as KPC-2, VIM, NDM, and IMP) through mobile genetic elements like plasmids.^5,6 KPC is a serine protease of the molecular class A and is frequently found in K. pneumoniae.⁷ KPC-producing P. aeruginosa (KPC-PA) was initially reported in 2007, and identified for the first time in Zhejiang in 2011.⁸ Reports showed that KPC-PA has a prevalence of around 40% in the clinical CRPA population in East China, and ST463 is the predominant KPC-producing clone.⁹ ST463 is the predominant strain in cases of bloodstream infection caused by CRPA, accounting for 48.0% of the cases. A studyutilizing¹⁰ multivariate analysis identified three independent risk factors for a fatal outcome: KPC carriage (odds ratio [OR] 4.8; 95% CI 1.0–23.7; P = 0.05), Pitt bacteraemia score (OR 1.3; 95% CI 1.0–1.6; P = 0.02), and underlying hematological disease (OR 8.5; 95% CI 1.6–46.4; P = 0.01). Mortality associated with ST463 CRPA is significantly higher compared to non-ST463 CRPA.¹¹

Bla_AFM-1 CRPA strains are relatively rare. In 2018, a subclass B1b (Metallo-b-lactamase) (MBL) was recognized in an Alcaligenes faecalis strain and named AFM-1. Subsequently, AFM-1 has been found in Comamonas testosteroni, Stenotrophomonas maltophilia and Bordetella trematum. As a novel MBL, AFM alleles have not been widely disseminated in P. aeruginosa (Table 2).^12–17 Piaopiao Zhang¹⁶ first reported the identification of bla_AFM-1 in the chromosome of P. aeruginosa. In a study conducted by Xuefei Zhang,¹⁷,605 consecutive non-duplicate P. aeruginosa isolates were examined, and only three blaAFM-positive strains were identified. BlaAFM-1 is located within a conserved region of groEL-ΔfloR-blaAFM-1-ble-ΔtrpF-ΔISCR27n2-ΔISPme1 -msrB2- msrA- yhgU-corA.¹⁷ An ISCR27-like element, named ISCR29 by Li,¹² inserted into the chromosome of P. aeruginosa is a prerequisite for the insertion of the blaAFM-1-carrying mobile unit, which would enable it to be intrinsically resistant to β-lactam antibiotics, including carbapenems without being subjected to selective pressures. These ISCR29-blaAFM units are highly similar in most plasmids carrying blaAFM1-3, indicating that ISCR29 plays a crucial role in the dissemination of blaAFM. Studies also reported that the co-existence of KPC and MBL in P. aeruginosa can inactivate all b-lactams, making clinical treatment extremely difficult.

Table 2 Related Literatures of PA Strains Isolated Carrying blaAFM

Based on the succeeded treatment process of this patient, the following three points that contributed to his survival are summarized, to help clinicians in the future: a. patients with hematological disorders, especially those with granulocytopenia, have a low positive rate of blood culture after infection. Therefore, it is recommended to perform pathogenic mNGS detection immediately, and adjust antibiotics promptly after obtaining positive results, which will be beneficial for controlling infection; b. for severe infections caused by CRPA, early, adequate, and combination antibiotic therapy is recommended; c. if there is a local soft tissue infection, surgical debridement can be performed if conditions permit, combined with effective anti-infective treatment, which can achieve better results. In addition, we should also keep an eye on side effect of the long-term polymyxin B application. In our case, the patient developed acute renal failure on Day +24 ascribe to the combination of nephrotoxic medications (cyclosporine A, etc.), but fortunately recovered completely with a timely withdraw of polymyxin B and initiate dialysis, without any recurrence of infection. Nevertheless, in cases where patients exhibit renal impairment and are unable to tolerate polymyxin B therapy, a study¹⁸ has shown that cefiderocol exhibits promising efficacy against isolates harboring acquired carbapenemases or PDC variants. It may be considered for use in the treatment thus infections. Ultimately, the patient attained profound molecular remission following negative ETO fusion gene testing (0.00%) 19 months after HSCT.

Conclusion

This rare case highlights the importance of infection prevention in transplant recipients, and strengthening the monitoring of multidrug-resistant bacteria to reduce the incidence and mortality of infections. In conclusion, it is particularly important to identify microbes with rare resistant genes and manage CRPA, especially in immunocompromised patients, using mNGS or WGS. Notably, for CRPA, Polymyxin B is one of the few remaining options for the treatment.