Metallo-Beta-Lactamases: Epidemiological Profile and Clinical Outcome in Gram-Negative Bacteria

Introduction

Infectious diseases currently represent a major public health challenge, requiring health professionals to employ effective strategies for infection prevention and control¹. Among the pathogens associated with these diseases, Gram-negative bacteria stand out, as they are responsible for a significant proportion of healthcare-associated infections (HAIs), contributing to increased morbidity and mortality among hospitalized patients². Despite the effectiveness of antibiotics in controlling bacterial infections, the development of antimicrobial resistance by microorganisms has become a major challenge and has contributed to increased mortality among affected patients³.

Antibiotic resistance can be acquired through several mechanisms, notably the production of enzymes capable of inactivating antimicrobial agents. To overcome this issue, antibiotics that are more effective have been developed to act against resistant bacteria, such as carbapenems. Antibiotics belonging to this class are highly potent and can withstand the action of various inactivating enzymes, including extended-spectrum beta-lactamases (ESBLs ) ³.

However, resistance to carbapenems has become a reality and a challenge for healthcare professionals³. Carbapenemases are enzymes that hydrolyze carbapenems and can facilitate the emergence of pan-resistant bacteria⁴.

Carbapenemases are classified into three beta-lactamase classes (A, B, and D), with class B corresponding to metallo-beta-lactamases (MBLs). These enzymes require zinc ions at their active site to hydrolyze antibiotics⁵. MBLs are mainly found in some Enterobacterales species, Pseudomonas aeruginosa, and Acinetobacter spp. They are capable of hydrolyzing all beta-lactams except aztreonam. Consequently, infections caused by MBL-producing bacteria represent a major concern today⁶.

The detection of bacterial resistance and identification of the underlying resistance mechanism are of fundamental importance in hospital care, strengthening Antimicrobial Stewardship Programs (ASP). This understanding enables comprehensive monitoring of the dissemination of multidrug-resistant strains and the implementation of strategies to prevent outbreaks⁷˒⁸.

Furthermore, understanding resistance mechanisms allows for targeted and effective treatment, avoiding inappropriate and excessive antibiotic use that may otherwise increase selective pressure and resistance development⁷˒⁸.

In this context, the present study aimed to describe the clinical and epidemiological profile of patients infected with MBL-producing Gram-negative bacteria at the University Hospital Complex of the Federal University of Ceará, seeking to understand how these infections affect patient outcomes.

Materials and Methods

This descriptive and retrospective study analyzed the clinical and epidemiological profile of patients infected with metallo-beta-lactamase (MBL)-producing microorganisms treated at the Walter Cantídio University Hospital (HUWC/UFC), part of the Brazilian Hospital Services Company (EBSERH) network. The study was approved by the hospital’s Ethics Committee under protocol number 6,717,757, in accordance with Resolution No. 466 of December 12, 2012, of the Brazilian National Health Council⁹.

Samples were collected in the hospital environment and processed by the institution’s Microbiology Laboratory. Microorganism identification and antimicrobial susceptibility testing (AST) were performed using the automated Vitek-2® system (BioMérieux®, Marcy-l’Étoile, France), except for polymyxin B susceptibility, which was determined manually by broth microdilution. Results were interpreted according to the standards established by the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST, 2025). Data were collected and managed using the REDCap electronic data capture tool.

Patients treated between January 2022 and December 2024 were included. Data collected included sex, age, type of biological sample, hospital ward, isolated microorganism, presence and classification of carbapenemase, antimicrobial susceptibility profile, treatment regimen, and clinical outcome. Only one culture per patient was included, selected randomly.

All data were organized in spreadsheets using Microsoft Excel® 2013. Categorical variables were expressed as absolute and relative frequencies. Statistical analyses included the chi-square test, Student’s t-test, and binary logistic regression using IBM SPSS Statistics v.22, with p < 0.05 considered statistically significant.

Results

A total of 179 positive cultures for MBL-producing bacteria were identified, in addition to 15 bacteria producing dual carbapenemases (serine beta-lactamase and MBL genes). After removing duplicate cultures from the same patient, 133 reports remained, including 12 patients infected with dual-carbapenemase-producing strains and 121 infected with MBL-producing bacteria alone. Of these, 39 cases occurred in 2022, 52 in 2023, and 42 in 2024.

Among the 133 infected patients, 58 (43.6%) were female and 75 (56.4%) were male. Age ranged from 19 to 85 years, with a mean of 57.49 ± 17.13 years. Patients were divided into adults (≤60 years; n = 64, 48.1%) and elderly (>60 years; n = 69, 51.9%). A total of 75 patients (56.4%) were discharged, while 58 (43.6%) died. All of this data is represented in Table 1.

Patients were admitted to all HUWC units; however, the highest incidence of MBL-producing strains occurred in Medical Ward II B (22.6%) and the Intensive Care Unit (ICU) (18.8%). Blood (43.6%) and urine (28.6%) were the most frequent biological samples. Other wards that presented significant occurrences were Medical Ward II A (n = 20, 15.0%), medical ward I (n = 16, 12.0%), renal transplant surgical ward (n = 12, 9.0%), and operating room (n = 10, 7.5%). As for biological samples, the most prevalent were blood (n = 58, 43.6%), followed by urine (n = 38, 28.6%) and tracheal aspirate (n = 15, 11.3%). The complete distribution of patients by ward and sample is shown in Table 2.

Among isolates, 125 (93.9%) were Enterobacterales, with Klebsiella pneumoniae being the most representative (n = 99, 74.4%). The others were infected by Pseudomonas spp. species, with 7 (5.3%) being Pseudomonas aeruginosa and 1 (0.8%) being Pseudomonas mendocina. The NDM gene was detected in 103 (77.4%) cases, followed by IMP (n = 9, 6.8%) and VIM (n = 1, 0.8%) (Table 3).

Regarding antimicrobial sensitivity profiles, the results were analyzed for the main antibiotics used in the therapeutic protocol for patients infected with carbapenem-resistant bacteria, namely Ceftolozane/Tazobactam (CTZ), Ceftazidime/Avibactam (CZA), Amicacin, Gentamicin, Aztreonam, and Polymyxin B. Among the results presented, it was observed that 100% of patients were carriers of strains resistant to CTZ and CZA. In contrast, polymyxin B was the antimicrobial with the highest percentage of sensitivity (n= 101, 75.9%). All results are shown in Table 4.

After performing the chi-square test, it was observed that the clinical outcome was associated with gender (p = 0.044) and inpatient wards (p = 0.010), indicating a higher death rate in female patients and in the Intensive Care Unit (ICU) and Medical Ward II B (Table 5). The other variables were not statistically significant when compared with the outcome.

Table 5: Relationship between clinical outcome and gender and sector of origin of patients treated between January 2022 and December 2024 at the Hospital Complex of the Federal University of Ceará (HUWC/MEAC)..

²Chi-square test revealed p = 0.044 for association between gender and outcome, with 1 degree of freedom. ¹Chi-square test revealed p = 0.010 for association between sector and outcome, with 11 degrees of freedom. Source: Authors

A binary logistic regression was performed to compare Medical Ward II B and the Intensive Care Unit (ICU) with Medical Ward I, where hematological patients are hospitalized. The regression confirmed an increase in the odds of death in these sectors by approximately 5.7 (OR: 5.667, p = 0.019) and 13.7 (OR: 13.722, p = 0.001) times, respectively.

In addition, another significant association was found between the sector of origin and the treatment used (p = 0.010), indicating greater use of the combination Ceftazidime/Avibactam/Aztreonam (CZA + AZT) in patients from Renal Transplant and Medical Ward I (Table 6).

Chi-square test revealed p = 0.010 for association between sector and treatment, with 26 degrees of freedom. 1CZA + AZT: Ceftazidime/Avibactam + Aztreonam. 2PB + AMC: Polymyxin B + Amicacin. Source: Authors

Discussion

Between 2022 and 2024, 133 patients were identified as carriers of strains capable of producing MBLs. During this same period, the laboratory provided care to a total of 5,492 patients in the hospital and outpatient clinic, of whom 2,057 had positive cultures. In other words, 37.45% of patients were affected by infections, and among these, there was a prevalence of MBL-producing strains of 6.46%.

This rate was higher than that reported in similar studies, which found that 4.5% of the analyzed strains were resistant to carbapenems¹⁰. Other investigations in this field did not detect the presence of NDM among multidrug-resistant Gram-negative bacteria¹¹. Additionally, a multicontinental study reported an overall rate of 4.5% of carbapenem-resistant Enterobacteriaceae (CRE), with a higher prevalence of 5.8% observed in Latin America¹².

Despite the worrying result, it is plausible that this increase can be attributed to the passage of time, due to the significant increase in multidrug-resistant bacteria in recent years^13,14. A marked increase of approximately tenfold in NDM genes was observed between 2015 and 2020 in another study¹⁴. However, this observation was not maintained in the present study, since, although there was a significant increase in infections by MBL-producing strains from 2022 to 2023, there was a decrease from 2023 to 2024.

Infections predominantly affected male patients, representing 56.4% of the total. However, mortality was higher in females. These findings are consistent with other similar studies, which also indicate a greater predisposition of males to infections, but greater fragility in females. Another factor that has been shown to increase susceptibility and fragility to infections, according to studies, is age. However, the present study did not identify a significant difference in infections and death rates between populations under and over 60 years of age¹⁵.

The predominance of the NDM gene as the main resistance mechanism found among isolates corroborates the literature, which points to this enzyme as one of the main factors responsible for the global emergence of pan-resistant strains, with the MBL gene being the most common^3,5. The widespread dissemination of strains carrying this gene in the hospital environment is a cause for concern, given its capacity for horizontal transmission by plasmids, which increases the speed of resistance propagation among different bacterial species^5,16.

The predominance of Klebsiella pneumoniae (74.4%) as the main producer of MBLs is consistent with previous studies, which point to this bacterium as one of the major producers of MBLs^6,17.

Furthermore, the higher prevalence of these infections in the Medical Ward II B (22.6%) and ICU (18.8%) sectors is a cause for concern when combined with the results shown by binary logistic regression, in which the risk of death in these sectors was found to be higher. Given the above, it can be inferred that these data are consistent with other studies in the literature, which demonstrate that admission to the intensive care unit is a risk factor for the development of infections by resistant bacteria. Other risk factors include procedures such as mechanical ventilation and intubation, which are highly prevalent in Medical Ward II B, where patients with respiratory problems are concentrated^18,19.

The antimicrobial susceptibility profile showed 100% resistance to CTZ and CZA, which is consistent with the literature, since these antibiotics do not inhibit MBL^20,21. However, the combination with aztreonam restores the efficacy of CZA, making it a viable treatment option²².

Aminoglycosides, especially amicacin, are also a promising option for combating such strains, which makes them frequently used in the treatment of patients carrying multidrug-resistant bacteria. Despite this, the effectiveness of treatment may be related to the sensitivity of the strain to the antibiotics in question. Finally, polymyxin B, despite being the antibiotic that showed the greatest sensitivity, has been shown in other studies to have limited action in monotherapy regimens. However, it has synergistic activity with other antibiotics, making it an effective alternative when used in combination^3,23.

The correlation between the therapeutic plan employed and the sector of origin probably stems from the protocols established by the hospital, which provides priority care to hematological patients in critical condition. In this context, studies show that CZA-based therapies have shown superior results to polymyxin B-based therapies in the treatment of patients affected by infections caused by CREs, making it the preferred treatment for these patients^24,25. In addition, polymyxin B is known to be nephrotoxic, making treatment with this drug unfeasible in patients undergoing kidney transplantation²⁶.

Furthermore, it is important to note that, for this study, data were collected only from strains with phenotypically detected MBL production. This intrinsic limitation in the study restricts the generalization of the results, since MBL production by many strains of P. aeruginosa and, especially, Acinetobacter sp. strains could only be detected by molecular biology methods.

Moreover, all results presented in this study, derived from the routine of the hospital’s clinical microbiology laboratory, are used by the team as a basis for implementing effective PGA and clinical pharmacy actions, thus helping and guiding therapy in a more assertive manner, in addition to being useful for creating indicators as a way to assess the profile of infections in the hospital and the use of antibiotic therapy by hospitalized patients.

Conclusion

Given the above, it can be inferred that the problem of bacterial resistance is not limited to the treatment of infections but involves a complex and multifactorial challenge, ranging from epidemiological surveillance to the development of new antimicrobial therapies. Thus, this study seeks to contribute to the improvement of local knowledge about the resistance profile and may support

In the context of HUWC, the study indicates the need for more careful attention to patients admitted to the ICU and Medical Ward II B. In addition, Ceftazidime-avibactam was used in only 30 of the 133 patients, revealing a failure of public infrastructure, since this drug, despite showing greater efficacy, is a less accessible treatment.

References

1. Bayma JCS, et al. Epidemiologia de doenças infecciosas: estratégias de prevenção e controle em saúde coletiva. Revistaft. 2024;28(134). DOI: 10.5281/zenodo.11507428

2. Sousa JA, et al. Características microbiológicas e perfil clínico de infecções por Stenotrophomonas maltophilia em um hospital de ensino do nordeste brasileiro. Revista Brasileira de Farmácia Hospitalar e Serviços de Saúde. 2024;15(3):e1109. DOI: 10.30968/rbfhss.2024.153.1109

3. Codjoe FS, Donkor ES. Carbapenem Resistance: A Review. Medical sciences. 2017;6(1):1-28. DOI: 10.3390/medsci6010001

4. Oliveira JWA, Paula CC. Bactérias gram-negativas multirresistentes: revisão sobre os desafios e demais discussões. Caderno de Publicações Univag. 2021;1(11). DOI: https://doi.org/10.18312/cadernounivag.v0i11.1651

5. Hansen GT. Continuous Evolution: Perspective on the Epidemiology of Carbapenemase Resistance Among Enterobacterales and Other Gram-Negative Bacteria. Infectious Diseases and Therapy. 2021;10:75–92. DOI: 10.1007/s40121-020-00395-2

6. Reis HC, et al. Delineamento de infecções em um hospital universitário por bactérias produtoras de metalobetalactamases: mapeando o inimigo! Revista Brasileira de Farmácia Hospitalar e Serviços de Saúde. 2023;14(4):979. DOI: 10.30968/rbfhss.2023.144.0979

7. Gagetti P, et al. Evolución del desempeño de Laboratorios de Referencia de América Latina en la detección de mecanismos de resistencia a los antimicrobianos. Rev Panam Salud Publica. 2020;44:e42. DOI: https://doi.org/10.26633/RPSP.2020.42

8. Ramos JR, Galleymore PR. Programas de optimización de antibióticos en la unidad de cuidados intensivos en caso de infecciones por bacilos gramnegativos multiresistentes, Medicina Intensiva. 2023;47(2):99-107. DOI: 10.1016/j.medin.2022.07.007

9. Resolução n.º 466 de 12 de dezembro de 2012 do Conselho Nacional de Saúde.

10. Cai B, et al. Prevalence of Carbapenem-Resistant Gram-Negative Infections in the United States Predominated by Acinetobacter baumannii and Pseudomonas aeruginosa. Open Forum Infectious Diseases. 2017;16;4(3):ofx176. DOI: 10.1093/ofid/ofx176

11. Viegas DM, Soares VM. Prevalência de carbapenemases em enterobactérias com sensibilidade diminuída aos carbapenêmicos isoladas em um hospital de referência terciária. Jornal Brasileiro de Patologia e Medicina Laboratorial. 2018;54(2):95-98. DOI:  https://doi.org/10.5935/1676-2444.20180017

12. Sader HS, et al. Aztreonam/avibactam activity against clinical isolates of Enterobacterales collected in Europe, Asia and Latin America in 2019. Journal of  Antimicrobial Chemotherapy. 2021;76(3):659-666. DOI: 10.1093/jac/dkaa504

13. Ministério da Saúde. Nota técnica conjunta CGLAB, BrCAST e Anvisa. Nota técnica nº 74, 2022.

14. Pillonetto M, et al. CARBA-LAC Group. Carbapenemases producing gram-negative bacteria surveillance in Latin America and the caribbean: a retrospective observational study from 2015 to 2020. Lancet Reg Health Am. 2025;49:101185. DOI: 10.1093/jac/dkaa504

15. Lima SA, et al. Prevalência de New Delhi Metallo-β-Lactamase (NDM) em pacientes de um serviço público. Research, Society and Development. 2024;13(12): e194131247890 DOI:

16. Rios VM, Almeida MTG. Carbapenemases: Um problema em evolução. Academia de Ciência e Tecnologia. 2015. DOI: 10.33448/rsd-v13i12.47890

17. Tenorio CH, et al. The Challenge of Treating Infections Caused by Metallo‐β‐

Lactamase–Producing Gram-Negative Bacteria: A Narrative Review. Drugs. 2024;84:1519-1539. DOI: 10.1007/s40265-024-02102-8

18. Ulu AC, et al. Risk factors of carbapenem-resistant Klebsiella pneumoniae infection: a serious threat in ICUs. Medical Science Monitor. 2015;21:219-224. DOI: 10.12659/MSM.892516

19. Wang Z, et al. Risk Factors for Carbapenem-resistant Klebsiella pneumoniae Infection and Mortality of Klebsiella pneumoniae Infection. Chinese Medical Journal. 2018;131(1):56-62. DOI: 10.4103/0366-6999.221267

20. Mehwish A, Iftikhar I. Emergence of ceftazidime-avibactam resistance in enterobacterales and Pseudomonas aeruginosa. Pakistan Journal of Pathology. 2023;34(4):113-117. DOI: 10.55629/pakjpathol.v34i4.755

21. Estabrook MSM, et al. P-1507. In Vitro Activity of Ceftazidime-avibactam against Enterobacterales Isolates Producing Multiple β-lactamases Collected Globally as a Part of the ATLAS Global Surveillance Program from 2018-2022. Open Forum Infectious Diseases. 2025;12:ofae631.1676. DOI: 10.1093/ofid/ofae631.1676

22. Patel AM, et al. Use of Aztreonam Plus Ceftazidime/Avibactam Combination for In vitro Susceptibility Testing of CRO Isolates in a Tertiary Care Hospital – Our experience. Journal of Marine Medical Society. 2024;26(2):278-282. DOI: 10.4103/jmms.jmms_171_23

23. Smith NM, et al. Mechanistic Insights to Combating NDM- and CTX-M-Coproducing Klebsiella pneumoniae by Targeting Cell Wall Synthesis and Outer Membrane Integrity. Antimicrobial Agents and Chemotherapy. 2022;66(9):e00527-22. DOI: 10.1128/aac.00527-22

24. Soman R, et al. Is it time to move away from polymyxins?: evidence and alternatives. European Journal of Clinical Microbiology & Infectious Diseases. 2021;40:461-475. DOI: 10.1007/s10096-020-04053-w

25. Prayag, PS. Ceftazidime-avibactam with or without Aztreonam vs Polymyxin-based Combination Therapy for Carbapenem-resistant Enterobacteriaceae: A Retrospective Analysis. Indian Journal of Critical Care Medicine. 2023;27(6):444-450. DOI: 10.5005/jp-journals-10071-24481

26. Zheng G, et al. Ceftazidime/Avibactam-Based Versus Polymyxin BBased Therapeutic Regimens for the Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infection in Critically Ill Patients: A Retrospective Cohort Study. Infectious Diseases and Therapy. 2022;11:1917-1934. DOI: 10.1007/s40121-022-00682-0