Abstract
Despite aggressive antibiotic therapy and surgical debridement, Aeromonas necrotizing fasciitis (NF) can lead to high amputation and mortality rates. Our study compares the different antibiotic minimum inhibitory concentrations (MICs) via Epsilometer tests (E-tests) between non-survivors and survivors of Aeromonas NF of limbs. A prospective review of 16 patients with Aeromonas NF was conducted for 3.5 years in a tertiary coastal hospital. E-tests were conducted for 15 antimicrobial agents to determine the MIC value for Aeromonas species. These patients were divided into non-survival and survival groups. The clinical outcomes, demographics, comorbidities, presenting signs and symptoms, laboratory findings, and microbiological results between the two periods were compared. A total of four patients died, whereas 12 survived, resulting in a 25% mortality rate. A higher proportion of bloodstream infections (100% vs. 41.7%; p = 0.042), monomicrobial infections (100% vs. 33.3%; p = 0.021), shock (100% vs. 33.3%; p = 0.021), serous bullae (50% vs. 0%; p = 0.009), liver cirrhosis (100% vs. 25%; p = 0.009), chronic kidney disease (100% vs. 33.3%; p = 0.021), lower susceptibility to cefuroxime (25% vs. 83.3%; p = 0.028), and ineffective antibiotic prescriptions (75% vs. 16.7%; p = 0.029) was observed in non-survivors. Aeromonas NF is an extremely rare skin and soft-tissue infection that is associated with high mortality, bacteremia, antibiotic resistance, and polymicrobial infection. Therefore, antibiotic regimen selection is rendered very challenging. To improve clinical outcomes and irrational antimicrobial usage, experienced microbiologists can help physicians identify specific pathogens and test MIC.
Keywords: necrotizing fasciitis, Aeromonas, antimicrobial susceptibility, and minimum inhibitory concentrations
1. Introduction
Currently, antibiotic resistance remains one of the greatest threats, and Aeromonas species are no exception [1,2]. The genus Aeromonas is a Gram-negative rod found in freshwater, estuaries, and marine environments [1,2]. Necrotizing fasciitis (NF) caused by Aeromonas species is a rare but potentially life-threatening necrotizing skin and soft-tissue infection (NSSTI) characterized by rapidly spreading necrosis, particularly in the fascia [3,4,5]. Aeromonas NF has a high mortality (26.4–100%) [3,4,5,6,7] and amputation rate (26.4–50%) despite aggressive surgical and antibiotic treatment [3,4,5,6].
Chia-Yi Chang Gung Memorial Hospital (CGMH) is a tertiary coastal hospital located in West Taiwan. Often, residents of this region encounter raw seafood, seawater, brackish water, and soil. Thus, Aeromonas species and Vibrio species infections are relatively common [3,4,5,6,7,8,9,10]. Therefore, we set up the Vibrio NSSTI Treatment and Research (VTR) Group with professional staff from specialties such as emergency medicine, intensive care unit (ICU), orthopedic surgery, infectious diseases (ID), and plastic surgery, with a hyperbaric oxygen treatment center [5,6,11,12]. A protocol was developed to diagnose and treat NF with broad-spectrum antibiotics with a third-generation cephalosporin plus glycopeptide, and a hospital-wide computerized antimicrobial approval system (HCAAS) was implemented to ascertain appropriate antibiotic use [6].
Microbiologists can perform prompt identification of organisms and pathogen susceptibility patterns. Despite the existence of the VTR Group for many years, microbiologists with extensive experience are still lacking. In clinical microbiology laboratories, a disk-diffusion method for routine antimicrobial susceptibility testing (AST) measures drug efficacy against bacteria. The Epsilometer Test (E-test) is a quantitative method for determining the antimicrobial susceptibility and minimum inhibitory concentrations (MICs) of Gram-negative and Gram-positive aerobic bacteria. In this test device, antibiotic concentrations are measured along an exponential gradient and immobilized on a rectangular plastic strip. Additionally, we collected Aeromonas isolates and tested MICs using an E-test during this study period. This study aims to determine whether non-survivors of Aeromonas NF have different antibiotic susceptibility results than survivors.
2. Materials and Methods
2.1. Study Design and Setting
This is a prospective study of 25 Aeromonas species isolated in 16 patients with NF by the VTR Group from April 2015 to August 2018 at our institution. Patients admitted to the emergency department with skin and soft-tissue infections were enrolled. NF of the limbs was established through surgical or histopathologic findings, and Aeromonas species were identified using conventional biochemical tests, API-20E Systems, or MALDI-TOF MS. Only patients with Aeromonas species infection whose isolates can be re-tested for MIC by an E-test can join this study (Figure 1). Patients were categorized into survival and non-survival groups. The demographics, comorbidities, antimicrobial susceptibility, clinical outcomes, presenting signs and symptoms, and laboratory findings were compared between the two groups.
Figure 1.
A flowchart showing the clinical isolates of Aeromonas strains collected from 16 patients with necrotizing fasciitis.
2.2. Definitions
A diagnosis of Aeromonas NF is defined as follows: (i) histological or surgical examination findings, such as skin necrosis, subcutaneous fat, superficial fascia, or underlying muscles; (ii) Aeromonas spp. was isolated from soft-tissue lesions or blood during an emergency room (ER) visit or surgery [5,11,13]. Empirical antimicrobial regimens were defined to cover all infectious pathogens according to their antimicrobial susceptibilities [9,14]. MIC50 is the MIC that inhibited 50% of bacterial growth, and MIC90 is the MIC that inhibited 90% of bacterial growth.
2.3. Laboratory Procedures for Microbiology
The Aeromonas species consist of oxidase-positive, polar flagellated, glucose-fermenting, facultatively anaerobic, motile bacteria that do not grow in Gram-negative rods containing 6.5% NaCl. Conventional methods and API-20E Systems (bioMérieux Inc., Hazelwood, MO, USA) were used to identify all strains. For further verification, MALDI-TOF MS (Bruker Daltonik, Bremen, Germany) was used.
2.4. Antimicrobial Susceptibility Testing
The disk-diffusion method was used to routinely evaluate all strains for resistance against 10 antimicrobial agents, including cefuroxime, ceftriaxone, ceftazidime, gentamicin, amikacin, ertapenem, ciprofloxacin, levofloxacin, tetracycline, and sulfamethoxazole-trimethoprim. A MIC value for Aeromonas species was determined using the E-test method, performed for 15 antimicrobial agents, including ampicillin, ampicillin-sulbactam, cefuroxime, ceftriaxone, ceftazidime, cefepime, gentamicin, amikacin, ertapenem, imipenem, meropenem, ciprofloxacin, levofloxacin, tetracycline, and tigecycline purchased from AB BIODISK (Solna, Sweden) according to the CLSI M45 second edition [15].
2.5. Disk-Diffusion Method
Several Aeromonas colonies are selected with a sterile inoculating loop. The organism is suspended in 2 mL of Tryptic Soy Broth (TSB). A smooth suspension is created by vortexing the TSB tube. To match a 0.5 McFarland standard, turbidity is adjusted visually using TSB. Within 15 min of preparation, this suspension is used. A sterile cotton swab is placed in the inoculum tube. The dried surface of an MH agar plate is inoculated by streaking the swab three times across the entire agar surface. Disks are applied to the agar surface with a dispenser and then incubated for 16–18 h at 35℃ in an ambient air incubator. The inhibition zone diameters are then measured to interpret the susceptibility test results according to the CLSI M45 second edition.
2.6. E-Test
Several Aeromonas colonies are selected with a sterile inoculating loop. The organism is suspended in 2 mL TSB. A smooth suspension is created by vortexing the TSB tube. To match a 0.5 McFarland standard, turbidity is adjusted visually using TSB. Within 15 min of preparation, this suspension is used. A sterile cotton swab is placed in the inoculum tube. The dried surface of an MH agar plate is inoculated by streaking the swab three times across the entire agar surface. The strip is then placed with the “E end” at the edge of the plate and with the scale visible. Plates are then incubated at 35 °C for 16–20 h. The susceptibility test results are interpreted according to the CLSI M45 2nd edition.
2.7. Statistical Analysis
To determine predictors of non-survivors, a logistic regression model analysis was conducted. Chi-square tests were used to test categorical variables, and Student’s t-tests and ANOVA were used to test continuous variables. Statistical significance was determined by p-values < 0.05 with two-tailed tests. Statistical analysis was conducted using SPSS version 25.0 for Windows (Chicago, IL, USA).
3. Results
3.1. Patient Selection and Clinical Isolates
From April 2015 to August 2018, 22 patients admitted via the ER were diagnosed with Aeromonas NF of the limbs (Figure 1). Furthermore, 25 Aeromonas isolates were collected from 16 patients. We obtained 10 isolates from wounds, nine from blood, four from tissue, and two from pus. The MICs of Aeromonas strains isolated from 16 patients with NF of the limbs are shown in Table 1. Table 2 shows susceptibility, intermediate status, and resistance for 16 Aeromonas NF patients as determined by the disk-diffusion and E-test methods, as well as the MIC50, MIC90, and MIC range using the E-test method. Among the Aeromonas species, ampicillin resistance is the highest (50%), followed by cefuroxime (25%), using the E-test method. However, in the disk-diffusion method, only 12.5% showed cefuroxime resistance.
Table 1.
MIC a of Aeromonas strains isolated from 16 patients with necrotizing fasciitis of limbs to different antimicrobial agents.
| Case No. | AMP | SAM | CXM | CRO | CAZ | FEP | GM | AN | ETP | IMP | MEM | CIP | LVX | TC | TGC |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 128 | 16 | 1 | 1 | 0.064 | 0.032 | 0.19 | 0.75 | 4 | 4 | 0.032 | 0.004 | 0.012 | 0.75 | 0.032 |
| 2 | 256 | 16 | 0.75 | 0.5 | 0.096 | 0.047 | 0.25 | 2 | 4 | 4 | 0.032 | 0.008 | 0.016 | 1.5 | 1 |
| 3 | 2 | 2 | 3 | 0.023 | 0.19 | 1 | 0.125 | 1.5 | 0.008 | 0.5 | 0.047 | 0.094 | 0.25 | 0.5 | 0.064 |
| 4 | 256 | 32 | 1 | 0.5 | 0.064 | 0.047 | 0.19 | 2 | 0.125 | 0.38 | 0.094 | 4 | 0.38 | 24 | 0.38 |
| 5 | 256 | 64 | 1 | 1 | 0.125 | 0.094 | 0.19 | 1.5 | 8 | 0.38 | 0.032 | 0.006 | 0.016 | 1 | 0.5 |
| 6 | 256 | 24 | 1.5 | 1 | 0.19 | 0.064 | 0.25 | 1.5 | 0.38 | 0.38 | 0.094 | 0.006 | 0.016 | 1 | 0.38 |
| 7 | 1 | 1 | 2 | 0.016 | 0.19 | 0.5 | 1 | 4 | 0.002 | 0.5 | 0.012 | 0.016 | 0.032 | 1 | 0.032 |
| 8 | 1.5 | 2 | 2 | 0.023 | 0.19 | 1 | 0.25 | 1.5 | 4 | 0.5 | 0.047 | 0.125 | 0.19 | 0.5 | 0.064 |
| 9 | 2 | 1.5 | 32 | 4 | 0.125 | 1 | 0.125 | 1.5 | 0.008 | 0.75 | 0.032 | 0.125 | 0.25 | 0.5 | 0.047 |
| 10 | 256 | 24 | 1.5 | 0.023 | 0.19 | 0.094 | 0.25 | 1.5 | 0.008 | 0.5 | 0.047 | 0.125 | 0.032 | 0.5 | 0.047 |
| 11 | 256 | 2 | 32 | 4 | 16 | 0.5 | 0.38 | 1.5 | 0.38 | 0.5 | 0.047 | 0.006 | 0.19 | 0.5 | 0.38 |
| 12 | 2 | 2 | 1 | 1 | 0.125 | 0.5 | 0.125 | 1.5 | 0.008 | 0.75 | 0.032 | 0.125 | 0.25 | 0.5 | 0.047 |
| 13 | 2 | 2 | 3 | 0.023 | 0.19 | 1 | 0.125 | 1.5 | 0.38 | 0.75 | 0.047 | 0.006 | 0.25 | 0.5 | 0.38 |
| 14 | 2 | 1.5 | 16 | 0.032 | 0.125 | 1 | 0.125 | 1.5 | 0.008 | 0.5 | 0.032 | 0.125 | 0.25 | 0.5 | 0.047 |
| 15 | 2 | 2 | 3 | 0.023 | 0.19 | 1 | 0.38 | 1.5 | 0.008 | 0.75 | 0.047 | 0.125 | 0.25 | 0.5 | 0.047 |
| 16 | 256 | 64 | 32 | 4 | 16 | 1 | 8 | 1.5 | 0.008 | 0.75 | 0.047 | 0.125 | 0.25 | 16 | 0.047 |
Abbreviations: a MIC: minimum inhibitory concentration; AMP: ampicillin; SAM: ampicillin-sulbactam; CXM: cefuroxime; CRO: ceftriaxone; CAZ: ceftazidime; FEP: cefepime; GM: gentamicin; AN: amikacin; ETP: ertapenem; IMP: imipenem; MEM: meropenem; CIP: ciprofloxacin; LVX: levofloxacin; TC: tetracycline; TGC: tigecycline.
Table 2.
Susceptibilities of Aeromonas strains isolated from 16 patients by CLSI a breakpoint interpretation.
| Antimicrobial Agents | MIC Breakpoint (μg/mL) | MIC (ug/mL) | E-Test (%) | Zone Diameter Breakpoint (mm) | Disk Diffusion (%) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| S | I | R | MIC50 b | MIC90 c | S | I | R | S | I | R | S | I | R | |
| Ampicillin | ≤8 | 16 | ≥32 | 128 | 256 | 50 | 50 | ≥17 | 14–16 | ≤13 | ||||
| Unasyn d | ≤8/4 | 2 | ≥32/16 | 2 | 32 | 56.3 | 25 | 18.8 | ≥15 | 12–14 | ≤11 | |||
| Cefuroxime | ≤1 | 2 | ≥4 | 2 | 32 | 68.8 | 6.3 | 25 | ≥26 | 23–25 | ≤22 | 75 | 12.5 | 12.5 |
| Ceftriaxone | ≤1 | 2 | ≥4 | 0.032 | 4 | 81.3 | 18.8 | ≥23 | 20–22 | ≤19 | 81.3 | 18.8 | ||
| Ceftazidime | ≤4 | 8 | ≥16 | 0.125 | 0.19 | 87.5 | 12.5 | ≥21 | 18–20 | ≤17 | 87.5 | 12.5 | ||
| Cefepime | ≤8 | 16 | ≥32 | 0.5 | 1 | 100 | ≥18 | 15–17 | ≤14 | |||||
| Gentamicin | ≤4 | 8 | ≥16 | 0.19 | 1 | 93.8 | 6.3 | ≥15 | 13–14 | ≤12 | 93.8 | 6.3 | ||
| Amikacin | ≤16 | 32 | ≥64 | 0.75 | 2 | 100 | ≥17 | 15–16 | ≤14 | 100 | ||||
| Ertapenem | ≤2 | 4 | ≥8 | 0.125 | 4 | 75 | 12.5 | 12.5 | ≥19 | 16–18 | ≤15 | 68.8 | 18.8 | 12.5 |
| Imipenem | ≤4 | 8 | ≥16 | 0.5 | 0.75 | 87.5 | 12.5 | ≥16 | 14–15 | ≤13 | ||||
| Meropenem | ≤4 | 8 | ≥16 | 0.032 | 0.047 | 100 | ≥16 | 14–15 | ≤13 | |||||
| Ciprofloxacin | ≤1 | 2 | ≥4 | 0.016 | 0.125 | 93.8 | 6.3 | ≥21 | 16–20 | ≤15 | 93.8 | 6.3 | ||
| Levofloxacin | ≤2 | 4 | ≥8 | 0.19 | 0.25 | 100 | ≥17 | 14–16 | ≤13 | 100 | ||||
| Tetracycline | ≤4 | 8 | ≥16 | 0.5 | 16 | 87.5 | 12.5 | ≥15 | 12–14 | ≤11 | 87.5 | 12.5 | ||
| Tigecycline | ≤2 | 4 | ≥8 | 0.047 | 0.5 | 100 | ||||||||
| TMP-SMX e | ≤2/38 | - | ≥4/76 | ≥16 | 11–15 | ≤10 | 81.3 | 18.8 | ||||||
Abbreviations: a CLSI: Clinical and Laboratory Standards Institute; b MIC50: minimum inhibitory concentrations that inhibited 50% of the isolates; c MIC90: minimum inhibitory concentrations that inhibited 90% of the isolates; d Unasyn: Ampicillin-sulbactam; e TMP-SMX: Trimethoprim-Sulfamethoxazole. S, I, and R stand for susceptible, intermediate, and resistant, respectively.
3.2. Microbiological Analysis and Empiric Antibiotics
Aeromonas hydrophila accounted for 14 (87.5%) of the 16 Aeromonas NF patients, followed by one Aeromonas caviae (6.25%) and one Aeromonas veronii biovar sobria (6.25%). The data for 16 cases of Aeromonas necrotizing fasciitis are summarized in Table 3.
Table 3.
Summary of data for 16 cases of Aeromonas necrotizing fasciitis.
| Case No. | Age/ Gender |
Underlying Disease | Antibiotic Regimen at ER a | First Types of Surgery | Outcome |
|---|---|---|---|---|---|
| 1 | 63/M | Hepatitis B | Ceftriaxone, vancomycin | Fasciectomy | Recovered |
| 2 | 64/M | CKD b under H/D c, DM d, hepatitis C | Ceftriaxone | Debridement | Recovered |
| 3 | 57/M | Alcoholism, DM, hepatitis C | Ceftriaxone, doxycycline | Fasciectomy | Recovered |
| 4 | 76/M | DM, lymphoma, prostate cancer | Ceftriaxone, vancomycin | Fasciectomy | Recovered |
| 5 | 46/M | Alcoholism, DM, liver cirrhosis | Ceftriaxone, vancomycin, metronidazole | Fasciectomy | Recovered |
| 6 | 77/M | Not evident | Ceftriaxone, teicoplanin, metronidazole |
Fasciectomy | Recovered |
| 7 | 49/M | Alcoholism, CKD | Ceftriaxone, teicoplanin, doxycycline |
Fasciectomy | Recovered |
| 8 | 63/M | Alcoholism, DM | Cefazolin, doxycycline | Fasciectomy | Recovered |
| 9 | 52/M | Alcoholism, CKD, DM, hepatitis B, liver cirrhosis | Ceftriaxone, doxycycline | Fasciectomy | Died |
| 10 | 58/M | Not evident | Ceftriaxone, doxycycline | Fasciectomy | Recovered |
| 11 | 65/F | DM, hepatitis C | Ceftriaxone, metronidazole, doxycycline | Fasciectomy | Recovered |
| 12 | 54/M | Alcoholism, CKD under H/D, DM, hepatoma, liver cirrhosis | Ceftriaxone, metronidazole, doxycycline | Fasciectomy | Died, amputation |
| 13 | 47/M | Alcoholism, CKD under H/D, DM, hepatitis B, hepatoma, liver cirrhosis | Ceftriaxone, teicoplanin, doxycycline |
Fasciectomy | Died |
| 14 | 79/M | Colon cancer, CKD, DM, hepatitis B & C, liver cirrhosis |
Ceftriaxone, doxycycline | Fasciectomy | Recovered |
| 15 | 79/M | Alcoholism, CKD, hepatitis C, liver cirrhosis | Ceftriaxone, amikacin, doxycycline |
Fasciectomy | Recovered |
| 16 | 59/M | Alcoholism, CKD under H/D, liver cirrhosis | Ceftriaxone | Debridement | Died |
Abbreviations: a ER: emergency room; b CKD: chronic kidney disease; c H/D: hemodialysis; d DM: Diabetes mellitus.
The non-survival and survival group included 4 and 12 patients, respectively, resulting in a 25% mortality rate. Additionally, we found a higher proportion of bloodstream (100% vs. 41.7%; p = 0.042) and monomicrobial infections (100% vs. 33.3%; p = 0.021) in non-survivors (Table 4). In the Aeromonas susceptibility to cefuroxime, the non-survival group had a statistically significant lower susceptibility (25% vs. 83.3%; p = 0.028). In the ER, the non-survival group had a higher rate of ineffective antibiotic prescriptions (75% vs. 16.7%; p = 0.029) (Table 4). Three non-survivals were prescribed ceftriaxone at the ER, two were changed to ciprofloxacin because of their resistance, and one was re-adjusted to levofloxacin because of the co-infection of pneumonia and respiratory failure. Based on antibiotic susceptibility, two survivors were prescribed cefazolin at the ER and escalated to cefuroxime.
Table 4.
Microbiological results, antimicrobial susceptibilities, and antimicrobial usage for 16 patients between non-survival and survival groups.
| Variable | Non-Survival (n = 4) |
Survival (n = 12) |
p-Value |
|---|---|---|---|
| Bloodstream infection | 4 (100) | 5 (41.7) | 0.042 * |
| Monomicrobial infection | 4 (100) | 4 (33.3) | 0.021 * |
| Susceptibility (MIC interpretation) | |||
| Ampicillin | 3 (75) | 5 (41.7) | 0.248 |
| Ampicillin-sulbactam | 3 (75) | 6 (50) | 0.411 |
| Cefuroxime | 1 (25) | 10 (83.3) | 0.028 * |
| Ceftriaxone | 2 (50) | 11 (91.6) | 0.064 |
| Ceftazidime | 3 (75) | 11 (91.6) | 0.383 |
| Cefepime | 4 (100) | 12 (100) | - |
| Gentamicin | 3 (75) | 12 (100) | 0.074 |
| Amikacin | 4 (100) | 12 (100) | - |
| Ertapenem | 4 (100) | 8 (66.7) | 0.411 |
| Imipenem | 4 (100) | 10 (83.3) | 0.383 |
| Meropenem | 4 (100) | 12 (100) | - |
| Ciprofloxacin | 4 (100) | 11 (91.6) | 0.551 |
| Levofloxacin | 4 (100) | 12 (100) | - |
| Tetracycline | 3 (75) | 11 (91.6) | 0.383 |
| Tigecycline | 4 (100) | 12 (100) | - |
| Colistin | 0 (0) | 5 (41.7) | 0.126 |
| Ineffective empirical antimicrobial usage at ER | 3 (75) | 2 (16.7) | 0.029 * |
Data were presented as frequency (%). *: p-value < 0.05.
3.3. Data on Demographics, Characteristics, and Clinical Outcomes
Patients in the non-survival group required more ICU care (100% vs. 41.7%; p = 0.042) and postoperative intubation (100% vs. 25%; p = 0.009) and had higher rates of alcoholism (100% vs. 41.7%; p = 0.042), liver cirrhosis (100% vs. 25%; p = 0.009), and chronic kidney disease (100% vs. 33.3%; p = 0.021) than those in the survival group. Non-survival patients had a statistically longer ICU stay (days) (18.3 ± 15.0 vs. 1.1 ± 1.8; p = 0.001) (Table 5).
Table 5.
Demographic data, comorbidities, and clinical outcomes of 16 patients between non-survival and survival groups.
| Variable | Non-Survival (n = 4) |
Survival (n = 12) |
p-Value |
|---|---|---|---|
| Age (years) | 53.0 ± 5.0 | 64.7 ± 11.3 | 0.069 |
| Sex, male | 4 (100) | 11 (91.6) | 0.551 |
| Involved region | 0.182 | ||
| Lower extremities | 4 (100) | 8 (66.7) | |
| Disease severity | |||
| APACHE a II score | 20.0 ± 5.4 | 14.3 ± 5.9 | 0.109 |
| ICU b stay | 4 (100) | 5 (41.7) | 0.042 * |
| Postoperative intubation | 4 (100) | 3 (25) | 0.009 * |
| Underlying chronic diseases | |||
| Chronic liver dysfunction | 4 (100) | 9 (75) | 0.267 |
| Alcoholism | 4 (100) | 5 (41.7) | 0.042 * |
| Viral hepatitis | 2 (50) | 6 (50) | 1.000 |
| Liver cirrhosis | 4 (100) | 3 (25) | 0.009 * |
| Chronic kidney disease | 4 (100) | 4 (33.3) | 0.021 * |
| Cancer | 2 (50) | 2 (16.7) | 0.182 |
| Diabetes mellitus | 3 (75) | 7 (58.3) | 0.551 |
| Misdiagnosis at ER | 1 (25) | 2 (16.7) | 0.712 |
| Methods of the first operation | 0.383 | ||
| Fasciectomy | 3 (75) | 11 (91.6) | |
| Debridement | 1 (25) | 1 (8.3) | |
| Number of surgical operations | 2.5 ± 1.7 | 2.8 ± 1.4 | 0.769 |
| Clinical outcomes | |||
| Number of amputations | 1 (25) | 0 (0) | 0.074 |
| ICU stay (days) | 18.3 ± 15.0 | 1.1 ± 1.8 | 0.001 * |
| Hospital stays (days) | 19.0 ± 17.3 | 35.9 ± 15.4 | 0.086 |
Data were presented as mean (standard deviation) or frequency (%). *: p-value < 0.05. a APACHE: Acute Physiology and Chronic Health Evaluation, b ICU: intensive care unit.
3.4. Surgical Treatment
As for the first surgery, 14 (87.5%) and two (12.5%) patients received fasciotomy and debridement, respectively (Table 5). Between the two groups, no difference was found in the first surgical method and the number of surgical operations or amputations.
3.5. Clinical Presentations
No significant differences between the two groups were found in the symptom and sign duration, presence of fever (body temperature > 38 °C), tachycardia (heart rate > 100/min), or tachypnea (respiratory rate > 20/min). Also, no difference was found between the two groups in the proportion of patients with erythematous, swollen, painful lesions, hemorrhagic bullae, and skin necrosis. The proportion of patients presenting with shock (systolic blood pressure < 90 mmHg, 100% vs. 33.3%; p = 0.021) and serous bullae (50% vs. 0%; p = 0.009) was higher in the non-survival groups (Table 6).
Table 6.
Clinical presentations of 16 patients with Aeromonas necrotizing fasciitis between non-survival and survival groups.
| Variable | Non-Survival (n = 4) |
Survival (n = 12) |
p-Value |
|---|---|---|---|
| Duration of symptoms/signs (days) | 1.5 ± 1.0 | 1.5 ± 0.8 | 1.000 |
| Symptoms/signs of systemic disease | |||
| Shock a | 4 (100) | 4 (33.3) | 0.021 * |
| Tachycardia b | 4 (100) | 9 (75.0) | 0.276 |
| Tachypnea c | 2 (50) | 5 (41.7) | 0.771 |
| Fever d | 1 (25) | 3 (25) | 1.000 |
| Symptoms/signs of the limbs | |||
| Pain and tenderness | 4 (100) | 11 (91.6) | 0.551 |
| Swelling and erythema | 4 (100) | 9 (75.0) | 0.267 |
| Hemorrhagic bullae | 2 (50) | 4 (33.3) | 0.551 |
| Serous bullae | 2 (50) | 0 (0) | 0.009 * |
| Skin necrosis | 2 (50) | 1 (8.3) | 0.064 |
Data were presented as mean (standard deviation) or frequency (%).*: p-value < 0.05. a Shock: systolic blood pressure < 90 mmHg; b Tachycardia: heartbeat > 100/min; c Tachypnea: respiratory rate > 20/min; d Fever: body temperature > 38 °C.
3.6. Laboratory Findings
Hemoglobin < 10 g/dL was detected more frequently in the non-survival group (Table 7). Also, the non-survival group had a longer prothrombin time (p < 0.001), activated partial thromboplastin time (p = 0.002), and higher serum total bilirubin (p = 0.005).
Table 7.
Laboratory findings of 16 patients with Aeromonas necrotizing fasciitis between non-survival and survival groups.
| Variable | Non-Survival (n = 4) |
Survival (n = 12) |
p-Value |
|---|---|---|---|
| Total WBC a count | |||
| Leukocytosis (≥12,000/μL) | 1 (25) | 9 (75) | 0.074 |
| Leukopenia (≤4000/μL) | 1 (25) | 1 (8.3) | 0.383 |
| Differential count | |||
| Neutrophilia (>7500/μL) | 2 (50) | 9 (75) | 0.350 |
| Band forms (>10%) | 2 (50) | 4 (33.3) | 0.551 |
| Lymphocytopenia (<1000/μL) | 4 (100) | 7 (58.3) | 0.119 |
| Hemoglobin < 10 g/dL | 3 (75) | 1 (8.3) | 0.008 * |
| Thrombocytopenia (platelet count < 15 × 104/μL) | 4 (100) | 6 (50) | 0.074 |
| eGFR b < 30 c.c./min | 3 (75) | 3 (25) | 0.074 |
| C-reactive protein (mg/dL) | 93.8 ± 69.5 | 108.2 ± 124.1 | 0.831 |
| Albumin (mg/dL) | 2.2 ± 0.8 | 3.2 ± 0.9 | 0.054 |
| PT c (s) | 25.2 ± 9.4 | 11.9 ± 2.0 | <0.001 * |
| APTT d (s) | 62.7 ± 23.7 | 32.2 ± 8.2 | 0.002 * |
| Total bilirubin (mg/dL) | 6.0 ± 2.6 | 1.8 ± 1.7 | 0.005 * |
Data were presented as mean (standard deviation) or frequency (%).*: p-value < 0.05. Abbreviations: a WBC: white blood cell; b eGFR: estimated glomerular filtration rate; c PT: prothrombin time; d APTT: activated partial thromboplastin time.
4. Discussion
In the family Aeromonadaceae of Gram-negative bacteria, aeromonads are rod-shaped, facultatively anaerobic, nonsporulating bacteria adapted to aquatic environments [1], including freshwater, seawater, sewage, and freshwater junctions. Currently, three major species are recognized as human pathogens: Aeromonas hydrophila, Aeromonas caviae, and Aeromonas veronii biovar sobria [1,16]. Conventional biochemical fermentation tests can only identify specific types of bacteria based on their metabolism, and they may not be suitable for identifying all bacterial strains. Ambiguous results can arise when different genera yield the same outcome. A total of five (31.3%) isolates from 16 patients were not able to be identified to species using conventional methods and API-20E systems. MALDI-TOF MS was used for further verification, and all isolates were successfully identified. In healthy participants, these bacteria can cause diarrhea, biliary tract infections, bloodstream infections, and skin and soft-tissue infections [5,6,17,18,19,20,21,22]. Immunocompromised patients, such as those with chronic liver disease, chronic kidney disease, diabetes mellitus, or malignant disease, are more susceptible to NF [4,5,13,22].
NF is a rare and life-threatening NSSTI [23]. Among the monomicrobial Gram-negative NF, Vibrio species and Aeromonas species are the most prevalent and fulminant pathogens [5,6,7,8,9,11,12,24]. Aeromonas bacteremia has a fatality rate of 30–70% [17,18,22]. Aeromonas NF can cause limb loss or even death if not diagnosed early and treated appropriately [3,4,5,6,7]. Aeromonas SSTIs are often polymicrobial [5,6,19] and exhibit high drug resistance [5,6], rendering it difficult to select an appropriate antimicrobial regimen. Despite aggressive surgical debridement and antimicrobial usage, initial ineffective antimicrobial treatments for Aeromonas NF still lead to high mortality [5,6]. NF diagnosis and treatment protocols were established by our VTR Group [6]. In Taiwan, HCAAS was implemented by the CGMH, which is an online antimicrobial control system [6,25,26]. HCAAS has been used by an experienced ID physician in CGMH-Chiayi to improve rational antibiotic use and clinical outcomes in ICU trauma and Aeromonas NF patients [6,26].
Before prescribing antibiotics, physicians should conduct infectious pathogen identification and AST. In vitro, ASTs can be performed accurately, reproducibly, and timely in clinical laboratories to ascertain clinical relevance. Qualitative disk diffusion, which is a traditional classification method [27], measures the inhibition zone diameter and quantitative dilution methods (broth or agar dilutions) [28], including the performance of the E-test [29,30], which determines the MIC of antibiotics. In microbiology laboratories, MICs are used to confirm microbe resistance [31,32]. Most clinical microbiology laboratories routinely test antimicrobial susceptibility using agar disk-diffusion testing, which was developed in 1940 [27]. Zones of inhibition are measured around paper disks containing antibiotics on agar culture dishes that have been evenly inoculated with bacteria. Antibiograms categorize bacteria as susceptible, intermediate, or resistant [33]. The advantages of disk-diffusion assays include simplicity, cost-efficiency, and the ability to test large numbers of microbes and antimicrobial agents. However, the agar disk-diffusion method is unsuitable for determining the MIC since it is impossible to quantify the amount of antimicrobial agent diffused into the medium.
In the past, reports have elucidated that antimicrobial resistance may develop during Aeromonas treatment [6,18]. As a second method to test drug susceptibility for the first isolated Aeromonas species, an E-test was used to re-evaluate the AST results. By placing a strip impregnated with antimicrobials on an agar plate, an E-test, an antimicrobial gradient method, can determine the MIC value. The procedure involves depositing an increasing concentration gradient of the antimicrobial agent on an agar surface inoculated with the microorganism being tested. A MIC value is determined when the strip intersects with the growth inhibition ellipse. However, E-test strips cost at least $2–3 (US dollars) each; therefore, this approach can be costly if numerous antibiotics are tested [33].
Resistance to penicillin and ampicillin is demonstrated by a majority of Aeromonas strains, while most are invariably susceptible to sulfa drugs, second- to fourth-generation cephalosporins, and carbapenems [34,35]. Aminoglycosides, fluoroquinolones, tetracyclines, and tigecycline are usually effective against the Aeromonas species [34,35,36]. Our study results are consistent with previous studies. The initial ineffective empirical antimicrobial usage is related to poor outcomes in patients with Aeromonas NF [5,6]. Aeromonas NF, which is cephalosporin-resistant, can have a mortality rate of 40% [5]. Culture-directed antimicrobial therapy should be aggressively administered to prevent antibiotic use delay in critically ill patients [7,26]. Ordering antibiotics with culture and susceptibility results is more appropriate than requesting them empirically [26]. In ER, oxacillin and gentamicin were prescribed empirically for NF infections before 2006, and third-generation cephalosporins were prescribed empirically for Vibrio and Aeromonas infections after 2006 [6]. Through HCAAS, an experienced ID physician can confirm an effective antimicrobial regimen against Aeromonas species [6]. Overall, 68.75% of Aeromonas species were susceptible to the disk-diffusion method, and in this study, cefuroxime susceptibility (25%) was significantly lower in the non-survival group. In this study, we found a two-fold cefuroxime resistance between the E-test and disk-diffusion methods. Therefore, after fulminant Aeromonas NF is established, re-testing an E-test for cefuroxime, ceftriaxone, and ertapenem should be conducted. Usually, these antibiotics have higher resistance rates and are used empirically; however, they are easily underestimated through disk diffusion.
According to a 30-year study, patients with bacteremia, septic shock, polymicrobial bacteremia, and inappropriate empirical antimicrobial treatment have a high risk of mortality [37]. Aeromonas NF combined with bloodstream infection significantly increased mortality [5,38]. It is estimated that 36.1–50% of deaths are caused by Aeromonas SSTI combined with bloodstream infection [5,6,21]. Conversely, monomicrobial Aeromonas NF infections are significantly more likely to result in death than polymicrobial infections [5]. In our study, all nonsurvivors had monomicrobial infections with Aeromonas and had bloodstream infections in addition to their NF.
Approximately 36–100% of patients with NF require ICU care [5,6,12,39] and 17–61% require postoperative intubation [6,12,39]. All non-survival patients in this study require ICU care and postoperative intubation. Patients with NF who also had chronic kidney disease, chronic liver dysfunction, diabetes mellitus, hematological diseases, or malignancy had a significantly higher mortality rate [4,5,13,21,38]. Approximately 27–33% of patients with Aeromonas NF also exhibited hepatic cirrhosis [4,5,6]. Compared with survivors, non-survivors had a higher rate of alcoholism (100%), liver cirrhosis (100%), chronic kidney disease (100%), and prolonged ICU stays.
The non-survival group exhibited a statistical tendency to manifest septic shock, anemia, and hyperbilirubinemia more than the survival group [5,38]. Some of the literature has reported that initial presentations of tachycardia and tachypnea were also predictors of poor outcomes in patients with NF [5,38]. In this study, neither tachycardia nor tachypnea were observed in the non-survival group, which may be attributed to the small sample size. Aeromonas NF patients frequently presented with hemorrhagic bullae and skin necrosis [3,4,5,7,12]. The mortality rate of patients with NF presenting with hemorrhagic bullae is higher than that of patients with serous-filled bullae or without bullae [12]. Skin necrosis was present in 28% of patients with Aeromonas NF, which was also a poor predictor of mortality [5,6]. Serous bullae were found in 12.5% of the patients. More patients in the non-survival group had more serous bullae than the survivor group. This is the first time Aeromonas NF has been reported with such an early clinical finding. In critically ill patients with fulminant NF, prompt fasciotomy combined with appropriate empiric antimicrobial therapy and the use of HCAAS by an experienced ID physician has been proven to save lives and limbs [6,7,13,26].
It has been reported that plasmid-encoded AmpC β-lactamases frequently cause β-lactam resistance in clinical strains. There are AmpC enzymes in the chromosomes of several bacterial genera, including Aeromonas. The ampC gene of Aeromonas may increase its responsiveness to antibiotic selection pressure [40]. AQU-1 and blaMOX, which are both Amp-C antibiotics, were detected in all Aeromonas caviae isolates and in 91.9% of Aeromonas dhakensis isolates, according to previous reports. Tigecycline, fluoroquinolones, and cefepime showed good anti-aeromonad activity in vitro [41]. Hospital-associated infections caused by carbapenemase-producing Aeromonas can be fatal. Despite being treated with multiple broad-spectrum antibiotic regimens to overcome various carbapenemase classes, these patients died from underlying comorbidities [42]. Several resistance determinants were found, including co-occurring KPC-3 and VIM-2, OXA-232, and chromosomal CphA-like carbapenemase genes, resulting in major treatment challenges [42].
This study has several limitations. The study was limited to only 16 patients, and 15 antimicrobial agents were tested using an E-test for MIC. Second, this study period was performed for only 3.5 years. Third, no AST test for Aeromonas species during treatment was performed during follow-up.
5. Conclusions
Aeromonas NF is a very rare NSSTI with high mortality, antibiotic resistance, and polymicrobial composition, rendering antibiotic treatment very challenging. AST performed by experienced microbiologists can identify the true pathogen causing NF and determine the appropriate antibiotic to be used.
Acknowledgments
Thanks to Chun-Yen Huang and Chun-Wei Huang for helping with the English modification.
Abbreviations
NF, necrotizing fasciitis; E-test, Epsilometer test; MIC, minimum inhibitory concentration; NSSTI, necrotizing skin, and soft-tissue infection.
Author Contributions
T.-Y.H. designed the study, collected the data, conducted the statistical analysis, and drafted the manuscript. Y.-H.T. and S.-F.K. collaborated on the study design and manuscript writing. K.-T.P., W.-H.H. and Y.-K.H. developed the study, performed the surgeries, and coordinated the research groups. Both J.-L.C. and Y.-Y.L. assisted with the surgical procedure and designed the study. H.-J.L. and C.-H.H. contributed to the design and revision of the study. C.-T.H. was involved in the study’s design and analysis. All authors have read and agreed to the published version of the manuscript.
Informed Consent Statement
Informed consent was obtained from all patients in this study, which was approved by the Institutional Review Board of Chang Gung Medical Foundation (Number: 102-5105B, 103-5112C, and 106-0752C).
Data Availability Statement
The datasets analyzed during the current study are not publicly available, due to confidentiality reasons, but are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare that they have no competing interest.
Funding Statement
A grant from the Chang Gung Medical Research Program Foundation supported this project [grants CMRPG6L0071, CMRPG6H0641, and CORPG6E0051~53].
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets analyzed during the current study are not publicly available, due to confidentiality reasons, but are available from the corresponding author upon reasonable request.