The Impact of Cefuroxime Susceptibility on Aeromonas Necrotizing Fasciitis Outcomes

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.

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 diskdiffusion 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.

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.

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 emer-gency 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.

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.

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°C 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.

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.

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).

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.

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.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 nonsurvivors (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.

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.

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).

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).

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].
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.
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 βlactam 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.

Figure 1 .
Figure 1.A flowchart showing the clinical isolates of Aeromonas strains collected from 16 patients with necrotizing fasciitis.

Figure 1 .
Figure 1.A flowchart showing the clinical isolates of Aeromonas strains collected from 16 patients with necrotizing fasciitis.

Table 1 .
MIC a of Aeromonas strains isolated from 16 patients with necrotizing fasciitis of limbs to different antimicrobial agents.

Table 2 .
Susceptibilities of Aeromonas strains isolated from 16 patients by CLSI a breakpoint interpretation.

Table 3 .
Summary of data for 16 cases of Aeromonas necrotizing fasciitis.
Abbreviations: a ER: emergency room; b CKD: chronic kidney disease; c H/D: hemodialysis; d DM: Diabetes mellitus.

Table 4 .
Microbiological results, antimicrobial susceptibilities, and antimicrobial usage for 16 patients between non-survival and survival groups.

Table 5 .
Demographic data, comorbidities, and clinical outcomes of 16 patients between non-survival and survival groups.

Table 6 .
Clinical presentations of 16 patients with Aeromonas necrotizing fasciitis between nonsurvival and survival groups.

Table 7 .
Laboratory findings of 16 patients with Aeromonas necrotizing fasciitis between non-survival and survival groups.