Independent Predictors of Mortality for Aeromonas Necrotizing Fasciitis of Limbs: An 18-year Retrospective Study

Necrotizing fasciitis (NF) of the limbs caused by Aeromonas species is an extremely rare and life-threatening skin and soft tissue infection. The purpose of this study was to evaluate the specific characteristics and the independent predictors of mortality in patients with Aeromonas NF. Sixty-eight patients were retrospectively reviewed over an 18-year period. Differences in mortality, demographics data, comorbidities, symptoms and signs, laboratory findings, microbiological analysis, empiric antibiotics treatment and clinical outcomes were compared between the non-survival and the survival groups. Twenty patients died with the mortality rate of 29.4%. The non-survival group revealed significant differences in bacteremia, monomicrobial infection, cephalosporins resistance, initial ineffective empiric antibiotics usage, chronic kidney disease, chronic hepatic dysfunction, tachypnea, shock, hemorrhagic bullae, skin necrosis, leukopenia, band polymorphonuclear neutrophils >10%, anemia, and thrombocytopenia. The multivariate analysis identified four variables predicting mortality: bloodstream infection, shock, skin necrosis, and initial ineffective empirical antimicrobial usage against Aeromonas. NF caused by Aeromonas spp. revealed high mortality rates, even through aggressive surgical debridement and antibacterial therapies. Identifying those independent predictors, such as bacteremia, shock, progressive skin necrosis, monomicrobial infection, and application of the effective antimicrobial agents against Aeromonas under the supervision of infectious doctors, may improve clinical outcomes.

Demographic data. No significant differences were observed within the parameters of age, gender, infective regions, or the number of amputations per patient between these two groups. The non-survival group was characterized by a significantly higher incidence of chronic kidney disease (CKD), chronic liver dysfunction, and ICU admission (Table 1). Meanwhile, the non-survival group had significantly associated with higher Acute Physiology and Chronic Health Evaluation II (APACHE a II) scores, fewer operations, and shorter hospitalization stays (Table 1).

Laboratory findings.
Within the non-survival group the following characteristics were observed more frequently than within the survival group: total white blood cell count <4000/uL, band leukocyte cells >10%, lymphocyte count of leukocytes <1000/uL, anemia (hemoglobin <10 g/dL), thrombocytopenia (platelet count <15 × 10 4 /uL), and estimated glomerular filtration rate <30 c.c./min (Table 5). In addition, the proportion of patients presenting with a lower albumin level was frequently observed and significantly higher in the non-survival group www.nature.com/scientificreports www.nature.com/scientificreports/ (p = 0.002). The prothrombin time (PT) and total bilirubin values for the non-survival group were significantly higher than those for the survival group (  1.190-17.597; p = 0.027), and a higher incidence of initial ineffective empirical antimicrobial usage (OR: 5.798; 95% CI: 1.247-26.951; p = 0.025) were the indicators of mortality (Table 6).

Discussion
Most Aeromonas SSTIs are associated with environmental exposure and are particularly related to traumatic occupational injuries or unexpected contact via recreational sporting activities 27,28 . This mode of acquisition results in soft tissue Aeromonas infections occurring more commonly during the summer season 25 . Our study was consistent with this finding.
Aeromonas SSTIs are often polymicrobial and frequently involve coinfections with other gram-negative rods or Clostridium species 25,27,30 . In studies of Aeromonas SSTIs or bacteremia, A. hydrophila was the most common pathogen isolated, and encompassed 47~69% Aeromonas infections 26,27,29 . In our results, A. hydrophila was also the most common infectious organism detected (67.6%), and Clostridium species were the most common coinfection pathogens.
According to past reports, Aeromonas and Clostridial necrotizing soft tissue infections were consistently associated with poor outcomes 7,19,31 . However, our patients exhibiting monomicrobial Aeromonas NF had a significantly higher mortality rate than those with polymicrobial Aeromonas NF. Another interesting phenomenon is that Aeromonas NF patients coinfected with Clostridial species also had better outcomes. On the other hand, we found Aeromonas NF combined with bloodstream infection (BSI) had significantly increased the mortality rate. Monomicrobial Aeromonas NF with bacteremia has commonly been reported to be associated with liver cirrhosis and malignancy that can rapidly impair the phagocytic activity of the reticuloendothelial system and result in septic shock and multiple organ failure 14,27,32 . Thus, we should pay more attention and aggressive treat those NF patients with Aeromonas bacteremia and monomicrobial infection.
Most Aeromonas strains are resistant to ampicillin, amoxicillin, and amoxicillin-clavulanate, whereas most are susceptible to sulfa drugs, fluoroquinolones, second-to fourth-generation cephalosporins, aminoglycosides, carbapenems, and tetracyclines 26,28,33,34 . Therefore, Aeromonas SSTIs may best be treated empirically with fluoroquinolones and/or a third-or fourth-generation cephalosporin or a carbapenem; however, a higher cephalosporins-resistance rate was found in the non-survival group ( www.nature.com/scientificreports www.nature.com/scientificreports/ therapy should be aggressively administered in such Aeromonas NF patients to avoid delayed use of appropriate antimicrobial therapy 11,13 .
A significantly higher mortality rate was observed in NF patients that also exhibited CKD, hepatic dysfunction, diabetes mellitus, and cancer 3,6,7,10,19,20,26 . Our Aeromonas NF patients with CKD or chronic hepatic dysfunction were present in statistically significantly higher numbers in the non-survival compared with the survival group. The non-survival group exhibited high severity of disease and 95% of patients required admission to the ICU. A delay in the first surgical intervention from symptom onset to the operating room of >24 h, as well as having advanced age, adversely affected survival outcome 3,20,26 . A delay in surgery of more than 24 h accounted for 30% of the patients within the non-survival group, and the mean age was >60 years old within these two groups.
In our study, the non-survival group contained a greater proportion of patients with hemorrhagic bullous lesions (60% vs. 29.2%) and skin necrosis (55% vs. 16.7%) than the surviving group, but multivariate logistic regression analysis revealed that non-survival patients presented associated with skin necrosis (p = 0.027). As the ischemic necrosis of the skin evolves, gangrene of the subcutaneous fat, dermis, and epidermis, manifesting progressively as bullae formation, ulceration and skin necrosis 14,35 . Hemorrhagic bullae and skin necrosis were also the late stage signals of necrotizing fasciitis 3,35,36 . Then, hemorrhagic bullae with skin necrosis appearance may increase the incidence of mortality (Fig. 2). Nonetheless, the emergence of hemorrhagic bullae would be considered a feature and an early independent predictor of mortality of Aeromonas NF.   www.nature.com/scientificreports www.nature.com/scientificreports/ In this study, the non-survival group exhibits a statistical tendency to have tachypnea and initially present with septic shock more than those within the survival group in univariate logistic analysis; however, multivariate logistic regression analysis bacteremia and shock revealed significant differences. Some literatures reported that initial presentations of tachypnea and hypotensive shock were also predictors for a poor outcome in NF patients 7,8,10,19 . This may explain to the fact that the death group presented more septicemia-related systemic inflammatory response symptoms to induce respiratory failure and shock.
Leucopenia, increased counts of banded leukocytes, thrombocytopenia, and severe hypoalbuminemia can be considered the clinical and laboratory risk indicators to initialize surgical intervention and to predict mortality for NF [3][4][5][6][7][8]15,20,21 . The non-survival group was associated with a higher rate of patients exhibiting leucopenia, leukocyte band cells >10%, lymphocytopenia, anemia, and thrombocytopenia compared with the survival group (Table 5). Lower serum albumin levels, prolonged PT, and higher total bilirubin levels were also noted within the non-survival group. These laboratory findings within Aeromonas NF infections are compatible with the aforementioned previous studies.
Multivariate logistic regression analysis revealed the initial ineffective empirical antimicrobial usage are related to poor outcomes for Aeromonas NF patients. Early prompt fasciotomy combined with appropriate antimicrobial therapy has been aggressively performed for patients highly suspected of having NF in our institution 8,13,14,17,19 . In our experience within the ICU, antimicrobial stewardship program and on-the-spot education by physicians specialized in infectious disease can potentially decrease sepsis-related and overall infection-related mortality rates by 54% and 41%, respectively 11 . The initial clinical presentation of Aeromonas NF is very similar to Vibrio NF, and especially within southern Taiwan, there may be a history of contact with dirty water or fish exposure. To treat these fulminate diseases, third-to fourth-generation cephalosporins combined with tetracycline or fluoroquinolones were commonly the empiric prescription before the infectious pathogens were identified 5,37 . In the non-survival group, we found 40% of Aeromonas species resistant to cephalosporins and 20% to tetracycline www.nature.com/scientificreports www.nature.com/scientificreports/ but only to 5% fluoroquinolones. So, we consider prescribing third-to fourth-generation cephalosporins combined with tetracycline and fluoroquinolones for highly suspected fulminate Vibrio or Aeromonas NF of limbs within the setting of our hospital. After the pathogens were identified, the antibiotic regimens were adjusted as necessary according to the patient's clinical condition and results of the antibiotics drug sensitivity tests.
The present study was limited by having only 68 patients in a period of 18 years. However, to our knowledge, these are the largest patient numbers within such a study that can be currently found within PubMed. Another limitation contained within is that due to the long study period, some cases, medical records, and laboratory data had been lost and were not able to be recovered.
In conclusion, Aeromonas spp. NF of limbs is very rare and exhibits resistance to multiple antibiotics. NF caused by Aeromonas spp. revealed high mortality rates, even through aggressive surgical debridement and antibacterial therapies. Identifying those independent predictors, such as bacteremia, shock, progressive skin necrosis, monomicrobial infection, and application of the effective antimicrobial agents against Aeromonas under the supervision of infectious doctors, may improve clinical outcomes.

Materials and Methods
Study design and setting. This is a retrospective study performed by the VTR Group at CGMH-Chiayi from December 2001 to November 2019. We analyzed those patients admitted to the emergency department (ED) that were diagnosed with NF of limbs with undergoing surgical intervention, and a total 68 of patients were surgically and pathologically confirmed to have Aeromonas NF of limbs were included.

Data availability
All datasets are available from the first author on reasonable request.
A 82 year-old male with a history of hepatitis B and old stroke had right low leg and foot pain on second day after a contused injury of toes. The right lower leg revealed patchy purpura, vesicles and skin necrosi with serous fluid soaking on the bed in the emergency room. After fasciotomy, the culture of blood and wound specimens confirmed Aeromonas hydrophila, however, this patient died on the 3rd day after admission owing to progressive septic shock and multiple organ failure.