Requiring Reconsideration of Differences of Aeromonas Infections Between Extra-Intestinal and Intestinal in Hospitalized Patients

Introduction

Aeromonas is a gram-negative, facultative anaerobic oxidase positive bacillus that ranges in size from 0.3 to 1.0 µm.¹ Intestinal infection is the most common type of infectious disease caused by Aeromonas,² but it can also result in infections of wound, liver and gallbladder, necrotizing fasciitis, sepsis, and other infectious diseases.³ However, it is important to be aware that there have been more instances of extra-intestinal infections in recent years.⁴ As an opportunistic pathogen, Aeromonas is more likely to infect and kill patients with low immunity, including those with malignant tumors, hematological tumors, cirrhosis, diabetes, and other diseases.^5–7 In 1986, Aeromonas separated from the vibrio family,⁸ which it had previously been a part of it. Since then, the group of bacteria known as Aeromonas has expanded. However, our capacity to identify them has been constrained by the use of common clinical microbe identification methods, including the VITEK2 Compact system, the VITEK MALDI-TOF system, and even 16S rRNA.^2,9,10 For precise identification, housekeeping genes like rpoD and gyrB can be sequenced.^11,12 It is also possible to use multilocus phylogenetic analysis (MLPA).³ Drug resistance in Aeromonas has been steadily rising in recent years as a result of the expanding use of antibiotics in both aquaculture and human healthcare.¹³

Exotoxin, extracellular protease, adhesion factor, and secretion system are the key components of Aeromonas’ virulence factors.¹⁴ This makes the harmful elements of this organism complicated and varied. Aeromonas is able to thwart the host’s immune system and spread infection thanks to a range of virulence factors.¹⁵

In this work, the characteristics of Aeromonas infection and mortality were examined. Comparing the virulence gene and drug susceptibility of intestinal isolates versus extra-intestinal isolates. A preliminary assessment of Aeromonas’ drug resistance mechanism was made.

Materials and Methods

Material

In the clinical microbiology database of the First Affiliated Hospital of Chongqing Medical University, a 3200-bed facility in southwest China, 188 strains of Aeromonas were gathered between January 2013 and September 2020. A total of 158 strains were obtained from patients with parenteral infection (39 strains in skin wound secretion, 28 strains in bile, 25 strains in pleural and peritoneal fluid, 24 strains in blood, 22 strains in urine, and 20 strains in others), while 30 strains were identified from patients with intestinal infection. The VITEK MALDI-TOF technology (bioMerieux, Marcy I ‘Etoile, France) was utilized by the microbiology lab to identify microorganisms. And 188 Aeromonas strains were kept at 80 °C in 15% glycerol.

Identification of Bacterial Strains

Housekeeping genes (rpoD, gyrB, and gyrA) sequencing were used to confirm the identity of all isolates. Primers were taken from prior publications.^16–18 The primers used for amplification and sequencing of the housekeeping genes are listed in Table 1.

Table 1 The Primers of Target Genes and PCR Conditions

Data Collection and Definition

In this study, a retrospective analysis was conducted. Only the first episode was considered in patients with more than one positive Aeromonas culture. The electronic medical records mainly covered the following contents: characteristics of infection, diagnosis and treatment, auxiliary examination, and prognosis. Sepsis with persistent hypotension needing vasopressors to keep MAP 65 mm Hg and having a blood lactate level >2 mmol/L (18 mg/dL) despite sufficient volume resuscitation are clinical indicators of patients with septic shock.¹⁹ Hemoglobin below 13 g/dL in adult males and 12 g/dL in adult females was considered anemia.²⁰ Serum albumin components were below 35 g, indicating hypoproteinemia.²¹ Serum potassium level <3.5 mmol/L was hypokalemia. Systemic steroid use was defined as oral or intravenous administration of at least 20 mg/day of a steroid (prednisone, hydrocortisone, methylprednisolone, or dexamethasone) within 1 month of infection.²² The result was death. The inappropriate antimicrobial therapy used improper drugs that are not susceptible to pathogens.

Drug Sensitivity Test

The microbroth dilution method was used to determine the antimicrobial susceptibility pattern of all isolates to some commonly used antimicrobial agents, including ciprofloxacin, levofloxacin, cefuroxime, ceftazidime, ceftriaxone, cefepime, aztreonam, imipenem, and meropenem. The breakpoints were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.²³ Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls for antibiotic susceptibility tests.

Polymerase Chain Reaction (PCR) Amplification of Drug Resistance and Virulence Genes

Touchdown PCR assays and sequencing were used to validate the presence of the carbapenemase genes (bla_CphA, bla_KPC, bla_IMP, bla_VIM, bla_NDM, and bla_OXA-48-like), extended-spectrum beta-lactamase (ESBL) genes (bla_CTX-M-1, bla_CTX-M-9, bla_{CTX-M-15-like}, bla_SHV, and bla_TEM), and Ampc (bla_ACC and bla_FOX).²⁴ A total of 12 virulence factor-encoding genes were also found using PCR, including act, ast, alt, aerA, hlyA, ela, gcaT, lip, lafA, fla, aexT, and ascF-G.^25–27 The amplification primer sequences are listed in Table 1.

Statistical Analysis

The data were analyzed using SPSS statistical software (version 25.0, IBM). Pearson’s chi-square test or Fisher’s exact test was used to analyze the categorical variables. Student’s t-test or Mann–Whitney U-test was utilized for continuous variables. To assess independent risk factors for Aeromonas mortality, univariate analysis was used to evaluate the putative variables, and multivariate logistic regression analysis was carried out for statistically significant variables in univariate analysis. Odds ratios (OR) were calculated with 95% confidence interval (CI). P<0.05 was deemed to statistically significant. The positive PCR amplicons were sequenced by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Using Seqman (DNAStar), the sequences were assembled, and the data were compared in the NCBI (http://www.ncbi.nlm.nih.gov) using BLAST.

Results

Identification of Bacteria

A total of 188 Aeromonas isolates were divided into 7 species based on the results of housekeeping genes. Aeromonas caviae (28.7%), Aeromonas hydrophila (26.1%), Aeromonas veronii (25%), and Aeromonas dhakensis (18.1%) were the four species of Aeromonas that were most common. Additionally, 2 isolates of Aeromonas jandaei, 1 isolate each of Aeromonas sobria and Aeromonas media were identified. Figure 1 depicts the spread of Aeromonas isolated from infections of the extra-intestinal and intestinal. Overall, intestinal isolates typically contained A. veronii (p=0.0001); and extra-intestinal isolates were mainly contained A. caviae and A. hydrophila (p=0.0001).

Figure 1 Distribution of various Aeromonas that have been isolated in and outside of the intestinal.

Characteristics of Infected Patients

A total of 188 patients with Aeromonas infection were found between 2013 and 2020. The patients were on average 53.9 years old, with a male-to-female ratio of 1.5 (114/74). Malignant neoplasms (solid tumors and hematologic malignancies), hepatobiliary diseases (posthepatitic cirrhosis and cholelithiasis), anemia, and hypoproteinemia were the most prevalent underlying illnesses among infected people (Table 2). Different comorbidities were seen in patients with infections both inside and outside the intestinal. Patients with age >80, hematological malignancies, and gastrointestinal diseases were more likely to have an intestine infection with Aeromonas.

Table 2 Clinical Characteristics of Patients with Aeromonas Infection

Twenty-seven individuals (14.4%) died from Aeromonas infection, 24 of whom had extra-intestinal infection. Hematological malignancies (P=0.006), cholelithiasis (P=0.028), septic shock (P=0.002), and surgical history within 6 months (P=0.011), were all risk factors for the nosocomial death of Aeromonas, according to a univariate analysis. Septic shock was the only independent risk factor for Aeromonas death (P=0.020), according to the multivariate logistic regression model’s results, which are displayed in Table 3. A connection between intestinal infection and death was not discovered. Most patients with intestinal infections had hematological malignancies, cholelithiasis, septic shock, chemoradiotherapy, systemic steroid use, and surgical history within 6 months (Table 4).

Table 3 Analysis of Risk Factors Associated with Hospital Death Caused by Aeromonas Infection

Table 4 Analysis of Risk Factors for Death from Intestinal and Extra-Intestinal Aeromonas Infection

Drug Sensitivity

According to Table 5, which summarizes the results of the antimicrobial susceptibility tests, more than 83% of the bacterial strains were susceptible to ciprofloxacin (83.5%), levofloxacin (89.4%), cefepime (87.2%), aztreonam (86.2%), imipenem (87.2%), and meropenem (93.6%). Ceftazidime and ceftriaxone each had a 79.8% and a 69.7% sensitivity rate for Aeromonas, respectively.

Table 5 Antimicrobial Susceptibilities of Aeromonas Isolates Determined by the Broth Microdilution Method and Aeromonas Isolates from Inside and Outside the Intestinal Were Compared for Drug Sensitivity

With the exception of imipenem, intestinal infection strains were more susceptible to other medications than extra-intestinal infection strains, and ceftazidime, ceftriaxone, and ciprofloxacin all demonstrated statistically significant sensitivity. However, compared to intestinal strains, extra-intestinal strains had a considerably higher susceptibility to imipenem (Table 5).

Drug Resistance and Virulence Genes

By using PCR, it was discovered that 42 Aeromonas strains solely carried the ESBLs and carbapenemase genes: 6 strains only carried bla_TEM; 31 strains only carried bla_CphA, including A. dhakensis, A. veronii, A. hydrophila and A. jandaei; and one A. caviae with bla_VIM. The remaining four Aeromonas strains possessed the following combinations of drug resistance genes: two A. hydrophila each had bla_CphA + bla_CTXM-M-9, and bla_CphA + bla_CTX-M-1 + bla_{CTX-M-15-like} + bla_TEM; two A. caviae each carried bla_NDM + bla_CTX-M-1 + bla_{CTX-M-15-like} + bla_TEM, and bla_NDM + bla_TEM. All of them were generated from extra-intestinal isolates, except for 7 strains with bla_CphA, and one with bla_VIM. 21 strains (63.64%) of the 33 strains with a positive CphA gene were discovered to be incompatible with the drug sensitivity of carbapenems. The ratios of act, ast, alt, aerA, hlyA, ela, lip, and gcaT were found to be 39.4%, 25.5%, 76.1%, 58%, 48.4%, 66%, 71.3%, and 88.3% respectively. Fla, lafA, aexT, and ascF-G were found in ratios of 72.3%, 21.8%, 20.2%, and 18.6%. The virulence genes act, aexT, and ascF-G were found in intestinal infection more frequently than extra-intestinal. Additionally, the detection rate of the virulence genes alt, hlyA, ela, and lip from extra-intestinal infection was considerably higher than that from intestinal infection (Table 6).

Table 6 Intestinal and Extra-Intestinal Infections Caused by Aeromonas Were Compared in Terms of Their Virulence Genes

Discussion

Aeromonas are easy to isolate; however, because of its heterogeneous phenotypic characteristics, species identification is difficult. In contrast to the use of the 16S rRNA gene and the VITEK MALDI-TOF method, nucleotide sequencing of housekeeping genes, such as rpoD and gyrB,^6,28 or multilocus phylogenetic analysis (MLPA),³ can enable a more precise identification of the species.

Despite the fact that Aeromonas infections are primarily intestinal infections,²⁹ there have been an increasing number of reports in recent years of extra-intestinal infections with Aeromonas.^30,31 While Qu et al³² found that Aeromonas extra-intestinal infection primarily came from blood, Fu et al⁴ reported that skin and soft tissue infection was the primary cause of Aeromonas extra-intestinal infection. In this study, extra-intestinal infection was the primary type of Aeromonas infection, while skin wound infection was the primary extra-intestinal infection type. There were statistically different Aeromonas species isolated from different infection sites. A. veronii was the most prevalent species of intestinal infection in the paper, despite earlier research³² suggesting that A. dhakensis was most frequently found in patients with intestinal infection. This study and that of Chen et al³³ are identical. In both cases, the extra-intestinal infections were mainly caused by A. caviae and A. hydrophila.

Similar to the findings of earlier studies,^2,6 the work revealed that men made up the majority of those infected with Aeromonas, and it was noted that people in their middle and later years made up the majority of those infected, which may be related to their pre-existing illnesses and lowered immune systems.⁷ According to this study, the majority of patients with Aeromonas infection also had underlying illnesses like malignancies (solid tumors, hematological malignancies), hepatobiliary diseases (posthepatitic cirrhosis, cholelithiasis), anemia and hypoproteinemia. Su et al⁶ reported that patients with liver cirrhosis have significant morbidity and mortality from Aeromonas infection, which may be caused by abnormal performance of the mononuclear macrophage system in patients with liver cirrhosis, and a decrease in the body’s immune defense mechanism.

In contrast to the 14.9% to 63% described in the literature,^5,34 the mortality rate with an Aeromonas infection (14.4%) was relatively low. According to Ji et al,⁵ the use of appropriate antimicrobial medicines, metastatic cancer and shock were linked to Aeromonas mortality (P<0.05). In the study, the following conditions increased the probability of death in patients with Aeromonas infection: hematological malignancy, cholelithiasis, septic shock, recent surgery, and transfusion. Immunosuppressive medications and intensive chemotherapy accelerate the immune system’s degeneration in hematological malignancy patients, making them more vulnerable to infection.³⁵ In a clinical study by Xu et al,³⁵ 42 people with leukemia complicated by an Aeromonas infection took part. Most Aeromonas infections were found to occur in leukemia patients who were experiencing granulocyte shortage, and granulocyte insufficiency lasting longer than 7 days was associated with patient mortality.

The 188 strains had higher susceptibility rates to ciprofloxacin, levofloxacin, cefepime, amtraenem, imipenem and meropenem, both of which were over 83%, similar to those reported in other regions.^6,29 And then, there are differences in the drug resistance rates of Aeromonas in different infection sites, and the drug resistance rate of strains with extra-intestinal infection is significantly higher than that of strains with intestinal infection, which is in line with the study results of Zhou et al.²⁹ However, intestinal infection strains were much less susceptible to imipenem than extra-intestinal strains, with a susceptibility rate of only 76.7%. Imipenem susceptibility ranged from 89.8% to 95% in earlier reports^29,36 using intestinal isolates. In order to do future research, we must increase the sample size.

The three primary lactamases found in Aeromonas are Ambler C cephalosporin enzymes, D penicillinases, and B metallic lactamases (MBLs).² The metalloenzyme CphA gene mainly exists in A. hydrophila, A. veronii, and A. jandaei, but not in A. caviae.³⁷ This experiment’s discovery of the CphA gene in A. dhakensis, A. veronii, A. hydrophila, and A. jandaei supported this assertion. In addition, imipenem and meropenem sensitivity was discovered in 22 of 33 CphA-positive Aeromonas strains. According to earlier research, the genotype and drug sensitivity phenotype of CphA were discordant because gene mutation altered the gene expression of the protein.³⁸ The research team will continue to look into what specific mechanism triggered this outcome. Two Aeromonas strains harboring the CphA gene and ESBLs were identified in this study, and the findings of the drug sensitivity tests were in line with the genotype expression. We need to pay more attention to the combined drug resistance of the CphA gene and the Aeromonas ESBLs, which has not yet been extensively explored. Additionally, two strains with both NDM and ESBLs were found, and NDM frequently co-existed with other drug resistance genes, which were transferred horizontally through plasmids and integrons in strains,³⁹ leading to its spread in different strains, which presented serious challenges to the prudent use of antibiotics.

The diversity and complexity of Aeromonas’s harmful nature were caused by a wide range of virulence factors. When relevant virulence factors were screened, it was discovered that 188 Aeromonas strains had detection rates of alt, aerA, ela, lip, gcaT and fla that were higher than 58%, comparable to those documented in the literature.⁴⁰ Extracellular proteases have the ability to break down different types of proteins, supply amino acids to bacteria, and directly cause significant tissue damage. This is a key pathogenic mechanism used by infected bacteria to get past the host’s defenses and propagate throughout the body.^2,41 In the study, it has been discovered that the extracellular protease detection rate of strains isolated from the intestinal was higher than that from the extra-intestinal, indicating a difference in pathogenicity between strains infected from the intestinal and those infected from the extra-intestinal.

Conclusion

In a word, Aeromonas in this region was mainly infected by extra-intestinal. Compared with intestinal strains, it was found that there were differences in species distribution, drug susceptibility, drug resistance and virulence gene distribution between the two types of infection.