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The First Report of the Antimicrobial Resistance of Acinetobacter baumannii Isolates from Seafood Samples

Acinetobacterbaumannii (A. baumannii) is one of the newly launched foodborne bacterium,which acts as an opportunistic pathogen originated from different infection sources, including contaminated water and environment and through human manipulation of seafood samples [3]. It is an aerobic, Gramnegative,rod and non-fermenting bacteriumthat colonizes the respiratory tract, skin, urinary and gastrointestinal systems[4]. The bacterium is also responsible for surgical infections, pneumonia, catheter-related blood circulatory, and urinary tract infection, mediastinitis, meningitis, cholangitis,and osteomyelitis [5].

According to the importance of A. baumannii as a newly-launched foodborne pathogen, the present investigation was addressed to assess the antimicrobial resistance of Acinetobacterbaumannii isolates from seafood samples.

Samples
From April to October 2020, fivehundred seafood samples, including fish (Scomberomoruscommerson) (n= 150), shrimp (Penaeus monodon) (n= 150), and lobster (Scalloped spiny) (n= 200) samples, were collected from shopping centers of Shiraz city, Iran. Each sample (100 g from the dorsal muscle) was collected separately in highly hygienic conditions using sterile tissue forceps in laboratory tubes containing a peptone water solution (Merck, Germany). These samples were healthy and fresh, and all were caught from the Persian Gulf, Iran. All samples were transferred to the laboratory using cool boxes at 4°C.

Bacterial isolation and identification
Twenty-five grams of each sample was blended in 225 nutrient broth medium (Merck, Germany) and incubated at 37 °C for 24 h. A loopful of the broth was cultured on both blood agar (BA) and MacConkey agar (MC, Merck, Germany) plates and incubated at 37 °C for 24 h. suspected colonies which were grown on BA (non-hemolytic, opaque and creamy) and MC agar (non-lactose fermenting) were identified according to various biochemical tests including Gram-staining, motility, catalase, coagulase, oxidase, sugar fermentation, indole, H2S production, Voges Proskauer, methyl Red, gelatin hydrolysis, nitrate reduction, and urease activity [8].

PCR confirmation of isolates
A. baumannii isolates were further confirmed by the PCR [9]. The isolates were sub-cultured innutrient broth and incubated at 37 °C for 24 h. DNA was extracted from A. baumannii broth cultures using DNA extraction kit (CinnaGen, Iran). The procedure was performed according to the manufacture's guidelines. The quality of extracted DNA was assessed using electrophoresis on the gel. The Nanodrop device (NanoDrop, Thermo Scientific, Waltham, USA) was used to assess the extracted DNA purity. Table 1 indicates primers and PCR conditions. PCR was performed using DNA thermo-cycler (Eppendorf 5330, Germany). PCR products were analyzed using gel electrophoresis (2% agarose gel stained with CYBR Green in 1X TBE buffer) (CinnaGen Co, Iran). Electrophoresis was done at 80 V for 30 min. All PCR materials and ingredients were purchased from CinnaGenCo, Iran [10][11][12].

Statistics
SPSS software and chi-square and Fisher tests were used for statistical examination. Differences in the prevalence and antimicrobial resistance of bacteria between diverse groups were considered. P-value < 0.05 was considered as significant level [17,18].

PCR amplification
PCR technique was used to detect the 16S-23S ribosomal DNA gene of the A. baumannii isolates from seafood samples. All isolates were confirmed by the PCR (Fig. 1).    Table 2 reveals the prevalence of A. baumannii in diverse kinds of seafood samples. Twenty-eight out of 500 (5.6%) seafood samples were contaminated with A. baumannii. Fish had the highest contamination rate with A. baumannii (10%), while lobster had the lowest (2.5%). A significant difference was obtained from the statistical view between the type of samples and prevalence of A. baumannii (P<0.05). Table 3 reveals the antimicrobial resistance pattern of A. baumannii isolates recovered from seafood samples. A. baumannii isolates showed the maximum rate of resistance against tetracycline (75%), gentamicin (71.4%), ciprofloxacin (64.2%), trimethoprim (57.1%), streptomycin (53.5%), and erythromycin (50%) antimicrobial agents.The lowest rate of resistance was found toward chloramphenicol (3.5%) and imipenem (7.1%) antimicrobial agents.A significant difference was obtained from the statistical view between the type of samples and the prevalence of antibiotic resistance (P<0.05).
Nevertheless, dissimilar to the common pathogens implicated in foodborne diseases, Acinetobacter species are infrequentlyaccompanied by diarrheal disease, possibly due to their hard isolation from food sources. Nonetheless, numerous species of Acinetobacter, particularlyA. baumannii, harborssome of the features associated with significant pathogens and show anextraordinaryaptitude to resist several antimicrobial agents. Some epidemiological informationregarding theantibiotic-resistant A. baumanniiin contaminated dairy products, together with raw fruit and vegetables and meat samples,establish extra-hospital reservoirs of this underrated pathogen, which may signify an augmented risk to immunocompromised individuals, elders, and young children in a hospital setting [26].
The present survey is the first report of the prevalence and antimicrobial resistance of A. baumanniiisolates from fish, shrimp, and lobster samples globally. Findings revealed the high prevalence of A. baumannii strains accompanied with the high prevalence of resistance toward tetracycline, gentamicin, ciprofloxacin, trimethoprim, streptomycin, and erythromycin antimicrobial agents. Transmission of resistantbacteria from the contaminated staff of the fishing ports and processing companies is the most probable reason for the findings. However, high, unauthorized, and illegal prescription of antimicrobial agents and using high doses and volumes of disinfectants are probable reasons for antimicrobial resistance in isolated bacteria. The filter-feeding activity of seafood, shrimp, and lobster in particular, maybe the main reason for the high prevalence of A. baumannii strains. Accumulation and survival of A. baumannii bacteria through the filter-feeding activity has occurred.
The present investigation was limited to the low numbers of isolated bacteria in seafood samples and the lack of additional research on other seafood samples presented in Iran. Additionally, the absence of significant literature-based documents is another important limitation of the current survey. However, this is a preliminary and first report of the presence of antibiotic-resistant A. baumannii among seafood samples globally.

Conclusion
The present research findings revealed the role of seafood samples, mainly fish, shrimp, and lobster, as important reservoirs of the A. baumannii strains in the environment. The high prevalence of A. baumannii was accompanied by the high prevalence of resistance toward routinely applied antimicrobials. Well-cooking of fish, shrimp, and lobster before consumption can diminish the risk of diarrheal illnesses due to the A. baumannii strains.Complete cooking of seafood samples before consumption can reduce the risk of antibiotic-resistant A. baumannii related infections.