NaCl Concentration-Dependent Aminoglycoside Resistance of Halomonas socia CKY01 and Identification of Related Genes

Abstract

Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.

Keywords: Halomonas strain; antibiotic resistance; genome sequencing; salt tolerance.

Fig. 1. Antibiotic resistance test under different salinity conditions.

(A) Growth observation in 1% NaCl after 72 h of cultivation at 30°C in the presence of 50 μg/ml kanamycin (Km), 35 μg/ml chloramphenicol (Cm), 100 μg/ml ampicillin (Amp), 6 μg/ml oxacillin (Oxa), or 100 μg/ml carbenicillin (Car). (B) Growth observed in the presence of 10% NaCl under the same conditions as (A). (C) Growth observed in the presence of 1% NaCl, 25 μg/ml gentamicin (Gm), and 50 μg/ml apramycin (Apra). (D) Growth observed in the presence of 10% NaCl and under the same conditions as (C). (E) Growth observed in the presence of 1% NaCl, 0.5 μg/ml tetracycline (Tetra), and 100 μg/ml spectinomycin (Spec). (F) Growth observed in the presence of 10% NaCl and under the same conditions as (E).

Fig. 2. Growth of H. socia CKY01 after adding kanamycin and under different salinity conditions.

(A) Growth observed with and without 50 μg/ml kanamycin (Km) after 24 h of cultivation. (B) Growth observed after 48 h under the same conditions as (A). (C) Growth observed with and without 60 mM proline in the presence of 50 μg/ml kanamycin after 24 h of cultivation. (D) Growth observed after 48 h under the same conditions as (C).

Fig. 3. Time-dependent semi-RT PCR expression results.

The gene expressions of kanamycin resistance-related genes of H. socia CKY01 were observed after adding kanamycin to LB medium containing 1% and 10% NaCl after culturing for 48 h.

Fig. 4. Results of the MIC test performed by treating constructed E. coli strains with kanamycin, gentamicin and apramycin.

(A) Kanamycin MIC result of E. coli strains after 24 h cultivation at 30°C in the presence of 100 μg/ml ampicillin and 0.05 mM IPTG. Paper discs contained 0, 1, 2, 4, 6, 8, and 10 μg kanamycin. (B) Gentamicin MIC result of E. coli strains under the same conditions as (A). (C) Apramycin MIC result of E. coli strains under the same conditions as (A).