Phenotypic Characteristics and Antibiotic Resistance Profiles of Proteolytic Halotolerant Bacillus Species Isolated from Curing Salts Used in Leather Industry and Their Inactivation by 1 Amp Direct Electric Current

Abstract

Proteolytic halotolerant Bacillus species that may adversely affect leather quality are the predominant bacteria found in the curing salt samples used in the leather industry. Therefore, this study aims to examine the growth of 83 proteolytic halotolerant Bacillus isolates (B. amyloliquefaciens, B. atrophaeus, B. halotolerans, B. licheniformis, B. mojavensis, B. paralicheniformis, B. pumilus, B. safensis, B. siamensis, B. subtilis, B. tequilensis, B. velezensis) recovered from 30 salt samples at different temperatures, pH values, enzymatic activities, utilization of carbon and amino acid sources, antibiotic resistance profiles of these  species against medically important antimicrobials and the inactivation effect of 1 A (amp) direct electric current (DC) on a mixed culture of proteolytic and lipolytic halotolerant Bacillus isolates in a brine solution with 0.85% NaCl. All Bacillus isolates grew between 20-45°C and pH 6-7. Most isolates grew at pH values ranging from 5 to 10. None of the Bacillusisolates grew at pH 4, 5°C, and 65°C. All salt samples contained Bacillus isolates with multiple hydrolytic enzymes. All test isolates produced caseinase enzyme. Although most Bacillus isolates produced cellulase, amylase, β-galactosidase, and xylanase, some Bacillus isolates produced urease, lipase, deoxyribonuclease, and pullulanase enzymes. While all Bacillus isolates utilized glucose, sucrose, and L-methionine, most isolates used ribose, L-cysteine, L-hydroxyproline, L-alanine, L-aspartic acid, L-isoleucine, glycerol, lactose, L-histidine, L-arginine, L-glycine, L-valine, L-leucine, L-glutamic acid, L-phenylalanine, and galactose. In addition, L-lysine, L-proline, L-tyrosine, L-serine, and L-threonine were used by some test isolates. Amino acid utilization test results showed that when the proteolytic enzyme of Bacillus isolates breaks down the collagens into amino acids, these isolates can use amino acids as nutrients to grow and subsequently damage salted skins. Multidrug-resistant Bacillus isolates, exhibiting resistance to three to six antibiotics, were common in the curing salt samples. Resistances of Bacillus  isolates against vancomycin (5 µg) and erythromycin (15 µg) were found to be high. Resistances against clindamycin (2 µg), norfloxacin (10 µg), ciprofloxacin (5 µg), and levofloxacin (5 µg) of some isolates were detected. A 1A DC treatment killed the mixed culture of multidrug-resistant proteolytic halotolerant Bacillus species with high catabolic activity at a voltage level of 10.9 V within 25 minutes. In conclusion, the examination of phenotypic characteristics, and antibiotic resistances of proteolytic halotolerant Bacillus species revealed potential characteristics of Bacillus species that can cause adverse effects on skin quality. Therefore, the extermination of the multidrug-resistant proteolytic and lipolytic halotolerant Bacillus  species with high catabolic activities via the 1A DC process is very important to prevent both contamination of hides and skins with these Bacillus isolates during salt curing processes and the development of antimicrobial resistance and spread on salted skins and hides in the leather industry.