Screening and Potential of Gram Negative Bacterial Isolates for their Extracellular Enzymatic Activities Isolated from the Hospital Aquatic Environment (2024)

Citation: Pharmaceutics, Industrial pharmacy, Medicinal chemistry,

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Bacteria can resist an antimicrobial agent by producing extracellular enzymes that eradicate or inactivate the antibiotics. Majority of drug/metal resistant Gram negative bacterial isolates showed amylase, β-Lactamase, protease, lipase, gelatinase and urease enzymatic activity. In isolates from site-1, maximum 96%, 74% and 72% of the total isolates showed amylase, lipase and catalase activity, whereas only 46%, 34%, 24% and 20% exhibited β-Lactamase, urease, protease and gelatinase activity respectively. In site-II, 88%, 84%, 74%, 68%, 58% and 56% of the isolates demonstrated lipase, protease, amylase, catalase, gelatinase and urease activity respectively, while only 48% of the isolates showed β-Lactamase activity. In case of site- III, maximum 86%, 74%, 68%, 62%, 56% and 52% of the total isolates exhibited lipase, amylase, β-Lactamase, catalase, urease and protease activity respectively. Whereas, only 46% of the isolates demonstrated gelatinase activity. In case of site-IV, maximum 96%, 80%, 66%, 62% of the isolates showed catalase, protease, β-Lactamase and gelatinase activity respectively. Whereas only 48% and 46% of the isolates demonstrated amylase, lipase and urease activity. In our observation most of the isolates from site I, II, III showing lipase activity its mean the hospital wastewater have been suitable source (carbon, nitrogen, salt and mineral) for growth and production of lipase activity. This result revealed that the presence of multi drug/metal resistance Gram negative bacteria from the hospital wastewater were showing various amylase, protease, lipase, catalase, betalactamase, gelatinase and urease, resistance mechanisms and this drug resistant strains may cause infections to the healthy living things. To reduce the spread of drug/metal resistant isolates from hospital to environment, and use good safety sterilization methods before release of waste materials to the environment or sewage.

Keywords

Drug resistance; extracellular enzymes; hospital wastewater; gram negative bacteria

Introduction

Bacteria can resist an antimicrobial agent by producingextracellular enzymes that eliminate or inactivate the antibiotics.As an example, penicillins, monobactams, carbapenems, andcephalosporin’s are identified chemically as beta-lactam antibiotics,and many bacteria happen to resistant to these drugs by producingvarious beta-lactamases that are capable to inactivate some forms ofthese antibiotics. Beta-lactamases break the beta-lactam ring of theantibiotics, thus demolishing the antimicrobials. Bacteria may developresistant to aminoglycosides (Streptomycin, netilmicin, neomycin,gentamicin, tobramycin, amikacin, etc.) by enzymatically adding newchemical groups to these antibiotics, thus inactivating the antibiotics.In Gram-negative bacteria, the antibiotics inactivating enzymesare frequently situated in the periplasmic space placed between thepeptidoglycan layers and the external membrane and interact with theantimicrobials. Gram-positive bacteria not have an external membraneand the antimicrobial inactivating enzymes are typically secreted fromthe bacteria and interact with the antibiotics. Amylases are starchdegrading enzymes. They are extensively spread in microorganism,plants and animals kingdoms. They degrade starch and associatedpolymers to yield products quality of individual amylolytic enzymes.[1] Tallur[2] characterized bacterial isolates, Bacillus toyonensis PNTB1,Lysinibacillus sphaericus PTB, Vibrio vulnificus PMD, Shewanella MPTDBS, and Pseudomonas chlororaphis PNTB for their tolerance tometal and antibiotics. Vibrio vulnificus PMD demonstrate maximumtolerance at higher concentration of metal than other bacteria. Thesebacterial isolates were test for the production of extracellular enzymessuch as lipase, pectinase, tannase, cellulase, chitinase, and l-glutaminase.Vibrio vulnificus exhibited higher production of l-glutaminaseenzyme. Bacillus toyonensis PNTB1 demonstrate lipase, CM-cellulaseand chitinase activities. Baral[3] reported that the extended spectrumb-lactamase (ESBL) production was detected in 55.2% of a subsetof MDR E. coli isolates. Rahman[4] screened extracellular enzymes(amylase, cellulase and xylanase) among the 52 bacterial isolates fromthe soil and water samples. Four isolates were found to be positive foramylase, 10 were found positive for xylanase, 7 were detected positivefor pullulanase and 2 for cellulase activities. Ong[5] studied enzymatictest in E. coli and reported that the all isolates were proteolyticphosphatase and lipolytic enzyme producers. Amylolytic activity didnot exist in all isolates. Protease, leucine arylamidase, alkaline and acidphosphatases, esterase lipase and phospholipase esterase were findout in all the isolates. In this study, we isolate and identify the Gramnegative bacteria from the different hospital wastewater. Furthermore,the antibiotic sensitivity and extracellular enzymes activity werecharacterized. The results will clarify the phenotypic characteristicsand, especially, the virulent traits of Gram negative bacteria to providethe theoretical basis in disease control.

Material and Methods

Sampling and sampling sites

Water samples were collected from three sites of hospitalwastewater along with King George’s Medical University Site (I),Sanjay Gandhi Post Graduate Institute of Medical Sciences (treated)Site (II), Sanjay Gandhi Post Graduate Institute of Medical Sciences(untreated) Site (III) and Dr. Ram Manohar Lohia Hospital Site(IV) at Lucknow city as shown in Figure 1. Samples were collectedin sterile 250-ml polypropylene bottles, according to internationally recommended methodology.[6] Samples were kept at 4°C until theirarrival to laboratory.

Isolation and identification of metal tolerant gramnegative bacteria

Isolation of metal resistant Gram negative bacteria fromwastewater samples were done on metal amended Mac conky agarplates at varying concentration (25-1200 µg/ml). Serial dilutions of thewastewater samples were plated by spreading 0.1 ml on medium for thecount of total metal resistant Gram negative bacteria. Plates incubatedat 37°C for 24 hours and Gram negative bacterial counts includingpink and colorless colonies were expressed as CFU/ml on Mac conkyagar medium. These isolates were finally identified on the basis ofmorphological, cultural and biochemical characteristics includingIMVic tests (indole, methyl red, voges proskeur and citrate utilizationtests, carbohydrate test, hydrogen sulphid test etc.[7]

Determination of antibiotic resistance

The antibiotic resistance was determined by a standard discdiffusion technique using Mueller-Hinton agar (Difco) according tothe recommendations of National Committee for Clinical LaboratoryStandards (NCCLS 2008) including Escherichia coli ATCC 25922 asa control strain. The antimicrobial drugs tested and their sensidiskconcentrations were: Amoxicillin (AMX) 25 µg, Nalidixic acid(NA) 30 µg, Neomycin (N) 30 µg, Kanamycin (K) 30 µg, Ampicillin(AMP) 10 µg, Gentamycin (GEN) 30 µg, Nitrofurazole (NR) 100 µg,Chloramphenicol (CHL) 30 µg, Polymixine B (PB) 300 µg, Methicillin(M) 5 µg, Penicillin (P) 10 µg, Ciprofloxacin (CIP) 5 µg, Erythromycin(ERY) 15 µg, Oflaxicin (OF) 5 µg and Tetracycline (TET) 10 µg. Within15 min of the application of the discs, the plates were inverted andincubated at 37°C. After 24 h of incubation, the plates were examined,and the diameters of the zones of complete inhibition to the nearestwhole millimetre were measured. The zone diameter for individualantimicrobial agents was then translated into sensitive and resistantcategories.[8]

Beta lactamase production

For detection of beta lactamase producing bacteria a loop full ofgrown culture will be transferred into small tube containing 1 ml ofpenicillin G solution and incubated at 37°C for 30 min. 0.5 ml of iodine solution will be added and mixed for 2-3 min. Change in colour tocolourless, indicates as positive result.[9]

Amylase test

Starch degrading activity of the cultures was screened by hydrolysisof starch on a medium (g/l) containing 5 g peptone, 3 g beef extract, 2g starch (soluble), and 20 g agar. Overnight Gram negative bacterialgrowth on Petri dishes was flooded with an iodine solution.[9]Development of a pale yellow zone around a colony in an otherwiseblue medium has indicated starch degrading activity. The isolates wereconsidered positive for amylase production.

Protease test

Protease producing enzymatic activity of the cultures was screened;single streak inoculation of the test microbe was done on agar surface.Incubate the plates at 37°C for 24-48 hours in an inverted position.Presence of clear area around the line of growth was observed, indicatesthe positive result.

Lipase test

Gram negative bacterial isolates were inoculated on tributyrin agarmedium (g/l) containing 5 g peptone, 3 g beef extract, 10 tributyrin and15 g agar, pH 6.5. After incubation at 28°C for 7 days the developmentof a clear halo zone around the colony indicated lipase activity.[10]

Catalase test

Catalase producing enzymatic activity of the cultures was screened;trypticase soy agar medium slants were prepared. Inoculation of testmicrobe was done and slants were kept at 37°C for 24-48 hours. Then,pour 3-4 drops of H2O2 gas bubbles were released it shows the positiveresult.

Gelatinase test

Test to detect gelatin hydrolysis is the Nutrient Gelatin platemethod. In this method, heavy inoculums of an 18-24 hour old testbacteria is stab-inoculated onto culture plates pre-filled with NutrientGelatin (23 g/L Nutrient Agar; 8 g/L 275 bloom gelatin). InoculatedNutrient Gelatin plates are incubated at 35°C for 24 hours. Gelatinhydrolysis is indicated by clear zones around Gelatinase-positivecolonies. In some cases, plates are flooded with mercuric chloridesolution to precipitate unhydrolyzed gelatin making the clear zones easier to see. Results are often observed within 5-10 minutes afterflooding with mercuric chloride solution.[11]

Urease test

Agar plate containing peptone 1.0 g (g/L), sodium chloride 5.0 g(g/L), potassium monohydrogen (or dihydrogen) phosphate 2.0 g (g/L),agar 20 g (g/L). Heat to boil and to dissolve the medium completely,adjust the pH to 6.8. Sterilize by autoclaving at 15 lbs pressure (121°C)for 15 minutes and cool it to 50°C, then add glucose 1.0 g and phenolred (0.2% solution) 6.0 ml and add molten base and steam for I hour,cool to 50°C add 20% aqueous solution 100.0 ml mix well and distributeinto sterile containers. Examine the slants/broth as their color for thepresence of urease (red color) and non-urease (yellow color).[11]

Results

The heavy metal and antibiotics resistance among the Gramnegative bacterial population varied considerably in different metal/antibiotic and water sampling sites. Gram negative bacteria showedlower metal resistant viable count range 4.01 × 10⁴-1.3 × 10³ at 50-100µg/ml in site-IV as compared to 11.03 × 10⁵-1.03 × 10⁴, 12.02 × 10⁵-1.4× 10³ and 12.33 × 10⁵-2.7 × 10³ in site-I, II and III against all metaltested, respectively. Of the 200 Gram negative bacterial isolates tested,marked antibiotic resistances (over 90%) were observed for amoxicillin,methicillin, ampicillin, nalidixic acid and penicillin depending uponthe sampling sites. All Gram negative bacterial isolates also showedmultiple resistance patterns (2-13 antibiotics) in different combinationof antibiotics. Majority of drug/metal resistant Gram negative bacterialisolates showed amylase, ß-Lactamase, protease, lipase, gelatinaseand urease enzymatic activity. In isolates from site-1, maximum 96%,74% and 72% of the total isolates showed amylase, lipase and catalaseactivity, whereas only 46%, 34%, 24% and 20% exhibited ß-Lactamase,urease, protease and gelatinase activity respectively. In site-II, 88%,84%, 74%, 68%, 58% and 56% of the isolates demonstrated lipase,protease, amylase, catalase, gelatinase and urease activity respectively,while only 48% of the isolates showed ß-Lactamase activity. In caseof site- III, maximum 86%, 74%, 68%, 62%, 56% and 52% of thetotal isolates exhibited lipase, amylase, ß-Lactamase, catalase, ureaseand protease activity respectively. Whereas, only 46% of the isolatesdemonstrated gelatinase activity. In case of site-IV, maximum 96%,80%, 66%, 62% of the isolates showed catalase, protease, ß-Lactamaseand gelatinase activity respectively. Whereas only 48% and 46% of theisolates demonstrated amylase, lipase and urease activity. Maximum74.5%, 73% and 60% of the isolates showed catalase, amylase and lipaseand protease activity, whereas, only 45% isolates showed urease activityfrom all the sites tested respectively [Figure 2].

Species based screening of extra cellular enzymatic activitieswere also determined from entire sites collectively. Majority of Gramnegative bacteria including Proteus vulgaris, E. coli, Enterobacteraerogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Citrobacter amalonaticus and Serratia marcescens isolatesproduced amylase, ß-Lactamase, protease, lipase, gelatinase and ureaseenzymatic activity. In Proteus vulgaris Maximum, 98%, 84% and 80%of the total isolates showed urease, gelatinase and amylase and lipaseactivity respectively. 71.1% and 60% of E. coli isolates demonstratedcatalase and amylase and ß-Lactamase activity, while 77.4% and 67.7%,of Enterobacter aerogenes isolates exhibited catalase, amylase and lipaseactivity, respectively. Maximum 82.1% and 78.5 of the Pseudomonasaeruginosa showed lipase and catalase activity respectively. While92.3%, 88.4% and 76.9% of the Klebsiella pneumoniae showed urease,gelatinase and lipase activity respectively. In case of Salmonella typhimaximum, 63.6% of isolates exhibited amylase activity. While in caseof Citrobacter amalonaticus, 81.8% isolates showed ß-Lactamase andlipase activity. 100% of the Serratia marcescens showed lipase andgelatinase activity [Table 1].

Table 1: Extracellular enzymatic activities in various species of Gram negative bacteria from the hospital wastewater (Proteus vulgaris, E. coli, Enterobacter aerogenes and Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Citrobacter amalonaticus and Serratia marcescens).

Discussion

Environmental pollution is one of the most serious problem of themodern time specially water pollution in India due to the dangerouslyhigh levels of antibiotics/metal and other pollutants. Studies haveshown that the release of wastewater from hospitals was associated withan increase in the prevalence of antibiotic resistance. Production ofextracellular photolytic enzymes is a property shared by non-infectiousand infectious microorganisms. These enzymes are crucial factors intheir life cycles and may be mortal to the host when created by infectiousbacteria.[12] The role of proteases in pathogenesis is not defined, butit appeard that they are concerned in colonization and offensiveduring host-infection interaction, apart from provide nutrients forthe microorganism. Nailah[13] reported that all the bacterial isolatesproduces amylase and protease, while only few strain of Gram negativebacteria could produce lipase enzymes. Bacteria produces over 1300diverse enzymes called ß-lactamases which exist as four classes: A, B,C and D. Class D ß-lactamase enzymes, also known as oxacillinasesor OXA enzymes, are able of deactivating a broad range of ß-lactamantibiotics, including “last resort” drugs such as the carbapenems.OXA carbapenemases are mainly find out in Gram-negative bacteriaand are potentially contributory to the resistance of carbapenems inclinical isolates. Their host species include Acinetobacter baumannii and A pittii, which are becoming vital human infections due generallyto the incident of ß-lactam resistance. Carbapenem resistance hasenlarged due to the multiply of such OXA type ß-lactamases.[14,15]Carbapenemase-producing Enterobacteriaceae (CPE) are Gramnegativebacteria that are nearly resistant to the last line Carbapenemclass of antibiotics and have appeared in the UK over the past decade.Bacterium may also attain resistance to antimicrobial agents duringsome mechanisms including production of ß-lactamases, up regulationof efflux pumps, and alteration or down regulation of outer membraneporins.[16] In our observation, maximum 74.5%, 73% and 60% of theisolates showed catalase, amylase, lipase and protease activity whereasonly 45% isolates showed urease activity from all the sites tested respectively. Our study supported by some other researcher.[17-19] In ourobservation most of the isolates from site-I, II, III showing lipase activityits mean the hospital wastewater have been suitable source (carbon,nitrogen, salt and mineral) for growth and production of lipase activity.Similar study reported by others.[20,21] In our observation maximumnumber of Gram negative bacterial isolates produces amylase, betalactamase,protease and lipase while just few bacteria could producegelatinase and urease enzymatic activity. Similar study also reportedby many other workers.[22,23] Nicoloff [24] confirmed IR in the existenceof ß-lactam antibiotics (mecillinam and ampicillin). They examinedthat the bacteria rapid antibiotic-modifying or -degrading enzymesEre(A), Tet(X2) or CatA1 caused IR in the incidence of macrolides(clarithromycin and erythromycin), tetracycline’s (tigecycline andtetracycline) and chloramphenicol respectively. Rahman (3) screenedextracellular enzymes (xylanase, amylase and cellulase) among the52 isolates from various water and soil samples. Four isolates werefound to be positive for amylase, 10 were positive for xylanase, 7 werepositive for pullulanase and 2 for cellulase activities. Representativeisolates was preliminarily known genera of Anoxybacillus, Geobacillus and Bacillus sp. The results demonstrate that hot springs in Malaysiaare rich in microbial life from the Bacillaceae family, and are dynamicas a source of biocatalysts. Albertson[25] reported that the aquaticbacteria produce many extracellular enzymes. Isolates 59 and a Vibriosp. Isolates S14 increase extracellular enzyme production under themexamined DNase, lipase, chitinase, amylase, and several proteases.The Sl4 isolates produces a DNase, chitinase, lipase and proreasesbetween isolates. Biswas[26] screened Halococc for the production ofextracellular enzymes such as glutaminase, amylase, aspa raginase,gelatinase, inulinase, xylanase, cellulasecaseinase, pectinase, urease andlipase. Among these hydrolyt ic enzymes, glutamine and asparaginehydrolytic activities were main, although lipid and casein degradingactivities were not lesser. However, amylase and gelatinase productionwere uncommon. None of the halophiles was competent to degradecellulose, inulin, pectin and xylan and only one isolate was capable ofhydrolyzing urea. Ong (5) studied enzymatic test in E. coli and reportedthat the all isolates were proteolytic phosphatase and lipolytic enzymeproducers. Amylolytic activity did not exist in all isolates. Protease,leucine arylamidase, alkaline and acid phosphatases, esterase lipaseand phospholipase esterase were find out in all the isolates. Khalil[27]screened clinical isolates for their extracellular activities. OF 95.2%isolates showed haemolytic activity followed by 83.7%, 81% and 73.3%protease, lipases and gelatinase, respectively. Our findings were also inagreement with other reports[28,29] that the role of enzymes in antibioticresistance is valuable. Vila[30] reported that the antibiotic resistanceis mostly relevant in infectious Aeromonas species in which, besidesthe classical resistance to ß-lactam antibiotics, multi resistance hasbeen usually identified. Baral[4] reported that the extended spectrumb-lactamase (ESBL) production was detected in 55.2% of a subsetof MDR E. coli isolates. John et al. studied Aeromonas sp for theirextra cellular enzymatic activities. All the isolates from both fish andwater samples produced gelatinase and nuclease but the capability toproduce lipase, caseinase and haemolysins was found to be differentamong isolates from various samples. Among the 15 antibiotics towhich the isolates were tested, all the isolates were found to be sensitiveto ciprofloxacin, gentamicin and chloramphenicol and resistant toamoxycillin. Present study revealed that the presence of multi drug/metal resistance Gram negative bacteria from the hospital wastewaterwere showing various amylase, protease, lipase, catalase, betalactamase,gelatinase and urease, resistance mechanisms and this drug resistantstrains may cause infections to the healthy living things.[31]

Conclusion

In our work we analyzed the enzymatic activity among metaltolerance and antibiotic resistance bacteria from hospital effluents.Bacterial pigmentation and enzymatic activities such as, protease,amylase, lipase, betalactamase, catalase, gelatinase and urease werethe characteristics chosen to study these effects. We find it relevant toevaluate the possible interactions between antibiotic and metal tolerantbacteria in regard to the physiological and metabolic alterationsderived from this contact. We suggest to minimize the spread of drugresistant isolates from hospital to environment is crucial, so good safetysterilization methods to be adopted before release of waste materials tothe environment or sewage.

Competing Interest

The authors declare that there are no competing interests.

Author Contribution

Each author should have participated sufficiently in the work to takepublic responsibility for appropriate portions of the content. Collectionof sample, experimental work and supervision of the research group.Manzar Alam carried out the all experimental studies and drafted themanuscript, Mohd Imran participated in the design and oversee thestudy. All authors read and approved the final manuscript.

Acknowledgements

We are thankful to Prof. SW Akhtar, Vice Chancellor, IntegralUniversity, for providing the necessary facility to conduct this research.The authors also thank the HOD, Department of Biosciences andBioengineering, Integral University, Lucknow for guidance and theircooperation with regard the research work.

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