Metallo-β-Lactamases (MBLs) and Extended Spectrum β-Lactamses (ESBLs) have emerged world-wide as a significant source of β-lactam resistance. The emergence of MBLs and ESBLs encoded on plasmids among Gram-negative pathogens in hospital dumpsites was investigated. Soils of different government and private hospitals were collected and processed following standard bacteriological techniques. Antimicrobial susceptibility testing was carried out by the disk-diffusion technique using Ceftazidime (30 μg), Cefuroxime (30 μg), Cefotaxime (30 μg), Cefixime (5 μg), Trimethprim-sulfamethoxazole (25 μg), Gentamycin (100 μg) Amoxicillin-Clavunalate (30 μg), Ciprofloxacin (5 μg), Ofloxacin (5 μg), Nitrofurantoin (300 μg) and Imipenem (10 μg). The role of plasmids in resistance was evaluated by subjecting isolates to curing using Sodium Dodecyl Sulfate (SDS). ESBLs production by Double-Disk Synergy Test (DDST) was carried out. Isolates resistant to Imipenem were subjected to a confirmatory test using Modified Hodge’s test and to MBLs production by DDST. Eighty-two Gram-negative isolates comprising of 32 (39.02%) Escherichia coli, 20 (24.39%) Serratia marcescens, 14 (17.07%) Klebsiella pneumonia, 10 (12.28%) Proteus mirabilis and 6 (7.32%) Enterobacter aerogenes were obtained. Susceptibility results revealed a 100% resistance of all isolates to Ceftazidime, Cefuroxime, Cefixime, Amoxycillin-clavulanate and Cefotaxime. A total of 66 (80.48%) isolates harboured plasmids out of which 26 (31.71%) isolates were ESBL producers. MBLs production was observed in 8 (25.00%) E. coli, 2 (2.41%) Klebsiella pneumonia and 2 (2.41%) Proteus mirabilis isolates. All MBLs producing isolates were ESBLs producers. The finding of highly resistant isolates producing ESBLs and MBLs in a hospital environment is quite disturbing and should be addressed urgently.
Mismanaged hospital wastes contribute to the ever increasing resistant bacteria in our environment. Most of the substances such as pharmaceuticals and disinfectants used in hospitals for treatment and disinfection end up in dumpsites without any prior treatment. Antimicrobial resistance is usually found in hospital environment. Synthesis of β-lactamase is one of the major mechanisms by which bacteria develop antibiotic resistance [
The emergence of carbapenems, broad spectrum antibiotics with remarkable stability towards β-lactamases especially ESBLs was a huge relief to clinicians. However, this relief was short lived, because infections with carbapenemase-producing enterobacteriaceae quickly emerged. The carbapenemases hydrolyse not only carbapenem but also all hydrolysable β-lactams and are resistant against inhibition by β-lactamase inhibitors. Carbapenemases belong to two major groups distinguished by the hydrolytic mechanisms at the active site. The first group (those that belong to A, C and D) utilizes serine at the active site to carry out its hydrolytic ability; the second group uses at least one zinc atom at the active site to promote hydrolysis, establishing them as metalloenzymes (metallo β-lactamases) [
Metallo β-lactamase genes are carried on mobile genetic elements, thus promoting the rapid dissemination of resistant genes [
A total of eighty two bacterial isolates of the family enterobacteriaceae were obtained from soils of different government and private hospital dump sites in Delta State, Nigeria.
Susceptibilities to antimicrobial agents were determined by standard disk diffusion method on Mueller-Hinton Agar (MHA) according to Clinical Laboratory Institute (CLSI) guidelines [
ESBL production was detected phenotypically by the Double Disc Synergy Test (DDST) method, in accordance with Clinical Laboratory Standard Institute, CLSI guidelines [
A 0.5 McFarland suspension of the test isolate was prepared and inoculated onto Muller Hinton Agar (MHA) plates. Amoxicillin-clavulanic acid disc (30 µg) was placed at the center of the MHA plate inoculated with the test isolate, a ceftazidime disc (30 µg) and a cefotaxime disc were each placed 15mm apart from the center disk. The plates were incubated at 37˚C for 24 hrs. Enhancement of zone diameters of cefotaxime or ceftazidime toward the amoxicillin-clavulanic acid disc were recorded as ESBL positive.
Screening of isolates for carbapenemases were first carried out according to the guideline of CLSI. In this method, an imipenem disc was placed on the surface of an inoculated Mueller Hinton Agar using a sterile forcep. This was incubated at 37˚C for 24 hrs. Isolates that showed reduced susceptibility of ≤23 mm were subjected to a confirmatory test by the modified Hodges test.
In the modified Hodges test, a 0.5 McFarland suspension of E. coli ATCC 25922 was evenly inoculated with a sterile cotton swab on the surface of MHA plates. An imipenem disc (10 µg, Oxoid England) was placed on the surface of the MHA using a sterile inoculating wire loop, the test isolate was streaked on a straight line from the edge of the imipenem disc to the edge of the plate. The plates were incubated at 37˚C for 24 hrs.
A clover lead type indentation or flattening at the intersection of the test organism and E. coli ATCC 25922 within the zone of inhibition of the imipenem disc is considered positive for carbapenemase production.
Plasmid curing experiment was carried out according to the procedures described by [
Beta-lactamase producing bacteria have properties of enzyme production and are currently increasing worldwide in the environment. A cocktail of different active compounds may be present in lower concentrations in hospital dumpsite than they are in therapy. Consistent, excessive dumping of these active compounds exerts pressure on bacterial evolution, creating minor modifications or mutations on an already existing gene leading to new genes that will further be a challenge to therapy.
Eighty-two gram negative bacteria (
Most studies have shown that the β-lactam antibiotics are the most common treatment for bacterial infections in hospital today [
To detect if resistance pattern was plasmid mediated, plasmid curing was carried out using sodium dodecyl sulfate (SDS). Result indicated that over 80% of the isolates harboured plasmids. The presence of antibiotic resistance genes on plasmids of pathogenic bacteria isolates has further helped in the fast spread of resistance among bacteria, causing diversity in plasmid profile, resulting in multi and pan drug resistant bacteria. This limits
Isolate | No. of isolate (%) |
---|---|
E. coli Serratia marcescens Klebsiella pneumonia Proteus mirabilis Enterobacter aerogenes Total | 32 (39.02) 20 (24.39) 14 (17.07) 10 (12.20) 6 (7.32) 82 |
therapeutic options, puts a challenge for the present day clinician, and demands the development of more antibiotics which sadly are no longer readily produced. To overcome this situation, there should be real change. There should be proper policy for preventive medicine. Appropriate ways of managing hospital wastes should be adopted and enforced.
ESBLs production was observed in 31.71% while 14.63% of isolates carried MBLs genes (
It is difficult to give valid epidemiological information on carbapenemases in different parts of the world. This information is dependent on investigators. Countries with active investigators have large number of reports though their countries may not necessarily have higher prevalence than others. However, high prevalence has been found in Greece [
Currently carbapenemases in Enterobacteriaceae are mainly found in K. pneumonia and E. coli. This makes the whole situation more worrying. In this study, 25.00% of E. coli, 14.29% of Klebsiella pneumonia and 10.00% Proteus mirabilis isolates were MBLs producers. These isolates harboured plasmids. Most MBL genes are often associated with transferable plasmids and some of them constitute cassettes in class 1 integrons (VIM-, IPM- and GIM-) [
Isolates (No.) | E. coli (32) | S. marcescens (20) | K. pneumonia (14) | P. mirabilis (10) | E. aerogenes (6) |
---|---|---|---|---|---|
NIT CPR CAZ CRX GEN OFL AMX-CLA CXM SXT CTX IMI | 18.75 6.25 100.00 100.00 31.25 0.00 100.00 100.00 56.25 100.00 46.88 | 50.00 20.00 100.00 100.00 50.00 10.00 100.00 100.00 70.00 100.00 15.00 | 14.29 78.52 100.00 100.00 28.5 14.29 100.00 100.00 57.14 100.00 42.86 | 20.00 20.00 100.00 100.00 20.00 0.00 100.00 100.00 40.00 100.00 0.00 | 33.33 0.00 100.00 100.00 66.67 33.33 100.00 100.00 33.33 100.00 0.00 |
Nitrofurantoin (NIT), Ciprofloxacin (CPR), Ceftazidine (CAZ), Cefuroxime (CRX), Gentamicin (CN), Ofloxacin (OFL). Amoxicillin-Clavulanic acid (Amx-ClA), Cefixime (CXM), Cefotaxime (CTX), Trimethoprime -Sulfamethoxazole (SXT), Imipenem (IMI).
Isolate | No. positive for ESBLs | No. of isolates resistant to imipenem (zone of inhibition ≤ 23 mm) | No positive for MBLs |
---|---|---|---|
E. coli (32) S. marcescens (20) k. pneum (14) P. mirabilis (10) E. aerogenes (6) Total 82 | 10 (31.25) 8 (40.00) 4 (28.57) 2 (20.00) 2 (33.33) 26 (31.71) | 8 (25.00) 0 (0.00) 12 (100.00) 8 (60.00) 0 (0.00) 28 (34.15) | 8 (25.00) 0 (0.00) 2 (14.29) 2 (10.00) 0 (0.00) 12 (14.63) |
The potential of the Klebsiella pneumoniae carbapenemase (kpc)-positive Enterobacteriaceae and VIM producers isolates to spread outside hospital settings and to circulate in the community has been reported [
In the past, all ESBLs were sensitive to carbapenem such as imipenem or meropenem. It was actually used to define ESBLs. Carpabenems were the last resort for infections caused by ESBLs. This is changing as revealed in this study. Though a very low prevalence figure of ESBL resistant to the carbapenem, imipenem was observed, it is emerging. The control of spread is now or never because cumulative experience with ESBL supports the idea that once the prevalence goes beyond a critical level, their eradication from bacterial communities is nearly impossible. Bearing the frightening properties of metallo-enzymes in mind, it is important that a great effort should be made in pursuance of preventive medicine
It is necessary to consider the hospital immediate environment outside the hospital in dealing with issues bordering on nosocomial infection. Nosocomial infections should not only be looked at as infections whose development is enhanced by the inside of a hospital environment. A patient hospitalized for some time and later discharged still has a weak immune system and as such can be easily re-infected on discharge, if the immediate environment outside the hospital is polluted with MDR isolates. This kind of infection should be included as nosocomial infections. This may be the reason for many discharged patient to return back to the hospital after a couple of days. A better health care quality and minimizing nosocomial infections is achievable if we also pay attention to the immediate environment outside the hospital. Information on the health implications of improperly managed healthcare wastes should be provided. Additionally, the implementation of measures that will check the importation of carbapenemases from endemic countries is of utmost importance. It is equally important that all involved in the management of health; from medical clinicians to cleaners play a part in hospital waste management. There should be behavioural change by healthcare givers, patients and visitors. The cleaners in particular have fundamental roles to play in providing a hygienic and clinically clean hospital environment.
The irresponsible dumping of wastes around the dust bin was observed more in government owned hospital than in the private hospitals. This underlines the need to make governments, as well as governmental bodies on health matters aware of this irresponsibility, and the implications so that they work on it. This should be a matter of priorities. Local governments where these government hospitals are located should allocate more financial resources for effective disposal of hospital wastes. The development of a conducive atmosphere in a hospital environment is imperative.
Olivia Sochi Egbule, (2016) Antimicrobial Resistance and β-Lactamase Production among Hospital Dumpsite Isolates. Journal of Environmental Protection,07,1057-1063. doi: 10.4236/jep.2016.77094