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Research article
Evaluation and Molecular Characterization of Colistin-Resistant Isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae from the Infected Wounds of Hospitalized Patients
1Department of Microbiology, University of Central Punjab, Lahore 54000, Pakistan
2Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Ajman University, Ajman 346, United Arab Emirates
J. Microbiol. Biotechnol. 2024; 34(10): 1981-1987
Published October 28, 2024 https://doi.org/10.4014/jmb.2407.07005
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Antimicrobial resistance refers to the ineffectiveness of antimicrobials [1]. This is a significant issue since a resistant illness has the potential to cause death and may be transmitted to others at substantial cost to people and society [2]. Researchers have been working for years to improve data comparability and enhance understanding of highly drug-resistant bacteria, particularly those for which there are no effective treatment options available.
Polymyxins, a collection of polypeptide antibiotics comprising five different chemical entities (polymyxins A-E), were first identified in 1947 [4]. Polymyxin B and polymyxin E (colistin) are the two antibiotics that have been primarily used in clinical settings [4]. Polymyxins exert activity against certain gram-negative bacteria, including
Polymyxin E, also known as colistin, is a cationic polypeptide. This antibiotic kills gram-negative bacteria by targeting the lipopolysaccharide molecules of the cell membrane to exert its pharmacological effect [6]. In the past, genetic mutations in the organism’s chromosomes were considered responsible for colistin resistance (CR). These mutations arise at considerable cost to the organism’s overall fitness and cannot be transferred to other organisms. Nonetheless, there are various drawbacks of colistin as an antibiotic moiety. For example, it produces neurotoxicity and nephrotoxicity. Despite these undesired effects, colistin remains one of the last effective therapeutic choices for invasive bloodstream infections due to carbapenemase-producing gram-negative rods [7].
In recent years, excessive use of colistin has resulted in the development of resistance to this antibiotic [8] in several bacterial species, including
The gene
The CDC has developed guidelines aimed at adopting practical methods to stop the spread of MDR and extensively drug resistant (XDR) gram-negative bacteria.
In this study, we aimed to uncover and analyze the distribution of CR genes among gram-negative bacteria isolated from clinical wound samples collected at Jinnah Hospital and Lahore General Hospital Lahore, Pakistan.
Material and Methods
Selective agars such as mannitol salt agar (Sigma-Aldrich, USA), cetrimide agar (HiMedia Laboratories, India), and MacConkey agar (HiMedia Laboratories) were purchased from various companies. Mueller-Hinton Agar (Thermo Fisher Scientific, UK), nutrient agar, and LB agar from Sigma-Aldrich was purchased through local vendors, Antibiotic disks were purchased from Scientific Laboratory, Lahore, Pakistan. The reverse and forward primers were purchased from SYNBIO Technologies, Italy. A Thermo Scientific Gene JET Plasmid Miniprep Kit was used for plasmid extractions, and the Bio-Rad T100 Thermal Cycler (Singapore) was used for polymerase chain reaction (PCR).
Clinical Sample Collection
Following a basic cleaning of the wounds using normal saline, samples were obtained from 50 patients (
Bacterial Strains
Fifty gram-negative strains (14
Sample Processing
To determine the presence of CR gram-negative bacteria, all clinical wound samples underwent bacteriological processing in accordance with the appropriate recommendations [1]. The samples were appropriately marked with the patient's ID number, lab ID number, date, time, and mode of collection. They were then transported to the laboratory in an insulated cold box with ice packs, in accordance with the recommendations set by the World Health Organization (WHO) [2]. However, samples that were not well marked, inadequately transported with evident indications of contamination, and lacking full patient information, were not included.
Standard Microbiological Testing
All clinical samples were processed using standard microbiological procedures, which included culture, microscopy, biochemical testing, and observation of colonial characteristics of bacteria on selected culture medium. The analysis was performed by streaking the samples on blood agar and nutrient agar plates. The identification was done by observing the typical features of colonies on selective medium. The colonies were transferred onto nutrient agar from the Gram stain slides in the incubator, where the temperature was set at 37°C for 24 h, which is shown in normal medium. The isolates from cetrimide selective agar and MacConkey agar were verified as
Antibiotic Sensitivity Testing
The Kirby Bauer disc diffusion technique was used to test for antibiotic susceptibility in all isolates on Mueller-Hinton agar using antibiotics recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines. The antibiotics used in the study were amikacin (AK, 30 μg), ciprofloxacin (CIP, 5 μg), imipenem (IPM, 10 μg), piperacillin-tazobactum (TZP, 110 μg), colistin (CT, 10 μg), cefepime (FEP, 30), cotrimoxazole (SXT, 25 μg), and ceftriaxone (CRO, 30 μg). Antibiotic resistance was reported in three groups or more idepressant categories and the isolates therein are classified as multiple-drug resistant strains (MDRS) [15].
Testing of Minimal Inhibitory Concentration (MIC)
We assessed colistin resistance in this study using a broth microdilution technique incorporating colistin sulfate powder (Sigma Aldrich) and 96-well, round-bottom microtiter plates [12]. To make the bacterial suspension, four to six colonies from bacterial growths were inoculated into cation-adjusted Mueller Hinton Broth (Sigma Aldrich). The bacterial suspension was calibrated to a 0.5 McFarland standard, which corresponded to a bacterial concentration of 1 - 2 × 108 CFU/ml. The bacterial solution was diluted at a ratio of 1:20 to get a final consultation of bacterial growth of 1 × 108 CFU/ml. A stock solution of colistin sulfate was produced using sterile distilled water. In addition, colistin was diluted in a microtiter plate well using a 2-fold serial dilution method. The dilutions ranged from 0.25 μg/ml to 128 μg/ml and were prepared in 100 μl of sterile CAMHB solution. A bacterial solution of 50 μl was added to each well, except the negative control. The positive control was generated by adding a bacterial suspension with the highest concentration. The lowest concentration of MIC [14] did not result in any growth. The microtiter plates were placed in an incubator and kept at 37°C for 24 h. Following incubation, the growth in each well was quantified using a microplate reader configured to measure the optical density (OD) at the specific wavelength of 600 nm. The MIC data were analyzed based on the CLSI guidelines [14]. The forthcoming MIC values on the microplate reader were calculated by estimating the growth inhibition rate (GIR) using the following method:
The GIR is expressed as a percentage. The OD of the control group is denoted as Vo, whereas the OD of the test group is denoted as V1 [6].
Preparation of Bacterial Cultures and Extraction of mcr-1 Plasmids from CR Strains
The investigation was done on
The plasmids were examined for the presence of the
Statistical Analysis
Findings were compared through Student’s
Results
Phenotypic Analysis
On blood agar,
Distribution of P. aeruginosa and K. pneumoniae among Different Body Sites
A total of 50 isolates of
-
Fig. 1. Distribution of
P. aeruginosa andK. pneumoniae among different body sites, with most of the CR strains being recovered from the burn unit (46%), and the lowest rate of strains recovered from the surgical unit (24%), while the diabetic unit showed a 30% rate.
Demographic Data
The average age of the patients included in the research was 43.1 years. The largest number (22) of strains were obtained from patients aged 20 to 40, whilst the smallest number (6) of strains were obtained from individuals aged 40 to 60. Out of 50 isolates, 40 isolates (35 isolates of
Susceptibility Testing
Among the collected wound samples, 50 gram-negative bacterial strains were obtained as follows:
-
Table 1 . The antibiotic susceptibility and resistance profile of carbapenem-resistant bacteria was analyzed in a sample size of 40.
Antibiotics Pseudomonas aeruginosa (n = 35)Klebsiella pneumoniae (n = 5)S (%) R (%) S (%) R (%) Amikacin 5 (14) 30 (86) 1(20) 4 (80) Ciprofloxacin 5 (14) 30 (86) 1(20) 4 (80) Imipenem 1 (2) 34 (97) 0 (0) 5 (100) Piperacillin/tazobactum 8 (23) 27 (77) 1(20) 4 (80) Cefepime 1 (2) 34 (97) 0 (0) 5 (100) Cotrimoxazole 2 (6) 33(94) 0 (0) 5 (100) Ceftriaxone 0 (0) 35 (100) 0 (0) 5 (100)
Testing of MIC
In this study, we analyzed the antibiotic susceptibility profiles of
-
Fig. 2. MIC values of the CR strains of
P. aeruginosa andK. pneumoniae . Among the 35P. aeruginosa isolates tested in this study, the majority (27 isolates) exhibited an MIC = 8 μg/ml. However, 8 isolates ofP. aeruginosa showed a higher MIC of 128 μg/ml. ForK. pneumoniae , out of the 5 isolates examined, 3 isolates displayed an MIC of 8 μg/ml. OneK. pneumoniae isolate demonstrated a higher MIC of 32 μg/ml, while another isolate showed a lower MIC of 2 μg/ml.
Determination of mcr-1 Gene
In this study, genetic detection of the
-
Table 2 . Detection of the
mcr-1 gene by PCR in bacterial isolates.Strains Total No. of Isolates Mcr-1 Gene No. of Isolates P. aeruginosa 35 + 4 K. pneumoniae 5 + 2
-
Fig. 3.
mcr-1 gene detected in colistin-resistant isolates. Genetic detection of themcr-1 gene was conducted using conventional PCR across a total of 40 bacterial isolates. Among these six isolates, 15% were found positive for themcr-1 gene. (Note: The letter ‘P’ representsP. aeruginosa . In addition, P5, P12, P14, and P40 are the isolate numbers ofP. aeruginosa ).
Discussion
In our study, we evaluated the antibiotic susceptibility profiles of
To provide a comprehensive analysis of antibiotic susceptibility, we included normal control isolates that exhibited typical antibiotic susceptibility profiles. These control isolates were sourced from non-infected patients and underwent the same MIC and antibiotic tests as the CR isolates. Our comparison showed that the normal control isolates of both
Comparing our findings with the recent studies, data from global surveillance and research publications highlight the ongoing challenges with antibiotic resistance in both
According to our research, conventional PCR was used to detect the gene
The presence of the
Study Area and Ethical Approval
The research was conducted between August 2023 and October 2023 at the Microbiology Laboratories of COMSATS University Islamabad (CUI), Lahore Campus, and the University of Central Punjab (UCP), Lahore, Pakistan. The Institutional Ethical Review Board (IREB) of UCP granted ethical approval with reference number FOST/DERC/2023/02. Prior to obtaining written informed permission from the patient/attendant, the patients were provided with comprehensive information about the nature and objective of the research. Due to the absence of further specimens and the lack of personal information received from patients, informed permission was not necessary for this investigation. The techniques were carried out in complete adherence to the criteria of the World Medical Association Declaration of Helsinki about the ethics for conducting medical research involving human subjects and identifiable human material/data.
Funding and Acknowledgement
We extend our appreciation to the Deanship of Graduate Studies at Ajman University, Ajman, United Arab Emirates, for their support for payment of publication charges.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Nation RL, Li J, Turnidge JD, Milne RW, Coulthard K, Rayner CR,
et al . 2006. Colistin: the re-emerging antibiotic for multidrugresistant gram-negative bacterial infections.Lancet Infect. Dis. 6 : 589-601. - World Health Organization (WHO). 2018. Global Antimicrobial Resistance Surveil- lance System (GLASS) report: early implementation 2016–2017. Geneva, Switzerland: WHO, 2018.
- Tang SS, Apisarnthanarak A, Hsu LY. 2014. Mechanisms of β-lactam antimicrobial resistance and epidemiology of major community- and healthcare-associated multidrug-resistant bacteria.
Adv. Drug Deliv. Rev. 78 : 3-13. - Falagas ME, Kasiakou SK, Saravolatz LD. 2005. Colistin: the revival of polymyxins for the management of multidrug-resistant Gramnegative bacterial infections.
Clin. Infect. Dis. 40 : 1333-1341. - Kumar A, Vimal A, Kumar A. 2023. Inhibitory interaction and pharmacological analyses of berries phenolics against
Listeria monocytogenes virulent protein internalin B.World J. Tradit. Chin. Med. 9 : 71-80. - Gregoire N, Aranzana-Climent V, Magreault S, Marchand S, Couet W. 2017. Clinical pharmacokinetics and pharmacodynamics of colistin.
Clin. Pharmacokinet. 56 : 1441-1460. - Kanazawa K, Sato Y, Ohki K, Okimura K, Uchida Y, Shindo M,
et al . 2009. Contribution of each amino acid residue in polymyxin B (3) to antimicrobial and lipopolysaccharide-binding activity.Chem. Pharm. Bull. 57 : 240-244. - Kempf I, Fleury MA, Drider D, Bruneau M, Sanders P, Chauvin C,
et al . 2013. What do we know about resistance to Colistin inEnterobacteriaceae in avian and pig production in Europe?Int. J. Antimicrob Agents 42 : 379-383. - Gunn JS. 2008. The Salmonella PmrAB regulon: lipopolysaccharide modifications, antimicrobial peptide resistance and more.
Trends Microbiol. 6 : 284-290. - Cannatelli A, D'Andrea MM, Giani T, Di Pilato V, Arena F, Ambretti S,
et al . 2013. In vivo emergence of Colistin resistance inKlebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator.Antimicrob. Agents Chemother. 57 : 5521-5526. - Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J,
et al . 2016. Emergence of plasmid-mediated Colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Zurfuh K, Poirel L, Nordmann P, Nüesch-Inderbinen M, Hächler H, Stephan R. 2016. Occurrence of the plasmid-borne
mcr-1 Colistin resistance gene in extended-spectrum β-lactamase-producingEnterobacteriaceae in river water and imported vegetable samples in Switzerland.Antimicrob. Agents Chemother. 60 : 2594-2595. - Malhotra-Kumar S, Xavier BB, Das AJ, Lammens C, Hoang HT, Pham NT,
et al . 2016. Colistin-resistantEscherichia coli harbouringmcr-1 isolated from food animals in Hanoi, Vietnam.Lancet Infect. Dis. 16 : 286-287. - Clinical and Laboratory Standard Institute (CLSI). M100 Performance Standards for Antimicrobial Susceptibility Testing 30th ed. An Informational Supplement; CLSI: Wayne, PA, USA 2020. Available from: https://www.nih.org.pk/wp-content/uploads/2021/02/CLSI-2020.pdf
- Sherry N, Howden B. 2018. Emerging Gram-negative resistance to last-line antimicrobial agents fosfomycin, Colistin and ceftazidime avibactam-epidemiology, laboratory detection and treatment implications.
Expert Rev. Anti Infect. Ther. 16 : 289-306. - Talbot GH, Bradley J, Edwards JE Jr, Gilbert D, Scheld M, Bartlett JG,
et al . 2006. Bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the infectious disease's society of America.Clin. Infect. Dis. 42 : 657-668. - Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H,
et al . 2016. Identification of a novel plasmid-mediated colistin-resistance gene, mcr2, inEscherichia coli , Belgium.Euro. Surveill. 21 : 130-280. - Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S,
et al . 2016. MCR-1.2, a new mcr variant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producingKlebsiella pneumoniae strain of sequence type 512.Antimicrob. Agents Chemother. 60 : 5612-2615. - Yang YQ, Li YX, Song T, Yang YX, Jiang W, Zhang A-Y,
et al . 2017. Colistin resistance genemcr-1 and its variant inEscherichia coli isolates fromchickens in China .Antimicrob Agents Chemother. 61 : e01204-e01205. - Bilal H, Hameed F, Khan M A, Khan S, Yang X, Rehman TU. 2020. Detection of
mcr-1 gene in extended-spectrum β-lactamaseproducingKlebsiella pneumoniae from human urine samples in Pakistan.Jundishapur. J. Microbiol. 13 : 236-376. - Cheesebrough M. 2006. District laboratory practice in tropical countries. Part 2, 2nd Edition, Cambridge University Press, New York. 30–71. Available on: https://www.me
- Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Gisake CG,
et al . 2011. Multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.Clin. Microb. Infect. 18 : 268-281. - Zubair KU, Shah AH, Fawwad A, Sabir R, Butt A. 2019. Frequency of urinary tract infection and antibiotic sensitivity of uropathogens in patients with diabetes.
Pak J. Med. Sci. 35 : 1664-1899. - Liu J, Zhang CC, Zhang SQ, Wang JH, Xu RR, Yang SL,
et al . 2023. Clinical factors affecting platelet growth in the treatment of aplastic anemia by tonifying kidney and generating blood.World J. Tradit. Chin. Med. 9 : 438-446. - Liu Y, Wang Y, Walsh T, Yi L, Zhang R, Spencer J,
et al . 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Wang J, Zhou Jy, Qu Tt, Shen P, Wei Zq, Yu Ys,
et al . 2021. Molecular epidemiology and mechanisms of carbapenem resistance inPseudomonas aeruginosa isolates from Chinese hospitals.Int. J. Antimicrob. Agents Chemother. 35 : 486-491. - Peirano G, Pitout D. 2022. Extended-spectrum β-lactamase-producing
Enterobacterales : update on molecular epidemiology and treatment options.Drugs 82 : 1477-1494. - Li R, Lu X, Wang Y,
et al . 2021. Occurrence of plasmid-mediated colistin resistance genemcr-1 in clinical isolates ofKlebsiella pneumoniae from China.Antimicrob. Agents Chemother. 65 : 89-150. - Mendes RE, Castanheira M, Wang X,
et al . 2023. First report ofmcr-1 -mediated colistin resistance inPseudomonas aeruginosa isolates from South America.Antimicrob. Agents Chemother. 67 : 45-52. - European Centre for Disease Prevention and Control (ECDC). 2022. Surveillance of antimicrobial resistance in Europe – Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). Stockholm: ECDC.
- Liu YY, Wang Y, Walsh TR, Ling-Xian Yi, Rong Zhang, James Spencer,
et al . 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Poirel L, Jayol A, Nordmann P. 2017. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes.
Clin. Microbiol. Rev. 30 : 557-596. - Mohamed Abd El-Gawad El-Sayed Ahmed, Lan-Lan Zhong, Cong Shen, Yongqiang Yang, Yohei Doi, Guo-Bao Tian. 2020. Colistin and its role in the Era of antibiotic resistance: an extended review (2000-2019).
Emerg. Microbes Infect. 9 : 868-885. - Li XY, Liang LN, Zhang WJ,
et al . 2024. Mechanism ofMingjing granules in treating wet age-related macular degeneration based on network pharmacology and experimental verification.World J. Tradit. Chin. Med. 10 : 22-32.
Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2024; 34(10): 1981-1987
Published online October 28, 2024 https://doi.org/10.4014/jmb.2407.07005
Copyright © The Korean Society for Microbiology and Biotechnology.
Evaluation and Molecular Characterization of Colistin-Resistant Isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae from the Infected Wounds of Hospitalized Patients
Munaza Ijaz1*, Madiha Khan1, and Haya Yasin2*
1Department of Microbiology, University of Central Punjab, Lahore 54000, Pakistan
2Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Ajman University, Ajman 346, United Arab Emirates
Correspondence to:Munaza Ijaz, munazza3murtaza@gmail.com
H Yasin, haya.yasin@gmail.com
Abstract
This study was planned to determine the colistin-resistant (CR) gene distribution among two species of gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae. In total, 50 isolates of K. pneumoniae (14 isolates, 28%) and P. aeruginosa (36 isolates, 72%) were isolated between August 2023 and October 2023 from clinical wound samples at Jinnah Hospital and Lahore General Hospital Lahore, Pakistan. To determine the resistance genes linked to CR and assess antimicrobial susceptibility, all isolates were kept at -80°C in 15% glycerol broth. Using the right primer sets, a polymerase chain reaction (PCR) was utilized to identify the CR-associated mcr-1 gene of the gram-negative isolates. Out of 50, 40 isolates (80%) showed resistance against colistin with MICs of 8 and 128 μg/ml. The majority (97%) of P. aeruginosa CR strains were considered multidrug resistant (MDR). All K. pneumoniae isolates were resistant to cefepime, cotrimoxazole, ceftriaxone, and imipenem. The clinical CR isolates of P. aeruginosa were highly resistant to ceftriaxone, imipenem, cefepime, cotrimoxazole, ciprofloxacin, amikacin, and piperacillin/tazobactum. The antibiotic resistance pattern was terrifyingly high among both bacterial species. According to the PCR results, CR was prevalent among the gram-negative samples, and the mcr-1 gene was positive in 6/40 (15%) of the CR isolates, including four P. aeruginosa and two K. pneumoniae strains. The high CR (80%) reported in this research is cause for concern and underscores an urgent need to use colistin in a limited and logical manner, similar to other antibiotics.
Keywords: Antibiotic resistance, antibacterial susceptibility, colistin, PCR, resistance gene
Introduction
Antimicrobial resistance refers to the ineffectiveness of antimicrobials [1]. This is a significant issue since a resistant illness has the potential to cause death and may be transmitted to others at substantial cost to people and society [2]. Researchers have been working for years to improve data comparability and enhance understanding of highly drug-resistant bacteria, particularly those for which there are no effective treatment options available.
Polymyxins, a collection of polypeptide antibiotics comprising five different chemical entities (polymyxins A-E), were first identified in 1947 [4]. Polymyxin B and polymyxin E (colistin) are the two antibiotics that have been primarily used in clinical settings [4]. Polymyxins exert activity against certain gram-negative bacteria, including
Polymyxin E, also known as colistin, is a cationic polypeptide. This antibiotic kills gram-negative bacteria by targeting the lipopolysaccharide molecules of the cell membrane to exert its pharmacological effect [6]. In the past, genetic mutations in the organism’s chromosomes were considered responsible for colistin resistance (CR). These mutations arise at considerable cost to the organism’s overall fitness and cannot be transferred to other organisms. Nonetheless, there are various drawbacks of colistin as an antibiotic moiety. For example, it produces neurotoxicity and nephrotoxicity. Despite these undesired effects, colistin remains one of the last effective therapeutic choices for invasive bloodstream infections due to carbapenemase-producing gram-negative rods [7].
In recent years, excessive use of colistin has resulted in the development of resistance to this antibiotic [8] in several bacterial species, including
The gene
The CDC has developed guidelines aimed at adopting practical methods to stop the spread of MDR and extensively drug resistant (XDR) gram-negative bacteria.
In this study, we aimed to uncover and analyze the distribution of CR genes among gram-negative bacteria isolated from clinical wound samples collected at Jinnah Hospital and Lahore General Hospital Lahore, Pakistan.
Material and Methods
Selective agars such as mannitol salt agar (Sigma-Aldrich, USA), cetrimide agar (HiMedia Laboratories, India), and MacConkey agar (HiMedia Laboratories) were purchased from various companies. Mueller-Hinton Agar (Thermo Fisher Scientific, UK), nutrient agar, and LB agar from Sigma-Aldrich was purchased through local vendors, Antibiotic disks were purchased from Scientific Laboratory, Lahore, Pakistan. The reverse and forward primers were purchased from SYNBIO Technologies, Italy. A Thermo Scientific Gene JET Plasmid Miniprep Kit was used for plasmid extractions, and the Bio-Rad T100 Thermal Cycler (Singapore) was used for polymerase chain reaction (PCR).
Clinical Sample Collection
Following a basic cleaning of the wounds using normal saline, samples were obtained from 50 patients (
Bacterial Strains
Fifty gram-negative strains (14
Sample Processing
To determine the presence of CR gram-negative bacteria, all clinical wound samples underwent bacteriological processing in accordance with the appropriate recommendations [1]. The samples were appropriately marked with the patient's ID number, lab ID number, date, time, and mode of collection. They were then transported to the laboratory in an insulated cold box with ice packs, in accordance with the recommendations set by the World Health Organization (WHO) [2]. However, samples that were not well marked, inadequately transported with evident indications of contamination, and lacking full patient information, were not included.
Standard Microbiological Testing
All clinical samples were processed using standard microbiological procedures, which included culture, microscopy, biochemical testing, and observation of colonial characteristics of bacteria on selected culture medium. The analysis was performed by streaking the samples on blood agar and nutrient agar plates. The identification was done by observing the typical features of colonies on selective medium. The colonies were transferred onto nutrient agar from the Gram stain slides in the incubator, where the temperature was set at 37°C for 24 h, which is shown in normal medium. The isolates from cetrimide selective agar and MacConkey agar were verified as
Antibiotic Sensitivity Testing
The Kirby Bauer disc diffusion technique was used to test for antibiotic susceptibility in all isolates on Mueller-Hinton agar using antibiotics recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines. The antibiotics used in the study were amikacin (AK, 30 μg), ciprofloxacin (CIP, 5 μg), imipenem (IPM, 10 μg), piperacillin-tazobactum (TZP, 110 μg), colistin (CT, 10 μg), cefepime (FEP, 30), cotrimoxazole (SXT, 25 μg), and ceftriaxone (CRO, 30 μg). Antibiotic resistance was reported in three groups or more idepressant categories and the isolates therein are classified as multiple-drug resistant strains (MDRS) [15].
Testing of Minimal Inhibitory Concentration (MIC)
We assessed colistin resistance in this study using a broth microdilution technique incorporating colistin sulfate powder (Sigma Aldrich) and 96-well, round-bottom microtiter plates [12]. To make the bacterial suspension, four to six colonies from bacterial growths were inoculated into cation-adjusted Mueller Hinton Broth (Sigma Aldrich). The bacterial suspension was calibrated to a 0.5 McFarland standard, which corresponded to a bacterial concentration of 1 - 2 × 108 CFU/ml. The bacterial solution was diluted at a ratio of 1:20 to get a final consultation of bacterial growth of 1 × 108 CFU/ml. A stock solution of colistin sulfate was produced using sterile distilled water. In addition, colistin was diluted in a microtiter plate well using a 2-fold serial dilution method. The dilutions ranged from 0.25 μg/ml to 128 μg/ml and were prepared in 100 μl of sterile CAMHB solution. A bacterial solution of 50 μl was added to each well, except the negative control. The positive control was generated by adding a bacterial suspension with the highest concentration. The lowest concentration of MIC [14] did not result in any growth. The microtiter plates were placed in an incubator and kept at 37°C for 24 h. Following incubation, the growth in each well was quantified using a microplate reader configured to measure the optical density (OD) at the specific wavelength of 600 nm. The MIC data were analyzed based on the CLSI guidelines [14]. The forthcoming MIC values on the microplate reader were calculated by estimating the growth inhibition rate (GIR) using the following method:
The GIR is expressed as a percentage. The OD of the control group is denoted as Vo, whereas the OD of the test group is denoted as V1 [6].
Preparation of Bacterial Cultures and Extraction of mcr-1 Plasmids from CR Strains
The investigation was done on
The plasmids were examined for the presence of the
Statistical Analysis
Findings were compared through Student’s
Results
Phenotypic Analysis
On blood agar,
Distribution of P. aeruginosa and K. pneumoniae among Different Body Sites
A total of 50 isolates of
-
Figure 1. Distribution of
P. aeruginosa andK. pneumoniae among different body sites, with most of the CR strains being recovered from the burn unit (46%), and the lowest rate of strains recovered from the surgical unit (24%), while the diabetic unit showed a 30% rate.
Demographic Data
The average age of the patients included in the research was 43.1 years. The largest number (22) of strains were obtained from patients aged 20 to 40, whilst the smallest number (6) of strains were obtained from individuals aged 40 to 60. Out of 50 isolates, 40 isolates (35 isolates of
Susceptibility Testing
Among the collected wound samples, 50 gram-negative bacterial strains were obtained as follows:
-
Table 1 . The antibiotic susceptibility and resistance profile of carbapenem-resistant bacteria was analyzed in a sample size of 40..
Antibiotics Pseudomonas aeruginosa (n = 35)Klebsiella pneumoniae (n = 5)S (%) R (%) S (%) R (%) Amikacin 5 (14) 30 (86) 1(20) 4 (80) Ciprofloxacin 5 (14) 30 (86) 1(20) 4 (80) Imipenem 1 (2) 34 (97) 0 (0) 5 (100) Piperacillin/tazobactum 8 (23) 27 (77) 1(20) 4 (80) Cefepime 1 (2) 34 (97) 0 (0) 5 (100) Cotrimoxazole 2 (6) 33(94) 0 (0) 5 (100) Ceftriaxone 0 (0) 35 (100) 0 (0) 5 (100)
Testing of MIC
In this study, we analyzed the antibiotic susceptibility profiles of
-
Figure 2. MIC values of the CR strains of
P. aeruginosa andK. pneumoniae . Among the 35P. aeruginosa isolates tested in this study, the majority (27 isolates) exhibited an MIC = 8 μg/ml. However, 8 isolates ofP. aeruginosa showed a higher MIC of 128 μg/ml. ForK. pneumoniae , out of the 5 isolates examined, 3 isolates displayed an MIC of 8 μg/ml. OneK. pneumoniae isolate demonstrated a higher MIC of 32 μg/ml, while another isolate showed a lower MIC of 2 μg/ml.
Determination of mcr-1 Gene
In this study, genetic detection of the
-
Table 2 . Detection of the
mcr-1 gene by PCR in bacterial isolates..Strains Total No. of Isolates Mcr-1 Gene No. of Isolates P. aeruginosa 35 + 4 K. pneumoniae 5 + 2
-
Figure 3.
mcr-1 gene detected in colistin-resistant isolates. Genetic detection of themcr-1 gene was conducted using conventional PCR across a total of 40 bacterial isolates. Among these six isolates, 15% were found positive for themcr-1 gene. (Note: The letter ‘P’ representsP. aeruginosa . In addition, P5, P12, P14, and P40 are the isolate numbers ofP. aeruginosa ).
Discussion
In our study, we evaluated the antibiotic susceptibility profiles of
To provide a comprehensive analysis of antibiotic susceptibility, we included normal control isolates that exhibited typical antibiotic susceptibility profiles. These control isolates were sourced from non-infected patients and underwent the same MIC and antibiotic tests as the CR isolates. Our comparison showed that the normal control isolates of both
Comparing our findings with the recent studies, data from global surveillance and research publications highlight the ongoing challenges with antibiotic resistance in both
According to our research, conventional PCR was used to detect the gene
The presence of the
Study Area and Ethical Approval
The research was conducted between August 2023 and October 2023 at the Microbiology Laboratories of COMSATS University Islamabad (CUI), Lahore Campus, and the University of Central Punjab (UCP), Lahore, Pakistan. The Institutional Ethical Review Board (IREB) of UCP granted ethical approval with reference number FOST/DERC/2023/02. Prior to obtaining written informed permission from the patient/attendant, the patients were provided with comprehensive information about the nature and objective of the research. Due to the absence of further specimens and the lack of personal information received from patients, informed permission was not necessary for this investigation. The techniques were carried out in complete adherence to the criteria of the World Medical Association Declaration of Helsinki about the ethics for conducting medical research involving human subjects and identifiable human material/data.
Funding and Acknowledgement
We extend our appreciation to the Deanship of Graduate Studies at Ajman University, Ajman, United Arab Emirates, for their support for payment of publication charges.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
Fig 2.
Fig 3.
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Table 1 . The antibiotic susceptibility and resistance profile of carbapenem-resistant bacteria was analyzed in a sample size of 40..
Antibiotics Pseudomonas aeruginosa (n = 35)Klebsiella pneumoniae (n = 5)S (%) R (%) S (%) R (%) Amikacin 5 (14) 30 (86) 1(20) 4 (80) Ciprofloxacin 5 (14) 30 (86) 1(20) 4 (80) Imipenem 1 (2) 34 (97) 0 (0) 5 (100) Piperacillin/tazobactum 8 (23) 27 (77) 1(20) 4 (80) Cefepime 1 (2) 34 (97) 0 (0) 5 (100) Cotrimoxazole 2 (6) 33(94) 0 (0) 5 (100) Ceftriaxone 0 (0) 35 (100) 0 (0) 5 (100)
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Table 2 . Detection of the
mcr-1 gene by PCR in bacterial isolates..Strains Total No. of Isolates Mcr-1 Gene No. of Isolates P. aeruginosa 35 + 4 K. pneumoniae 5 + 2
References
- Nation RL, Li J, Turnidge JD, Milne RW, Coulthard K, Rayner CR,
et al . 2006. Colistin: the re-emerging antibiotic for multidrugresistant gram-negative bacterial infections.Lancet Infect. Dis. 6 : 589-601. - World Health Organization (WHO). 2018. Global Antimicrobial Resistance Surveil- lance System (GLASS) report: early implementation 2016–2017. Geneva, Switzerland: WHO, 2018.
- Tang SS, Apisarnthanarak A, Hsu LY. 2014. Mechanisms of β-lactam antimicrobial resistance and epidemiology of major community- and healthcare-associated multidrug-resistant bacteria.
Adv. Drug Deliv. Rev. 78 : 3-13. - Falagas ME, Kasiakou SK, Saravolatz LD. 2005. Colistin: the revival of polymyxins for the management of multidrug-resistant Gramnegative bacterial infections.
Clin. Infect. Dis. 40 : 1333-1341. - Kumar A, Vimal A, Kumar A. 2023. Inhibitory interaction and pharmacological analyses of berries phenolics against
Listeria monocytogenes virulent protein internalin B.World J. Tradit. Chin. Med. 9 : 71-80. - Gregoire N, Aranzana-Climent V, Magreault S, Marchand S, Couet W. 2017. Clinical pharmacokinetics and pharmacodynamics of colistin.
Clin. Pharmacokinet. 56 : 1441-1460. - Kanazawa K, Sato Y, Ohki K, Okimura K, Uchida Y, Shindo M,
et al . 2009. Contribution of each amino acid residue in polymyxin B (3) to antimicrobial and lipopolysaccharide-binding activity.Chem. Pharm. Bull. 57 : 240-244. - Kempf I, Fleury MA, Drider D, Bruneau M, Sanders P, Chauvin C,
et al . 2013. What do we know about resistance to Colistin inEnterobacteriaceae in avian and pig production in Europe?Int. J. Antimicrob Agents 42 : 379-383. - Gunn JS. 2008. The Salmonella PmrAB regulon: lipopolysaccharide modifications, antimicrobial peptide resistance and more.
Trends Microbiol. 6 : 284-290. - Cannatelli A, D'Andrea MM, Giani T, Di Pilato V, Arena F, Ambretti S,
et al . 2013. In vivo emergence of Colistin resistance inKlebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator.Antimicrob. Agents Chemother. 57 : 5521-5526. - Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J,
et al . 2016. Emergence of plasmid-mediated Colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Zurfuh K, Poirel L, Nordmann P, Nüesch-Inderbinen M, Hächler H, Stephan R. 2016. Occurrence of the plasmid-borne
mcr-1 Colistin resistance gene in extended-spectrum β-lactamase-producingEnterobacteriaceae in river water and imported vegetable samples in Switzerland.Antimicrob. Agents Chemother. 60 : 2594-2595. - Malhotra-Kumar S, Xavier BB, Das AJ, Lammens C, Hoang HT, Pham NT,
et al . 2016. Colistin-resistantEscherichia coli harbouringmcr-1 isolated from food animals in Hanoi, Vietnam.Lancet Infect. Dis. 16 : 286-287. - Clinical and Laboratory Standard Institute (CLSI). M100 Performance Standards for Antimicrobial Susceptibility Testing 30th ed. An Informational Supplement; CLSI: Wayne, PA, USA 2020. Available from: https://www.nih.org.pk/wp-content/uploads/2021/02/CLSI-2020.pdf
- Sherry N, Howden B. 2018. Emerging Gram-negative resistance to last-line antimicrobial agents fosfomycin, Colistin and ceftazidime avibactam-epidemiology, laboratory detection and treatment implications.
Expert Rev. Anti Infect. Ther. 16 : 289-306. - Talbot GH, Bradley J, Edwards JE Jr, Gilbert D, Scheld M, Bartlett JG,
et al . 2006. Bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the infectious disease's society of America.Clin. Infect. Dis. 42 : 657-668. - Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H,
et al . 2016. Identification of a novel plasmid-mediated colistin-resistance gene, mcr2, inEscherichia coli , Belgium.Euro. Surveill. 21 : 130-280. - Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S,
et al . 2016. MCR-1.2, a new mcr variant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producingKlebsiella pneumoniae strain of sequence type 512.Antimicrob. Agents Chemother. 60 : 5612-2615. - Yang YQ, Li YX, Song T, Yang YX, Jiang W, Zhang A-Y,
et al . 2017. Colistin resistance genemcr-1 and its variant inEscherichia coli isolates fromchickens in China .Antimicrob Agents Chemother. 61 : e01204-e01205. - Bilal H, Hameed F, Khan M A, Khan S, Yang X, Rehman TU. 2020. Detection of
mcr-1 gene in extended-spectrum β-lactamaseproducingKlebsiella pneumoniae from human urine samples in Pakistan.Jundishapur. J. Microbiol. 13 : 236-376. - Cheesebrough M. 2006. District laboratory practice in tropical countries. Part 2, 2nd Edition, Cambridge University Press, New York. 30–71. Available on: https://www.me
- Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Gisake CG,
et al . 2011. Multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.Clin. Microb. Infect. 18 : 268-281. - Zubair KU, Shah AH, Fawwad A, Sabir R, Butt A. 2019. Frequency of urinary tract infection and antibiotic sensitivity of uropathogens in patients with diabetes.
Pak J. Med. Sci. 35 : 1664-1899. - Liu J, Zhang CC, Zhang SQ, Wang JH, Xu RR, Yang SL,
et al . 2023. Clinical factors affecting platelet growth in the treatment of aplastic anemia by tonifying kidney and generating blood.World J. Tradit. Chin. Med. 9 : 438-446. - Liu Y, Wang Y, Walsh T, Yi L, Zhang R, Spencer J,
et al . 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Wang J, Zhou Jy, Qu Tt, Shen P, Wei Zq, Yu Ys,
et al . 2021. Molecular epidemiology and mechanisms of carbapenem resistance inPseudomonas aeruginosa isolates from Chinese hospitals.Int. J. Antimicrob. Agents Chemother. 35 : 486-491. - Peirano G, Pitout D. 2022. Extended-spectrum β-lactamase-producing
Enterobacterales : update on molecular epidemiology and treatment options.Drugs 82 : 1477-1494. - Li R, Lu X, Wang Y,
et al . 2021. Occurrence of plasmid-mediated colistin resistance genemcr-1 in clinical isolates ofKlebsiella pneumoniae from China.Antimicrob. Agents Chemother. 65 : 89-150. - Mendes RE, Castanheira M, Wang X,
et al . 2023. First report ofmcr-1 -mediated colistin resistance inPseudomonas aeruginosa isolates from South America.Antimicrob. Agents Chemother. 67 : 45-52. - European Centre for Disease Prevention and Control (ECDC). 2022. Surveillance of antimicrobial resistance in Europe – Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). Stockholm: ECDC.
- Liu YY, Wang Y, Walsh TR, Ling-Xian Yi, Rong Zhang, James Spencer,
et al . 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect. Dis. 16 : 161-168. - Poirel L, Jayol A, Nordmann P. 2017. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes.
Clin. Microbiol. Rev. 30 : 557-596. - Mohamed Abd El-Gawad El-Sayed Ahmed, Lan-Lan Zhong, Cong Shen, Yongqiang Yang, Yohei Doi, Guo-Bao Tian. 2020. Colistin and its role in the Era of antibiotic resistance: an extended review (2000-2019).
Emerg. Microbes Infect. 9 : 868-885. - Li XY, Liang LN, Zhang WJ,
et al . 2024. Mechanism ofMingjing granules in treating wet age-related macular degeneration based on network pharmacology and experimental verification.World J. Tradit. Chin. Med. 10 : 22-32.