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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 3  |  Page : 360-366

Determination of In vitro Antimicrobial activity of copper on the clinical isolates of Acinetobacter spp


Department of Microbiology, Stamford University Bangladesh, Dhaka, Bangladesh

Date of Submission24-May-2022
Date of Decision29-Jun-2022
Date of Acceptance20-Jul-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Md Shahidul Kabir
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217
Bangladesh
Nila Begum
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217
Bangladesh
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_129_22

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  Abstract 


Background: Acinetobacter spp., emerging pathogens equipped with the competence to establish multitudinous severe infections in immunocompromised hosts, are grievous threats to human health. To tackle the enormous burden of disease caused by Acinetobacter spp., the headlong discovery and the advancement of novel therapies are of the essence at this juncture. The present study attempted to determine the antimicrobial effects of copper on the clinical isolates of Acinetobacter spp. (Iso-03 and Iso-04). Methods: The potential deployment of copper-based antibacterial strategies against Acinetobacter spp. was assessed by exposing the isolates to the increasing concentrations of CuSO4 (from 2.5 to 1.5 mM) in liquid culture (M9 minimal medium) for 6 h and also through the exposure of them on solid metal surfaces (stainless steel and copper coupons) for 75 min, wherein the copper sensitivity and resistance of the clinical isolates of Acinetobacter spp. were determined. Results: There was no interference with the growth of the isolates at the low concentrations, whereas the bacterial growth was affected by the high concentrations of CuSO4 at different levels. During the exposure on the solid metal coupons, no loss of viability of isolates was observed on stainless steel, however, the rapid death of isolates was discernible on copper surface, leading to a dramatic decrease in the number of colony-forming units (CFUs), eventually to the limit of detection (3 CFUs per coupon). Conclusion: This study substantiated that copper possesses antimicrobial properties which can be deployed in novel therapies for the prevention of the infections caused by Acinetobacter spp. and other emerging pathogens. Further studies on the sensitivity and resistance of Acinetobacter spp. to copper at the molecular genetics levels can open the door to better exploitation of this metal for the inhibition of the vigorous growth of drug-resistant Acinetobacter spp.

Keywords: Acinetobacter spp., copper sensitivity and resistance, metal surface


How to cite this article:
Begum N, Kabir MS. Determination of In vitro Antimicrobial activity of copper on the clinical isolates of Acinetobacter spp. Biomed Biotechnol Res J 2022;6:360-6

How to cite this URL:
Begum N, Kabir MS. Determination of In vitro Antimicrobial activity of copper on the clinical isolates of Acinetobacter spp. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Dec 8];6:360-6. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/360/356142




  Introduction Top


Acinetobacter spp., opportunistic human pathogens, have emerged globally with the dreadful aptness for establishing a multitude of life-threatening infections, especially in susceptible individuals with compromised immune system.[1] Through the modification in their metabolism and nutritional needs, they obtained the potential to withstand the hostile environments with their downright ability to give rise to numerous types of nosocomial infections, including pneumonia, urinary tract infections, bacteremia, skin and soft tissue infections, osteomyelitis, even meningitis in immunocompromised patients, and mortality in intensive care unit patients.[2] Acinetobacter spp. are becoming a grave threat to health-care systems on account of their attainment of antibiotic resistance genes (AdeA, AdeB, AdeC, and New Delhi-metallo-beta-lactamase-1) with the eventuality of an intolerable disease burden around the globe.[3] The Centers for Disease Control and Prevention classified Acinetobacter spp. as critical threat in the light of the high prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.[4] Being known as opportunistic infections, Acinetobacter spp. infections are highly prevalent in hospital settings, however, healthy individuals are mostly not at a high risk of such infections.[2] Transmission of Acinetobacter spp. can occur through direct contact with surfaces, objects, and the skin of contaminated people.[5] Importation of Acinetobacter spp. occurs through various surfaces and subsequent presence in hospitals.[6] Studies showed that ventilator-associated pneumonia cases are caused by Acinetobacter spp.[7] Besides, case studies were documented in countries around the world, including Australia,[8] Brazil,[9] Bangladesh,[3],[10] China,[11] Germany,[12] India,[13] South Korea,[14] the United Kingdom,[15] the United States,[16] and so on.

The World Health Organization paid heed to Acinetobacter spp. as the top critical pathogens based on the urgency of novel antibiotics against them.[17] The diseases caused by Acinetobacter spp. are yet to become rather complicated, possibly resulting in higher mortality rate by reason of the lack of currently available efficacious treatments against the antibiotic resistance strains.[18],[19] A worldwide upsurge in MDR, extensively drug resistance (EDR), and pandrug resistance strains of Acinetobacter spp. is badly complicating the current treatment options which emphasizes the urgent need for novel therapeutics.[18],[19] However, most antibiotics target vital functions for bacterial growth and survival for which metal homeostasis is an essential process and that could be used as potential new antibiotic targets. Despite the importance of copper for cellular function, e.g., for redox balance, and as an enzyme cofactor, copper ions show toxicity at high concentrations, for instance, these ions take part in Fenton chemistry for the formation of hydroxyl radicals that cause reaction and damage to essential biomolecules, but categorical bioreporter can play an important role in detecting copper in cells.[20],[21] Through the adhesion to sulfur and displacing iron, copper ions damage iron–sulfur cluster proteins.[22] The studies in Escherichia coli and Salmonella spp. achieved an incredible outcome, wherein as a consequence of placing the bacteria on copper surfaces, the outer membrane integrity was compromised and hydroxyl radicals were produced, respiration was inhibited, and DNA was degraded.[23] Besides, copper appeared to be employed by macrophages and neutrophils for its antibacterial activity within the host.[24],[25] In our study, we attempted to determine the copper sensitivity and resistance of Acinetobacter spp. with the aim to better understand the copper-based antimicrobial strategies against Acinetobacter spp.


  Materials and Methods Top


Sample collection and bacterial isolates

Clinical bacterial samples were collected in thermostable box from one of the tertiary-level hospitals in Dhaka, Bangladesh. The pure cultures were obtained in the Microbiology Research Laboratory of Stamford University, Bangladesh.

Biochemical identification

For the identification of bacterial isolates, all necessary and available biochemical tests were meticulously conducted, namely catalase test, oxidase test, indole production test, citrate utilization test, motility indole urea test, methyl red test, and Voges-Proskauer test.[26]

Media preparation and growth conditions

Bacterial isolates were grown overnight on lysogeny broth (Thermo Scientific) agar (1.5%) plates at 37°C. M9 minimal medium, commonly known as amino acid supplemented medium, was manually prepared and always supplemented with 0.1% beef extract. Inoculated media were incubated at 37°C with shaking at 190 rpm in water bath as liquid culture for the growth of Acinetobacter isolates. Luria Bertani broth (Thermo Scientific) supplemented with 20% glycerol was used for the maintenance of Acinetobacter spp. isolates as stock in the refrigerator at −20°C for future studies.[27]

Antibiotic sensitivity test

The antibiotic sensitivity test on Acinetobacter isolates was randomly carried out against eight available antibiotics in the laboratory such as ampicillin (10 μg), cefepime (30 μg), ceftriaxone (30 μg), imipenem (10 μg), gentamicin (10 μg), amikacin (30 μg), ceftazidime (30 μg), and tetracycline (30 μg), following the Kirby-Bauer antibiotic sensitivity test procedure.[28]

Determination of the sensitivity of Acinetobacter spp. isolates to copper in liquid culture

The assessment of the antimicrobial impact of copper on the growth of Acinetobacter spp. was determined in 10 ml of M9 minimal media containing various concentrations of CuSO4 solution ranging from low (2.5, 10, and 100 μM) to high (0.5, 1, and 1.5 mM) concentrations as well as without any CuSO4. An aliquot of each culture was serially diluted and plated at different time intervals (0, 2, 4, and 6 h) for the enumeration of growth as colony-forming unit (CFU) per ml.[27]

Determination of the sensitivity of Acinetobacter spp. isolates on metal coupons

For the purpose of this experiment, metal sheets (stainless steel and copper) were collected from a local metal shop and then cut into coupons (1 cm × 1 cm) which were later degreased by vortexing in acetone for 30 s. Prior to use, coupons were immersed in absolute ethanol, thoroughly flamed, and paced on sterile Petri dishes. A total of 5 μl of bacterial culture was used for spotting onto metal coupons and for dilution and plating to enumerate the CFUs, at 2.5 h (log phase), 6 h (early stationary phase), and 24 h (late stationary phase) following incubation at the room temperature for up to 75 min. The coupons spotted with 5-μl bacterial samples were also transferred to vials containing 3-ml phosphate-buffered saline (PBS) at specific times so as to allow the spotted samples to mix with the PBS by vortexing for 1 min. As a continuation of the process, the samples were serially diluted and plated for the enumeration of the surviving bacteria on each coupon.[27]

Ethical statements

The samples were collected from the research collaborative organization of Stamford University Bangladesh.


  Results Top


Biochemical identification

The identification of Acinetobacter spp. was done only up to the genus level due to the inadequate laboratory facilities.

Antibiotic sensitivity test

Acinetobacter isolates demonstrated susceptibility to seven antibiotics (cefepime [30 μg], ceftriaxone [30 μg], imipenem [10 μg], gentamicin [10 μg], amikacin [30 μg], ceftazidime [30 μg], and tetracycline [30 μg]) out of eight used in this study while both of them were resistant to ampicillin (10 μg) [Table 1].[28]
Table 1: Antibiotic sensitivity test on Acinetobacter isolates

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Copper sensitivity of Acinetobacter spp. isolates in liquid culture

At low concentrations (2.5, 10, and 100 mM), copper had no effect on the growth of Acinetobacter isolates [Figure 1] and [Figure 2]. In case of high concentrations (0.5, 100, and 1.5 mM) of CuSO4, the isolates were able to grow in 0.5-mM CuSO4, whereas 1-mM CuSO4 affected the growth of Iso-03 for up to 2 h only [Figure 3] and [Figure 4]. In contrast, the highest concentration (1.5 mM) of CuSO4 displayed destructive effects on the growth of both isolates (Iso-03 and Iso-04) for up to 2 h, but thereafter the recurrence growth of Iso-03 was observed and comparably, there was a constant delay in the growth of Iso-04 over the test periods [Figure 3] and [Figure 4].
Figure 1: Determination of copper sensitivity of Acinetobacter Iso-03 in liquid culture supplemented with low micromolar concentrations (2.5, 10, and 100 μM) of CuSO4. CFU: Colony-forming units

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Figure 2: Determination of copper sensitivity of Acinetobacter Iso-04 in liquid culture supplemented with low micromolar concentrations (2.5, 10, and 100 μM) of CuSO4. CFU: Colony-forming units

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Figure 3: Determination of copper sensitivity of Acinetobacter Iso-03 in liquid culture supplemented with high millimolar concentrations (0.5, 1, and 1.5 mM) of CuSO4. CFU: Colony-forming units

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Figure 4: Determination of copper sensitivity of Acinetobacter Iso-04 in liquid culture supplemented with high millimolar concentrations (0.5, 1, and 1.5 mM) of CuSO4. CFU: Colony-forming units

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Survival of Acinetobacter isolates on metal surfaces

Based on the deployment of copper as an environmental decontaminant in the latest studies, this experiment was conducted to determine the survival of Acinetobacter isolates (Iso-03 and Iso-4) on metal surfaces. Coupons of copper and stainless steel (negative control) were incubated with isolates grown to log phase (2.5 h) in order to examine the level of survival on copper surfaces. Concentration of the surviving bacteria was determined at 15-min intervals. Expectedly, both of the isolates expressed no loss of viability on stainless steel over the 75-min test periods regardless of the growth phase of the cells [Figure 5] and [Figure 6]. Conversely, rapid killing of Iso-03 and Iso-04 was found on the copper surfaces [Figure 7] and [Figure 8], and notably, a drastic decrease in the number of CFUs was observed for both Iso-03 and Iso-04 at log phase in particular [Figure 7] and [Figure 8]. The number of CFUs reached the limit of detection (3 CFUs per coupon of copper) in 60 min in log phase for Iso-04, whereas for Iso-04, it was at early stationary phase [Figure 7] and [Figure 8]. The comparative results that are found by the survival of Acinetobacter Iso-03 and Iso-04 on copper and stainless steels varied largely because of the dramatic killing of bacteria on copper surfaces but no loss of viability of bacteria on steel surfaces [Figure 9] and [Figure 10]. According to this study, copper sensitivity was comparatively intense in the log phase than that of the early stationary and the stationary phases. On the contrary, there was no considerable change in the viability of any of the strains used in this investigation on the surfaces of the stainless steel in different growth phases.
Figure 5: Survival of Acinetobacter Iso-03 on stainless steel coupons with the bacterial sample taken from log phase (2.5 h), early stationary phase (6 h), and stationary phase (24 h) cultures. CFU: Colony-forming units

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Figure 6: Survival of Acinetobacter Iso-04 on stainless steel coupons with the bacterial sample taken from log phase (2.5 h), early stationary phase (6 h), and stationary phase (24 h) cultures (The error bars are present in each graph, but they are too small to be seen). CFU: Colony-forming units

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Figure 7: Survival of Acinetobacter Iso-03 on copper coupons with the bacterial sample taken from log phase (2.5 h), early stationary phase (6 h), and stationary phase (24 h) cultures. CFU: Colony-forming units

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Figure 8: Survival of Acinetobacter Iso-04 on copper coupons with the bacterial sample taken from log phase (2.5 h), early stationary phase (6 h), and stationary phase (24 h) cultures. CFU: Colony-forming units

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Figure 9: Survival of Acinetobacter Iso-03 on copper (3 CFUs per coupon) and stainless steel surfaces. CFU: Colony-forming units

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Figure 10: Survival of Acinetobacter Iso-04 on copper (3 CFUs per coupon) and stainless steel surfaces. CFU: Colony-forming units

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  Discussion Top


Emergence of Acinetobacter spp. as highly perilous pathogens has become a mounting concern, especially for the immunocompromised hosts due to their association with nosocomial infections. Various infections caused by drug-resistant Acinetobacter spp. are alarmingly on the rise. Hence, novel therapeutics with the discovery and development of rational treatment options to fight against the infections associated with Acinetobacter spp. are essential at this point.

Although copper is an integral nutrient, it possesses toxicity at high concentrations.[20] In view of antimicrobial properties, the utilization of copper can favor the development of therapeutics against drug-resistant pathogens. A published study[27] that investigated the copper resistance level in Acinetobacter baumannii in liquid culture supplemented with 100-μM to 1.5-mM CuSO4 revealed that the more resistant Acinetobacter baumannii strains grew similarly to control cultures in up to 500-μM CuSO4, but the minimal inhibition of the growth of these strains was observed in 1-mM CuSO4. On the contrary, 1.5-mM copper delayed growth. However, no cell death was noticed.

Considering the antimicrobial effects of copper on Acinetobacter spp., our experiment was performed to determine copper sensitivity to the clinical isolates of Acinetobacter spp., therein we looked into M9 minimal media (supplemented with 0.1% beef extract) containing low (2.5, 10, and 100 μM) to high (0.5, 1, and 1.5 mM) concentrations CuSO4 along with zero concentration for comparative assessment for 6 h. In our study, no interference of copper with the growth of the bacterial isolates was observed at the low concentrations (2.5, 10, and 100 mM) [Figure 1] and [Figure 2]. On the other hand, at the high concentrations, the growth of the isolates occurred in 0.5-mM CuSO4, whereas the inhibition of the growth of Iso-03 by 1-mM CuSO4 was noticed only for up to 2 h [Figure 3] and [Figure 4]. On the contrary, the highest concentration (1.5 mM) of CuSO4 showed calamitous effects on the growth of both isolates (Iso-03 and Iso-04) for up to 2 h, but Iso-03 regained the potency to grow afterward, and a dramatically constant delay in the growth of Iso-04 was observed later over the test periods. Several published studies revealed the antimicrobial effects of copper-containing metal coupons on Acinetobacter spp. and other MDR pathogens.[27],[29],[30] In our study, we also aimed to examine the survival of Acinetobacter isolates on metal surfaces (stainless steel and copper in the form of coupons) after the exposure for 75 min. There was no loss of viability of isolates on stainless steel [Figure 5] and [Figure 6], but the rapid death of isolates took place on copper surface with the copper sensitivity at the peak in log phase, and there was a dramatic decrease in the number of CFUs which eventually reached the limit of detection (3 CFUs per coupon) [Figure 7] and [Figure 8].

In addition to the potential use of copper in novel therapeutics, copper is also being examined to be incorporated in environmental decontamination. Recent clinical trials revealed that if getting high touch surfaces in hospitals unexposed with copper is possible, it will more likely to remarkably descend the bacterial load resulting in subsiding the number of nosocomial infections by more than 50%.[31],[32],[33] Several studies opened a new realm of possibilities of utilizing copper and copper alloy surfaces as passive antimicrobial sanitizing agents.[34] A number of recent studies[35] proved that copper ions released by copper alloy surfaces are the vital reasons behind the contact-based killing of microorganisms. Numerous laboratory investigations culminated with the demonstrations of efficient and rapid killing of bacteria, fungi, and viruses upon exposure to surfaces consisting of copper or copper-containing alloys but not stainless steel.[34] Several studies revealed that copper is incorporated by the human immune system in a number of ways to combat infections, and in macrophage phagosomes, the concentrations of copper were measured upward of 500 μM.[36],[37] Some investigations are launched in the techniques of increasing the concentrations of copper in the body to improve its antimicrobial effects.[38],[39] This suggests that future discoveries of the different mechanisms of Acinetobacter spp. and other infectious bacteria in the presence of copper will lead to the way of the advancement of new therapeutic treatments against bacterial infections.


  Conclusion Top


The analytical results showed the antimicrobial effects of copper on two experimentally used clinical isolates of Acinetobacter spp. through the demonstration of their copper sensitivity both in liquid and on solid metal surface in vitro. However, the future identification of resistance and virulence mechanisms of Acinetobacter spp. and other drug resistance pathogens at the molecular genetics levels along with their reactions toward copper can pave the way for the discovery as well as advancement of efficacious and novel copper-based therapeutics for the emerging and untreatable infections.

Limitations of the study

This study was conducted only on two clinical isolates of Acinetobacter spp. due to the limited access to such pathogenic bacterial samples. The virulence of the bacteria was not examined in this experiment. The concentration of copper ions in medium exposed to metal surfaces was not measured. Performing the research at molecular genetics levels, such as RNA extraction, c-DNA sequence, quantitative reverse transcription–polymerase chain reaction, identification of putative copper-related genes, and copper resistance systems in Acinetobacter spp. was quite unattainable by reason of the substantial inadequacy of the laboratory facilities. For the same reason, the current study failed to identify the specific protein functions associated with copper resistance which could be incorporated in the discovery of novel antibiotics by targeting those functions, resulting in the accentuation of the antimicrobial properties of copper.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
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